CORD-19:ee0a7632371a08727d41e55aad7d621daeceef7d JSONTXT 8 Projects

Annnotations TAB TSV DIC JSON TextAE

Id Subject Object Predicate Lexical cue
TextSentencer_T1 0-13 Sentence denotes GENETICS G01.
TextSentencer_T2 14-28 Sentence denotes WITHDRAWN G02.
TextSentencer_T3 29-37 Sentence denotes ClinVar:
TextSentencer_T4 38-118 Sentence denotes A Centralized Database for Interpretations of Both Germline and Somatic Variants
TextSentencer_T5 120-128 Sentence denotes Abstract
TextSentencer_T6 131-172 Sentence denotes solid organ post-transplant surveillance.
TextSentencer_T7 173-437 Sentence denotes We have validated a method based on targeted amplification and next-generation sequencing (NGS) that does not require genotyping of the donor or recipient and is applicable to all single solid organ transplants except those from an identical twin donor (AlloSure).
TextSentencer_T8 438-543 Sentence denotes Selection criteria for the 266 targeted SNPs included high minor allele frequency and low fixation index.
TextSentencer_T9 544-730 Sentence denotes Quantification of dd-cfDNA for this method is based on the degree of genetic similarity between the donor and recipient, the extent of which differs based on their familial relationship.
TextSentencer_T10 731-806 Sentence denotes Other factors, such as HLA matching, may also inform on genetic similarity.
TextSentencer_T11 807-979 Sentence denotes Here we evaluated the AlloSure SNP allele distribution in kidney transplant patients with no mismatches at the HLA A, B, and DR loci as determined by low resolution typing.
TextSentencer_T12 980-988 Sentence denotes Methods:
TextSentencer_T13 989-1270 Sentence denotes Kidney transplant recipients with unrelated donors were selected for 0, 5, or 6 mismatches at the HLA A, B, and DR loci. cfDNA purified from plasma was amplified and sequenced to determine the percent dd-cfDNA and also the relative contribution of each of two alleles for each SNP.
TextSentencer_T14 1271-1493 Sentence denotes The number of SNPs determined to be homozygous for the same allele in both donor and recipient was calculated by determining the number of SNPs with only one allele present in the plasma in a set of high %dd-cfDNA samples.
TextSentencer_T15 1494-1564 Sentence denotes The results were compared to expected proportions in unrelated donors.
TextSentencer_T16 1565-1573 Sentence denotes Results:
TextSentencer_T17 1574-1750 Sentence denotes Of SNPs that are homozygous in the recipients, the average number of SNPs expected to be homozygous for the same allele in the donor is 25% based on the SNP selection criteria.
TextSentencer_T18 1751-1925 Sentence denotes A set of 97 samples was identified through which the number of 'homozygous-same' SNPs was not impacted by low numbers of donor molecules in the plasma (above 0.65% dd-cfDNA).
TextSentencer_T19 1926-2116 Sentence denotes For unrelated donor-recipient pairs with 5 or 6 HLA mismatches, the mean percentage of homo-same SNPs is 31%, with a 90% contained within a range of 20% to 43% (n=94 samples, 61 recipients).
TextSentencer_T20 2117-2256 Sentence denotes In the 3 unrelated, 0 mismatch donor-recipient pairs, the homozygous recipient SNPs were also homozygous in the donor at 24%, 25%, and 26%.
TextSentencer_T21 2257-2377 Sentence denotes Conclusions: SNPs used in the AlloSure assay were selected based on data indicating near random population distribution.
TextSentencer_T22 2378-2488 Sentence denotes To optimize successful transplant outcomes, kidney transplants are often matched at the HLA A, B, and DR loci.
TextSentencer_T23 2489-2567 Sentence denotes Only low resolution typing is available due to limited ischemia time required.
TextSentencer_T24 2568-2725 Sentence denotes Patients lacking low resolution mismatches (0 of 6 potential mismatches) had the expected number of donor homozygous alleles matching an unrelated recipient.
TextSentencer_T25 2726-2828 Sentence denotes Therefore, the AlloSure assay is not expected to be impacted by the level of low resolution HLA match.
TextSentencer_T26 2829-3020 Sentence denotes An even smaller number of these drug labels include recommendations for PGx testing, that can be required or recommended prior to drug prescription; or associated therapeutic recommendations.
TextSentencer_T27 3021-3201 Sentence denotes NIH Medical Genetics Summaries (MGS) provide free, comprehensive reviews on the currently available information about genetic impact on drug responses for use in clinical settings.
TextSentencer_T28 3202-3297 Sentence denotes Methods: MGS is available on the NCBI Bookshelf located at www.ncbi.nlm.nih.gov/books/NBK61999.
TextSentencer_T29 3298-3385 Sentence denotes The articles are prepared in a standardized format and include a formal review process.
TextSentencer_T30 3386-3624 Sentence denotes MGS articles combine actionable, evidence-based, and often nonoverlapping information from authoritative sources, including professional societies such as the Clinical Pharmacogenetics Implementation Consortium (CPIC) and FDA drug labels.
TextSentencer_T31 3625-3699 Sentence denotes In 2016, there are 23 MGS that review genetic variants and drug responses.
TextSentencer_T32 3700-3886 Sentence denotes Additionally, NCBI features and links MGS content in related resources, such as the NIH Genetic Testing Registry (GTR; www.ncbi.nlm.nih.gov/gtr) and MedGen (www.ncbi.nlm.nih.gov/medgen).
TextSentencer_T33 3887-4025 Sentence denotes GTR currently lists over 35,000 clinical and research genetic tests, of which over 300 are PGx tests for approximately 161 drug responses.
TextSentencer_T34 4026-4121 Sentence denotes Results: MGS contain recommendations on genetic testing, sometimes not included in drug labels.
TextSentencer_T35 4122-4262 Sentence denotes For example, the warfarin drug label recommends initial anti-coagulation dose ranges in a dosing table based on CYP2C9 and VKORC1 genotypes.
TextSentencer_T36 4263-4445 Sentence denotes However, MGS stress the importance of using a specific online PGx algorithm that takes into account both genetic and non-genetic factors to give a more accurate prediction of dosing.
TextSentencer_T37 4446-4501 Sentence denotes MGS exposes discrepancies in available PGx information.
TextSentencer_T38 4502-4582 Sentence denotes For example, tamoxifen is used in the treatment and prevention of breast cancer.
TextSentencer_T39 4583-4660 Sentence denotes At this time, the FDA drug label does not discuss genetic testing for CYP2D6.
TextSentencer_T40 4661-4825 Sentence denotes The National Comprehensive Cancer Network (NCCN) does not recommend CYP2D6 testing nor do the 2010 guidelines from the American Society of Clinical Oncology (ASCO).
TextSentencer_T41 4826-4957 Sentence denotes In contrast, the Dutch Pharmacogenetics Working Group (DPWG) makes recommendations for tamoxifen therapy based on CYP2D6 genotypes.
TextSentencer_T42 4958-5185 Sentence denotes MGS incorporates their recommendation to consider using aromatase inhibitors for postmenopausal women (for both poor/intermediate metabolizers), and that intermediate metabolizers avoid the concomitant use of CYP2D6 inhibitors.
TextSentencer_T43 5186-5298 Sentence denotes Conclusions: MGS are filling a critical knowledge gap in PGx testing and associated therapeutic recommendations.
TextSentencer_T44 5299-5463 Sentence denotes As PGx testing expands, MGS will also continue to grow and provide the evidence-based information healthcare providers need in this ever-evolving field of medicine.
TextSentencer_T45 5464-5516 Sentence denotes impact of individual mutations on MMR gene function.
TextSentencer_T46 5517-5525 Sentence denotes Methods:
TextSentencer_T47 5526-5685 Sentence denotes We evaluated somatic and germline mutations in 87 tumors with IHC staining patterns suggestive of Lynch syndrome tested using the clinical ColoSeq-Tumor assay.
TextSentencer_T48 5686-5851 Sentence denotes Paired tumornormal samples were used to identify somatic variants and germline variants, and determine normalized variant read allele ratios (VARs) across the tumor.
TextSentencer_T49 5852-6014 Sentence denotes VARs were used to differentiate between putative driver mutations, those likely to be responsible for IHC patterns, and putative passenger mutations in MMR genes.
TextSentencer_T50 6015-6158 Sentence denotes We evaluated distributions of VARs normalized to tumor percentage to determine Bayes factors for pathogenicity of variants identified in tumor.
TextSentencer_T51 6159-6293 Sentence denotes We used intronic mutations in MMR genes to define passenger mutation characteristics and compared these to known pathogenic mutations.
TextSentencer_T52 6294-6302 Sentence denotes Results:
TextSentencer_T53 6303-6419 Sentence denotes Comparing normalized VARs, 35% of normalized tumor reads was an optimal allele fraction cutoff for driver mutations.
TextSentencer_T54 6420-6522 Sentence denotes We found the Bayes factor for pathogenicity for nonsynonymous variants seen above this cutoff was 4.5.
TextSentencer_T55 6523-6640 Sentence denotes Somatic tumor mutations are independent events, so Bayes factors may be combined from events seen in multiple tumors.
TextSentencer_T56 6641-6653 Sentence denotes Conclusions:
TextSentencer_T57 6654-6882 Sentence denotes Evaluation of the mutational spectrum of MMR genes in tumor samples with loss of MMR protein expression by IHC from patients with negative germline results has been shown to alleviate concerns of Lynch syndrome in many patients.
TextSentencer_T58 6883-7135 Sentence denotes Interrogation of the observed mutations in MMR genes in this patient population may aid in the identification and characterization of mutations that lead to absence if MMR expression by IHC and may reduce the need for high throughput functional assays.
TextSentencer_T59 7136-7138 Sentence denotes Z.
TextSentencer_T60 7139-7148 Sentence denotes Fan, C.A.
TextSentencer_T61 7149-7161 Sentence denotes Stolle, J.R.
TextSentencer_T62 7162-7173 Sentence denotes Murrell, A.
TextSentencer_T63 7174-7185 Sentence denotes Santani, M.
TextSentencer_T64 7186-7248 Sentence denotes Luo The Children's Hospital of Philadelphia, Philadelphia, PA.
TextSentencer_T65 7249-7262 Sentence denotes Introduction:
TextSentencer_T66 7263-7477 Sentence denotes Germline inactivating variants in the VHL gene cause dominantly inherited von Hippel-Lindau syndrome characterized by hemangioblastomas, renal cell carcinoma, pancreatic cysts, pheochromocytomas and paragangliomas.
TextSentencer_T67 7478-7569 Sentence denotes Approximately 72% of the pathogenic variants are sequence variants and 28% large deletions.
TextSentencer_T68 7570-7645 Sentence denotes Nearly all individuals with a VHL mutation will develop symptoms by age 65.
TextSentencer_T69 7646-7812 Sentence denotes Due to age dependent penetrance, variable expression, incomplete family segregation information and somatic occurrence, VHL variant classification can be challenging.
TextSentencer_T70 7813-7951 Sentence denotes The purpose of this study is to use newly established guidelines to reclassify variants detected by our clinical laboratory over 14 years.
TextSentencer_T71 7952-8053 Sentence denotes This extensive dataset is used to update the mutation spectrum and aid future VHL variant assessment.
TextSentencer_T72 8054-8062 Sentence denotes Methods:
TextSentencer_T73 8063-8176 Sentence denotes A retrospective review of 3,205 cases submitted to our laboratory for VHL gene analysis since 2002 was conducted.
TextSentencer_T74 8177-8394 Sentence denotes These cases were previously tested by Sanger sequencing for sequence variants and Southern blot analysis, relative quantitative PCR or multiplex ligation-dependent probe amplification for large deletions/duplications.
TextSentencer_T75 8395-8530 Sentence denotes Variants were reassessed using our laboratory's criteria modified from the American College of Medical Genetics and Genomics guideline.
TextSentencer_T76 8531-8723 Sentence denotes Resources used for reclassifications included published literature, databases, and family segregation information focusing on the collection of new evidence that emerged since last assessment.
TextSentencer_T77 8724-8732 Sentence denotes Results:
TextSentencer_T78 8733-8863 Sentence denotes Out of 3,205 cases, 861 had a test finding with 21.6% having partial or whole VHL gene deletions and 78.4% with sequence variants.
TextSentencer_T79 8864-9049 Sentence denotes Reclassification of 186 unique sequence variants yielded 62.3% pathogenic, 21% likely pathogenic, 13% variant of uncertain significance (VOUS), 3.2% likely benign and 0.5% benign calls.
TextSentencer_T80 9050-9250 Sentence denotes Of these, 42 novel variants were identified in our patients that have not been published or reported in public databases and an additional 14 germline variants were previously reported in COSMIC only.
TextSentencer_T81 9251-9374 Sentence denotes Classification of novel variants yielded 55.4% pathogenic, 8.9% likely pathogenic, 26.8% VOUS and 8.9% likely benign calls.
TextSentencer_T82 9375-9548 Sentence denotes Among a total of 155 pathogenic and likely pathogenic variants, 47.1% were missense, 31.6% frameshift, 13.5% nonsense, 5.2% splicing, and 2.6% in-frame deletions/insertions.
TextSentencer_T83 9549-9561 Sentence denotes Conclusions:
TextSentencer_T84 9562-9688 Sentence denotes This study utilized our laboratory's database to reclassify VHL variants using established guidelines and updated information.
TextSentencer_T85 9689-9835 Sentence denotes This knowledge database can be used for future classification of VHL variants, which will be of benefit to clinicians who care for these families.
TextSentencer_T86 9836-9913 Sentence denotes Therefore this information was submitted to ClinVar for public dissemination.
TextSentencer_T87 9914-10026 Sentence denotes The experience gained from this study can also help variant classification in other cancer-predisposition genes.
TextSentencer_T88 10027-10029 Sentence denotes T.
TextSentencer_T89 10030-10049 Sentence denotes Spenlinhauer 1 , C.
TextSentencer_T90 10050-10066 Sentence denotes Zimmerman 2 , J.
TextSentencer_T91 10067-10079 Sentence denotes Stone 2 , R.
TextSentencer_T92 10080-10094 Sentence denotes Winegar 2 , S.
TextSentencer_T93 10095-10109 Sentence denotes Nesbitt 1 , S.
TextSentencer_T94 10110-10121 Sentence denotes Kaul 1 , J.
TextSentencer_T95 10122-10139 Sentence denotes Keierleber 1 , T.
TextSentencer_T96 10140-10155 Sentence denotes Laughlin 3 , J.
TextSentencer_T97 10156-10171 Sentence denotes Starbuck 4 , C.
TextSentencer_T98 10172-10186 Sentence denotes Rundell 1 , J.
TextSentencer_T99 10187-10361 Sentence denotes Gordon 1 1 Maine Molecular Quality Controls Inc, Saco, ME; 2 MRIGlobal, Palm Bay, FL 3 University of Rochester Medical Center, Rochester NY; 4 Cleveland Clinic, Cleveland OH.
TextSentencer_T100 10362-10375 Sentence denotes Introduction:
TextSentencer_T101 10376-10529 Sentence denotes Next-generation sequencing (NGS), provides an opportunity for clinicians to identify multiple mutations in a highly efficient and high-throughput manner.
TextSentencer_T102 10530-10651 Sentence denotes However, this technology requires a complex multistep procedure which presents many challenges for clinical laboratories.
TextSentencer_T103 10652-10888 Sentence denotes Each application and platform is unique and involves challenging, time-consuming, and expensive optimization and validation of 3 complex components -the sequencing platform, the specific assay/test panel and the bioinformatics analysis.
TextSentencer_T104 10889-11108 Sentence denotes Here we demonstrate a practical solution for monitoring the identification of many mutations using a multiplex, synthetic control panel created to monitor the analytical performance of molecular cystic fibrosis testing.
TextSentencer_T105 11109-11347 Sentence denotes The multiple insertions, deletions, and homopolymers of varying lengths and composition in the control make it potentially useful for monitoring the ability of the NGS systems to correctly identify variants found in genes other than CFTR.
TextSentencer_T106 11348-11356 Sentence denotes Methods:
TextSentencer_T107 11357-11566 Sentence denotes Synthetic DNA composed of all 27 CFTR gene exons plus intronic borders containing CF associated variants were designed in silco, ligated into MMQCI vectors and transformed to create stable frozen clone stocks.
TextSentencer_T108 11567-11823 Sentence denotes Multiple plasmids were created with various mutations to represent 186 CFTR variants and mixed to create either heterozygous or homozygous alleles for each variant and diluted to have equivalent copy numbers of the targeted gene as extracted human samples.
TextSentencer_T109 11824-12021 Sentence denotes The plasmid mixes were suspended in buffers and stabilizers, with and without proprietary matrix and tested either as an extractable (with matrix) or non-extractable (without matrix) control panel.
TextSentencer_T110 12022-12112 Sentence denotes Initial studies were performed to determine optimal concentrations for subsequent testing.
TextSentencer_T111 12113-12268 Sentence denotes The extractable samples were extracted by various methods (QiaAmp DNA Blood Mini kit, QiASymphony, and SPRI-TE) and processed the same as a patient sample.
TextSentencer_T112 12269-12383 Sentence denotes The non-extractable panel was tested using 10ul of each sample added to 5uL of Oligo pool and 35uL, of HYB buffer.
TextSentencer_T113 12384-12457 Sentence denotes All samples were tested with the Illumina's MiSeqDX CF 139-Variant Assay.
TextSentencer_T114 12458-12466 Sentence denotes Results:
TextSentencer_T115 12467-12530 Sentence denotes All samples resulted in a valid run with a call rate of 99.26%.
TextSentencer_T116 12531-12668 Sentence denotes The extractable controls, resulted in 99.6% concordance with expected calls across multiple testing sites and various extraction methods.
TextSentencer_T117 12669-12747 Sentence denotes The non-extractable controls resulted in 100% concordance with expected calls.
TextSentencer_T118 12748-12900 Sentence denotes When compared to human gDNA and Coriell samples, relative coverage of the target regions appear similar and generated roughly 1,000X to 1,700X coverage.
TextSentencer_T119 12901-12912 Sentence denotes Conclusion:
TextSentencer_T120 12913-13129 Sentence denotes A highly multiplexed, synthetic and well-characterized molecular CFTR QC reference material can be used as a reliable control to monitor highly complex NGS panels for verification, validation or as a routine control.
TextSentencer_T121 13130-13349 Sentence denotes jmd.amjpathol.org ■ The Journal of Molecular Diagnostics quality nucleic acid with optimal yield, with few steps for molecular applications is an asset and COPAN kit is for nucleic acid preservation for genetic testing.
TextSentencer_T122 13350-13429 Sentence denotes COPAN Kit is CE marked and currently available in the US for research use only.
TextSentencer_T123 13430-13639 Sentence denotes The objective of this study was to validate the performance of the COPAN Kit, an hDNA-free FLOQSwabs and 1 ml tube of eNAT medium, for the collection and storage of buccal specimens for human DNA preservation.
TextSentencer_T124 13640-13796 Sentence denotes Methods: in this study buccal swab samples were collected in duplicate, one using the COPAN Kit and another with Epicentre Catch-All-Sample-Collection-Swab.
TextSentencer_T125 13797-14062 Sentence denotes The entire Epicenter swab and only a portion of 400ul eNAT medium were used to extract genomic DNA using the Thermo Fisher MagMAX DNA Multi-Sample Ultra Kit and the Roche MagNA Pure; DNA yield from both devices was recorded using Thermo Fisher Qubit dsDNA HS Assay.
TextSentencer_T126 14063-14295 Sentence denotes Genomic-DNA from both systems was tested on the current comprehensive panel at Patients Choice Laboratories with Applied Biosystems 7900HT-Fast-Real-Time-PCR System for pharmacogenetics, coagulation factors mutations and genotyping.
TextSentencer_T127 14296-14389 Sentence denotes Data were analyzed with TaqMan Genotyper Software and results for both systems were compared.
TextSentencer_T128 14390-14398 Sentence denotes Results:
TextSentencer_T129 14399-14548 Sentence denotes Optimal quality and concentration of genomic-DNA was obtained from 400ul of eNAT compared to the Epicenter entire swab with the Qubit dsDNA HS Assay.
TextSentencer_T130 14549-14694 Sentence denotes Results generated with eNAT-extracted genomic-DNA showed good amplification and confident calls, all 47 assays of 17 genes showed 100% agreement.
TextSentencer_T131 14695-14869 Sentence denotes Conclusions: COPAN hDNA-free FLOQSwabs and 1ml eNAT medium kit is a convenient device for buccal sample collection, storage, preservation and transportation of nucleic acids.
TextSentencer_T132 14870-14998 Sentence denotes Direct lysis in the eNAT medium inactivates bacterial proliferation, reduces sample processing steps and facilitates automation.
TextSentencer_T133 14999-15100 Sentence denotes The genomic-DNA yielded from a 400ul portion of eNAT medium is pure and adequate for genetic testing.
TextSentencer_T134 15101-15222 Sentence denotes Moreover, the leftover eNAT medium can be stored for additionalrepeat confirmatory testing, avoiding sample recollection.
TextSentencer_T135 15223-15236 Sentence denotes Introduction:
TextSentencer_T136 15237-15476 Sentence denotes For the past three years our laboratory has participated in a study focused on carrier screening in a pre-conception population using whole genome sequencing (WGS), as part of the Clinical Sequencing Exploratory Research (CSER) Consortium.
TextSentencer_T137 15477-15485 Sentence denotes Methods:
TextSentencer_T138 15486-15758 Sentence denotes Approximately 720 genes/condition pairs for autosomal recessive (AR) and X-linked (XL) disorders were selected for an expanded carrier screening panel and were divided into categories of: lifespan limiting, serious, mild, unpredictable outcome, and adult-onset conditions.
TextSentencer_T139 15759-16042 Sentence denotes Participants, recruited from Kaiser Permanente Northwest (KPNW), consented to receive results for lifespanlimiting conditions but could choose whether or not to receive results for the other categories as well as additional medically actionable findings (~120 genes/condition pairs).
TextSentencer_T140 16043-16156 Sentence denotes Our protocol is sequential screening, with females tested first, and if positive, the partner is offered testing.
TextSentencer_T141 16157-16330 Sentence denotes All WGS is performed at Illumina, and sequence alignment and variant annotations were performed at the University of Washington using the SeattleSeq bioinformatics pipeline.
TextSentencer_T142 16331-16429 Sentence denotes Our laboratory filtered, classified, and confirmed carrier variants, and issued a clinical report.
TextSentencer_T143 16430-16497 Sentence denotes We use the ACMG and AMP recommendations for variant classification.
TextSentencer_T144 16498-16672 Sentence denotes Additionally, we perform functional analysis for classification of novel splice variants and have developed an in-house algorithm for classification and confirmation of CNVs.
TextSentencer_T145 16673-16733 Sentence denotes Only pathogenic or likely pathogenic variants were reported.
TextSentencer_T146 16734-16793 Sentence denotes Both pre-and post-test genetic counseling was done at KPNW.
TextSentencer_T147 16794-16802 Sentence denotes Results:
TextSentencer_T148 16803-16939 Sentence denotes Most (~92%) of participants requested all categories of carrier disorders returned, as well as additional medically actionable findings.
TextSentencer_T149 16940-17246 Sentence denotes To date over 150 participants have had results reported with approximately 70% carrying at least one pathogenic or likely pathogenic variant (ranging up to 5 variants) in the selected set of carrier genes and ~1% to 2% having a pathogenic variant in a gene associated with a medically actionable condition.
TextSentencer_T150 17247-17443 Sentence denotes All types of variants have been detected, including copy number variants (CNVs), although the majority (>50%) are missense variants, which present the greatest challenge in variant classification.
TextSentencer_T151 17444-17456 Sentence denotes Conclusions:
TextSentencer_T152 17457-17552 Sentence denotes All variants we reported from WGS would also have been detected by exome or gene panel testing.
TextSentencer_T153 17553-17810 Sentence denotes Our experience suggests such an expanded carrier screening panel for AR and XL conditions, including rare and ultra-rare disorders, using a gene panel approach, or as additional findings in WES, may be an appropriate consideration for clinical laboratories.
TextSentencer_T154 17811-17815 Sentence denotes P.R.
TextSentencer_T155 17816-17828 Sentence denotes Reynolds, J.
TextSentencer_T156 17829-17839 Sentence denotes Abbott, P.
TextSentencer_T157 17840-17848 Sentence denotes Hauk, S.
TextSentencer_T158 17849-17862 Sentence denotes Dirscherl, M.
TextSentencer_T159 17863-17876 Sentence denotes Salfinger, R.
TextSentencer_T160 17877-17888 Sentence denotes Harbeck, E.
TextSentencer_T161 17889-17932 Sentence denotes Gelfand National Jewish Health, Denver, CO.
TextSentencer_T162 17933-17946 Sentence denotes Introduction:
TextSentencer_T163 17947-18167 Sentence denotes In a tertiary referral center with a program in immunodeficiency diagnosis and treatment, it is not uncommon to find patients with overlapping clinical and laboratory phenotypes resulting from distinct genetic mutations.
TextSentencer_T164 18168-18389 Sentence denotes A number of recent studies have associated certain immunodeficiency symptoms with small groups of genes; however, analyzing small numbers of candidate genes via Sanger sequencing has rarely uncovered a causative mutation.
TextSentencer_T165 18390-18477 Sentence denotes Therefore, a panel of 347 genes was created for analysis by next-generation sequencing.
TextSentencer_T166 18478-18590 Sentence denotes All genes have an established relationship to Primary Immune Deficiencies, including complement pathway defects.
TextSentencer_T167 18591-18599 Sentence denotes Methods:
TextSentencer_T168 18600-18723 Sentence denotes Genomic coordinates for all exons (+/-100 nucleotides) of each of the 347 genes were obtained from the UCSC Genome Browser.
TextSentencer_T169 18724-18942 Sentence denotes Coordinates were submitted to Agilent for design and synthesis of a custom QXT library kit (Sure Select Target Enrichment System), which includes Biotin-labeled RNA oligonucleotides for capturing sequences of interest.
TextSentencer_T170 18943-19005 Sentence denotes Libraries were created according to manufacturer's directions.
TextSentencer_T171 19006-19153 Sentence denotes Quality and quantity of each library was established on an Agilent Bioanalyzer, and a MiSeq (Illumina) was loaded at 12 pM total for all libraries.
TextSentencer_T172 19154-19178 Sentence denotes Typical runs yielded ca.
TextSentencer_T173 19179-19302 Sentence denotes 7 Gbase of sequence data, resulting in coverages of 50x-300x for nearly all targets, and 99% overall coverage of the panel.
TextSentencer_T174 19303-19374 Sentence denotes Sequence data was analyzed for variants by NextGENe (softgenetics.com).
TextSentencer_T175 19375-19683 Sentence denotes Variants with "Damaging" predictions via programs such as SIFT and Polyphen and low or zero frequency in 1000 genomes were investigated further by checking on frequency in the EXaC database, reports in the literature and/or OMIM of known or similar mutations, and fit of symptoms/phenotype with mutated gene.
TextSentencer_T176 19684-19692 Sentence denotes Results:
TextSentencer_T177 19693-19769 Sentence denotes A number of disease-causing mutations were identified, both known and novel.
TextSentencer_T178 19770-19885 Sentence denotes The genes containing the mutations include ATM, BTK, CARD11, CTLA4, FOXN1, FOXP3, LIG1, LIG4, NOD2, STAT1, and TAP.
TextSentencer_T179 19886-20297 Sentence denotes Examples: a novel AIRE mutation in a youngster with diabetes and family history of autoimmunity; a CARD11 mutation in a patient with severe eczema and herpes infections; a LIG4 in a newborn with T cell lymphopenia, a NOD2 mutation in a 2-year-old boy with severe arthritis in all joints, FOXP3 mutation in a 12-year-old boy with autoimmune enteropathy, and a FOXN1 mutation in an infant with T cell lymphopenia.
TextSentencer_T180 20298-20310 Sentence denotes Conclusions:
TextSentencer_T181 20311-20448 Sentence denotes With a rapid turnaround time, this panel provides an excellent adjunct to the newborn screening programs developed for statewide testing.
TextSentencer_T182 20449-20649 Sentence denotes Since laboratory testing may be indicative but not diagnostic of a PIDD, genetic testing is now becoming an essential clinical tool since it defines the diagnosis and serves to guide specific therapy.
TextSentencer_T183 20650-20663 Sentence denotes Introduction:
TextSentencer_T184 20664-20868 Sentence denotes Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is an autosomal recessive disorder that results in defective fatty acid metabolism by preventing the conversion of medium-chain fatty acids to energy.
TextSentencer_T185 20869-20938 Sentence denotes The disease is typically diagnosed during infancy or early childhood.
TextSentencer_T186 20939-21081 Sentence denotes Symptoms may include vomiting, lethargy, hypoglycemia, seizures, breathing difficulties, liver problems, brain damage, coma, and sudden death.
TextSentencer_T187 21082-21191 Sentence denotes The symptoms are commonly triggered by prolonged periods of fasting or by illnesses such as viral infections.
TextSentencer_T188 21192-21300 Sentence denotes Patients are usually identified during newborn screen while final confirmation is made by mutation analysis.
TextSentencer_T189 21301-21457 Sentence denotes The disease manifestations are managed mainly by strict avoidance of fasting, avoidance of medium chain triglycerides and carnitine supplementation in diet.
TextSentencer_T190 21458-21547 Sentence denotes The ACADM gene coding for MCAD deficiency maps to chromosome 1p band 31 and has 12 exons.
TextSentencer_T191 21548-21757 Sentence denotes The most common mutation in the ACADM gene that is found in about 80% of MCAD patients is c.985A>G (p.K329E) and the second most common mutation occurring in approximately 15% of patients is c.199T>C (p.Y67H).
TextSentencer_T192 21758-21848 Sentence denotes In this study we report a 10-month old infant girl who was positive on the newborn screen.
TextSentencer_T193 21849-21942 Sentence denotes Besides the abnormal new born screen, she does not show clinical symptoms of MCAD deficiency.
TextSentencer_T194 21943-21990 Sentence denotes The patient has been metabolically stable at 10
TextSentencer_T195 21991-22159 Sentence denotes The Journal of Molecular Diagnostics ■ jmd.amjpathol.org population, summarize the phenotypic findings, and determine the frequency of cooccurring copy number variants.
TextSentencer_T196 22160-22168 Sentence denotes Methods:
TextSentencer_T197 22169-22494 Sentence denotes Internal databases of our clinical SNPbased chromosomal microarray (CMA) testing that are used for quality assurance purposes were searched to determine the total number of these tests ordered at our institution between August 2012 and April 2016 and to determine the total number of cases with the 15q11.2 BP1-BP2 deletions.
TextSentencer_T198 22495-22649 Sentence denotes The deletions found in parents or other family members of 15q11.2 BP1-BP2 deletion probands were excluded from the final count of cases with the deletion.
TextSentencer_T199 22650-22658 Sentence denotes Results:
TextSentencer_T200 22659-22886 Sentence denotes During a forty-five month period we detected the 15q11.2 BP1-BP2 deletion in eighteen probands out of approximately 1352 total CMA tests performed, a frequency of 1.3% among patients referred for CMA testing at our institution.
TextSentencer_T201 22887-23184 Sentence denotes The primary clinical indications for study in these individuals included autism (3/18, 16.7%), epilepsy (1/18, 5 .6%), developmental delay (5/18, 27.8%), learning disabilities (2/18, 11.1%), congenital anomaly or anomalies (4/18, 22.2%), dysmorphic features (1/18, 5.6%) , and other (2/18, 11.1%).
TextSentencer_T202 23185-23318 Sentence denotes Under our filtering and reporting criteria, one or more co-occurring copy number variants were reported in 12/18 individuals (66.7%).
TextSentencer_T203 23319-23507 Sentence denotes Three of these cooccurring CNVs fulfilled our criteria for pathogenic (1) or likely pathogenic (2) calls, five were of uncertain clinical significance, and eight were deemed likely benign.
TextSentencer_T204 23508-23577 Sentence denotes One individual was noted to have 4.4% homozygosity across the genome.
TextSentencer_T205 23578-23590 Sentence denotes Conclusions:
TextSentencer_T206 23591-23724 Sentence denotes The 15q11.2 BP1-BP2 deletion was enriched in our patient populations compared to published frequencies in normal control populations.
TextSentencer_T207 23725-23809 Sentence denotes Emerging data continue to support the pathogenicity of the 15q11.2 BP1-BP2 deletion.
TextSentencer_T208 23810-23964 Sentence denotes The incomplete penetrance and phenotypic variability of this deletion may be related to the presence of additional genetic variation in affected patients.
TextSentencer_T209 23965-23967 Sentence denotes S.
TextSentencer_T210 23968-23982 Sentence denotes Lincoln 1 , M.
TextSentencer_T211 23983-23995 Sentence denotes Cline 2 , S.
TextSentencer_T212 23996-24007 Sentence denotes Yang 1 , Y.
TextSentencer_T213 24008-24024 Sentence denotes Kobayashi 1 , S.
TextSentencer_T214 24025-24038 Sentence denotes Topper 1 , D.
TextSentencer_T215 24039-24054 Sentence denotes Haussler 2 , B.
TextSentencer_T216 24055-24067 Sentence denotes Paten 2 , R.
TextSentencer_T217 24068-24162 Sentence denotes Nussbaum 1 1 Invitae, San Francisco CA; 2 University of California Santa Cruz, Santa Cruz, CA.
TextSentencer_T218 24163-24176 Sentence denotes Introduction:
TextSentencer_T219 24177-24301 Sentence denotes As increasing numbers of laboratories offer genetic tests, the potential for inconsistent variant classifications increases.
TextSentencer_T220 24302-24421 Sentence denotes Classification differences between public databases that laboratories may use have been raised as a particular concern.
TextSentencer_T221 24422-24605 Sentence denotes Although real, the clinical impact of these differences is not clear, because experienced and responsible lab directors should never simply copy classifications from public databases.
TextSentencer_T222 24606-24720 Sentence denotes Instead, they critically evaluate underlying evidence and determine classifications following rigorous guidelines.
TextSentencer_T223 24721-24953 Sentence denotes Our recent study (Lincoln et al., JMD 2015) demonstrated high (99.8%) concordance of 975 BRCA1/2 tests classified following ACMG/AMP guidelines and using only public data, compared to tests that also utilized non-public information.
TextSentencer_T224 24954-25078 Sentence denotes Here, we sought to similarly evaluate concordance of BRCA1/2 variant classifications in a larger, inter-laboratory data set.
TextSentencer_T225 25079-25087 Sentence denotes Methods:
TextSentencer_T226 25088-25236 Sentence denotes Over 5000 ClinVar submissions of classified variants from 6 established laboratories (Ambry, Counsyl, Emory, GeneDx, Invitae, and Myriad) were used.
TextSentencer_T227 25237-25304 Sentence denotes Myriad data were submitted by the Sharing Clinical Reports Project.
TextSentencer_T228 25305-25536 Sentence denotes Clinically significant differences were those that would substantially change actionability, i.e., between pathogenic (including likely pathogenic), versus VUS, benign or likely benign; otherwise results were considered concordant.
TextSentencer_T229 25537-25545 Sentence denotes Results:
TextSentencer_T230 25546-25622 Sentence denotes Counting each variant separately, concordance between pairs of labs is high:
TextSentencer_T231 25623-25637 Sentence denotes 98.5% overall.
TextSentencer_T232 25638-25746 Sentence denotes However this calculation greatly underestimates the much higher concordance observed on a per-patient basis.
TextSentencer_T233 25747-25854 Sentence denotes Most discordant classifications are in rare variants that, by definition, are present in very few patients.
TextSentencer_T234 25855-25939 Sentence denotes Moreover, classifications of most rare variants agree (sometimes, concordantly VUS).
TextSentencer_T235 25940-26093 Sentence denotes Based on clinical and population prevalence, we calculate that 99.8% of patients receive net concordant reports, similar to our previous study's results.
TextSentencer_T236 26094-26270 Sentence denotes Even after detailed examination of all evidence underlying the remaining disagreements, the maximally correct classification under current guidelines sometimes remains unclear.
TextSentencer_T237 26271-26283 Sentence denotes Conclusions:
TextSentencer_T238 26284-26377 Sentence denotes Classification concordance needs to be measured carefully to avoid over-counting differences.
TextSentencer_T239 26378-26543 Sentence denotes Whereas substantial differences are seen in few patients, it is important to resolve these collaboratively, not competitively, as is done in other areas of medicine.
TextSentencer_T240 26544-26724 Sentence denotes Thorough peer review of classifications is enabled by public databases like ClinVar and both supports laboratory quality control efforts and helps improve critical care guidelines.
TextSentencer_T241 26725-26727 Sentence denotes R.
TextSentencer_T242 26728-26737 Sentence denotes Kolhe, C.
TextSentencer_T243 26738-26749 Sentence denotes Pundkar, A.
TextSentencer_T244 26750-26760 Sentence denotes Mondal, W.
TextSentencer_T245 26761-26768 Sentence denotes Lee, A.
TextSentencer_T246 26769-26809 Sentence denotes Chaubey Augusta University, Augusta, GA.
TextSentencer_T247 26810-26823 Sentence denotes Introduction:
TextSentencer_T248 26824-26957 Sentence denotes A correct diagnosis is critical in separating spontaneous pregnancy loss and RPL and for determining appropriate therapeutic options.
TextSentencer_T249 26958-27079 Sentence denotes Traditionally, karyotyping has been used in molecular analysis of RPL and IF and been overall very helpful in RPL workup.
TextSentencer_T250 27080-27319 Sentence denotes Unfortunately Karyotype analysis also has multiple limitations such as low diagnostic yield, long turnaround time, missing cryptic changes (<5MB), required trained people to perform analysis and is a subjective method prone to human error.
TextSentencer_T251 27320-27328 Sentence denotes Methods:
TextSentencer_T252 27329-27533 Sentence denotes We performed an internal audit at our institution (2012 to 2015) and out of the 578 samples sent to cytogenetics lab for karyotype analysis only few (< 6%) cases had information which helped patient care.
TextSentencer_T253 27534-27597 Sentence denotes This led us to re-examine our RPL cases (FFPE samples) on SNPM.
TextSentencer_T254 27598-27654 Sentence denotes Archival blocks with slides were retrieved and reviewed.
TextSentencer_T255 27655-27737 Sentence denotes Clinical information was obtained from patient charts under approved IRB protocol.
TextSentencer_T256 27738-27866 Sentence denotes H&E slides were examined and chorionic villi (fetal tissue) was identified and marked for DNA isolation by surgical pathologist.
TextSentencer_T257 27867-28021 Sentence denotes The whole genome SNPM was performed on the DNA isolated from FFPE specimens following manufacturer's protocol (OncoScan FFPE Assay Kit, Affymetrix, Inc.).
TextSentencer_T258 28022-28140 Sentence denotes The raw data was analyzed in Chromosome Analysis Suite 3.0 software and were matched to in silico FFPE reference sets.
TextSentencer_T259 28141-28287 Sentence denotes This platform consists of 274,000 probes including 74 somatic mutations from 9 genes (BRAF, KRAS, EGFR, IDH1, IDH2, PTEN, PIK3CA, NRAS and TP53) .
TextSentencer_T260 28288-28355 Sentence denotes The IF cases were examined on Cytoscan (Affymetrix, Inc.) on blood.
TextSentencer_T261 28356-28364 Sentence denotes Results:
TextSentencer_T262 28365-28499 Sentence denotes All the RPL and IF cases (n=10) resulted in substantial information helping the diagnosis and patient prognosis on SNPM. e.g., Case 1:
TextSentencer_T263 28500-28590 Sentence denotes 38 YO female with history of 7 miscarriages with failed multiple attempts for karyotyping.
TextSentencer_T264 28591-28716 Sentence denotes Her SPMN analysis demonstrated mosaic trisomies of chromosomes 21 (~75% mosaic gain) and 22 (~70% mosaic gain) were observed.
TextSentencer_T265 28717-28724 Sentence denotes Case 2:
TextSentencer_T266 28725-28795 Sentence denotes 32 YO male with infertility/testicular failure with 46XY on karyotype.
TextSentencer_T267 28796-28845 Sentence denotes SNPM analysis showed Y chromosome micro deletion.
TextSentencer_T268 28846-28857 Sentence denotes Conclusion:
TextSentencer_T269 28858-29094 Sentence denotes There is high failure rate in accurately karyotyping products of conceptions (POC) for multiple reasons (Blinded sampling error in OR by D&C, overgrowth of maternal cells resulting in 46, XX, culture failure, microbial overgrowth etc.).
TextSentencer_T270 29095-29274 Sentence denotes SNPM technology has a remarkably higher resolution than conventional cytogenetics (karyotyping and FISH) in identifying cryptic deletions and duplications within the human genome.
TextSentencer_T271 29275-29448 Sentence denotes Having any additional genetic information from POC is very comforting to the patient and extremely beneficial to the reproductive medicine clinic to plan the next pregnancy.
TextSentencer_T272 29449-29601 Sentence denotes We anticipate that this approach of obtaining high-resolution data from an FFPE sample will facilitate studies of RPL which was not previously possible.
TextSentencer_T273 29602-29604 Sentence denotes I.
TextSentencer_T274 29605-29613 Sentence denotes King, M.
TextSentencer_T275 29614-29626 Sentence denotes Zariwala, K.
TextSentencer_T276 29627-29635 Sentence denotes Chao, K.
TextSentencer_T277 29636-29647 Sentence denotes Michael, K.
TextSentencer_T278 29648-29714 Sentence denotes Weck University of North Carolina at Chapel Hill, Chapel Hill, NC.
TextSentencer_T279 29715-29728 Sentence denotes Introduction:
TextSentencer_T280 29729-29885 Sentence denotes Primary Ciliary Dyskinesia (PCD) is an inherited disease causing defects in motile cilia, which manifests primarily with lung and upper airway difficulties.
TextSentencer_T281 29886-30007 Sentence denotes Specialized management of PCD patients has been able to improve outcomes greatly, as lung damage is in PCD is cumulative.
TextSentencer_T282 30008-30218 Sentence denotes However, most PCD patients do not receive a definitive diagnosis because the current gold standard diagnostic methods (electron microscopy and video analysis of cilia) require highly specialized interpretation.
TextSentencer_T283 30219-30363 Sentence denotes We have validated a comprehensive genetic diagnostic panel that will enable higher yield genetic diagnosis for patients suspected of having PCD.
TextSentencer_T284 30364-30372 Sentence denotes Methods:
TextSentencer_T285 30373-30475 Sentence denotes A custom hybridization capture reagent was developed for the Nextera Rapid Capture process (Illumina).
TextSentencer_T286 30476-30629 Sentence denotes This reagent incorporates capture probes targeting all exons of 33 clinically identified PCD genes, as well as one deep intronic site in the CCDC39 gene.
TextSentencer_T287 30630-30757 Sentence denotes All exons of two genes associated with inherited immune disorders that can phenocopy PCD (RAG1 and GAS2L2), were also included.
TextSentencer_T288 30758-30880 Sentence denotes All exons of CFTR were included, as cystic fibrosis is the major differential diagnosis in patients suspected to have PCD.
TextSentencer_T289 30881-31055 Sentence denotes In addition to these clinically reportable genes, all exons of 65 genes hypothesized to contribute to ciliary function were included as capture targets for research purposes.
TextSentencer_T290 31056-31064 Sentence denotes Results:
TextSentencer_T291 31065-31240 Sentence denotes Sequencing under anticipated clinical conditions (multiplexing up to 12x per MiSeq flowcell) yielded coverage of > 0.2x mean coverage for >95% of targets in 5 validation runs.
TextSentencer_T292 31241-31355 Sentence denotes Sporadic target dropout (defined as <20x coverage) was observed, but no one target was consistently under covered.
TextSentencer_T293 31356-31481 Sentence denotes A validation cohort was assembled from 21 PCD patient samples with known mutations from an ongoing PCD research study at UNC.
TextSentencer_T294 31482-31674 Sentence denotes With variants interpreted under a strictly autosomal recessive model of inheritance, no false positive results were obtained, whereas known mutations were correctly identified in all 21 cases.
TextSentencer_T295 31675-31837 Sentence denotes Variants identified by sequencing were filtered for population frequency, alleleic fraction, and to remove known sequencing artifacts and pseudogene interference.
TextSentencer_T296 31838-31932 Sentence denotes With this filtering strategy no more than 5 variants in each sample required characterization.
TextSentencer_T297 31933-32162 Sentence denotes Additionally, one patient, who also suffers from Cri-du-chat syndrome and carries a 5p deletion, was found to have a previously unidentified variant in DNAH5, which provides a potential explanation for the patient's PCD symptoms.
TextSentencer_T298 32163-32175 Sentence denotes Conclusions:
TextSentencer_T299 32176-32370 Sentence denotes This validation has demonstrated the technical and clinical utility of an expanded sequencing panel for PCD, and has identified a potential cause of PCD in one patient with a clinical diagnosis.
TextSentencer_T300 32371-32373 Sentence denotes M.
TextSentencer_T301 32374-32390 Sentence denotes Cleveland 1 , L.
TextSentencer_T302 32391-32405 Sentence denotes Borshuk 1 , J.
TextSentencer_T303 32406-32417 Sentence denotes Zook 1 , M.
TextSentencer_T304 32418-32430 Sentence denotes Salit 2 , P.
TextSentencer_T305 32431-32571 Sentence denotes Vallone 3 1 National Institute of Standards and Technology, Gaithersburg, MD; 2 National Institute of Standards and Technology, Stanford CA.
TextSentencer_T306 32572-32585 Sentence denotes Introduction:
TextSentencer_T307 32586-32691 Sentence denotes The Genome in a Bottle (GIAB) Consortium aims to highly characterize a small number of benchmark genomes.
TextSentencer_T308 32692-32843 Sentence denotes These genomes have been sequenced by several sequencing technologies, each with different biases in detection of indels, SNPs, and structural variants.
TextSentencer_T309 32844-32950 Sentence denotes These highly characterized genomes can thus be used as benchmarks to evaluate new sequencing technologies.
TextSentencer_T310 32951-33122 Sentence denotes As of June 2016, NIST has one highly characterized genome, NA12878, sold as RM 8398, and there are several more genomes in the pipeline to become NIST reference materials.
TextSentencer_T311 33123-33234 Sentence denotes The GIAB consortium would ultimately like to offer more clinically relevant benchmark genomes to the community.
TextSentencer_T312 33235-33398 Sentence denotes Targeted sequencing panels, which cover hundreds of genes, may be utilized to help identify and characterize additional genomes with clinically relevant mutations.
TextSentencer_T313 33399-33407 Sentence denotes Methods:
TextSentencer_T314 33408-33741 Sentence denotes In this project, we use two different targeted sequencing panels (the Illumina Inherited Disease Panel* and the Ion Torrent Ampliseq Inherited Disease Panel*) to examine jmd.amjpathol.org ■ The Journal of Molecular Diagnostics seven different genomes in triplicate (NA12878, GM24385, GM24149, GM24143, GM24694, GM24695, and GM24631).
TextSentencer_T315 33742-33840 Sentence denotes The Illumina Inherited Disease Panel (IDP) covers 550 genes and the Ampliseq IDP covers 328 genes.
TextSentencer_T316 33841-33979 Sentence denotes We examine the concordance of calls between the two inherited disease panels and the agreement of the sequencing with the GIAB base calls.
TextSentencer_T317 33980-33988 Sentence denotes Results:
TextSentencer_T318 33989-34055 Sentence denotes The Illumina IDP and the Ampliseq lDP have an overlap of 96 genes.
TextSentencer_T319 34056-34146 Sentence denotes Of those 96 genes, 29 are 100% covered in the current GIAB High Confidence regions (HCRs).
TextSentencer_T320 34147-34243 Sentence denotes Seven genes (CTNS, F8, HEXA, PEX5, WAS, GBA, GAA) are covered at less than 80% in the GIAB HCRs.
TextSentencer_T321 34244-34482 Sentence denotes There are 8 genes on the Illumina IDP only which are not covered at all in the GIAB HCR (CYP11B1, CYP21A2, HBA1, SBDS, SLC6A8, TUBA1A) and 4 genes on the Ampliseq IDP that are not covered at all in the GIAB HCR (FRG1, HBA2, KRT14, MAPT) .
TextSentencer_T322 34483-34495 Sentence denotes Conclusions:
TextSentencer_T323 34496-34620 Sentence denotes We have demonstrated how the GIAB highly characterized genomes may be used to evaluate different targeted sequencing panels.
TextSentencer_T324 34621-34829 Sentence denotes Targeted sequencing panels may be a cost effective way to characterize a high number of clinically important genomic regions. *Certain commercial products are identified in this paper to foster understanding.
TextSentencer_T325 34830-35062 Sentence denotes Such identification does not imply recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the materials or equipment identified are necessarily the best available for the purpose.
TextSentencer_T326 35063-35188 Sentence denotes To avoid these hypersensitivity reactions, HLA-B*57:01 genotyping prior to ABC administration is considered standard of care.
TextSentencer_T327 35189-35406 Sentence denotes Several HLA-B*57:01 genotyping techniques have been developed; however, most of them have limited specificity and sensitivity for HLA-B*57:01 detection, require special instruments/techniques, and are labor intensive.
TextSentencer_T328 35407-35510 Sentence denotes Here we are reporting a novel HLA-B*57:01 genotyping method using hydrolysis probe-based real-time PCR.
TextSentencer_T329 35511-35519 Sentence denotes Methods:
TextSentencer_T330 35520-35630 Sentence denotes Eleven fully HLA genotyped genomic DNA samples were purchased from the Coriell Institute for Medical Research.
TextSentencer_T331 35631-35714 Sentence denotes Thirty previously-tested patient samples were obtained from a reference laboratory.
TextSentencer_T332 35715-35908 Sentence denotes Primer and probes were designed based on published sequence variations in exon 3 of HLA-B between ABC-sensitive (e.g. HLA-B*57:01) and ABCinsensitive alleles (e.g. HLA-B*57:03 and HLA-B*58:01).
TextSentencer_T333 35909-35952 Sentence denotes There are two polymorphism sites in exon 3.
TextSentencer_T334 35953-36045 Sentence denotes The first one is close to the 5' end of the exon and the other is in the middle of the exon.
TextSentencer_T335 36046-36119 Sentence denotes The first polymorphic site was used for allelespecific PCR amplification.
TextSentencer_T336 36120-36260 Sentence denotes PCR primers were designed to specifically amplify HLA-B*57:01 and its closely related alleles such as HLA-B*57:03 directly from genomic DNA.
TextSentencer_T337 36261-36341 Sentence denotes Most of the unrelated alleles would not produce any product in the PCR reaction.
TextSentencer_T338 36342-36494 Sentence denotes Our hydrolysis probes enable differentiation of HLA-B*57:01 from the other amplified, but ABC-insensitive, ABC alleles based on the second polymorphism.
TextSentencer_T339 36495-36603 Sentence denotes As confirmatory measures, additional hydrolysis probes/primer and PCR-RFLP markers were designed and tested.
TextSentencer_T340 36604-36612 Sentence denotes Results:
TextSentencer_T341 36613-36785 Sentence denotes Our real-time PCR demonstrated 100% accuracy in distinguishing HLA-B*57:01 positive (n=2) and negative (n=9) samples among the genomic DNA samples with known HLA genotypes.
TextSentencer_T342 36786-36938 Sentence denotes Additionally, thirty samples previously tested with a separate HLA-B*57:01 real-time PCR method in a reference laboratory were evaluated with our assay.
TextSentencer_T343 36939-37047 Sentence denotes We found concordant results with all but one apparent false-negative and one apparent false-positive result.
TextSentencer_T344 37048-37195 Sentence denotes Additional real-time PCR with different primer and probe sets and PCR-RFLP showed that the discordant genotypes were correctly called by our assay.
TextSentencer_T345 37196-37208 Sentence denotes Conclusions:
TextSentencer_T346 37209-37355 Sentence denotes The hydrolysis probe-based real-time PCR for HLA-B*57:01 genotyping showed better accuracy compared to previously developed genotyping techniques.
TextSentencer_T347 37356-37521 Sentence denotes Our newly developed test will allow clinical laboratories with real-time PCR capabilities to determine the HLA-B*57:01 genotype in a timely and costeffective manner.
TextSentencer_T348 37522-37526 Sentence denotes A.G.
TextSentencer_T349 37527-37535 Sentence denotes Hadd, S.
TextSentencer_T350 37536-37547 Sentence denotes Morales, A.
TextSentencer_T351 37548-37560 Sentence denotes Anderson, H.
TextSentencer_T352 37561-37570 Sentence denotes Rey, G.J.
TextSentencer_T353 37571-37605 Sentence denotes Latham Asuragen, Inc., Austin, TX.
TextSentencer_T354 37606-37619 Sentence denotes Introduction:
TextSentencer_T355 37620-37812 Sentence denotes Database accessibility to next-generation sequencing (NGS) data from thousands of individuals has substantially increased our understanding of human DNA variation across different populations.
TextSentencer_T356 37813-37946 Sentence denotes However, many SNPs in publically-available databases are invalidated and thus could reflect intrinsic error rates of large-scale NGS.
TextSentencer_T357 37947-38100 Sentence denotes One SNP, rs111485627, reportedly overlaps the FAMlabeled reverse primer of the AmplideX PCR/CE FMR1 Kit, a commonly-used reagent set for FMR1 genotyping.
TextSentencer_T358 38101-38234 Sentence denotes This study addresses testing and secondary analysis of samples reported with this SNP and discusses the implications of the findings.
TextSentencer_T359 38235-38243 Sentence denotes Methods:
TextSentencer_T360 38244-38359 Sentence denotes A total of 20 unique cell-line genomic DNA, 12 female and 8 male, were obtained from the Coriell Cell Repositories.
TextSentencer_T361 38360-38466 Sentence denotes These samples were identified with rs111485627 in either Phase I or Phase III of the 1000Genomes database.
TextSentencer_T362 38467-38631 Sentence denotes DNAs were analyzed using AmplideX FMR1 PCR (Asuragen) using standard primers, and a single-offset base primer designed to avoid the putative SNP, in duplicate PCRs.
TextSentencer_T363 38632-38732 Sentence denotes The primer binding region for each cell-line DNA was independently assessed using Sanger sequencing.
TextSentencer_T364 38733-38869 Sentence denotes Finally, a synthetic construct of the CGG region designed with the SNP was prepared, sequenced verified and analyzed using AmplideX PCR.
TextSentencer_T365 38870-38878 Sentence denotes Results:
TextSentencer_T366 38879-39087 Sentence denotes All 20 samples reported with the SNP (5 from) were successfully PCR-amplified in the FMR1 triplet repeat region and genotyped without loss of signal compared to samples of comparable genotype lacking the SNP.
TextSentencer_T367 39088-39205 Sentence denotes Amplification with a single-base offset primer yielded similar signal intensity and the expected genotyping profiles.
TextSentencer_T368 39206-39324 Sentence denotes Importantly, Sanger sequencing unequivocally showed the wild-type sequence at this location in both males and females.
TextSentencer_T369 39325-39476 Sentence denotes In addition, a synthetic construct containing the G>T substitution was successfully genotyped compared to a wild-type construct and source genomic DNA.
TextSentencer_T370 39477-39489 Sentence denotes Conclusions:
TextSentencer_T371 39490-39624 Sentence denotes A putative SNP in the fragile X gene demonstrated no interference or loss of signal across 20 different samples reported with the SNP.
TextSentencer_T372 39625-39689 Sentence denotes The SNP failed independent verification using Sanger sequencing.
TextSentencer_T373 39690-39808 Sentence denotes Moreover, even if present, constructs with the reported SNP were successfully genotyped for FMR1 CGG repeat sequences.
TextSentencer_T374 39809-39893 Sentence denotes The SNP may be a consequence of error rates in NGS reported for GC-rich DNA regions.
TextSentencer_T375 39894-40066 Sentence denotes These findings provide a cautionary note regarding the accuracy of variants reported in public databases, and their potential impact or lack thereof on nucleic acid assays.
TextSentencer_T376 40067-40069 Sentence denotes D.
TextSentencer_T377 40070-40082 Sentence denotes Vendrone, M.
TextSentencer_T378 40083-40095 Sentence denotes Pieretti, L.
TextSentencer_T379 40096-40107 Sentence denotes Medling, A.
TextSentencer_T380 40108-40149 Sentence denotes Gonzalez BayCare Laboratories, Tampa, FL.
TextSentencer_T381 40150-40163 Sentence denotes Introduction:
TextSentencer_T382 40164-40249 Sentence denotes Clinical mutation assays commonly used to assess predisposition to thrombophilia are:
TextSentencer_T383 40250-40387 Sentence denotes Factor V Leiden (c.1691G>A), Prothrombin c.20210G>A and Methylenetetrahydrofolate Reductase (MTHFR) c.665C>T and c.1286A>C gene mutation.
TextSentencer_T384 40388-40600 Sentence denotes Our laboratory has offered clinical assays for these mutations for several years, but we sought to implement new versions requiring less hands-on, shorter detection, and no extraction to increase cost efficiency.
TextSentencer_T385 40601-40609 Sentence denotes Methods:
TextSentencer_T386 40610-40766 Sentence denotes We chose Focus ASRs designed as Scorpion Primers (fluorescently labeled sequence specific); and enzymes mixes optimized for common thermocycling conditions.
TextSentencer_T387 40767-40889 Sentence denotes Fluorescence thresholds, valid Ct values and other cutoff criteria are not provided and were established during the study.
TextSentencer_T388 40890-41040 Sentence denotes For each assay we used a "training set" of known patient samples to set criteria, and a "validation set" of distinct patient samples to validate them.
TextSentencer_T389 41041-41121 Sentence denotes All patient samples were previously characterized with Hologic Invader-Plus IVD.
TextSentencer_T390 41122-41291 Sentence denotes We established criteria for both whole blood specimens and extracted nucleic acids, intending to use whole blood as the primary method and nucleic acids only as back-up.
TextSentencer_T391 41292-41354 Sentence denotes Fluorescence thresholds for each mutation were chosen so that:
TextSentencer_T392 41355-41450 Sentence denotes 1) The background fluorescence of the negative target would not cross the positivity threshold.
TextSentencer_T393 41451-41557 Sentence denotes 2) For heterozygous samples, the delta Ct value (mutant -wild-type) would be as close as possible to zero.
TextSentencer_T394 41558-41738 Sentence denotes For each mutation, Ct mean and 2 standard deviation values from the "training sets" were used to establish Ct and delta Ct cutoffs for wild-type, homozygous and heterozygous calls.
TextSentencer_T395 41739-41747 Sentence denotes Results:
TextSentencer_T396 41748-42037 Sentence denotes Eighty-nine whole blood samples (20 for Factor V, 27 for Prothrombin and 42 for MTHFR) were used as the "training sets." After all cutoffs were established, an additional 128 samples (32 for Factor V, 33 for Prothrombin and 63 for MTHFR) were analyzed to verify the established parameters.
TextSentencer_T397 42038-42169 Sentence denotes Eighty extracted DNA samples (20 for each assay) were used as "training sets" for the extracted nucleic acid version of the assays.
TextSentencer_T398 42170-42304 Sentence denotes After thresholds and cutoffs were established, an additional 40 samples (10 each assay) were run to verify the established parameters.
TextSentencer_T399 42305-42384 Sentence denotes For both versions and all assays, correlation with the Hologic assays was 100%.
TextSentencer_T400 42385-42580 Sentence denotes Precision, interference studies and DNA concentration/dilution studies, as well as controls' range studies were also performed to further establish the analytical characteristics of these assays.
TextSentencer_T401 42581-42593 Sentence denotes Conclusions:
TextSentencer_T402 42594-42832 Sentence denotes We established Laboratory Developed Tests (LDTs) for Factor V Leiden, Prothrombin and MTHFR gene mutations assays using Diasorin ASR reagents; these tests are frequently used to assess patients risk and management of thrombophilic events.
TextSentencer_T403 42833-42897 Sentence denotes Using Anchored Multiplex PCR and Next-Generation Sequencing B.P.
TextSentencer_T404 42898-42913 Sentence denotes Culver 1 , L.M.
TextSentencer_T405 42914-42930 Sentence denotes Griffin 1 , M.T.
TextSentencer_T406 42931-43003 Sentence denotes Hardison 2 1 ArcherDX, Inc., Boulder, CO; 2 BabyGenes, Inc., Golden, CO.
TextSentencer_T407 43004-43017 Sentence denotes Introduction:
TextSentencer_T408 43018-43151 Sentence denotes Cystic Fibrosis (CF) is an autosomal recessive disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene.
TextSentencer_T409 43152-43264 Sentence denotes CF is characterized by the build-up of thick mucus resulting in chronic lung infections and airway inflammation.
TextSentencer_T410 43265-43385 Sentence denotes As such, early diagnosis and treatment interventions are crucial to help prevent airway obstruction and lung infections.
TextSentencer_T411 43386-43512 Sentence denotes Therefore, carrier identification and newborn screening have significant implications in the overall prognosis of CF patients.
TextSentencer_T412 43513-43612 Sentence denotes Unfortunately, there is a selection bias in CF diagnosis of white compared to nonwhite populations.
TextSentencer_T413 43613-43813 Sentence denotes This ethnic disparity in CF diagnosis is primarily attributed to differences in underlying CFTR mutations, which were recently shown to vary significantly across ethnic groups by Iris Schrijver et al.
TextSentencer_T414 43814-43978 Sentence denotes Current CFTR genotyping assays detect mutations highly prevalent in white individuals, yet fail to detect mutations that are more prevalent in nonwhite individuals.
TextSentencer_T415 43979-44126 Sentence denotes We present a rapid, cost-effective assay for comprehensive detection of CFTR mutations for pan-ethnic carrier identification and newborn screening.
TextSentencer_T416 44127-44135 Sentence denotes Methods:
TextSentencer_T417 44136-44355 Sentence denotes BabyGenes, Inc, in partnership with ArcherDX, Inc., has developed a targeted next-generation sequencing (NGS) assay based on Anchored Multiplex PCR (AMP) to detect all mutations currently reported in the CFTR2 database.
TextSentencer_T418 44356-44596 Sentence denotes AMP is a library preparation method for NGS that uses unidirectional gene-specific primers (GSPs) and molecular barcoded adaptors ligated to random start sites to enrich for both known and unknown mutations across a panel of target regions.
TextSentencer_T419 44597-44605 Sentence denotes Results:
TextSentencer_T420 44606-44633 Sentence denotes The BabyGenes Introduction:
TextSentencer_T421 44634-44810 Sentence denotes Multiplex PCR and capillary electrophoresis (CE)-based T cell receptor gene rearrangement assay has been widely used as an ancillary test for the diagnosis of T cell lymphomas.
TextSentencer_T422 44811-45038 Sentence denotes Identifying clonal peaks of identical sizes in different specimens from the same patient helps to improve test specificity; however, discrepant results can occasionally be encountered and pose challenges in test interpretation.
TextSentencer_T423 45039-45319 Sentence denotes Next-generation sequencing (NGS)-based clonality analysis reveals the sequences of individual T cell clonotypes in a quantitative way, and can potentially reconcile the differences among the specimens and overcome the challenges associated with fragment size-based testing method.
TextSentencer_T424 45320-45328 Sentence denotes Methods:
TextSentencer_T425 45329-45512 Sentence denotes A total of 10 specimens (4 peripheral blood and 6 tissue) from 4 patients with mycosis fungoides (MF) and discrepant CE-based T cell clonality test results were included in the study.
TextSentencer_T426 45513-45611 Sentence denotes DNA was extracted followed by BIOMED-2 primer-based TCRG and TCRB sequencing library construction.
TextSentencer_T427 45612-45719 Sentence denotes The amplicon libraries with TruSeq adaptors incorporated were subsequently sequenced on the MiSeq platform.
TextSentencer_T428 45720-45879 Sentence denotes After the paired-end reads were joined and the primer sequences were removed, the clonotypes were submitted to IMGT/HighV-Quest for TCR rearrangement analysis.
TextSentencer_T429 45880-45948 Sentence denotes The NGS results were then analyzed to correlate with the CE results.
TextSentencer_T430 45949-45957 Sentence denotes Results:
TextSentencer_T431 45958-46119 Sentence denotes The tissue specimens with clonal patterns identified by the CE method were also demonstrated by the NGS method to harbor clonotypes with predominant frequencies.
TextSentencer_T432 46120-46270 Sentence denotes Clonal peaks of the same sizes identified by CE in different tissue specimens from the same patients were confirmed to be identical clonotypes by NGS.
TextSentencer_T433 46271-46410 Sentence denotes The tumor clonotype present in tissue specimens was also detected by NGS in the blood specimen of a patient with suspected Sézary syndrome.
TextSentencer_T434 46411-46650 Sentence denotes A clonal pattern demonstrated by the CE method in the blood specimen of one patient was not supported by the polyclonal NGS results, suggestive of a false positive result by CE as a result of different clonotypes of the same amplicon size.
TextSentencer_T435 46651-46851 Sentence denotes The tumor clonotype sequences identified in the diagnostic tissue specimens could be used to definitively confirm the presence or absence of tumor clones in blood as well as in other tissue specimens.
TextSentencer_T436 46852-46864 Sentence denotes Conclusions:
TextSentencer_T437 46865-47116 Sentence denotes Compared to the fragment size-based method, NGS-based T cell clonality analysis makes it possible to not only determine the overall clonality pattern, but also sensitively and specifically detect the tumor clonotype in specimens from patients with MF.
TextSentencer_T438 47117-47268 Sentence denotes The discrepancies between the CE-based tissue and blood clonality results can be better interpreted and potentially resolved by the NGS-based approach.
TextSentencer_T439 47269-47415 Sentence denotes The NGS-based method also provides an opportunity to more accurately monitor response to therapy and minimal residual disease in patients with MF.
T35185 47416-47418 Sentence denotes R.
T1670 47419-47431 Sentence denotes Kolhe 1 , W.
T88888 47432-47442 Sentence denotes Lee 2 , C.
T49904 47443-47457 Sentence denotes Pundkar 2 , A.
T14572 47458-47471 Sentence denotes Mondal 2 , J.
T70849 47472-47486 Sentence denotes Chaffin 2 , A.
T86895 47487-47503 Sentence denotes Kornfield 2 , R.
T71196 47504-47610 Sentence denotes Kolhe 2 1 Medical College of Georgia at Augusta Univeristy, Augusta, GA; 2 Augusta University, Augusta GA.
T71113 47611-47726 Sentence denotes Introduction: AML is the most common form of acute leukemia in adults with an incidence of approximately 4/100,000.
T22133 47727-47825 Sentence denotes Upon diagnosis, patients receive induction chemotherapy aiming to achieve complete remission (CR).
T18038 47826-47973 Sentence denotes It known that despite successful induction therapy and completed consolidation therapy, relapse occurs in 60% to 70% of patients within five years.
T14457 47974-48147 Sentence denotes In the first relapse, the likelihood of achieving a renewed CR is lower than at onset of disease, and the duration the second CR is almost invariably shorter than the first.
T86788 48148-48310 Sentence denotes Relapse is a significant reason why the overall prognosis of adult patients with AML remains poor with a 20% to 30% likelihood of 5-year survival after diagnosis.
T25534 48311-48319 Sentence denotes Methods:
T75725 48320-48455 Sentence denotes Archival blocks with slides were retrieved, reviewed and clinical information obtained from patient charts under approved IRB protocol.
T91536 48456-48625 Sentence denotes Several patient/disease characteristics were identified including age, sex, race, body mass index (BMI), and cytogenetics with or without mutational analysis were noted.
T57937 48626-48749 Sentence denotes Theraphy indicators such as treatment received, transplantation status, remission status and relapse status was also noted.
T60546 48750-49133 Sentence denotes Seven al RNA was extracted from ( n=6 with > 5 years of survival) & n=7 with < 6 months of survival) and analyzed with nanoString nCounter using PanCancer Immune Profiling Panel designed to quantitate 770 genes associated with identifying immune cells, assessing immunological function (e.g., immune checkpoint regulation), identifying tumor-specific antigens and housekeeping genes.
T67309 49134-49311 Sentence denotes For mechanistic studies 7 Leukemia cell lines (KU812, HL-60, THP-1, K0562, RS4, MOLT-4, and CCRF-CEM) were obtained from ATCC along with three non-leukemia human controls cells.
T78925 49312-49320 Sentence denotes Results:
T32750 49321-49485 Sentence denotes The gene expression profiles of early relapse vs no-relapse, showed significant (>3 times, p<0.05) upregulation of a set of 8 genes, and down regulation of 8 genes.
T77361 49486-49617 Sentence denotes Interestingly, gene IFI27 was upregulated 13X (p< 0.0012) in early relapse (<6months) patients as compared to >5yrs survival group.
T95348 49618-49762 Sentence denotes To further investigate the mechanism of IFI27 in cellular model RNA was isolated from 7 cell lines and real-time quantitative PCR was performed.
T47826 49763-49864 Sentence denotes THP-1 (acute monocytic leukemia) revealed the most notable upregulation of IFI27 (1,728 fold change).
T49553 49865-49877 Sentence denotes Conclusions:
T20538 49878-50046 Sentence denotes To the best of our knowledge, the current study represents the first gene expression studies investigating immune tolerance exploration of patients with relapse in AML.
T22192 50047-50151 Sentence denotes IFI27 is found to be upregulated in breast cancer, squamous cell carcinoma and hepatocellular carcinoma.
T66072 50152-50227 Sentence denotes Overexpression of IFI27 in ovarian cancer is associated with poor survival.
T98500 50228-50358 Sentence denotes This work is intriguing for the new information it provides about the possible role of IFI27 in AML relapse and treatment failure.
T91582 50359-50361 Sentence denotes M.
T30524 50362-50379 Sentence denotes Steenkamer 1 , A.
T73935 50380-50394 Sentence denotes Stuitje 1 , L.
T62907 50395-50411 Sentence denotes Atanesyan 1 , K.
T31170 50412-50426 Sentence denotes Stouten 2 , M.
T43631 50427-50440 Sentence denotes Werken 2 , R.
T13510 50441-50454 Sentence denotes Castel 2 , S.
T89805 50455-50552 Sentence denotes Savola Amsterdam, Netherlands; 2 Albert Schweitzer Hospital, Netherlands; Amsterdam, Netherlands.
T24656 50553-50566 Sentence denotes Introduction:
T64079 50567-50706 Sentence denotes Myeloproliferative neoplasms (MPNs) are a group of hematopoietic stem cell disorders, characterized by clonal proliferation of blood cells.
T52254 50707-50809 Sentence denotes Relevant diagnostic molecular markers in MPN are recurrent mutations in CALR, JAK2, KIT and MPL genes.
T91217 50810-50994 Sentence denotes Multiplex Ligation-dependent Probe Amplification (MLPA) is a widely used technique for gene copy number detection, while it allows simultaneous identification of known point mutations.
T86146 50995-51255 Sentence denotes However, the sensitivity of standard MLPA is limited to a mutant allele burden of a high sensitivity MLPA assay enabling detection of an allele burden as low as 1% jmd.amjpathol.org ■ The Journal of Molecular Diagnostics for the most frequent mutations in MPN.
T74395 51256-51388 Sentence denotes Our aim was to develop and to demonstrate the feasibility of a modified MLPA assay to artificial positive DNA and cell line samples.
T79245 51389-51470 Sentence denotes Validation of this assay was further performed on diagnostic MPN patient samples.
T43925 51471-51479 Sentence denotes Methods:
T49374 51480-51885 Sentence denotes Novel Salsa MLPA MPN probemix was designed and optimized to detect, in a single allele burden of the eight most frequent mutations in MPNs: CALR, 52-bp deletion and 5-bp insertion in exon 9 (L367fs*46 and K385fs*47), JAK2, deletions in exon 12 (N542_E543del and E543-D544del), JAK2, substitution in exon 14 (V617F), KIT, substitution in exon 17 (D816V) and MPL, substitutions in exon 10 (W515L and W515K).
T62556 51886-52089 Sentence denotes The analytic sensitivity and specificity of this newly developed MLPA assay was determined on artificial positive DNA samples, cell-line titration series (UKE-1) and on healthy human DNA samples (n=143).
T27751 52090-52214 Sentence denotes The assay was further validated using commercial reference DNA with 1% allelic burden of JAK2 V617F and KIT D816V mutations.
T8696 52215-52330 Sentence denotes Validation of the novel probemix was performed in a single-blind setting on diagnostic MPN patient samples (n=167).
T69930 52331-52339 Sentence denotes Results:
T41692 52340-52470 Sentence denotes Results obtained with this high sensitivity MLPA assay were concordant with an allele-specific PCR results in MPN patient samples.
T75547 52471-52661 Sentence denotes Moreover, 2 novel cases with 1.4 % to 5% JAK2 V617F burden, which were not detected by allele-specific PCR, were identified with our novel assay and confirmed by the Ipsogen MutaQuant assay.
T37155 52662-52769 Sentence denotes Furthermore, no false positive calls for mutations were obtained when testing on healthy human DNA samples.
T32983 52770-52782 Sentence denotes Conclusions:
T61263 52783-53017 Sentence denotes Our results demonstrate that our novel high sensitivity MLPA assay is a reliable technique for simultaneous detection of eight frequent mutations in MPNs, even when the mutant allele burden of the patient DNA sample is low (1% to 5%).
T98246 53018-53148 Sentence denotes These results merit further consideration of MLPA as a possible alternative for mutation testing for newly diagnosed MPN patients.
T59010 53149-53151 Sentence denotes Y.
T87174 53152-53159 Sentence denotes Cho, S.
T75586 53160-53168 Sentence denotes Jang, E.
T64530 53169-53176 Sentence denotes Seo, J.
T74358 53177-53184 Sentence denotes Lee, J.
T25946 53185-53192 Sentence denotes Lee, K.
T99962 53193-53200 Sentence denotes Lee, C.
T25972 53201-53310 Sentence denotes Park University of Ulsan, College of Medicine and Asan Medical Center, Seoul, Republic of Korea Introduction:
T8111 53311-53424 Sentence denotes Mutational status and bone marrow histopathology are essential for the diagnosis of myeloproliferative neoplasms.
T68664 53425-53576 Sentence denotes Specifically, bone marrow histopathology is the key to differentiation of essential thrombocythemia (ET) from prefibrotic primary myelofibrosis (PMF) .
T15400 53577-53682 Sentence denotes The aim of this study is to assess the correlation of bone marrow histopathology with mutational profile.
T59408 53683-53691 Sentence denotes Methods:
T72011 53692-53844 Sentence denotes We rereviewed the bone marrow biopsies of 128 patients with either ET (n=69) or PMF (n=59) without knowledge of mutational status or original diagnoses.
T5523 53845-53981 Sentence denotes JAK2 V617F mutations were positive in 68 patients, CALR exon 9 mutations were positive in 31, and MPL W515 mutations were positive in 3.
T30259 53982-53990 Sentence denotes Results:
T71532 53991-54250 Sentence denotes Among the ET group, patients with the CALR exon 9 mutation more frequently showed a higher number of megakaryocytes (P=0.043), dense clusters of megakaryocytes (P=0.021), small megakaryocytes (P=0.003), and bulbous nuclei (P<0.001) than those with JAK2 V617F.
T82316 54251-54407 Sentence denotes A re-review of bone marrow biopsies revealed that 10 of 69 (14.5%) patients originally diagnosed as ET had histopathological features of PMF rather than ET.
T73606 54408-54499 Sentence denotes This finding might indicate that these patients should be re-classified as prefibrotic PMF.
T42831 54500-54613 Sentence denotes However, the histologic diagnoses of patients originally diagnosed as PMF were not changed, even after re-review.
T46361 54614-54828 Sentence denotes Among the PMF patients, including both previous cases and ET cases showing histologic features of PMF, mutational status was not associated with significant differences in overall survival or leukemiafree survival.
T71382 54829-55002 Sentence denotes Instead, patients negative for all three mutations had shorter overall survival (P<0.001) and leukemia-free survival (P=0.009) than patients with one of the three mutations.
T14646 55003-55015 Sentence denotes Conclusions:
T27092 55016-55241 Sentence denotes Patients with JAK2 V617F mutation exhibited a heterogeneous feature in bone marrow histopathology, whereas CALR mutationpositive ET patients showed megakaryocyte abnormalities and exhibited a tendency towards prefibrotic PMF.
T87931 55242-55244 Sentence denotes T.
T14788 55245-55256 Sentence denotes Yang 1 , A.
T28811 55257-55267 Sentence denotes Box 2 , C.
T27194 55268-55359 Sentence denotes Hill 1 1 Emory University, Atlanta, GA; 2 Emory University School of Medicine, Atlanta, GA.
T81425 55360-55373 Sentence denotes Introduction:
T29799 55374-55611 Sentence denotes The diagnosis of myelodysplastic syndrome (MDS) is very challenging in that nonspecific findings, such as mild trilineage dysplastic changes, although important features for the diagnosis of MDS, can be seen in numerous other conditions.
T42351 55612-55763 Sentence denotes Genetic abnormalities, if detected, can provide clonal evidence of myeloid neoplasm and significant prognostic information to guide patient management.
T76245 55764-55994 Sentence denotes With the discovery of MDS-associated mutations, next-generation sequencing (NGS) has become one of the most sensitive methods to provide critical information to assist in the diagnosis of MDS, especially in low grade MDS patients.
T50131 55995-56123 Sentence denotes In this study, we have analyzed the utility of NGS myeloid neoplasm panel (NGS-MNP) to facilitate a definitive diagnosis of MDS.
T11843 56124-56132 Sentence denotes Methods:
T28455 56133-56206 Sentence denotes This study was performed under Emory institutional review board protocol.
T36112 56207-56311 Sentence denotes Clinical data of patients with NGS-MNP testing performed from September 2015 to March 2016 was reviewed.
T25910 56312-56394 Sentence denotes Pre-NGS, post-NGS diagnosis, gene mutations and allele frequencies were tabulated.
T90054 56395-56599 Sentence denotes The data was filtered for patients with a post-NGS diagnosis of MDS, acute myeloid leukemia with myelodysplasia-related changes or if bone marrow examination was performed for a clinical suspicion of MDS.
T63533 56600-56811 Sentence denotes The NGS-MNP was considered to have altered the diagnosis if any MDS-associated mutation was identified and used to confirm the diagnosis, or if no mutation was identified and the result was used to rule out MDS.
T91328 56812-56820 Sentence denotes Results:
T24255 56821-56893 Sentence denotes Between Sept 2015 and March 2016, NGS-MNP was performed on 214 patients.
T51962 56894-56983 Sentence denotes Thirty-one percent (66 cases) of the panels were performed for suspected/presumptive MDS.
T1537 56984-57095 Sentence denotes Of these, 58% (38 cases) of the initial diagnoses were altered based upon the results of the myeloid NGS panel.
T92037 57096-57242 Sentence denotes In cases where the post-NGS diagnosis of MDS was altered, 34% (13 cases) of the cases had no mutations and this result assisted in ruling out MDS.
T80840 57243-57389 Sentence denotes The most common mutations identified in MDS cases with NGS altered diagnoses were TET2 (11; 17%), TP53 (6; 9%), DNMT3A (6; 9%), and SF3B1 (5; 8%).
T17875 57390-57500 Sentence denotes In the 5 cases of low risk MDS, the most commonly identified mutations were SF3B1 (3; 60%) and RUNX1 (2; 40%).
T13291 57501-57614 Sentence denotes A myeloproliferative neoplasm-associated mutation was seen in 8 cases: JAK2 V617F (4; 6%) or CALR in/dels 4; 6%).
T46851 57615-57626 Sentence denotes Conclusion:
T48571 57627-57887 Sentence denotes Myeloid panel NGS testing resulted in a clinically significant change in diagnosis in 58% of the suspected/presumptive MDS cases received at the Emory Molecular diagnostic Laboratory and represent a rapidly expanding and valuable tool for the diagnosis of MDS.
T21211 57888-57958 Sentence denotes Tanzania; 6 University of Utah School of Medicine, Salt Lake City, UT.
T82391 57959-57972 Sentence denotes Introduction:
T10571 57973-58166 Sentence denotes Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common genetic abnormality known to predispose to acute hemolytic anemia (AHA), which can be triggered by certain drugs or by infection.
T52493 58167-58317 Sentence denotes However, the commonest trigger is the ingestion of fava beans (Vicia faba), causing AHA (favism), which may be lifethreatening especially in children.
T88257 58318-58441 Sentence denotes G6PD deficiency is genetically highly heterogeneous, as nearly 200 different mutations have been observed in the G6PD gene.
T89996 58442-58450 Sentence denotes Methods:
T45234 58451-58702 Sentence denotes We have investigated the hematological and genetic features of acute favism in the Palestinian Gaza community utilizing 131 children hospitalized for G6PD deficiency induced AHA and comparing the findings with indices from the general Gaza population.
T9765 58703-58711 Sentence denotes Results:
T40228 58712-58847 Sentence denotes We discovered the polymorphic coexistence of three different G6PD deficiency genes (G6PD A-, G6PD Cairo, G6PD Med) in the Gaza society.
T61577 58848-59058 Sentence denotes We have found by comparison to the general population (485 adults and 466 newborns) that children with favism, in terms of relative frequency, G6PD A-was under-represented, whereas G6PD Med was overrepresented.
T43365 59059-59379 Sentence denotes We also found that the severity of anemia was significantly greater with G6PD Med and G6PD Cairo than with G6PD A-; and with G6PD Cairo, compared to the other two variants, there was greater hyperbilirubinemia, as well as persistence of mild anemia and reticulocytosis for as long as 4 months after recovery from favism.
T28422 59380-59391 Sentence denotes Conclusion:
T51425 59392-59613 Sentence denotes We conclude that children with G6PD A-deficiency are also susceptible to AHA, but demonstrate in direct comparison within this same population that G6PD Med and G6PD Cairo are more severe forms of deficiency than G6PD A-.
T27833 59614-59729 Sentence denotes Further, we show that the heretofore poorly studied G6PD Cairo may be associated with low-level, chronic hemolysis.
T72685 59730-59924 Sentence denotes This study illustrates favism is a significant public health problem in Gaza due to fava beans as a staple in the diet and the coexistence of polymorphic G6PD deficiency variants in the society.
T70725 59925-60052 Sentence denotes Favism is an easily preventable and manageable genetic disorder with the proper awareness, intervention and education programs.
T59911 60053-60362 Sentence denotes 6 1 Rutgers University, Piscataway, NJ; 2 Rutgers Robert Wood Johnson Medical School, New Brunswick, NJ; 3 Rutgers University, New Brunswick, NJ; 4 Providence Heath and Services, Portland, OR; 5 Moffitt Cancer Center, Tampa, FL; 6 Dartmouth Hitchcock Medical Center and Geisel School of Medicine, Lebanon, NH.
T44234 60363-60376 Sentence denotes Introduction:
T85810 60377-60567 Sentence denotes Next-generation sequencing of cancer tissue is becoming a mainstream technique in clinical laboratories because of its potential to contribute to the selection of patient-specific therapies.
T49659 60568-60806 Sentence denotes Here, we describe the validation of the ThunderBolts (TB) Myeloid Panel from RainDance Technologies for the detection of sequence variants in DNA isolated from bone marrow and peripheral blood samples from patients with myeloid neoplasms.
T73285 60807-60815 Sentence denotes Methods:
T30977 60816-61166 Sentence denotes We received 72 previously tested blood or bone marrow DNA samples from three CLIA-certified and CAP-accredited laboratories: i) Molecular Genomics Laboratory, Providence Heath and Services; ii) Molecular Diagnostics Laboratory, Moffitt Cancer Center; and iii) CGAT, Department of Pathology and Laboratory Medicine, Dartmouth Hitchcock Medical Center.
T4068 61167-61274 Sentence denotes All three labs used the TruSight Myeloid Panel and the MiSeq instrument from Illumina for variant analysis.
T63316 61275-61409 Sentence denotes A minimum of 40 ng DNA was analyzed using the TB panel and PCR libraries from up to 12 samples were pooled and sequenced on the MiSeq.
T57450 61410-61508 Sentence denotes DNA from three well-characterized human cell lines was also used to validate the TB myeloid panel.
T95193 61509-61594 Sentence denotes Nucleotide sequence data were analyzed using the NextGENe software from SoftGenetics.
T68952 61595-61603 Sentence denotes Results:
T92217 61604-61750 Sentence denotes We observed an average of 4,886 reads per analytical sensitivity and specificity of the assay, tested using DNA from three cell lines, were 98.1%.
T67591 61751-61864 Sentence denotes The reproducibility of the assay, determined by analyzing two patient samples in three different runs, was 92.8%.
T39757 61865-61998 Sentence denotes We determined the lower limit of this assay for the detection of sequence variants by mixing cell line DNAs in different proportions.
T58637 61999-62074 Sentence denotes We were able to detect sequence variants at the 2% level in these mixtures.
T92920 62075-62148 Sentence denotes Sequence data were available for 46 samples from the three external labs.
T65678 62149-62329 Sentence denotes These labs did not report any pathogenic variants or variants of unknown significance in 12 of these samples and variants in 5 samples could only be detected by the TruSight panel.
T39729 62330-62384 Sentence denotes The corresponding genes were not part of the TB panel.
T24556 62385-62461 Sentence denotes The external labs reported 50 sequence variants in the remaining 29 samples.
T77202 62462-62534 Sentence denotes The TB panel detected 48 of these variants, giving a concordance of 96%.
T52127 62535-62667 Sentence denotes Our software did not detect 2 variants, but one variant was in the region of a gene (CEBPA) where the coverage depth was low (281X).
T96290 62668-62749 Sentence denotes When we examined this region in more detail, we were able to detect this variant.
T46857 62750-62869 Sentence denotes Including this variant, the concordance was 98%, suggesting excellent agreement between the TB and the TruSight panels.
T74065 62870-62882 Sentence denotes Conclusions:
T62459 62883-62982 Sentence denotes We have validated the TB myeloid panel for the detection of sequence variants in myeloid neoplasms.
T55972 62983-63075 Sentence denotes The assay has high accuracy and it can readily detect minor allele frequencies below the 5%.
T68383 63076-63149 Sentence denotes Myeloproliferative Neoplasia and their Relation with JAKV617F Mutation D.
T29066 63150-63169 Sentence denotes Aguilar León 1 , E.
T15592 63170-63193 Sentence denotes Martínez-Cordero 2 , G.
T99373 63194-63213 Sentence denotes Herrera Maya 1 , C.
T97140 63214-63467 Sentence denotes Lara Torres 3 1 Instituto Nacional de Ciencias Medicas y Nutrición "Salvador Zubirán," Mexico City, Mexico; 2 Instituto Nacional de Enfermedades Respiratorias, Mexico City, Mexico; 3 The American British Cowdray Medical Center, IAP, Mexico City, Mexico.
T43362 63468-63481 Sentence denotes Introduction:
T63716 63482-63559 Sentence denotes Cytokines play an important role controlling cell growth and immune response.
T22824 63560-63714 Sentence denotes Several of them function activating cell membrane associated receptors that rely on JAK2 to transduce signals and regulate transcription of various genes.
T30743 63715-63794 Sentence denotes In mammals JAK family of kinases have four members (JAK1, JAK2, JAK3 and TYK2).
T13351 63795-63952 Sentence denotes JAK2 is a tyrosine kinase that promotes growth of myeloid cell lines through constitutive activation of pathways related with JAK2, STAT5, PI3K, ERK and AKT.
T58825 63953-64117 Sentence denotes JAKV617F mutation may be present in myeloproliferative neoplasia such as; polycythaemia vera (PV), essential thrombocythaemia (ET), and primary myelofibrosis (PMF).
T33098 64118-64264 Sentence denotes This suggest that the immunologic environment may be a factor influencing the evolution of myeloproliferative neoplasia, although little is known.
T59385 64265-64273 Sentence denotes Methods:
T74079 64274-64493 Sentence denotes The study included 9 healthy donors of bone marrow stem cells (controls) and 21 pared samples (bone marrow and peripheral blood) of patients with diagnosis of myeloproliferative neoplasia according to WHO 2014 criteria.
T85554 64494-64634 Sentence denotes Extraction of mRNA was performed with RNA FFPE extraction kit (Promega) and cDNA synthetized with Omniscript Reverse Transcriptase (Qiagen).
T870 64635-64730 Sentence denotes Primers were design to amplify IL-17, TNF--18, IL-23 e IFNbased on sequences from ensemble.org.
T53815 64731-64866 Sentence denotes Assessment of mRNA expression using Quanti Tect Master Mix Kit SYBR Green (Qiagen) and copy number/ul determined using standard curves.
T84528 64867-64998 Sentence denotes JAK2V617F mutation was determined using PCR with melting curve analysis and conventional PCR with restriction endonuclease enzymes.
T71519 64999-65097 Sentence denotes Melting Mean and standard deviation were determined and the results compared using T-Student test.
T78455 65098-65106 Sentence denotes Results:
T8941 65107-65203 Sentence denotes The distribution of diagnosis was as follows: PV (28%, n=6), ET (58%, n=12), and PMF (14%, n=3).
T62668 65204-65319 Sentence denotes The age of the patients range from 56 to 63 years-old, without differences according to diagnostic group or gender.
T48981 65320-65400 Sentence denotes JAKV617F mutation was detected in 100% of PMF, 50% of ET and 50% of PV patients.
T62960 65401-65546 Sentence denotes The cytokine expression analysis of PV, ET and PMF showed no statistically significant differences between groups or sample types (BM versus PB).
T68314 65547-65684 Sentence denotes However, we identified high levels of IL-4 (both BM and PB) for the three conditions, as well as increased expression of IL-23 and IL-17.
T44702 65685-65831 Sentence denotes Of special interest, dividing the cases by JAK2V617F mutation status we found significant difference in the level of expression of TNF-Conclusion:
T60624 65832-66011 Sentence denotes We identified a possible role of T-reg (Th17/IL-17 expressing) lymphocytes on bcr-abl negative MPN as well as differences in cytokine expression according to JAK2 mutation status.
T40460 66012-66180 Sentence denotes PCR with melting curve analysis had a higher diagnostic yield for JAK2V617K mutation determination compared to PCR followed by digestion with restriction endonucleases.
T61350 66181-66183 Sentence denotes Y.
T90772 66184-66211 Sentence denotes Linnik, F.B. de Abreu, J.D.
T51669 66212-66226 Sentence denotes Peterson, S.A.
T94147 66227-66237 Sentence denotes Turner, P.
T12025 66238-66248 Sentence denotes Kaur, D.L.
T21762 66249-66263 Sentence denotes Ornstein, G.J.
T67048 66264-66279 Sentence denotes Tsongalis, E.Y.
T79951 66280-66391 Sentence denotes Loo Dartmouth Hitchcock Medical Center, Norris Cotton Cancer Center and Geisel School of Medicine, Lebanon, NH.
T93605 66392-66405 Sentence denotes Introduction:
T93521 66406-66550 Sentence denotes The cohesin protein complex forms a ring structure around sister chromatids and helps regulate chromosome separation during meiosis and mitosis.
T23416 66551-66714 Sentence denotes Genes belonging to this complex are recurrently mutated in myeloid neoplasms; but reported disease subtype associations have been varied and sometimes conflicting.
T69560 66715-66876 Sentence denotes We describe the cohesion-complex variants detected in all bone marrow biopsies at a single institution, concerning for a myeloid neoplasm during specimen triage.
T99405 66877-66885 Sentence denotes Methods:
T32504 66886-67027 Sentence denotes All newly diagnosed myeloid neoplasm cases were evaluated using the 54 gene Illumina TruSight Myeloid Sequencing Panel on the MiSeq platform.
T83600 67028-67150 Sentence denotes Basecalling and sequence alignment were performed using the MiSeq Reporter Software and analyzed using VariantStudio v2.1.
T37583 67151-67159 Sentence denotes Results:
T2398 67160-67432 Sentence denotes Seventy-nine cases were evaluated, and variants in cohesion-complex genes were seen in 6/14 (43%) secondary AML (s-AML) and 4/21 (19%) of morphologically non-neoplastic cases (NN); STAG2 (s-AML, n=6; NN, n=3), RAD21 (s-AML, n=1; NN, n=1), and SMC3 were found (s-AML, n=1).
T3193 67433-67552 Sentence denotes No cohesion-complex mutations were seen in our cohort of primary AML (n=16), MDS (n=19), MPN (n=4) , and MDS/MPN (n=5).
T10428 67553-67837 Sentence denotes For STAG2, variants were identified across the transcript; one case had a frame-shift with early termination and loss of the SCD region and remaining transcript, the remaining variants were mostly missense mutations involving non-named/non-conserved regions throughout the transcript.
T40946 67838-67923 Sentence denotes Both RAD21 missense variants occurred in the conserved C-terminalSMC1 binding domain.
T22484 67924-67989 Sentence denotes The sole SMC3 missense variant involved the protein hinge region.
T60106 67990-68069 Sentence denotes Two of six s-AML cases had co-mutation of two different cohesion-complex genes.
T96936 68070-68217 Sentence denotes TET2, ASXL1, and RUNX1 mutations were the three most frequently affected genes in our cohort seen in 27.8%, 20.2%, and 17.7% of cases respectively.
T70002 68218-68325 Sentence denotes RUNX1 was most frequently co-mutated with cohesin-complex genes; in 5/6 s-AML (83%) and 1/4 NN (25%) cases.
T65349 68326-68427 Sentence denotes Co-mutation with TET2 was seen in 3/6 s-AML cases and 1/4 NN cases, and withASXL1 in 3/6 s-AML cases.
T42771 68428-68440 Sentence denotes Conclusions:
T39731 68441-68558 Sentence denotes Our cohort shows cohesin mutations most frequently occurring in s-AML, and often in association with RUNX1 mutations.
T79524 68559-68830 Sentence denotes Prior reports have indicated that cohesion-complex genes were associated with denovo AML as well as with NPM1; but our findings refute this idea, and the discrepancy may be due to the fact that many of these studies included cohorts classified by the old FAB methodology.
T91377 68831-68976 Sentence denotes Our findings support the association of cohesion-complex mutations with s-AML and high-grade MDS, which has also been reported in the literature.
T91009 68977-69096 Sentence denotes Although no variants were detected in our MDS cohort, this may be due to a relatively low sampling of high-grade cases.
T16666 69097-69099 Sentence denotes Y.
T42322 69100-69107 Sentence denotes Kim, H.
T99874 69108-69116 Sentence denotes Kang, Y.
T32122 69117-69124 Sentence denotes Han, S.
T93571 69125-69132 Sentence denotes Kim, H.
T34608 69133-69141 Sentence denotes Yang, H.
T94731 69142-69150 Sentence denotes Yoon, T.
T54674 69151-69159 Sentence denotes Park, H.
T93069 69160-69230 Sentence denotes Lee Kyung Hee University School of Medicine, Seoul, Republic of Korea.
T82474 69231-69244 Sentence denotes Introduction:
T33342 69245-69346 Sentence denotes The t(3;12)(q26;p13) is a recurrent translocation in hematologic malignancy however, rarely observed.
T28352 69347-69546 Sentence denotes The ETV6 gene is located on 12q13 and the 33 translocation partner gene are reported, and the most frequent partner band is 3q26 where the MECOM (complex locus of two genes EVI1 and MDS1) is located.
T33706 69547-69724 Sentence denotes The fusion transcript of ETV6 to MECOM as a result of t(3;12)(q26;p13) is related with development and progression of malignancy by promotion of proliferative capacity of cells.
T63538 69725-69974 Sentence denotes Most cases of t(3;12)(q26;p13) were confirmed with fluorescence in situ hybridization (FISH) and/or RT-PCR however, to the best of our knowledge, only one case proved the fusion site with sequencing and with hemi-nested PCR using two set of primers.
T96316 69975-70123 Sentence denotes In this case, we describe a case of therapy related AML with t(3;12)(q26;p13)ETV6/MECOM, which was confirmed by sequencing using single set primers.
T82824 70124-70196 Sentence denotes A 60-year-old man visited our hospital due to a cough, fever and sputum.
T61690 70197-70324 Sentence denotes He had osteosarcoma, and was treated with four cycles of cisplatin and doxorubicin, and two cycles of ifosfamide and cisplatin.
T98049 70325-70480 Sentence denotes Initial complete blood count showed pancytopenia; hemoglobin level of 6.0 g/dL, white blood cell count of 4.45× 10 9 /L, and platelet count of 68× 10 9 /L.
T44727 70481-70566 Sentence denotes The peripheral blood film smear showed 16% of immature cells among white blood cells.
T98739 70567-70692 Sentence denotes Bone marrow (BM) aspiration showed 26% of leukemic blasts and 36.8% of monocytes, and focal fibrosis were observed in biopsy.
T16325 70693-70807 Sentence denotes Metaphase cytogenetic analysis using BM aspirate revealed 46, XY, t(3;12)(q26;p13),-7 in 16 of 20 metaphase cells.
T21812 70808-70843 Sentence denotes We suspected the ETV6/MECOM fusion.
T35582 70844-70852 Sentence denotes Methods:
T8664 70853-70996 Sentence denotes To further characterize, RT-PCR was performed with primer set; ETV6 F1 (5'-CCTCCAGAGAGCCCAGTGCCGAGT-3') and EVI1R (5'-CTGATCATAACAGCCAGCGA-3').
T72978 70997-71049 Sentence denotes The second PCR was performed using the same primers.
T20314 71050-71264 Sentence denotes The PCR products were purified and sequenced to further characterize the fusion product using BigDye (R) Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and ABI PRISM 3730XL Analyzer (Applied Biosystems).
T22377 71265-71273 Sentence denotes Results:
T8444 71274-71431 Sentence denotes The RT-PCR showed 212 bp size band, and the sequencing analysis using RT-PCR product showed a fusion between exon 2 of the ETV6 gene and exon 2 of EVI1 gene.
T33027 71432-71469 Sentence denotes The patient was diagnosed with t-AML.
T34193 71470-71675 Sentence denotes A follow up BM study was performed after induction therapy with standard-dose cytarabine and idarubicin however, 38.4% of cells were leukemic blasts and ETV6/MECOM fusion gene was still detected in RT-PCR.
T90693 71676-71688 Sentence denotes Conclusions:
T11002 71689-71835 Sentence denotes Our case of t-AML withETV6/MECOM clearly showed the fusion site with simplified RT-PCR, and the patient showed poor response to induction therapy.
T63670 71836-71959 Sentence denotes Further studies will be necessary for better diagnosis and treatment of ETV6/MECOM fusion related hematologic malignancies.
T36870 71960-71962 Sentence denotes Y.
T99542 71963-71970 Sentence denotes Cho, S.
T32143 71971-71979 Sentence denotes Jang, E.
T2663 71980-71987 Sentence denotes Seo, J.
T83724 71988-71995 Sentence denotes Lee, J.
T5553 71996-72003 Sentence denotes Lee, K.
T82482 72004-72011 Sentence denotes Lee, C.
T15810 72012-72108 Sentence denotes Park University of Ulsan, College of Medicine and Asan Medical Center, Seoul, Republic of Korea.
T93299 72109-72280 Sentence denotes Introduction: FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) mutations are associated with poor prognosis in patients with acute myeloid leukemia (AML).
T52625 72281-72447 Sentence denotes We performed an analysis of the clinical and prognostic impact of FLT3-ITD mutations according to mutant to wild-type ratio (MR) and duplicated fragment length (DFL).
T22938 72448-72456 Sentence denotes Methods:
T95846 72457-72536 Sentence denotes A total of 122 patients with AML harboring FLT3-ITD were enrolled in the study.
T92171 72537-72604 Sentence denotes GeneScan followed by sequencing was used to analyze the MR and DFL.
T69208 72605-72686 Sentence denotes The MR was determined by dividing the peak area of mutation by that of wild-type.
T47106 72687-72695 Sentence denotes Results:
T10201 72696-72789 Sentence denotes Among 744 consecutive AML patients, FLT3-ITD mutations were observed in 122 (16.4%) patients.
T41405 72790-72887 Sentence denotes FLT3-ITD was found equally in both the FAB classification and World Health Organization subtypes.
T82153 72888-72936 Sentence denotes The MR ranged from 0.01 to 23.31 (median 0.545).
T15249 72937-72981 Sentence denotes The DFL ranged from 15 to 173 (median 45.5).
T27226 72982-73072 Sentence denotes The distribution of these parameters was not different between FAB M3 and non-M3 patients.
T39586 73073-73181 Sentence denotes In the non-M3 group (n=10 significantly higher leukocyte count (P=0.005) and lower platelet count (P=0.009).
T7311 73182-73475 Sentence denotes There were no significant differences with respect to AML subtypes, cytogenetic risk groups, nucleophosmin (NPM1), CCAAT-enhancer binding protein, alpha (CEBPA), jmd.amjpathol.org ■ The Journal of Molecular Diagnostics and mixed lineage leukemia-partial tandem duplication (MLL-PTD) mutations.
T24284 73476-73608 Sentence denotes We observed worse overall survival or relapse-free survival for patients with MR 1.0 or higher (P=0.005 and P<0.0001, respectively).
T99067 73609-73681 Sentence denotes However, the DFL did not provide any additional prognostic significance.
T78531 73682-73787 Sentence denotes In the M3 group (n=17), neither the MR nor the DFS showed clinical difference or prognostic significance.
T61003 73788-73912 Sentence denotes The MR of patients with hematological relapse was significantly higher than that of patients at initial diagnosis (P<0.001).
T76689 73913-74048 Sentence denotes Conclusions: DFL did not confer clinical significance, whereas the MR conferred significant prognostic difference in patients with AML.
T68172 74049-74165 Sentence denotes Therefore, the MR is helpful for performing more detailed risk stratification in patients with FLT3-ITDpositive AML.
T63280 74166-74264 Sentence denotes A higher MR at relapse may indicate selective survival of a FLT3-ITDpositive chemoresistant clone.
T1411 74265-74278 Sentence denotes Introduction:
T82364 74279-74372 Sentence denotes Ph-like ALL is a subtype of B-ALL associated with poor outcomes in children and young adults.
T718 74373-74491 Sentence denotes These patients have a Ph-like gene expression profile similar to Ph+ ALL, but lack the characteristic BCR-ABL1 fusion.
T71423 74492-74662 Sentence denotes Additional chromosome fusions have recently been identified in Ph-like patients ALL that respond to ABL class tyrosine kinase inhibitors in vitro analogously to BCR-ALB1.
T29367 74663-74901 Sentence denotes These clinically actionable fusions are currently being detected using combined methods of multiplex PCR, kinome capture, or full RNA sequencing, but these assays are laborious, expensive, and time consuming to be useful on a large scale.
T34935 74902-75054 Sentence denotes We report the validation of two additional technologies that are cost effective, accurate, and faster for identifying both known and novel gene fusions.
T90495 75055-75063 Sentence denotes Methods:
T27391 75064-75227 Sentence denotes Two technologies were assessed across three laboratories using retrospective samples from consented patients enrolled on Children's Oncology Group clinical trials.
T8518 75228-75546 Sentence denotes The Nanostring nCounter Leukemia Fusion Gene Expression targeted assay and the Archer FusionPlex comprehensive fusion detection assay were used at both Nationwide Children's Hospital (NCH) and Children's Hospital of Philadelphia (CHOP), whereas Brigham and Women's Hospital (BWH) used the Nanostring assay exclusively.
T74968 75547-75721 Sentence denotes The Nanostring assay uses six fluorescent labeled RNA molecules with barcodes bound to target-specific oligonucleotide probes to potentially detect 972 sequences of interest.
T53635 75722-75924 Sentence denotes The Archer assay uses an anchored multiplex technology with primers flanking genes involved in known leukemia fusions and next-generation sequencing (NGS) to detect known and unknown 5' and 3' partners.
T3460 75925-75933 Sentence denotes Results:
T91454 75934-76123 Sentence denotes Pilot testing of Ph-like ALL cases with and known genomic lesions using the Nanostring assay identified all but one case 46/47 (97.9%) with ABL class, JAK2, NTRK3 or TSLP fusions correctly.
T80010 76124-76276 Sentence denotes Testing was performed in parallel at NCH and Brigham and Women's Hospital with 26/27 cases identified correctly and 27/27 cases correctly, respectively.
T53365 76277-76440 Sentence denotes For comparison, a panel of 29 Ph-like ALL cases with defined genetic lesions was tested in parallel at NCH and CHOP, with 27/29 (93%) fusions identified correctly.
T89880 76441-76453 Sentence denotes Conclusions:
T77918 76454-76622 Sentence denotes The Nanostring nCounter Leukemia Fusion Gene Expression assay performs well and is a fast and efficient method for detecting currently known Ph-like fusion transcripts.
T66889 76623-76768 Sentence denotes The Archer FusionPlex assay is more expensive due to its NGS requirement, but is good for identifying novel fusions involving known kinase genes.
T47746 76769-76993 Sentence denotes Whereas neither assay has the capacity of unbiased RNA sequencing for discovering alterations with novel kinases, both appear to be promising platforms for determining the presence of known, targetable fusions in Phlike ALL.
T75387 76994-76996 Sentence denotes V.
T27684 76997-77010 Sentence denotes Ortega 1 , C.
T17642 77011-77026 Sentence denotes Mendiola 1 , M.
T30482 77027-77040 Sentence denotes Khalil 2 , Y.
T64852 77041-77052 Sentence denotes Qian 2 , G.
T1652 77053-77192 Sentence denotes Velagaleti 1 1 University of Texas Health Science Center San Antonio, San Antonio, TX; 2 University of Texas Medical Branch, Galveston, TX.
T17222 77193-77206 Sentence denotes Introduction:
T15896 77207-77370 Sentence denotes Mantle cell lymphoma (MCL) is a lymphoid tumor derived from naïve CD5+ cells with the cytogenetic hallmark t(11;14) resulting in over-expression of the CCND1 gene.
T11045 77371-77527 Sentence denotes The presence of multiple chromosome abnormalities in addition to t(11;14) is known to be associated with blastoid variants (BMCL) and poor prognosis in MCL.
T64964 77528-77668 Sentence denotes High proportion of these tumors also show deletion of chromosome 11q22-23 or loss of heterozygosity; a region where the ATM gene is located.
T34122 77669-77894 Sentence denotes MCL tumors with bi-allelic ATM inactivation show significantly higher chromosome imbalances compared to MCLs with wild-type ATM alleles suggesting that loss of ATM alleles increases chromosomal instability of the tumor cells.
T15898 77895-77903 Sentence denotes Methods:
T78879 77904-78094 Sentence denotes We report a unique case of BMCL in a 52-year-old male who presented with worsening dyspnea, fever/chills, diffuse lymphadenopathy, splenomegaly and leukocytosis with blasts per differential.
T38531 78095-78205 Sentence denotes CT scan demonstrated extensive retroperitoneal, pelvic and inguinal lymphadenopathy with massive splenomegaly.
T17152 78206-78411 Sentence denotes Peripheral blood and bone marrow morphological, immunohistochemical and flow cytometry studies and bone marrow chromosome, FISH and high resolution microarray studies were performed using standard methods.
T70063 78412-78420 Sentence denotes Results:
T84501 78421-78505 Sentence denotes The blood smear showed leukocytosis due to the presence of "blasts" appearing cells.
T44377 78506-78577 Sentence denotes Bone marrow aspirate showed about 40% abnormal "blast" appearing cells.
T24202 78578-78740 Sentence denotes Bone marrow biopsy revealed remarkable lymphoid infiltrate in interstitial pattern and large lymphoid aggregates, contributing to about 40% of entire cellularity.
T32315 78741-78809 Sentence denotes The abnormal cells were immunoreactive to CD20, PAX-5 and Cyclin D1.
T70375 78810-78843 Sentence denotes Scattered were a few CD3 T-cells.
T43907 78844-79098 Sentence denotes Flow cytometry from the bone marrow aspirate detected a population (about 50% of total events) of lambda monoclonal B-cell population expressing CD19, CD22, CD20, and FMC-7 with coexpression of CD5, but were negative for CD10, CD200, CD43, CD34 and CD38.
T70747 79099-79159 Sentence denotes Based on these results, the patient was diagnosed with BMCL.
T61730 79160-79350 Sentence denotes Conventional cytogenetic analysis showed multiple chromosome abnormalities including t (11;14) and, contrary to published reports, bi-allelic amplification of ATM by FISH and SNP microarray.
T7773 79351-79580 Sentence denotes Apart from several characteristic gains, losses and regions of LOH, SNP microarray also showed high complexity abnormalities suggestive of chromoanagenesis involving chromosomes 7 and 11, especially involving the ATM gene region.
T35195 79581-79593 Sentence denotes Conclusions:
T21677 79594-79690 Sentence denotes Although Bi-allelic ATM mutations are common, gene amplification has never been reported in MCL.
T36092 79691-79949 Sentence denotes Focal chromothripsis as a mechanism of inactivating tumor suppressor genes was postulated in Retinoblastoma and we propose that chromoanagenesis inactivated both ATM alleles in our patient leading to multiple chromosome abnormalities and development of BMCL.
T77089 79950-80019 Sentence denotes Droplet PCR for the Molecular Assessment of Acute Myeloid Leukemia Q.
T62587 80020-80027 Sentence denotes Wei, N.
T22492 80028-80039 Sentence denotes Miltgen, M.
T73785 80040-80088 Sentence denotes Lovell Children's Hospital Colorado, Aurora, CO.
T53638 80089-80102 Sentence denotes Introduction:
T34671 80103-80194 Sentence denotes Library preparation methods are the backbone of any next-generation sequencing (NGS) assay.
T17051 80195-80385 Sentence denotes Specifically with targeted gene panels, the process of generating a library relies on multiplex primer-based enrichment chemistries to generate hundreds of discreet amplicons simultaneously.
T64683 80386-80551 Sentence denotes Currently, a handful of commercially available library preparation kits provide mutational analysis of acute myeloid leukemia (AML)-specific molecular abnormalities.
T84628 80552-80746 Sentence denotes Whereas targeted panel library preparation paired with next-generation sequencing (NGS) technologies have emerged as a powerful duo in the clinical setting, these methods have their limitations.
T72861 80747-80886 Sentence denotes Not surprisingly, it is difficult to optimize or "fine tune" the amplification efficiency for individual amplicons in multiplex PCR assays.
T16756 80887-81011 Sentence denotes Single molecule droplet PCR is a robust methodology for generating NGS libraries with superior amplicon coverage uniformity.
T72086 81012-81020 Sentence denotes Methods:
T26579 81021-81419 Sentence denotes Fifty-one bone marrow samples previously characterized with NGS methods from patients with acute myeloid leukemia/myeloproliferative neoplasm were compared in a double blind fashion to the results obtained using an AML gene panel with 548 amplicons covering clinical relevant regions in 49 genes using single molecule droplet PCR (ThunderBolts Myeloid Panel, RainDance Technologies; Billerica, MA).
T99940 81420-81493 Sentence denotes Sequencing was performed using a MiSeq bench top sequencer (Illumina Inc.
T59258 81494-81641 Sentence denotes San Diego, CA), and bioinformatic analysis was perform using NextGENe 2 nd generation sequence analysis software (SoftGenetics; State College, PA).
T3078 81642-81650 Sentence denotes Results:
T13974 81651-81776 Sentence denotes The panel successfully called all single nucleotide variants (SNV) and small insertions/deletions (indels) in all 51 samples.
T90239 81777-81936 Sentence denotes FLT3-internal tandem duplications were not initially detected with NextGENe software and an indel detection algorithm is needed for their successful detection.
T48880 81937-82027 Sentence denotes Both mean and median amplicon coverage (read depth) of CEBPA gene was greater than 2,500X.
T14386 82028-82162 Sentence denotes Dilution of AML patient DNA showed this panel could detect an allelic frequency down to 5% with a high level of assay reproducibility.
T31222 82163-82174 Sentence denotes Conclusion:
T51077 82175-82569 Sentence denotes Utilizing RainDance's patented droplet technology to ensure single occupancy of DNA templates targeted enrichment allows for the detection of clinically relevant somatic mutations using a simplified workflow with a 7-day turn-around time, requires only 75 ng of patient DNA, and results in superior coverage uniformity, especially in difficult to amplify GC-rich regions such as the CEBPA gene.
T86629 82570-82593 Sentence denotes Bone Marrow Biopsies L.
T71190 82594-82596 Sentence denotes N.
T59587 82597-82622 Sentence denotes Toth, F.B. de Abreu, J.D.
T92645 82623-82637 Sentence denotes Peterson, S.A.
T88744 82638-82648 Sentence denotes Turner, P.
T5766 82649-82659 Sentence denotes Kaur, D.L.
T46106 82660-82674 Sentence denotes Ornstein, G.J.
T81413 82675-82690 Sentence denotes Tsongalis, E.Y.
T66859 82691-82802 Sentence denotes Loo Dartmouth Hitchcock Medical Center, Norris Cotton Cancer Center and Geisel School of Medicine, Lebanon, NH.
T51269 82803-82816 Sentence denotes Introduction:
T71818 82817-83060 Sentence denotes The recently described acquisition of somatic mutations in the absence of abnormal cell counts or dysplasia has been termed Clonal Hematopoiesis of Indeterminate Potential (CHIP), and can reportedly be found in ~10% of persons >65 year of age.
T4237 83061-83169 Sentence denotes Being asymptomatic, these patients would theoretically never be identified as they would not require biopsy.
T40780 83170-83395 Sentence denotes Here, we characterize the cases of clonal hematopoiesis identified in patients who underwent bone marrow biopsy for workup of potential myeloid neoplasm, but were ultimately found to be morphologically non-neoplastic samples.
T38393 83396-83404 Sentence denotes Methods:
T6827 83405-83700 Sentence denotes All cases of suspected myeloid neoplasm during initial bone marrow triage were evaluated using the 54 gene Illumina TruSight Myeloid Sequencing Panel on the MiSeq platform.Base-calling and sequence alignment were performed using the MiSeq Reporter Software and analyzed using VariantStudio v2.1.
T32988 83701-83709 Sentence denotes Results:
T23726 83710-83851 Sentence denotes Of the 79 evaluated cases (36 F, 43 M); 21 biopsies (12 F, 9 M) did not show morphologic criteria for a diagnosis of dysplasia or malignancy.
T29205 83852-84026 Sentence denotes Ten of these 21 cases (~48%) carried at least one mutation in a gene recurrently mutated in myeloid neoplasms (mean 1.8/case, range 1-4), with 16 different variants detected.
T33066 84027-84095 Sentence denotes The average age of those with detected variants was 48 years (range:
T1116 84096-84142 Sentence denotes 17 to 76); all had normal cytogenetic studies.
T39886 84143-84333 Sentence denotes The most frequently reported gene was TET2 (n=4 cases) followed by STAG2 (n=3); other mutations including RUNX1, CUX1, ASXL1, EZH2, PHF6, ZRSR4, MPL, and RAD21 occurred at lower frequencies.
T85936 84334-84600 Sentence denotes All 10 of the ten cases with an identifiable clonal variant had clinical resolution of the issue prompting bone marrow biopsy, either with spontaneous resolution of the issue prompting biopsy (n=2) or identification of a separate exogenous cause for the issue (n=8).
T90683 84601-84722 Sentence denotes Only one of the 11 negative cases continued to have persistent cytoses of undetermined etiology at nine months follow-up.
T19095 84723-84734 Sentence denotes Conclusion:
T93837 84735-84980 Sentence denotes About a quarter of all marrows concerning for a myeloid neoplasm at initial triage were ultimately given a non-neoplastic morphologic diagnosis, and nearly half of these cases showed evidence of clonal hematopoiesis of undetermined significance.
T56394 84981-85084 Sentence denotes Although there is some selection bias, the detection of clonal hematopoiesis was greater than expected.
T2336 85085-85255 Sentence denotes With a mean of about 1 year of clinical follow-up, the detected variants appear to be unrelated to the underlying etiology necessitating bone marrow biopsy in most cases.
T52249 85256-85475 Sentence denotes The long-term consequence of these variants is yet to be determined, but suggest that more extensive studies are necessary before the medical community can reliably determine clinically significant clonal hematopoiesis.
T71630 85476-85489 Sentence denotes Introduction:
T62374 85490-85706 Sentence denotes Whereas the JAK-2V617F allele has been implicated in several of the myeloproliferative disorders, a significant proportion of patients with primary myelofibrosis (MF), or thrombocythemia (ET) are JAK-2V617F negative.
T63042 85707-85863 Sentence denotes One of the factors implicated in these cases is the constitutive activation of JAK-STAT pathway through mutations in the thrombopoietin receptor (MPL) gene.
T15743 85864-86111 Sentence denotes Specifically, mutations at the W515 position are present in approximately 5% of patients with primary myelofibrosis (MF), and in 1% of patients with ET, whereas mutations at the S505 position are common in patients with hereditary thrombocythemia.
T91521 86112-86233 Sentence denotes Mutations of MPL at the S505 and W515 positions have been tentatively linked to severity of myeloproliferative disorders.
T25870 86234-86385 Sentence denotes For this reason, we analyzed MPL sequences of 8700 clinical specimens from JAK-2V617F negative patients suspected to have myeloproliferative disorders.
T39432 86386-86394 Sentence denotes Methods:
T49422 86395-86542 Sentence denotes Clinical specimens were obtained and DNA was sequenced from 8700 patients suspected of having myeloproliferative disorders over an 18 month period.
T57858 86543-86706 Sentence denotes Purified DNA was then amplified by PCR on the ABI 9700 Thermalcycler using primers covering a 667 bp region of MPL that contained both the S505 and W515 mutations.
T52575 86707-86839 Sentence denotes The amplified products were subsequently sequenced using on the ABI 3100 or3730 sequencer in both directions for the covered region.
T8250 86840-86906 Sentence denotes These data were analyzed for mutations at S505 and W515 positions.
T65885 86907-86915 Sentence denotes Results:
T93936 86916-87028 Sentence denotes Of the 8700 patients' DNA analyzed, 104 had identified mutations at S505, W515 or within in the adjacent region.
T66146 87029-87145 Sentence denotes The majority of individuals with a variant sequence carried the mutations in at the W515 position (n=85/104, 81.7%).
T88626 87146-87335 Sentence denotes The majority of these individuals carried the W515L mutation (n=67, 64.4%), with the remainder carrying W515K (n=11/104, 10.6 %), W515A (n=3, 2.9 %), W515S (n=3, 2.9%) and W515R (n=1, <1%).
T68377 87336-87467 Sentence denotes A smaller number of individuals (n=12/104, 11.5%) had mutations at the S505 position, with all of them carrying the S505N mutation.
T46793 87468-87631 Sentence denotes Lastly, a small subset of individuals (n=7/104, 6.7 %) carried previously identified variants of R514S (n=1, ~1%), R514K (1, %), V507E (n=1, ~1%) and 501A (n=2,%).
T63271 87632-87818 Sentence denotes Of greatest interest was identification of two novel indels at position 515 in two different patients: c.1543_1552delinsAAAA, p.W515_PdelinsKT and c.1543_1559delinsAAAACTGCCAC, p.W515FS.
T84682 87819-87830 Sentence denotes Conclusion:
T54364 87831-87948 Sentence denotes This study examines the MPL mutations in JAK-2V617F negative patients suspected to have myeloproliferative disorders.
T23250 87949-88138 Sentence denotes We identified 104 individuals carrying variant sequences in MPL, predominantly at positions W515 (81.7%) and S505 (11.6%) and in adjacent regions (6.7%) in a population of 8700 individuals.
T70695 88139-88252 Sentence denotes Importantly, we have identified two novel indels at position 515 and further studies needed for the significance.
T92249 88253-88257 Sentence denotes A.F.
T90747 88258-88272 Sentence denotes Brown 1,3 , T.
T34767 88273-88287 Sentence denotes Parnell 2 , P.
T93281 88288-88305 Sentence denotes Szankasi 3 , J.A.
T52407 88306-88323 Sentence denotes Schumacher 3 , W.
T113 88324-88335 Sentence denotes Shen 3 , K.
T44410 88336-88351 Sentence denotes Frizzell 3 , S.
T18765 88352-88369 Sentence denotes Sorrells 3 , J.L.
T84847 88370-88384 Sentence denotes Patel 1 , T.W.
T68556 88385-88527 Sentence denotes Kelley 1,3 1 University of Utah, Salt Lake City, UT; 2 Huntsman Cancer Institute, Salt Lake City, UT; 3 ARUP Laboratories, Salt Lake City, UT.
T98365 88528-88686 Sentence denotes Introduction: FLT3 internal tandem duplication (ITD) mutations are a marker of poor prognosis in cytogenetically normal acute myeloid leukemia (AML) patients.
T87095 88687-88754 Sentence denotes The overall mutation burden has additional prognostic significance.
T34681 88755-88929 Sentence denotes The FLT3-ITD allelic ratio (AR), derived from a PCR and capillary electrophoresis (CE)-based assay, is the current gold standard measurement of FLT3-ITD mutation frequencies.
T65579 88930-89088 Sentence denotes However, much of the testing of AML prognostic markers has moved away from single gene PCR/CE-type assays to next-generation sequencing (NGS) mutation panels.
T30924 89089-89202 Sentence denotes It is unclear if a FLT3-ITD variant allele frequency (VAF) from NGS is comparable to an AR, as currently defined.
T85386 89203-89283 Sentence denotes Here we sought to compare VAFs to ARs in a large set of FLT3-ITD positive cases.
T97415 89284-89292 Sentence denotes Methods:
T44568 89293-89347 Sentence denotes This study was approved by the University of Utah IRB.
T89875 89348-89441 Sentence denotes DNA was extracted from blood or bone marrow from patients with myeloid malignancies (n=1153).
T70451 89442-89593 Sentence denotes A NGS library was prepared and subjected to enrichment for genes recurrently mutated in myeloid malignancies using SureSelect (Agilent) capture probes.
T12308 89594-89692 Sentence denotes Sequencing was performed on Illumina platforms to identify cases that harbored FLT3-ITD mutations.
T11297 89693-89863 Sentence denotes The VAF of FLT3-ITDs and the insert size was evaluated using two informatics algorithms (Pindel and Scalpel) for detecting insertions and deletions (in/dels) in NGS data.
T49032 89864-89975 Sentence denotes FLT3-ITD positive cases were identified (n=63) and 47 were subsequently analyzed by CE and ARs were calculated.
T74758 89976-90111 Sentence denotes Among the 1153 total samples subjected to NGS, PCR/CE was performed on 77 consecutive cases to confirm that NGS did not miss FLT3-ITDs.
T74485 90112-90120 Sentence denotes Results:
T74929 90121-90222 Sentence denotes Initially using Pindel, we identified 63 cases that were positive for a total of 70 FLT3-ITDs by NGS.
T82568 90223-90284 Sentence denotes 21 of these cases were also subjected to analysis by Scalpel.
T45331 90285-90470 Sentence denotes Among the 77 consecutive cases analyzed by PCR/CE, 4 were FLT3-ITD positive (all confirmed by NGS with Pindel) and 73 were FLT3-ITD negative by PCR/CE (all also negative by NGS Pindel).
T30728 90471-90518 Sentence denotes ITD size by NGS and CE were in close agreement.
T8114 90519-90586 Sentence denotes All FLT3-ITD mutations detected by Pindel were confirmed by PCR/CE.
T12376 90587-90727 Sentence denotes However, Scalpel failed to detect 2 of 21 (n=9.5%) large (159 bp and 163 bp) FLT3-ITDs that were detected by Pindel and confirmed by PCR/CE.
T90781 90728-90846 Sentence denotes We observed that the NGS-derived VAFs from both Pindel and Scalpel were consistently lower than the corresponding ARs.
T74533 90847-90895 Sentence denotes This was the case for VAFs from both algorithms.
T22170 90896-91000 Sentence denotes Overall, Pindel showed better correlation with CE compared to Scalpel (r 2 =0.733 vs. 0.351; p=<0.0001).
T61652 91001-91129 Sentence denotes Conclusions: NGS is sensitive for the detection of FLT3-ITDs but the VAF may underestimate the overall FLT3-ITD mutation burden.
T48305 91130-91254 Sentence denotes We hypothesize that this may be due to loss of mutant reads in the informatics analysis, possibly during the alignment step.
T5165 91255-91268 Sentence denotes Introduction:
T21780 91269-91450 Sentence denotes Chromosomal abnormalities in myelodysplastic syndromes (MDS) and acute myeloid leukemias (AML) provide an important diagnostic and prognostic value, assisting in therapeutic choice.
T75834 91451-91651 Sentence denotes Unbalanced chromosomal abnormalities (chromosomes 5, 7, 8, 11, 13, 17, 20 and 21) are often related, but due to karyotype (KT) low resolution it may bypass clinically important smaller rearrangements.
T75685 91652-91746 Sentence denotes As a confirmation technique, FISH is performed for specific regions, however, it is expensive.
T84475 91747-91899 Sentence denotes MLPA, on the other hand, is less costly, evaluates more genomic regions and is performed with genomic DNA, simplifying biological material availability.
T56152 91900-92036 Sentence denotes The aim of this study was to correlate the results of unbalanced chromosomal abnormalities detection in MDS/AML patients by KT and MLPA.
T76638 92037-92045 Sentence denotes Methods:
T11744 92046-92102 Sentence denotes Twentynine MDS/AML patients were tested for KT and MLPA.
T28633 92103-92238 Sentence denotes G-banding KTs were performed in cultured cells, and divided in 3 subclasses: normal (n=5), 1-2 abnormalities (n=12) and complex (n=12).
T99811 92239-92324 Sentence denotes MLPA was performed with genomic bone marrow DNA using MRC-Holland kits P144 and P145.
T944 92325-92577 Sentence denotes Outcomes were classified in: i) "concordant", if MLPA detected KT abnormalities; ii) "partially concordant", if MLPA detected previous KT abnormalities and extra anomalies; iii) "discordant", if abnormalities detected by KT were not reproduced by MLPA.
T34349 92578-92586 Sentence denotes Results:
T42738 92587-92724 Sentence denotes Overall failure rate for MLPA was 20.68%, which may be explained by DNA heterogeneity, as failures correlate with low DNA quantification.
T41124 92725-92862 Sentence denotes Normal KT was discordant with MLPA in one case (20%), in which MLPA detected a deletion of RUNX1 gene (chromosome 21), confirmed by FISH.
T23873 92863-92990 Sentence denotes Alterations in regions -5; del5q, -7, del7q, +8, del11q, del13q, del17p, del20p, del20q and del21q were most commonly detected.
T59932 92991-93188 Sentence denotes For KTs with 1-2 abnormalities, MLPA was partially concordant in two cases (22.2%): (i) it revealed that the translocation t(6;7)(q25;q22) was unbalanced, as a deletion was detected in region 7q22.
T59749 93189-93281 Sentence denotes 1, and (ii) it showed an amplification in 11q23.3 in addition to the loss of del(5)(q13q31).
T6516 93282-93436 Sentence denotes In complex KTs, MLPA was partially concordant in 3 cases (42.8%), and discordant in one case (14.2%), in which a deletion of 20q was not detected by MLPA.
T72087 93437-93472 Sentence denotes Overall ratios of concordance were:
T18636 93473-93536 Sentence denotes 68.1% concordant, 22.7% partially concordant and 9% discordant.
T17377 93537-93771 Sentence denotes Conclusions: MLPA is a powerful and less expensive technique for the detection of high resolution genomic unbalanced abnormalities in MDS and AML patients, frequently adding more information to KT results, especially in complex cases.
T600 93772-93785 Sentence denotes Introduction:
T45110 93786-93960 Sentence denotes Acute lymphoblastic leukemia (ALL), the most common cancer in children, is a leading cause of cancer mortality among young adults, and portends a dismal prognosis for adults.
T76672 93961-94290 Sentence denotes Philadelphia-like (Ph-like) ALL is a recently described and high-risk subset constituting 10-15% of ALL whose hallmark features are 1) similarity by gene expression to BCR-ABL1+ ALL and 2) frequent activating ABLand JAK-family kinase fusions that are known or suspected to be responsive to FDAapproved tyrosine kinase inhibitors.
T36229 94291-94520 Sentence denotes However, current diagnostic algorithms for detecting these genetic lesions are multimodal, time-intensive, and not optimally designed to identify the diversity of fusions between kinases and partner genes present in this disease.
T23445 94521-94690 Sentence denotes Therefore, the development of a robust, timely, and efficient method for detecting known and novel actionable lesions in Ph-like ALL would address a major clinical need.
T77219 94691-94699 Sentence denotes Methods:
T47807 94700-95051 Sentence denotes In collaboration with ArcherDX, we designed the Heme Fusion Assay, a single-reaction RNA and DNA next-generation sequencing (NGS) assay targeting kinase fusions and point mutations known to jmd.amjpathol.org ■ The Journal of Molecular Diagnostics occur in Ph-like ALL, as well as genes important for the subclassification of ALL and myeloid neoplasms.
T38454 95052-95233 Sentence denotes This assay, composed of over 800 probes for loci in 58 genes, employs Anchored Multiplex PCR to target relevant kinase domains and identify fusions to known and novel partner genes.
T26267 95234-95424 Sentence denotes Fresh and archived tissue obtained from 25 B-ALL cases at MGH and DFCI/BCH were included in this study, using clinical molecular genetic data or transcriptome sequencing data as a benchmark.
T63242 95425-95632 Sentence denotes Sequencing libraries were constructed according to manufacturer specifications (Archer Universal RNA Reagent Kit v2 for Illumina-8), and 12-15 barcoded sample libraries were sequenced per Illumina MiSeq run.
T1051 95633-95712 Sentence denotes Novel fusion transcripts and gene expression findings were confirmed by RT-PCR.
T66010 95713-95721 Sentence denotes Results:
T44033 95722-95940 Sentence denotes In 25 cases of B-ALL with prior molecular characterization, the Heme Fusion Assay identified all known fusions (n=10, involving ABL1, CRLF2, JAK2, and PDGFRB) and point mutations (n=3, involving CRLF2, JAK2, and KRAS).
T33117 95941-96223 Sentence denotes Unexpected findings within this cohort included the identification and confirmation by RT-PCR of single cases revealing a novel TCF3-FLI1 fusion and markedly elevated PDGFRB mRNA expression without evidence of a fusion event, suggesting an alternate mechanism for kinase activation.
T55445 96224-96236 Sentence denotes Conclusions:
T99253 96237-96347 Sentence denotes Here we present a novel NGS assay designed for detection of kinase fusions and point mutations in Ph-like ALL.
T38476 96348-96499 Sentence denotes In validation cases benchmarked to RNA-seq or clinical molecular genetic data, the Heme Fusion Assay identified all previously detected kinase lesions.
T91589 96500-96608 Sentence denotes Novel findings within this cohort highlight the utility of this assay for targeted discovery in Ph-like ALL.
T35862 96609-96775 Sentence denotes Introduction: NPM1 mutation, typically involving a net insertion of four base pairs in exon 11, is a common molecular lesion in acute myeloid leukemia (AML) patients.
T30534 96776-96900 Sentence denotes Mutated NPM1 confers a favorable prognosis in cytogenetically normal AML patients who lack FLT3 internal tandem duplication.
T5872 96901-97072 Sentence denotes One-third to one-half of AML patients harbor an NPM1 mutation with the most common form being a TCTG insertion designated Type A that is seen in 80% of NPM1-mutated cases.
T93498 97073-97349 Sentence denotes Recent studies show that minimal residual disease (MRD) monitoring of AML patients after chemotherapy provides important prognostic information which is independent of other risk factors and may guide clinical decision making regarding hematopoietic stem cell transplantation.
T65730 97350-97461 Sentence denotes NPM1 mutation is a stable marker of disease in AML and represents a desirable target for MRD assay development.
T89733 97462-97562 Sentence denotes Real-time quantitative PCR is an established method well-suited for detection of low-level variants.
T4245 97563-97723 Sentence denotes Digital droplet PCR (ddPCR) is a relatively new technique that uses allele-specific PCR in single molecule oil-water emulsions to amplify target(s) of interest.
T99796 97724-97848 Sentence denotes Here we describe a comparison study intended to assess the feasibility of ddPCR in the detection of NPM1 Type A transcripts.
T29863 97849-97857 Sentence denotes Methods:
T87326 97858-97935 Sentence denotes This study was approved by the University of Utah Institutional Review Board.
T17724 97936-98066 Sentence denotes OCI-AML3 cells, expressing the NPM1 Type A mutation, were serially diluted 10-fold into Jurkat cells which express wild-type NPM1.
T19138 98067-98286 Sentence denotes RNA was isolated, reverse-transcribed into cDNA, and analyzed in a multiplex assay measuring NPM1 Type A with allele-specific primers and ABL1 transcripts with fluorescent-labeled probes for measuring product formation.
T40133 98287-98426 Sentence denotes For ddPCR, samples were analyzed on the RainDance RainDrop ddPCR system in singlet on two separate runs with varied reagent concentrations.
T77137 98427-98480 Sentence denotes Data was analyzed using RainDrop Analyst II software.
T18324 98481-98619 Sentence denotes For RT-qPCR, samples were analyzed on the Roche LightCycler 480 real-time PCR instrument and data analyzed using LightCycler 480 software.
T72505 98620-98628 Sentence denotes Results:
T49105 98629-98756 Sentence denotes We successfully detected NPM1 Type A transcripts in serial dilutions of OCI-AML3 cells using both RT-qPCR and ddPCR techniques.
T55713 98757-98873 Sentence denotes Preliminary results show that the sensitivity of RT-qPCR (0.01% to 0.001%) exceeds that of ddPCR by 1 log to 2 logs.
T41041 98874-98886 Sentence denotes Conclusions:
T90353 98887-99035 Sentence denotes Both RT-qPCR and digital droplet PCR are viable methods that may be used to detect NPM1 Type A transcripts in RNA samples derived from AML patients.
T77698 99036-99232 Sentence denotes Although ddPCR has been reported to be a highly sensitive technique for detection of low level variants, our preliminary experiments suggest that RT-qPCR remains the gold standard for sensitivity.
T67273 99233-99396 Sentence denotes Further optimization of the ddPCR technique is necessary to provide assay sensitivity at the level required for clinical MRD testing and these studies are ongoing.
T49174 99397-99410 Sentence denotes Introduction:
T50096 99411-99530 Sentence denotes Current comprehensive genetic testing of hematologic malignancies requires multiple testing strategies with high costs.
T89148 99531-99756 Sentence denotes Our previous proof-of-concept study demonstrated the feasibility of using the same NGS data to simultaneously detect both somatic mutations and copy number variants (CNVs) in the targeted genomic regions (Shen et al., 2016) .
T79506 99757-99874 Sentence denotes However, this targeted panel-based approach is limited in its ability to detect CNVs outside of the targeted regions.
T57220 99875-100006 Sentence denotes The limited number of SNPs in the targeted regions also prevented detection of copy number neutral loss of heterozygosity (CN-LOH).
T34861 100007-100209 Sentence denotes Here, we hypothesize that implementation of a genome-wide SNP sequencing backbone will allow for sensitive and comprehensive detection of genome-wide CNVs and CN-LOH by NGS for hematologic malignancies.
T83199 100210-100218 Sentence denotes Methods:
T74521 100219-100367 Sentence denotes This study was conducted in accordance with protocols approved by the University of Utah Institutional Review Board and the Declaration of Helsinki.
T9977 100368-100452 Sentence denotes Genomic DNA was prepared and sequenced as previously described (Shen et al., 2016) .
T70289 100453-100579 Sentence denotes For genome-wide CNV detection, we designed a SNP backbone with 22,762 SNP regions evenly distributed across the entire genome.
T37293 100580-100705 Sentence denotes These SNP regions were selected based on GC content close to 50%, unique mappability and population minor allele frequencies.
T37119 100706-100820 Sentence denotes The read depth of the SNP regions were normalized and converted to a log2 ratio as described in Shen et al., 2016.
T15375 100821-100964 Sentence denotes To detect CN-LOH, SNP genotypes and B allele frequencies were analyzed by UnifiedGenotyper (GATK version 3.4, Broad Institute, Cambridge, MA) .
T24636 100965-101154 Sentence denotes For detection of targeted somatic mutations, sixty-three frequently mutated genes with clinical significance in hematologic malignancies were sequenced simultaneously with the SNP backbone.
T64240 101155-101163 Sentence denotes Results:
T29515 101164-101338 Sentence denotes The normalized read depth of regions in the SNP backbone was highly consistent across different normal reference samples, which allowed for the detection of genome-wide CNVs.
T44266 101339-101399 Sentence denotes The B allele frequencies were accurate for CN-LOH detection.
T72663 101400-101556 Sentence denotes In the limited number of positive cases sequenced thus far, we have detected genome wide CNVs and CN-LOH by NGS in complete concordance with SNP microarray.
T36245 101557-101569 Sentence denotes Conclusions:
T33559 101570-101807 Sentence denotes Our on-going study suggests that the strategy of combining the SNP backbone and targeted mutation NGS panel allows for comprehensive genetic profiling of CNV, CN-LOH and somatic mutations in hematologic malignancies using a single assay.
T19314 101808-101821 Sentence denotes Introduction:
T93109 101822-102090 Sentence denotes Recent advancements of technologies including next-generation sequencing (NGS) have led to discovery of molecular pathogenesis of malignant diseases including hematologic malignancies, and these discoveries are now enabling the beginning of molecular targeted therapy.
T89941 102091-102329 Sentence denotes The MLL-rearranged fusion gene is among the main leukemogenic mutation which is found in both acute myeloid leukemia (AML) and acute lymphoblastic leukemia with a frequency of about 5-10%, and is associated with a poor clinical prognosis.
T80597 102330-102547 Sentence denotes Cooperative mutation study among MLL-rearranged leukemia based on NGS technology has mainly been conducted in Caucasian or western population, and has yet been performed in Asian ethnicity including Korean population.
T2276 102548-102556 Sentence denotes Methods:
T88579 102557-102713 Sentence denotes This study includes total of 24 MLLrearranged AML patients who visited two separate tertiary care hospitals between the period of January 2009 and May 2014.
T75299 102714-102836 Sentence denotes The number of each MLL fusion genes are as follows; MLL/MLLT3 n=12; MLL/MLLT4 n=6; MLL/ELL n=2; other MLLfusion genes n=4.
T41588 102837-103103 Sentence denotes Mutation profile study for 19 candidate genes for cooperative mutation (TET2, DNMT3A, IDH1, IDH2, NPM1, FLT3, CEBPA, ASXL1, BRAF, CBL, KIT, KRAS, NRAS, PTPN11, RUNX1, TP53 , WT1, SETD2, JAK2) was performed using Miseq sequencing equipment (Illumina, San Diego, CA).
T3512 103104-103112 Sentence denotes Results:
T67873 103113-103246 Sentence denotes Among the 24 MLL-rearranged patients, 7 patients (29.2%) had positive results for more than 1 gene mutations which were analyzed for.
T56173 103247-103386 Sentence denotes Positive gene mutations found were in the order of frequency; ASXL1 (n=4), FLT3 (n=2), CEBPA (n=2), KRAS (n=1), NRAS (n=1) andPTPN11 (n=1).
T55766 103387-103487 Sentence denotes Interestingly, 4 out of 6 patients harboring MLL/MLLT4 were found to have additional gene mutations.
T49566 103488-103595 Sentence denotes Two patients had multiple gene mutations whereas one patient had four gene mutations concurrently detected.
T3951 103596-103608 Sentence denotes Conclusions:
T70481 103609-103733 Sentence denotes This is the first cooperative mutation study on MLLrearranged AML patients of Asian ethnicity using targeted NGS technology.
T93319 103734-103982 Sentence denotes Despite small number of cases, a higher incidence of gene mutation among MLL/MLLT4 patient group (4/6) and relatively higher incidence of ASXL1mutation (4/24) being found could be carefully suspected as an ethnical difference in the disease of AML.
T19468 103983-104116 Sentence denotes Further study requires a larger number of MLL-rearranged MLL patients and additional mutation profile study with ethnical comparison.
T70817 104117-104137 Sentence denotes Resistance in CLL J.
T69523 104138-104145 Sentence denotes Lee, S.
T53737 104146-104156 Sentence denotes Sharma, N.
T6398 104157-104169 Sentence denotes Galanina, A.
T64144 104170-104177 Sentence denotes Guo, S.
T65028 104178-104187 Sentence denotes Kadri, C.
T43879 104188-104206 Sentence denotes Van Slambrouck, B.
T12103 104207-104215 Sentence denotes Long, W.
T14191 104216-104224 Sentence denotes Wang, M.
T28295 104225-104235 Sentence denotes Ming, L.V.
T97254 104236-104249 Sentence denotes Furtado, J.P.
T52109 104250-104259 Sentence denotes Segal, W.
T69975 104260-104269 Sentence denotes Stock, G.
T49039 104270-104286 Sentence denotes Venkataraman, W.
T58177 104287-104295 Sentence denotes Tang, P.
T4619 104296-104302 Sentence denotes Lu, Y.
T14332 104303-104343 Sentence denotes Wang University of Chicago, Chicago, IL.
T2293 104344-104357 Sentence denotes Introduction:
T2970 104358-104552 Sentence denotes Ibrutinib (ibr), a first-in-class Bruton tyrosine kinase (BTK) inhibitor, has demonstrated high response rates in both relapsed/refractory and treatment naïve chronic lymphocytic leukemia (CLL).
T2185 104553-104697 Sentence denotes However, about 25% of patients discontinue ibrutinib therapy at a median follow-up of 20 months, usually due to leukemia relapse or progression.
T24986 104698-104839 Sentence denotes Treatment options for these patients are limited and outcomes are dismal with mortality rate exceeding 75% and a median survival of 3 months.
T46452 104840-104935 Sentence denotes Our groups and others have identified the first mutation that drives ibr resistance, BTK C481S.
T66174 104936-105075 Sentence denotes We have also shown that the mechanism of resistance is through disruption of the covalent binding between ibr and BTK at the C481 residual.
T98522 105076-105220 Sentence denotes Recently, additional BTK mutations (C481F/Y/R, T474I, and L528W) associated with ibr resistance have also been reported in ibr refractory cases.
T65476 105221-105393 Sentence denotes All the BTK mutations identified so far are located within the kinase domain of the protein and are predicted to weaken or hinder ibr docking to the BTK ATP-binding pocket.
T8907 105394-105526 Sentence denotes Here we report a CLL patient who relapsed on ibr and evolved a structurally novel BTK mutation that is outside of the kinase domain.
T75283 105527-105535 Sentence denotes Methods:
T89465 105536-105666 Sentence denotes Serial samples were collected from a Richter transformed CLL patient who were treated with ibrutinib, responded and then relapsed.
T31829 105667-105787 Sentence denotes The samples were analyzed using Onco1K, a 1,200-gene next-gen sequencing panel with an average sequencing depth of 420x.
T62244 105788-105902 Sentence denotes The uncovered novel mutation was then validated with Sanger sequencing and characterized with structural modeling.
T86528 105903-106091 Sentence denotes The role of the mutation was further functionally defined with cell transfection followed by assays for cell-proliferation, BrdU-incorporation, and intracellular B-cell receptor signaling.
T74770 106092-106100 Sentence denotes Results:
T58716 106101-106191 Sentence denotes A structurally novel mutation of BTK was identified which was associated with CLL relapse.
T89766 106192-106343 Sentence denotes Functionally, cells carrying the novel mutant BTK show resistance to ibrutinib at both cellular and molecular levels to a similar extent as BTK C481S .
T67694 106344-106355 Sentence denotes Conclusion:
T17077 106356-106577 Sentence denotes Our study lends further insight into the diverse mechanisms of ibrutinib resistance that has important implications for the development of next-generation BTK inhibitors as well as mutation detection in relapsed patients.
T45224 106578-106582 Sentence denotes H25.
T81856 106583-106724 Sentence denotes Optimised, One-Day Hybridization-Based NGS Protocol Yields 1% Sensitivity in MPN Samples, as Quickly and Cost-Effectively as Multiplex PCR G.
T4173 106725-106739 Sentence denotes Speight 1 , E.
T78080 106740-106751 Sentence denotes Chin 2 , L.
T56363 106752-106768 Sentence denotes Georgieva 3 , D.
T66921 106769-106925 Sentence denotes Cook 3 1 Oxford Gene Technology, Begbroke, Oxford, England; 2 Oxford Gene Technology, Tarrytown, NY; 3 Oxford Gene Technology, Oxford, Oxfordshire, England.
T73328 106926-106939 Sentence denotes Introduction:
T13551 106940-107118 Sentence denotes Myeloproliferative neoplasms (MPNs) are a group of diseases that affect normal blood cell production in the bone marrow resulting in the overproduction of one or more cell types.
T84734 107119-107324 Sentence denotes The key MPN driver mutations involve the JAK2, MPL and CALR genes, namely JAK2 V617F, which has an occurrence of 50% to 98% depending on the MPN sub-type; JAK2 exon 12; MPL W515K/L; and CALR exon 9 indels.
T67189 107325-107490 Sentence denotes The SureSeq Core MPN Panel allows NGS-based detection of the above driver mutations and is designed for research into the diagnosis, aetiology and prognosis of MPNs.
T49443 107491-107646 Sentence denotes The aim of this study is to evaluate the SureSeq Core MPN Panel in conjunction with a new streamlined hybridisation-based NGS library preparation protocol.
T46240 107647-107855 Sentence denotes The protocol offers a similar turn-around time to amplicon-based protocols, without the associated disadvantages, such as PCR bias, allelic bias (indels) and drop-outs, as well as poor uniformity of coverage.
T68677 107856-107864 Sentence denotes Methods:
T83284 107865-108097 Sentence denotes The SureSeq Core MPN Panel was validated with the JAK2 V617F Genotyping Sensitivity Panel from the National Institute for Biological Standards and Control (NIBSC) to confirm the lower levels of analytical sensitivity and confidence.
T55111 108098-108301 Sentence denotes The panel was then used to confirm a broader set of variants in 14 clinical samples containing variants for each of the targeted regions, in particular the indels associated with exon 9 of the CALR gene.
T72506 108302-108349 Sentence denotes Sequencing was conducted on a MiSeq (Illumina).
T51363 108350-108358 Sentence denotes Results:
T92987 108359-108508 Sentence denotes The SureSeq Core MPN Panel was shown to accurately detect alleles down to 1% variant allele fraction (VAF) in JAK2 (V617F) at a read depth of >1000x.
T57748 108509-108725 Sentence denotes Analysis in clinical research samples has shown that the panel was able to reliably detect not only single nucleotide variants but also 5 bp insertions in JAK2 (exon 12) and deletions of up to 52 bp in CALR (exon 9).
T88925 108726-108856 Sentence denotes In this region, the uniformity of coverage of the panel allowed key CALR indels to be identified, facilitating MPN stratification.
T35374 108857-109121 Sentence denotes The introduction of an alternative enzymatic fragmentation step and a rapid hybridisation step of just 30 minutes has reduced the overall length of the protocol by almost 6 hours, offering a streamlined, single-day sequencing workflow from DNA sample to sequencer.
T47806 109122-109134 Sentence denotes Conclusions:
T19227 109135-109274 Sentence denotes To detect alleles that contribute only a small percentage of the reads at any locus, a highly uniform and sensitive enrichment is required.
T53862 109275-109433 Sentence denotes We have utilised an optimised 1-day protocol in combination with the SureSeq Core MPN Panel to reliably and routinely detect somatic mutations down to 1% VAF.
T25689 109434-109546 Sentence denotes Researchers can now generate hybridisation-quality NGS data as quickly and as cost-effectively as multiplex PCR.
T86173 109547-109549 Sentence denotes P.
T62567 109550-109560 Sentence denotes Velu, C.C.
T22120 109561-109571 Sentence denotes Hsiung, K.
T22333 109572-109584 Sentence denotes Salafian, S.
T91066 109585-109594 Sentence denotes Luger, A.
T4543 109595-109603 Sentence denotes Bagg, J.
T39940 109604-109661 Sentence denotes Morrissette University of Pennsylvania, Philadelphia, PA.
T40477 109662-109675 Sentence denotes Introduction:
T41062 109676-109844 Sentence denotes Routine use of clinical cancer next-generation sequencing (NGS) in diagnosis and disease monitoring has resulted in sequential mutation profiles of individual patients.
T40858 109845-109981 Sentence denotes Clonal myeloid malignancies such as AML are often characterized and treated based on specific mutation profiles identified at diagnosis.
T97679 109982-110102 Sentence denotes On NGS profiling at interval follow-ups, mutations identified at diagnosis may change, or shift, through disease course.
T85986 110103-110225 Sentence denotes Here we track the AML mutational landscape in the context of therapy, disease progression, clinical course, and pathology.
T37951 110226-110234 Sentence denotes Methods:
T71026 110235-110396 Sentence denotes Patients whose blood or marrow specimens were sequenced using the Penn hematological-NGS panel and found on at least 2 occasions to have mutations were included.
T31606 110397-110473 Sentence denotes An R script was written to track mutations and allele frequencies over time.
T52688 110474-110623 Sentence denotes These data were integrated with corresponding pathology and clinical data to compare mutation timelines to disease progression and therapy timelines.
T53833 110624-110675 Sentence denotes The institutional review board approved this study.
T5322 110676-110684 Sentence denotes Results:
T96434 110685-110828 Sentence denotes Review of clinical NGS data identified 92 AML patients with samples characterized as de novo, recurrent, transformed from MDS, or in remission.
T11108 110829-111000 Sentence denotes NGS data revealed high variability in mutation profiles between patients, but also specific types of mutational shifts between diagnosis, recurrence, and remission of AML.
T68584 111001-111061 Sentence denotes These included no change, gain, loss, and all new mutations.
T21968 111062-111142 Sentence denotes There were several mutations that shifted rarely or never during disease course.
T72343 111143-111267 Sentence denotes Tracking of allele frequencies (>10% change) revealed changes in populations of multiple subclones at different time points.
T35311 111268-111428 Sentence denotes At recurrence, many patients had increased allele frequencies in mutations that were previously present at low levels and/or displayed completely new mutations.
T23905 111429-111686 Sentence denotes Decrease in allele frequency or loss of NPM1 and FLT3 mutations found at diagnosis did not necessarily correlate with remission, as this was associated with the emergence of pathogenic previously low level or undetectable subclones with different mutations.
T4403 111687-111804 Sentence denotes Loss of FLT3 mutations was often associated with FLT3-inhibitor therapy and emergence of an NRAS mutation at relapse.
T77266 111805-111915 Sentence denotes In patients with an initial NPM1 mutation, half lost that mutation but had new NPM1 mutation(s) at recurrence.
T48765 111916-111928 Sentence denotes Conclusions:
T41330 111929-112114 Sentence denotes Monitoring of AML patients by NGS rather than single gene assays is crucial due to the frequent finding of mutational shift and emergence of new disease associated clones at recurrence.
T46124 112115-112219 Sentence denotes Diagnostic mutational profiles can change with divergent allele frequencies, suggesting multi-clonality.
T17341 112220-112295 Sentence denotes The mutations that rarely or never shift are early or initiating mutations.
T94870 112296-112457 Sentence denotes Replacement of FLT3 mutations with NRAS mutations in patients on FLT3 inhibitors is suggestive of AML dependence on mutations in these kinase-signaling pathways.
T38250 112458-112462 Sentence denotes H27.
T52282 112463-112569 Sentence denotes Analytical Evalutation and Applications of the nCounter Vantage RNA:Protein Immune Cell Profiling Panel N.
T43291 112570-112584 Sentence denotes Riccitelli, A.
T36702 112585-112594 Sentence denotes Beams, I.
T68959 112595-112605 Sentence denotes Summit, R.
T24979 112606-112637 Sentence denotes Pollner Genoptix, Carlsbad, CA.
T45910 112638-112651 Sentence denotes Introduction:
T75732 112652-112801 Sentence denotes The NanoString RNA:Protein panel offers the ability to simultaneously examine the expression of 770 RNA targets and 31 proteins in a single reaction.
T11150 112802-113031 Sentence denotes Herein, we examine the analytical validity of the RNA:Protein Immune panel in fresh and frozen human peripheral blood mononuclear cell (PBMC) isolates from healthy donors, characterized cell lines, and chemically-induced samples.
T97279 113032-113158 Sentence denotes Furthermore, we outline a data analysis strategy to maximize interpretable information from low input or poor quality samples.
T76488 113159-113327 Sentence denotes Overall, the RNA:Protein panel exhibits exceptional accuracy and precision, providing an unparalleled level of information simultaneously from precious patient samples.
T64993 113328-113573 Sentence denotes Methods: RNA and protein expressions of both fresh and frozen, as well as phytohaemagglutinin-induced and non-induced human PBMCs, commercial PBMCs, and cell lines were measured using the nCounter Vantage RNA:Protein Immune Cell Profiling panel.
T83528 113574-113714 Sentence denotes Protein fold-change levels observed with the RNA:Protein panel were correlated with flow cytometry measurements for confirmation of results.
T25806 113715-113822 Sentence denotes Correlations between RNA and protein of induced PBMCs were evaluated for consistency with established data.
T68097 113823-113907 Sentence denotes NanoString measurements were compared across multiple runs for precision assessment.
T1058 113908-114005 Sentence denotes A relative count system was established to allow for normalization of RNA and protein expression.
T87363 114006-114014 Sentence denotes Results:
T16900 114015-114128 Sentence denotes Robust correlations were observed between flow cytometry results and NanoString protein fold-change measurements.
T87618 114129-114273 Sentence denotes For induced PBMCs, protein expression by NanoString was increased for expected markers, and correlated well with the respective mRNA expression.
T78617 114274-114472 Sentence denotes Negative and positive control data (n>30) indicate a high degree of reproducibility for the NanoString protein workflow while maintaining exceptional accuracy across independent sample preparations.
T47905 114473-114485 Sentence denotes Conclusions:
T25081 114486-114651 Sentence denotes By combining RNA and protein analysis into one straightforward workflow, the NanoString RNA:Protein panel maximizes the utility of frequently limited sample amounts.
T60732 114652-114795 Sentence denotes Furthermore, robust detection of protein targets was observed for both fresh and frozen PBMC sample types at clinically relevant sample inputs.
T92830 114796-115185 Sentence denotes Whereas the single-cell resolution of flow cytometry will remain critical in many applications, the ability to multiplex over 30 protein targets, as well as hundreds of mRNA analytes, from a single blood draw using the NanoString RNA:Protein panel offers tremendous advantages to researchers and clinicians looking to obtain actionable data and patients seeking to minimize medical visits.
T21538 115186-115251 Sentence denotes Lymphoproliferative Disorders by Massively Parallel Sequencing P.
T44486 115252-115267 Sentence denotes Szankasi 1 , A.
T39330 115268-115282 Sentence denotes Bolia 1 , E.P.
T12841 115283-115295 Sentence denotes Gee 1 , J.A.
T4671 115296-115315 Sentence denotes Schumacher 1 , J.L.
T32550 115316-115330 Sentence denotes Patel 2 , T.W.
T93920 115331-115422 Sentence denotes Kelley 2 1 ARUP Laboratories, Salt Lake City, UT; 2 University of Utah, Salt Lake City, UT.
T3865 115423-115436 Sentence denotes Introduction:
T76146 115437-115545 Sentence denotes Balanced translocations occur in many malignancies and lead to the deregulation of one of the partner genes.
T70242 115546-115677 Sentence denotes Typically, these rearrangements lead to fusion transcripts that can be detected with high sensitivity by reverse transcription-PCR.
T56062 115678-115847 Sentence denotes B-cell lymphoproliferative disorders (LPDs) often harbor translocations between the strong enhancers of the immunoglobulin (Ig) loci and oncogenes, such as BCL2, or MYC.
T3010 115848-115951 Sentence denotes This results in over-expression of the oncogene without the formation of an abnormal fusion transcript.
T68297 115952-116089 Sentence denotes These events are more difficult to detect by classical PCR assays due to the wide distribution of breakpoints over hundreds of kilobases.
T31444 116090-116280 Sentence denotes Here we present a method employing solution capture and massively parallel sequencing that allows comprehensive detection of translocations in DNA isolated from FFPE tissue from B-cell LPDs.
T43645 116281-116289 Sentence denotes Methods:
T76192 116290-116344 Sentence denotes This study was approved by the University of Utah IRB.
T92017 116345-116488 Sentence denotes Genomic DNA was isolated from FFPE specimens and subjected to NGS library preparation and target enrichment using SureSelect capture (Agilent).
T7427 116489-116740 Sentence denotes Capture probes were targeted to all known RSS sites flanking the V, D and J elements of the IGH, IGK and IGL loci, the IGH class switch regions, the IGK deleting elements, and known breakpoint clusters in BCL2, BCL6, MYC, CCND1, MALT1, and AIP2/BIRC3.
T44561 116741-116831 Sentence denotes In addition, 19 genes commonly mutated in B-cell LPDs were captured for mutation analysis.
T25392 116832-116892 Sentence denotes Paired-end sequencing was performed on Illumina instruments.
T33632 116893-116946 Sentence denotes Reads were aligned to the reference genome using BWA.
T81697 116947-116999 Sentence denotes Translocations were identified using DELLY software.
T79286 117000-117054 Sentence denotes Point mutations were identified by the program LOFREQ.
T71292 117055-117063 Sentence denotes Results:
T64076 117064-117201 Sentence denotes We tested 27 patient samples with diagnoses of diffuse large B-cell lymphoma, follicular lymphoma or Burkitt lymphoma, and 10 cell lines.
T62070 117202-117416 Sentence denotes We were able to detect translocations confirmed by other validated methods (FISH and PCR) including t(14;18);IGH-BCL2 (10/10), t(3;14);IGH-BCL6 (3/3), t(11;14);IGH-CCND1 (2/2) and translocation involving MYC (5/6).
T12414 117417-117528 Sentence denotes One discrepant jmd.amjpathol.org ■ The Journal of Molecular Diagnostics sample had very low positivity by FISH.
T99013 117529-117601 Sentence denotes We also detected translocations between BCL6 and 6 non-Ig partner genes.
T48345 117602-117693 Sentence denotes Capture of only one partner gene was sufficient for successful detection of translocations.
T61429 117694-117830 Sentence denotes This allowed detection of translocations with a breakpoint several hundred kb away from MYC and between BCL6 and novel, non-Ig partners.
T54634 117831-117940 Sentence denotes Dilution studies with 4 cell lines indicated that translocations could be detected down to 5% positive cells.
T91598 117941-117953 Sentence denotes Conclusions:
T21859 117954-118104 Sentence denotes We describe a method for comprehensive detection of mutations and translocations using massively parallel sequencing of genomic DNA from FFPE samples.
T17691 118105-118274 Sentence denotes The method allows concurrent detection of multiple translocations in the same sample at 5% positive cells and represents a valuable adjunct to the workup of B-cell LPDs.
T45378 118275-118488 Sentence denotes The ability to detect cancer specific fusion genes is important not only in cancer research, but also increasingly in clinical settings to ensure that the correct diagnosis is made and optimal treatment is chosen.
T23715 118489-118693 Sentence denotes Current methodologies to detect such fusion genes are laborious and time consuming, whereas near patient/point of care testing (POCT) offers a more efficient approach to leukemia treatment and management.
T13005 118694-118854 Sentence denotes Here we report a feasibility study to develop a rapid real-time PCR (RT-qPCR) assay that can detect leukemia fusion genes with high sensitivity and specificity.
T25023 118855-118863 Sentence denotes Methods:
T22963 118864-119007 Sentence denotes The QuanDx ALL Q-Fusion Screening Kit is a novel multiplex RT-qPCR assay for the simultaneous detection of 16 fusion genes with 71 breakpoints.
T81183 119008-119167 Sentence denotes Fusion and control genes are co-amplified and identified with specific Yin-Yang Probes, a novel technology for nucleic acid detection and related applications.
T59444 119168-119302 Sentence denotes The Streck Philisa Thermal Cycler is an innovative high speed PCR instrument designed to simplify and expedite PCR-based applications.
T90626 119303-119596 Sentence denotes An armored RNA positive template control and GUSB internal control were reverse transcribed and used along with a negative template control in both the standard QuanDx RT-qPCR protocol as well as a Streck modified fast protocol to evaluate the feasibility of developing a rapid screen for ALL.
T34208 119597-119707 Sentence denotes These results were then compared with additional data from the same protocol using p190 and p210 DNA plasmids.
T29325 119708-119757 Sentence denotes The total run times and Cq values were evaluated.
T22652 119758-119766 Sentence denotes Results:
T10541 119767-119888 Sentence denotes No significant Cq differences were observed with the Streck modified protocol compared with the QuanDx standard protocol.
T15381 119889-120026 Sentence denotes The standard protocol run time of 140 minutes was reduced to 77 minutes with the modified fast protocol, or 55% of the original run time.
T21900 120027-120189 Sentence denotes The reduction in amplification time is due to the ramp rates of the Philisa instrument, as well as a reduction of touchdown PCR denature and extension hold times.
T41940 120190-120202 Sentence denotes Conclusions:
T95939 120203-120481 Sentence denotes The successful feasibility study for running a rapid multiplex PCR leukemia test with the QuanDx ALL Q-Fusion Screening Kit combined with a modified fast protocol on the Streck real-time Philisa instrument shows the potential for developing near patient/POCT leukemia screening.
T68621 120482-120626 Sentence denotes Rapid diagnosis of ALL and other leukemias would allow for immediate health counseling and treatment options to be put in place for the patient.
T8844 120627-120721 Sentence denotes Future studies are planned for rapid AML screening, and to further reduce the time to results.
T421 120722-120726 Sentence denotes H30.
T94821 120727-120837 Sentence denotes The ABL and JAK Tyrosine Kinase Pathways Are Co-Activated in BCR/ABL1 Positive Acute Lymphoblastic Leukemia S.
T46218 120838-120852 Sentence denotes Bhagavathi, H.
T79217 120853-120896 Sentence denotes Aviv Rutgers University, New Brunswick, NJ.
T12558 120897-120910 Sentence denotes Introduction:
T5945 120911-121121 Sentence denotes In newly discovered Philadelphia-like ALL or (Ph-like ALL) lacks the BCR/ABL1 fusion, driving event in chronic myeloid leukemia (CML), a subset of acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL).
T95196 121122-121270 Sentence denotes It is characterized by activation of signaling Tyrosine Kinases (TK) and can be responsive to TK inhibitors, similar to the BCR/ABL1 fusion protein.
T47131 121271-121394 Sentence denotes We explored the possibility that ALL with the BCR/ABL1 fusion harbors other driving mutations in the TK signaling pathways.
T55293 121395-121545 Sentence denotes To this end, we studied patients with BCR/ABL1 positive ALL for the co-incidence of other mutations that can be present in the JAK signaling pathways.
T52130 121546-121554 Sentence denotes Methods:
T82371 121555-121775 Sentence denotes From December 2013 to November 2014, all cases positive for BCR/ABL1 by fluorescent in-situ hybridization (FISH) in our database were identified and subjected to FISH analysis using the IGH dual color, break apart probe.
T11569 121776-121872 Sentence denotes Three cases negative for the IgH rearrangement were further analyzed with CRLF2 and JAK2 probes.
T76956 121873-121881 Sentence denotes Results:
T27679 121882-121944 Sentence denotes A total of 18 patients identified; 9 ALL, 7 had CML and 2 AML.
T23620 121945-122002 Sentence denotes Most ALL patients showed complex hyperdiploid karyotypes.
T23225 122003-122085 Sentence denotes The BCR/ABL1 fusion protein was p190 KDa and p210 KDa in all AML and CML patients.
T70227 122086-122232 Sentence denotes Of the 9 ALL patients; 6 showed p190 KDa fusion protein, 2 patients had p210 KDa fusion protein and one patient had no detectable BCR/ABL1 fusion.
T66465 122233-122393 Sentence denotes Whereas no CML or AML BCR/ABL1 positive case showed separation of the IGH gene probe, 6 of 9 ALL patients (4 p190 and 2 p210) demonstrated IGH probe separation.
T75562 122394-122507 Sentence denotes The IGH partner chromosomes were identified in 50% of patients as t(Y;14), t(X;14), and t(14;20), 1 patient each.
T29982 122508-122663 Sentence denotes Hybridization with probe for the CRLF2 gene on the remaining 3 IgH rearrangement negative patients demonstrated the CRLF2/P2RY8 gene fusion in one patient.
T72078 122664-122731 Sentence denotes Two of the 9 patients were negativefor IgH, CRLF2 and JAK2 fusions.
T33282 122732-122743 Sentence denotes Conclusion:
T31432 122744-122830 Sentence denotes We discovered that 6 of 9 Ph positive ALL cases harbor concomitant IGH translocations.
T96535 122831-122964 Sentence denotes The partner gene of these translocations was mostly the CRLF2 gene that resides in the pseudoautosomal region of the sex chromosomes.
T50946 122965-123022 Sentence denotes One additional patient had a CRLF2 and P2RY8 gene fusion.
T47342 123023-123113 Sentence denotes The CRLF2 gene is responsible for activating the JAK pathway of tyrosine kinase signaling.
T25029 123114-123447 Sentence denotes We postulate that ALL with the 9;22 translocation is less responsive to tyrosine kinase inhibitors because in addition to the ABL signaling pathway activation, the JAK signaling pathway is also activated and only a combination of drugs that inhibit both pathways will be effective in stopping progression of ALL with BCR/ABL1 fusion.
T8166 123448-123452 Sentence denotes J.T.
T23829 123453-123462 Sentence denotes Brown, I.
T67506 123463-123475 Sentence denotes Beldorth, W.
T783 123476-123495 Sentence denotes Laosinchai-Wolf, M.
T48419 123496-123505 Sentence denotes Fahey, J.
T42476 123506-123516 Sentence denotes Hedges, B.
T24662 123517-123546 Sentence denotes Andruss Asuragen, Austin, TX.
T43576 123547-123560 Sentence denotes Introduction:
T63829 123561-123680 Sentence denotes Detection of BCR-ABL1 e13a2/e14a2 fusion transcripts (major breakpoint, M-BCR) of t(9;22) assesses tumor burden in CML.
T35887 123681-123759 Sentence denotes The International Scale (IS) standardized reporting against a common baseline.
T50143 123760-123950 Sentence denotes As newer TKI therapies create deeper responses, analytical sensitivity has become a critical topic in investigations into TKI discontinuation, where researchers require an assay that MR4.5).
T961 123951-124079 Sentence denotes This has led to various reporting formats, creating non-contiguous monitoring terms: baseline, 10%IS, 1%IS, MMR, MR4, and MR4.5.
T70022 124080-124390 Sentence denotes We describe analytical and clinical validation of a multiplex system reporting continuous MR values via automated analysis, clinical accuracy, analytical sensitivity of MR4.7, and direct traceability to the WHO Primary BCR-ABL1 materials without requiring establishment and revalidation of a conversion factor.
T23554 124391-124399 Sentence denotes Methods:
T37804 124400-124529 Sentence denotes Armored RNA Quant molecules form a blend of nuclease-resistant BCR-ABL1 and ABL1 transcripts to calibrate and control the system.
T71137 124530-124807 Sentence denotes A single 4-point curve using ARQ blends mimics the WHO Primary BCR-ABL1 reference materials and accounts for the relative batch run-specific efficiency of the RT step. cDNA generation and qPCR were optimized, including allowance of high mass of nucleic acid without inhibition.
T80198 124808-124880 Sentence denotes Residual clinical RNAs were tested to estimate LOD at minimum RNA input.
T15395 124881-125001 Sentence denotes Software includes a floating, traceable logic algorithm to ensure that sufficient ABL1 was detected to protect this LOD.
T58013 125002-125059 Sentence denotes A multi-site clinical outcome study included 139 samples.
T54057 125060-125202 Sentence denotes Monitoring was assessed by EFS at 32-40 months against test results at 12-18 months on TKI as estimated by the Kaplan Meier survival function.
T37392 125203-125211 Sentence denotes Results:
T90719 125212-125336 Sentence denotes We generated 1680 measurements across 28 low analyte levels, yielding an LOD estimate of 95% positivity of MR4.7 (0.002%IS).
T9984 125337-125353 Sentence denotes LOQ was similar.
T37005 125354-125452 Sentence denotes Despite deep analytical sensitivity, this system maintains analytical specificity (true negative).
T29226 125453-125510 Sentence denotes Linearity was at least MR0.3 (50%IS) to MR4.7 (0.002%IS).
T75537 125511-125562 Sentence denotes Single-site precision (lot, -site was demonstrated.
T14413 125563-125589 Sentence denotes The EFS difference between
T43587 125590-125602 Sentence denotes Conclusions:
T175 125603-125737 Sentence denotes The BCR-ABL1 test improves workflow with its streamlined reagent formulation, multiplex assay format, and automated software analysis.
T41509 125738-125961 Sentence denotes It facilitates assessment on the IS without conversion (via integrated ARQ materials traceable to the WHO Primary), reports results on a continuous scale (as both MR and %IS values), and quantifies deep molecular responses.
T55705 125962-126015 Sentence denotes It is clinically validated at MR3 using an EFS model.
T6727 126016-126029 Sentence denotes Introduction:
T55429 126030-126221 Sentence denotes Gene fusions resulting from chromosomal translocations play an important role in driving tumorigenesis in hematologic malignancies, and provide critical diagnostic and prognostic information.
T77716 126222-126380 Sentence denotes Real-time PCR (RT-PCR) or Fluorescent in-situ hybridization (FISH) can be used to detect fusions, but these methods are limited to known fusion gene partners.
T45247 126381-126505 Sentence denotes Advances in next-generation sequencing (NGS) are removing the technical challenges to detecting these critical gene fusions.
T73133 126506-126696 Sentence denotes Anchored Multiplex PCR (AMP) uses molecular barcoded adaptors for universal priming and gene-specific primers, enabling sensitive NGS-based detection of novel fusions from low-input samples.
T47868 126697-126873 Sentence denotes We evaluated the AMP-based Archer FusionPlex system for NGS-based detection of gene fusions in hematological malignancies through identification of fusion breakpoint sequences.
T30666 126874-127054 Sentence denotes Methods: RNA was evaluated from 17 hematologic malignancy blood or bone marrow samples with known or suspected gene fusions previously analyzed by FISH, karyotyping, and/or RT-PCR.
T56833 127055-127114 Sentence denotes All samples were run on the Archer FusionPlex Heme v1panel.
T22152 127115-127163 Sentence denotes Sequencing was done on the Illumina NextSeq 500.
T948 127164-127222 Sentence denotes All samples were analyzed by ArcherAnalysis v3.0 software.
T83645 127223-127231 Sentence denotes Results:
T74868 127232-127300 Sentence denotes Sequencing detected gene fusions in a total of 14 of the 17 samples.
T81488 127301-127421 Sentence denotes Five of these detected fusions were consistent with RT-PCR results, and 6 were consistent with dual-fusion FISH results.
T1216 127422-127626 Sentence denotes Importantly, sequencing enabled the identification of fusion partners in 3 samples where break-apart FISH probes detected a likely gene rearrangement event, but without information as to the partner gene.
T57791 127627-127673 Sentence denotes Fusions were not detected by NGS in 3 samples.
T43585 127674-127835 Sentence denotes The first of these sequence-negative samples had a very low level BCR-ABL1 e1a2 minor translocation (0.47% by RT-PCR), which is below the detection limit of NGS.
T42978 127836-128084 Sentence denotes Two additional sequence-negative samples had FISH and karyotyping results suggesting a chromosomal translocation, but the gene partners were not defined, and a gene fusion event could not be confirmed by sequencing using the original reagent panel.
T81270 128085-128292 Sentence denotes However, an updated prototype version of the fusion panel enabled detection of a novel KMT2A-MLLT4 fusion in one of these initially false-negative cases and suggested a KAT6A-CREBBP fusion in the other case.
T7016 128293-128424 Sentence denotes These latter two fusion events were not initially detected because these genes were not included in the first version of the panel.
T16523 128425-128531 Sentence denotes However these genes are now included in the updated version of this fusion gene panel, FusionPlex Heme v2.
T37762 128532-128544 Sentence denotes Conclusions:
T18746 128545-128682 Sentence denotes The Archer FusionPlex Heme panel v1 performs well and provides a streamlined workflow for successful NGS-based detection of gene fusions.
T79124 128683-128790 Sentence denotes Improvements in target gene coverage are ongoing based on the ever-changing knowledge base of gene fusions.
T56108 128791-128804 Sentence denotes Introduction:
T1081 128805-128900 Sentence denotes Mastocytosis is a clonal proliferation of mast cells accumulating in one or more organ systems.
T26575 128901-129111 Sentence denotes Systemic mastocytosis, which includes involvement of the bone marrow, can be either indolent or aggressive based on the presence or absence of "C-symptoms." There is currently no cure for systemic mastocytosis.
T9276 129112-129277 Sentence denotes Mastocytosis is a systemic disease, and the microenvironment shaped by mast cells may differ in many aspects from that of anatomically localized, solid malignancies.
T98214 129278-129453 Sentence denotes Whereas those with indolent systemic mastocytosis (ISM) have a normal life expectancy, those with aggressive systemic mastocytosis have historically survival only a few years.
T46767 129454-129595 Sentence denotes We sought to investigate potential biomarkers for both differentiating between ISM and ASM as well as identify potential therapeutic targets.
T97339 129596-129604 Sentence denotes Methods:
T8100 129605-129712 Sentence denotes Archival blocks with slides were retrieved, reviewed and clinical information obtained from patient charts.
T60380 129713-129764 Sentence denotes re used with >50% lesion, n=4 (ASM), and n=3 (ISM).
T13321 129765-130042 Sentence denotes RNA was extracted from pre-treatment FFPE bone marrow slides and analyzed with nanoString nCounter using the nCounter PanCancer Progression Panel designed to quantitate 770 genes from four major biological processes that contribute to increased tumor growth and aggressiveness.
T35821 130043-130051 Sentence denotes Results:
T5759 130052-130265 Sentence denotes The gene expression profiles of ASM versus ISM, show significant (>3 times, p<0.05) upregulation of a set of 4 genes (FN1, SFRP2, PTGS2, and POSTN) and down regulation of 4 genes (TMPRSS6, EPCAM, IFNG, and CLDN3).
T75063 130266-130349 Sentence denotes Interestingly, gene FN1 was upregulated 6.5X (p< 0.0047) in ASM as compared to ISM.
T16220 130350-130507 Sentence denotes Of the four ASM patients, three had an associated hematological clonal non-mast cell disorder (AHNMD) as identified by biopsies performed at our institution.
T14727 130508-130619 Sentence denotes All ASM patients demonstrated significant weight loss and palpable splenomegaly with evidence of hypersplenism.
T25862 130620-130723 Sentence denotes Two patients also showed hepatomegaly with liver dysfunction and osteolytic lesions on skeletal survey.
T5543 130724-130778 Sentence denotes Of the IDM patients, none demonstrated any C-symptoms.
T24443 130779-130791 Sentence denotes Conclusions:
T59489 130792-130934 Sentence denotes To the best of our knowledge, the current study represents the first gene expression studies profiling exploration of aggressive Mastocytosis.
T39114 130935-131073 Sentence denotes Of the upregulated genes, two play a role in wound healing and fibrosis including FN1 (the most significantly upregulated gene) and POSTN.
T5132 131074-131231 Sentence denotes Three of the downregulated genes (TMPRSS6, EPCAM, and CLDN3) encode transmembrane proteins, two of which (EPCAM and CLDN3) participate in cell-cell adhesion.
T99850 131232-131378 Sentence denotes FN1 is upregulated in many cancers, associated with aggressive phenotype in ovarian cancer, and is associated with poor survival in breast cancer.
T20420 131379-131548 Sentence denotes This work is intriguing for the new information it provides about the possible role of FN1 in mast cell migration and aggressiveness in Aggressive Systemic Mastocytosis.
T94272 131549-131553 Sentence denotes H34.
T31133 131554-131695 Sentence denotes Development of a PCR Assay for Detection of JAK2 Exon 14 (V617F), JAK2 Exon 12 and CALR Exon 9 Mutations in Myeloproliferative Disorders D.C.
T65668 131696-131707 Sentence denotes Maxwell, S.
T45191 131708-131719 Sentence denotes Grenier, B.
T39769 131720-131733 Sentence denotes Nwachukwu, M.
T98808 131734-131744 Sentence denotes Jensen, W.
T35394 131745-131759 Sentence denotes Morrison, T.L.
T28439 131760-131772 Sentence denotes Stockley, S.
T13589 131773-131787 Sentence denotes Kamel-Reid, C.
T46551 131788-131870 Sentence denotes Wei Toronto General Hospital, University Health Network, Toronto, Ontario, Canada.
T7069 131871-131884 Sentence denotes Introduction:
T65300 131885-132003 Sentence denotes Myeloproliferative neoplasms (MPNs) are clonal stem cell disorders characterized by proliferation of myeloid lineages.
T18521 132004-132125 Sentence denotes The classic non-CML MPNs include essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF).
T58595 132126-132271 Sentence denotes JAK2 exon 12 and 14 mutations are frequently observed in PV, and mutations in CALR are seen in 67% of ET cases and 88% of PMF cases respectively.
T13786 132272-132498 Sentence denotes We developed a multiplex molecular assay to detect JAK2 exon 14 V617F, JAK2 exon 12 K539L and indels, and CALR exon 9 indels in a single test to expedite the diagnosis, prognosis and management of myeloproliferative disorders.
T70736 132499-132507 Sentence denotes Methods:
T88361 132508-132648 Sentence denotes Genomic DNA extracted from peripheral blood (Qiagen M48) was used in multiplex PCR using custom primers and ABI AmpliTaq Gold PCR mastermix.
T96307 132649-132731 Sentence denotes PCR fragments were analyzed by capillary electrophoresis on an ABI 3500 sequencer.
T2264 132732-132877 Sentence denotes Custom primers were labeled with FAM fluorescent label (blue) for JAK2 exon 14 and CALR mutations, and VIC (green) for the JAK2 exon 12 mutation.
T56507 132878-132971 Sentence denotes Limits of detection were established at 5% for JAK2 exon 12 and CALR and 1% for JAK2 exon 14.
T27662 132972-133133 Sentence denotes Expected normal alleles sizes were 380bp to 381bp for CALR, and 280bp to 281bp for JAK2 exon 12, with mutant indels sizes occurring outside of the normal values.
T28881 133134-133248 Sentence denotes The expected normal allele size of JAK2 exon 12 K539L was 135bp to 136bp and 130bp to 131bp for the mutant allele.
T22274 133249-133345 Sentence denotes The expected normal allele size for JAK2 exon 14 V617F was 98bp and 103bp for the mutant allele.
T92166 133346-133354 Sentence denotes Results:
T3191 133355-133560 Sentence denotes Validation included 25 cases which were previously tested for JAK2 exon 12 mutation by Sanger sequencing and for JAK2 exon 14 V617F mutation and CALR exon 9 indels by multiplex PCR based fragment analysis.
T69172 133561-133645 Sentence denotes The results using the mulitplex assay showed 100% concordance with previous results.
T35294 133646-133762 Sentence denotes The inclusion of JAK2 exon 12 primers to the assay now allows 97% of reported JAK2 exon 12 mutations to be detected.
T97444 133763-133889 Sentence denotes The remaining 3% that are not detected are substitutions, which are either clinically non-significant or synonymous in COSMIC.
T43884 133890-134048 Sentence denotes We identified the co-existence of double mutations in JAK2 exon 12 and exon 14, which would be missed if algorithms are confined to JAK2 exon 14 testing only.
T90016 134049-134320 Sentence denotes Bleed through of FAM and VIC dyes may occur when peak height is exceedingly high, leading to interference with variant calling, so other complementary methodologies such as Sanger sequencing and fragment analysis are employed for further clarification in these scenarios.
T55943 134321-134333 Sentence denotes Conclusions:
T9415 134334-134428 Sentence denotes We present data summarizing development of a comprehensive multiplexed assay for use in MPN's.
TextSentencer_T440 134429-134593 Sentence denotes This type of simple multiplexed assay can be used as an alternative to other methods such as next-generation sequencing, when a shorter turnaround time is required.
TextSentencer_T441 134594-134654 Sentence denotes Multiplex Ligation-dependent Probe Amplification (MLPA) M.S.
TextSentencer_T442 134655-134665 Sentence denotes Hein, K.C.
TextSentencer_T443 134666-134677 Sentence denotes Swanson, P.
TextSentencer_T444 134678-134691 Sentence denotes Lundquist, L.
TextSentencer_T445 134692-134700 Sentence denotes Coon, D.
TextSentencer_T446 134701-134713 Sentence denotes Dawson, J.L.
TextSentencer_T447 134714-134728 Sentence denotes Oliveira, J.D.
TextSentencer_T448 134729-134762 Sentence denotes Hoyer Mayo Clinic, Rochester, MN.
TextSentencer_T449 134763-134776 Sentence denotes Introduction:
TextSentencer_T450 134777-134843 Sentence denotes Beta globin cluster large deletions are heterogeneous and complex.
TextSentencer_T451 134844-134977 Sentence denotes They are classified into groups with important phenotypic differences when present in combination with another beta cluster mutation.
TextSentencer_T452 134978-135004 Sentence denotes These classifications are:
TextSentencer_T453 135005-135206 Sentence denotes 1) epsilon gamma delta beta thalassemia (EGDBT); 2) gamma delta beta thalassemia (GDBT); 3) hereditary persistence of fetal hemoglobin (HPFH); 4) delta beta thalassemia (DBT) and beta thalassemia (BT).
TextSentencer_T454 135207-135364 Sentence denotes High genetic diversity in this genomic region decreases the applicability of routine GAP-PCR methodologies, as novel or less common mutations will be missed.
TextSentencer_T455 135365-135517 Sentence denotes We compared a higher resolution MLPA probe strategy to published deletion breakpoint data to assess improvement in the classification power of our test.
TextSentencer_T456 135518-135526 Sentence denotes Methods:
TextSentencer_T457 135527-135711 Sentence denotes Thirty five probe sets cover the beta globin gene locus and span from 29 kb upstream of the -locus control region (LCR) to 110 kb downstream of the 3' hypersensitivity region (3'HS-1).
TextSentencer_T458 135712-135906 Sentence denotes Probes 1 to 7 cover the LCR and -globin gene (HBE), 8 to 13 the -globin genes (HBG1& HBG2), 14 to 20 upstream and in the -globin gene (HBD), 21 to 35 encompass the -globin gene (HBB) and 3'HS-1.
TextSentencer_T459 135907-136003 Sentence denotes A gene dosage ratio was calculated using the median fluorescent intensity value (Luminex LX200).
TextSentencer_T460 136004-136080 Sentence denotes Case file review identified 301 routine clinical cases with large deletions.
TextSentencer_T461 136081-136150 Sentence denotes Higher resolution MLPA data was applied and phenotypically confirmed.
TextSentencer_T462 136151-136295 Sentence denotes We classified the cases into the 5 groups and projected our probe data to coordinates of 48 defined deletions available in the published record.
TextSentencer_T463 136296-136304 Sentence denotes Results:
TextSentencer_T464 136305-136420 Sentence denotes The 301 cases were able to be classified as follows: EGDBT (n=7), GDBT (n=34), DBT (n=97), HPFH (n=101), BT (n=52).
TextSentencer_T465 136421-136483 Sentence denotes Six cases could not be classified due to ambiguous phenotypes.
TextSentencer_T466 136484-136798 Sentence denotes In addition, our assay showed capacity to further differentiate specific deletion subtypes in 4 /4 EGDBT, 7/8 GDBT, 7/11 HPFH, 7/11 DBT and 8/9 BT cases studied and the ability to assign the Corfu delta gene deletion in 3/3 (100%) cases and identified crossover variants Hb Kenya and Hb Lepore (3 unique subtypes).
TextSentencer_T467 136799-136811 Sentence denotes Conclusions:
TextSentencer_T468 136812-136968 Sentence denotes Accurate classification is critically important in large beta cluster deletions due to the variance in protective phenotype among these groups of mutations.
TextSentencer_T469 136969-137059 Sentence denotes Identifying specific classification subtypes is interesting but less important clinically.
TextSentencer_T470 137060-137246 Sentence denotes By our higher resolution strategic MLPA assay alone we were able to classify deletions in 295/301 (98%) of our clinical cases and further subtype 37/48 (77%) according to published data.
TextSentencer_T471 137247-137483 Sentence denotes It is important to note that although it is possible to classify many deletions using this assay, many disorders have multiple mutations and correlation of phenotypic data remains crucial for accurate assessment of hemoglobin disorders.
T99451 137484-137486 Sentence denotes J.
T11526 137487-137496 Sentence denotes Gregg, M.
T46988 137497-137566 Sentence denotes Chen University of California Davis Medical Center, Sacramento, CA. .
T38088 137567-137580 Sentence denotes Introduction:
T7039 137581-137675 Sentence denotes Hairy cell leukemia (HCL) is a low grade B-cell malignancy with a distinctive immunophenotype.
T80189 137676-137776 Sentence denotes Purine analog therapy is highly effective, with most patients achieving durable complete remissions.
T75731 137777-138044 Sentence denotes The somatically acquired V600E mutation of the BRAF gene has recently been described as a molecular marker of HCL to distinguish it from other B-cell lymphomas with similar clinical and morphologic features, such as the HCL-variant and splenic marginal zone lymphoma.
T76774 138045-138224 Sentence denotes The rare aggressive cases of HCL that are resistant to standard therapy with BRAF oncoprotein inhibitors have prompted the search for additional new HCL associated gene mutations.
T72258 138225-138237 Sentence denotes Case report:
T49363 138238-138313 Sentence denotes Here we report a 67 year-old male with a ten-year history of recurrent HCL.
T88533 138314-138438 Sentence denotes A bone marrow biopsy and flow cytometry analysis revealed prototypical morphological and immunophenotypical features of HCL.
T98362 138439-138549 Sentence denotes The patient was resistant to conventional treatment with cladiribine, pentostatin, rituximab, and splenectomy.
T83938 138550-138692 Sentence denotes Computed tomography showed extensive lymphadenopathy involving cervical, mediastinal, portal hepatic, retroperitoneal, and pelvic lymph nodes.
T9580 138693-138774 Sentence denotes Right cervical lymph node biopsy showed persistent HCL involving the lymph nodes.
T1233 138775-138934 Sentence denotes No evidence of BRAF V600E gene mutation was detected by a genotyping qPCR assay for BRAF codon 600 mutations on formalinfixed, paraffin-embedded (FFPE) tissue.
T2110 138935-139184 Sentence denotes Methods: QClamp is a novel technology to screen for somatic mutations, which utilizes the sequence specific wild-type template xenonucleic acid "Clamp" to suppress amplification of wild-type DNA and selectively enhance mutant template amplification.
T57915 139185-139310 Sentence denotes We applied the DiaCarta QClamp BRAF codon-specific test kit to analyze the FFPE tissue, which resulted in indeterminate call.
T97877 139311-139508 Sentence denotes The BRAF exon 15 amplicons from QClamp qPCR reactions performed on DNA samples extracted from four separate FFPE slides of the cervical lymph nodes were sequenced by the conventional Sanger method.
T12060 139509-139517 Sentence denotes Results:
T96878 139518-139759 Sentence denotes All four jmd.amjpathol.org ■ The Journal of Molecular Diagnostics sequences are identical, which contain no mutation in V600 locus, but have a silent mutation in position A598 with nucleotide change from GCT to GCC (both coding for Alanine).
T28463 139760-139899 Sentence denotes There is also a c.1807C>T (substitution C to T in the R603* codon leading to a nonsense mutation from Arginine (CGA) to a stop codon (TGA).
T19961 139900-139912 Sentence denotes Conclusions:
T19518 139913-140084 Sentence denotes This study reports new BRAF mutation variants distinct from the common V600E in HCL, which is clinically more aggressive and resistant to the conventional therapy for HCL.
T3443 140085-140200 Sentence denotes These findings suggest possible existence of new BRAF mutations causing resistant HCL with prognostic implications.
T63573 140201-140366 Sentence denotes The genetic study of more cases of BRAF V600E negative HCL cases is necessary to further investigate the new BRAFmutants and their potential therapeutic application.
T87287 140367-140369 Sentence denotes S.
T51136 140370-140383 Sentence denotes Loghavi, M.J.
T47142 140384-140396 Sentence denotes Routbort, R.
T36907 140397-140419 Sentence denotes Kanagal-Shamanna, S.A.
T75680 140420-140430 Sentence denotes Wang, J.D.
T72377 140431-140441 Sentence denotes Khoury, C.
T93429 140442-140450 Sentence denotes OK, C.C.
T703 140451-140460 Sentence denotes Yin, R.R.
T44310 140461-140470 Sentence denotes Singh, Z.
T22563 140471-140480 Sentence denotes Zuo, C.E.
T79118 140481-140496 Sentence denotes Bueso-Ramos, L.
T11646 140497-140509 Sentence denotes Medeiros, R.
T60205 140510-140522 Sentence denotes Luthra, K.P.
T43877 140523-140588 Sentence denotes Patel University of Texas MD Anderson Cancer Center, Houston, TX.
T41170 140589-140602 Sentence denotes Introduction:
T76600 140603-140726 Sentence denotes Guanine Nucleotide Binding Protein Alpha Stimulating Activity Polypeptide (GNAS) is a component of the G signaling pathway.
T26055 140727-140789 Sentence denotes GNAS mutations (codon 201) have been reported in solid tumors.
T73162 140790-140995 Sentence denotes High-throughput sequencing of myelodysplastic syndromes (MDS) have revealed recurrent GNASmutations in <1% of cases, but information about GNAS mutations in other myeloid neoplasms remains largely lacking.
T37099 140996-141004 Sentence denotes Methods:
T2551 141005-141077 Sentence denotes We searched our archives for myeloid neoplasms with GNAS R201 mutations.
T63948 141078-141157 Sentence denotes Cases were classified according to the World Health Organization 2008 criteria.
T42504 141158-141211 Sentence denotes Conventional G-band karyotype analysis was performed.
T33877 141212-141396 Sentence denotes Mutation analysis was performed using DNA extracted from BM aspirates using next-generation sequencing-based mutation analysis of 53-gene hotspot genomic loci (Illumina, San Diego CA).
T99126 141397-141472 Sentence denotes Clinical and laboratory data were obtained from electronic medical records.
T21771 141473-141481 Sentence denotes Results:
T80701 141482-141622 Sentence denotes We identified 16 patients with GNAS p.R201 mutations including 10 men and 6 women with a median age of 67 years at diagnosis (range, 49-79).
T96431 141623-141984 Sentence denotes Cases were subclassified into primary myelofibrosis (PMF), n=4; refractory cytopenia with multilineage dysplasia, n=3; acute myeloid leukemia (AML) with myelodysplasia-related changes, n=3; refractory anemia with excess blasts-2, n=1; AML-not otherwise specified, n=1; therapy-related AML, n=1; blast phase of PMF, n=1; and chronic myelomonocytic leukemia, n=1.
T56722 141985-142080 Sentence denotes One case was a remission bone marrow in a patient with history of acute promyelocytic leukemia.
T54658 142081-142200 Sentence denotes The median bone marrow blast count was 7% (range, 0-79) and the median bone marrow cellularity was 75% (range, 20-100).
T96851 142201-142322 Sentence denotes Fourteen cases showed the p.R201H substitution, 2 cases showed p.R201C and 1 case had both in mutually exclusive alleles.
T47871 142323-142405 Sentence denotes The mutation was present at a median allelic frequency of 0.26 (range, 0.01-0.53).
T61267 142406-142550 Sentence denotes The most common co-mutated genes were JAK2 p.V617F (n=3); TP53 (n=3), IDH1 p.R132 (n=2); NPM1 p.W288fs(n=2); IDH2 p.R140Q (n=1); and KRAS (n=2).
T85628 142551-142670 Sentence denotes GNAS mutation was the sole identified mutation in 7/16 (44%) cases, of note 5 of these cases had non-diploid karyotype.
T42797 142671-142824 Sentence denotes Nine (56%) of cases had a diploid karyotype; 2 cases showed deletion/monosomy of chromosome 20; 2 cases showed trisomy 8 and one case showed deletion 5q.
T68235 142825-142890 Sentence denotes Other non-recurrent chromosomal abnormalities were also observed.
T77342 142891-142986 Sentence denotes Conclusions: GNAS R201 mutations are infrequent but recurrent alterations in myeloid neoplasms.
T35845 142987-143196 Sentence denotes Our data shows that these mutations may be observed in settings other than MDS, including myeloproliferative neoplasms, myelodysplastic/myeloproliferative neoplasms, as well as de novo and therapy-related AML.
T50642 143197-143322 Sentence denotes In our series of cases, the mutation frequently co-occurred with either cytogenetic abnormalities or other somatic mutations.
T45355 143323-143325 Sentence denotes S.
T6613 143326-143339 Sentence denotes Chaudhary, N.
T51950 143340-143350 Sentence denotes Rabade, S.
T22218 143351-143360 Sentence denotes Joshi, R.
T78991 143361-143376 Sentence denotes Mascerhenas, K.
T14130 143377-143389 Sentence denotes Kulkarni, P.
T982 143390-143402 Sentence denotes Tembhare, P.
T81825 143403-143418 Sentence denotes Subramanian, S.
T48978 143419-143429 Sentence denotes Gujral, N.
T46706 143430-143486 Sentence denotes Patkar Tata Memorial Centre, Mumbai, Maharashtra, India.
T81458 143487-143500 Sentence denotes Introduction:
T56877 143501-143677 Sentence denotes Patients with primary myelofibrosis (MF) carry mutations in the Janus kinase 2 gene (JAK2) or thrombopoietin receptor gene (MPL) in 50% to 60% and 5% to 10% cases respectively.
T61844 143678-143870 Sentence denotes Recently, the presence of somatic mutations in the calreticulin gene (CALR) has been described in JAK2 and MPL negative cases of essential thrombocythaemia (ET) and primary myelofibrosis (MF).
T9274 143871-143987 Sentence denotes Patients with mutated CALR had lower risk of thrombosis and longer overall survival than patients with mutated JAK2.
T39847 143988-144120 Sentence denotes It seems imminent that CALR mutation testing will become essential in the work up of Ph negative myeloproliferative neoplasms (MPN).
T13247 144121-144248 Sentence denotes Here, we describe a fragment length analysis based assay for the rapid diagnosis of CALR positive myeloproliferative neoplasms.
T37845 144249-144328 Sentence denotes This is the first data from India describing CALR mutations in Ph negative MPN.
T89034 144329-144337 Sentence denotes Methods:
T53829 144338-144487 Sentence denotes As somatic mutations in CALR are predominantly insertions and/or deletions we developed a PCR using primers that flanked the exon 9 of the CALR gene.
T79261 144488-144544 Sentence denotes Both blood and bone marrow were used as a sample source.
T31320 144545-144666 Sentence denotes The CALR assay was standardized using genomic DNA as a template and subjected to PCR with fluorescently labelled primers.
T76432 144667-144756 Sentence denotes PCR amplicons were subjected to capillary electrophoresis on an ABI3500 genetic analyser.
T58414 144757-144765 Sentence denotes Results:
T75388 144766-144856 Sentence denotes The assay sensitivity was found to be 5% to 7% and inter and intra run precision was 100%.
T84598 144857-144900 Sentence denotes A total of 21 CALR mutations were detected.
T44375 144901-144942 Sentence denotes Of these 19 patients had MF and 2 had ET.
T79284 144943-145078 Sentence denotes Patients who harboured these mutations had ages ranging between 18 years to 63 years (median 46 years) with a male: female ratio 3.2:1.
T32075 145079-145161 Sentence denotes CALR mutations were exclusively detected in JAK2 and MPL mutations negative cases.
T25651 145162-145238 Sentence denotes The commonest type of mutations were deletions (66.6%) rest were insertions.
T20469 145239-145306 Sentence denotes Type 1 CALR mutation was the commonest followed by type 2 mutation.
T42987 145307-145382 Sentence denotes Other deletions were 2bp, 18bp, 46bp as well as a 164bp and 155bp deletion.
T34317 145383-145439 Sentence denotes Insertions were predominantly of 5bp and 3bp insertions.
T17500 145440-145555 Sentence denotes In addition to these 22 mutations, we also detected a 9 bp CALR deletion that has been described as a polymorphism.
T31539 145556-145567 Sentence denotes Conclusion:
T387 145568-145695 Sentence denotes To summarize, we describe a rapid, sensitive fragment length analysis based method to detect CALR mutations in Ph negative MPN.
T61324 145696-145698 Sentence denotes G.
T92968 145699-145708 Sentence denotes Zheng, A.
T53525 145709-145726 Sentence denotes Pallavajjalla, L.
T23907 145727-145736 Sentence denotes Haley, M.
T13613 145737-145744 Sentence denotes Lin, J.
T70670 145745-145759 Sentence denotes Eshleman, C.D.
T91751 145760-145806 Sentence denotes Gocke Johns Hopkins University, Baltimore, MD.
T21785 145807-145820 Sentence denotes Introduction:
T16214 145821-145992 Sentence denotes Therapy-related myeloid neoplasm (T-MN) is a complication of cytotoxic chemotherapy and/or radiotherapy with distinct features including higher incidence of TP53 mutation.
T14800 145993-146334 Sentence denotes Subclonal mutations and clonal architecture in T-MN are important but not well-studied: whole exome or genome sequencing lacks sufficient sensitivity and accuracy to identify low-frequency subclonal mutations; a small gene panel achieving sufficient coverage to detect subclonal mutations is too limited to appreciate the mutation landscape.
T59429 146335-146459 Sentence denotes We employed a 643 gene panel with a bioinformatics pipeline designed for somatic mutations including low-frequency variants.
T28878 146460-146621 Sentence denotes With this platform we studied the prevalence of subclonal mutations in T-MN and de novo AML, and compared their mutation landscape and related clinical features.
T46032 146622-146630 Sentence denotes Methods:
T16834 146631-146705 Sentence denotes The study was approved by the IRB at the Johns Hopkins Medical Institutes.
T34345 146706-146931 Sentence denotes From the specimens submitted to the Molecular Diagnostics Laboratory at the Johns Hopkins Hospital between July 2015 and March 2016, we collected 10 consecutive patients with T-MN and 23 consecutive patients with de novo AML.
T79981 146932-147028 Sentence denotes Clinical, hematological, and cytogenetic data were collected from the electronic medical record.
T84535 147029-147135 Sentence denotes The custom panel was designed as a clinical leukemia panel, and covers 643 genes important in oncogenesis.
T30773 147136-147144 Sentence denotes Results:
T44696 147145-147268 Sentence denotes Although the limit of detection depends on variant position, mutations with as low as 1% VAF were detected by our platform.
T3694 147269-147531 Sentence denotes Mutations of a subset of genes appear to be enriched in T-MN cases: TP53 (30% versus 13%), MLL2 (20% versus 4.3%), and BCOR (30 versus 8.7%); whereas mutations of another subset of genes are underrepresented in T-MN cases: TET2 (0% versus 13%), NRAS (10% versus.
T66988 147532-147556 Sentence denotes 21.7%), FLT3 (0% versus.
T62090 147557-147584 Sentence denotes 17.4%), DNMT3A (10% versus.
T47695 147585-147614 Sentence denotes 21.7%), and MSH3 ( 0% versus.
T29437 147615-147622 Sentence denotes 17.4%).
T69648 147623-147739 Sentence denotes Subclonal mutations were found in all cases of T-MN (range of cases per patient: , and in 91% of de novo AML (range:
T20240 147740-147749 Sentence denotes 0 to 10).
T19432 147750-147852 Sentence denotes On average, T-MN has 7.1±7.4 subclonal mutations, whereas de novo AML has 3.3±2.3 mutations (P=0.027).
T27900 147853-148094 Sentence denotes Although mutations of NRAS and TP53 are considered driver mutations, our study identified 4 cases with low frequency clones harboring mutations in those genes: two de novo AML cases with NRAS mutations and two T-MN cases with TP53 mutations.
T44365 148095-148204 Sentence denotes One of the T-MN cases had 25 subclonal mutations, the patient is the only one who received radiation therapy.
T24543 148205-148338 Sentence denotes Conclusion: T-MNs have a different genomic mutation profile from de novo AMLs, and harbor more subclonal mutations than de novo AMLs.
T8403 148339-148460 Sentence denotes The complexity of the clonal architecture may partially contribute to the lack of response to treatment in T-MN patients.
T23158 148461-148622 Sentence denotes The study is limited in case numbers, but the findings will lay the groundwork for a larger study to assess the clinical significance of the subclonal mutations.
T57269 148623-148627 Sentence denotes H40.
T44701 148628-148784 Sentence denotes An Algorithmic Approach to the Diagnosis of Hermansky-Pudlak Syndromes by Using Both Platelet Electron Microscopy and Targeted Next-Generation Sequencing J.
T78532 148785-148787 Sentence denotes A.
T84883 148788-148801 Sentence denotes Majerus, L.M.
T60806 148802-148810 Sentence denotes Coon, E.
T76886 148811-148823 Sentence denotes Turley, W.L.
T18752 148824-148837 Sentence denotes Nichols, R.K.
T19733 148838-148848 Sentence denotes Pruthi, R.
T44465 148849-148855 Sentence denotes He, R.
T82623 148856-148864 Sentence denotes He, D.S.
T66402 148865-148879 Sentence denotes Viswanatha, D.
T9385 148880-148888 Sentence denotes Chen, J.
T13197 148889-148929 Sentence denotes Perez Botero Mayo Clinic, Rochester, MN.
T42458 148930-148943 Sentence denotes Introduction:
T54730 148944-149156 Sentence denotes Hermansky-Pudlak syndrome (HPS) is a heterogeneous group of rare autosomal recessive disorders characterized by oculocutaneous albinism and mild bleeding diathesis due to severe platelet dense granule deficiency.
T24622 149157-149227 Sentence denotes Some patients may present with chronic colitis and pulmonary fibrosis.
T34003 149228-149306 Sentence denotes Currentlythere are 9 HPS subtypes associated with mutations involving 9 genes.
T68079 149307-149453 Sentence denotes Most subtypes are associated with defects in biogenesis of lysosome-related organelle complexes (BLOCs) that participate in endosomal trafficking.
T20341 149454-149562 Sentence denotes Some subtypes (HPS-1, HPS-2, and HPS-4) are commonly associated with chronic colitis and pulmonary fibrosis.
T14843 149563-149663 Sentence denotes HPS-3, HPS5, and HPS-6 form a BLOC-2 complex, and their mutations usually result in milder symptoms.
T41036 149664-149795 Sentence denotes From limited available literature, HPS-5 appears to have a mild clinical course with a lower risk of developing pulmonary fibrosis.
T41357 149796-149907 Sentence denotes Since genotypic variations in HPS may predict clinical outcome, molecular testing is important in this setting.
T30554 149908-150101 Sentence denotes With a combination of platelet electron microscopy (PTEM) and targeted nextgeneration sequencing (NGS), we confirmed HPS-5 in a patient and identified a novel mutation in this rare form of HPS.
T31078 150102-150110 Sentence denotes Methods:
T24008 150111-150261 Sentence denotes A 65 y/o male patient presented with subtle ocular albinism, nystagmus, bleeding diathesis, without evidence of pulmonary fibrosis or chronic colitis.
T85089 150262-150308 Sentence denotes PTEM showed virtual absence of dense granules.
T88089 150309-150476 Sentence denotes A targeted NGS panel encompassing 9 HPS genes (HPS1, AP3B1, HPS3, HPS4, HPS5, HPS6, DTNBP1, BLOC1S3, and BLOC1S6) was designed using SureDesign (Agilent Technologies).
T27893 150477-150639 Sentence denotes DNA library preparation was performed using the SureSelect Target Enrichment System for Illumina Paired-End Multiplexed Sequencing Library (Agilent Technologies).
T35487 150640-150721 Sentence denotes The enriched indexed DNA sample was then sequenced on an Illumina MiSeq platform.
T18920 150722-150864 Sentence denotes All sequence variants were classified following the current American College of Medical Genetics and Genomics (ACMG) Standards and Guidelines.
T46194 150865-150873 Sentence denotes Results:
T23133 150874-150913 Sentence denotes A novel homozygous mutation in the HPS5
T70202 150914-151016 Sentence denotes The Journal of Molecular Diagnostics ■ jmd.amjpathol.org gene, c.1960A>T (p.Lys654X) , was identified.
T45644 151017-151147 Sentence denotes This mutation results in a premature protein truncation with loss of amino acids downstream of K654 to the c-terminal end of HPS5.
T82711 151148-151208 Sentence denotes The genetic variant was also confirmed by Sanger Sequencing.
T47615 151209-151316 Sentence denotes The truncatedHPS5 protein interrupts the BLOC-2 complex and likely disrupts platelet dense granule genesis.
T20172 151317-151413 Sentence denotes It was therefore interpreted as "likely pathogenic" following the ACMG Standards and Guidelines.
T73860 151414-151507 Sentence denotes The PTEM and NGS findings, along with the clinical history, confirmed the diagnosis of HPS-5.
T7222 151508-151520 Sentence denotes Conclusions:
T99422 151521-151763 Sentence denotes The identification of this novel mutation underscores the importance of the combination of PTEM and targeted NGS testing in accurately characterizing specific variants of HPS, which should lead to more informed anticipatory clinical guidance.
T10357 151764-151768 Sentence denotes A.E.
T23862 151769-151780 Sentence denotes Quesada, Z.
T17025 151781-151789 Sentence denotes Hu, M.J.
T613 151790-151804 Sentence denotes Routbort, K.P.
T79442 151805-151814 Sentence denotes Patel, R.
T80039 151815-151825 Sentence denotes Luthra, S.
T34550 151826-151837 Sentence denotes Loghavi, Z.
T15153 151838-151845 Sentence denotes Zuo, C.
T14913 151846-151853 Sentence denotes Yin, R.
T76595 151854-151875 Sentence denotes Kanagal-Shamana, S.A.
T89672 151876-151886 Sentence denotes Wang, J.L.
T34291 151887-151900 Sentence denotes Jorgensen, L.
T76299 151901-151915 Sentence denotes Medeiros, C.Y.
T81922 151916-151923 Sentence denotes Ok M.D.
T58127 151924-151960 Sentence denotes Anderson Cancer Center, Houston, TX.
T31591 151961-151974 Sentence denotes Introduction:
T8386 151975-152065 Sentence denotes Knowledge of the mutations that occur in mixed phenotype acute leukemia (MPAL) is limited.
T62633 152066-152249 Sentence denotes MPAL is an uncommon presentation of acute leukemia, and few studies which have assessed MPAL cases for genetic mutations; none have done so utilizing next-generation sequencing (NGS).
T94417 152250-152394 Sentence denotes The aim of this study is to further elucidate the genetic mutations that may occur in MPAL in the hope of finding potential therapeutic targets.
T64152 152395-152403 Sentence denotes Methods:
T27555 152404-152485 Sentence denotes We searched for all MPAL cases tested by a NGS panel over 4 years (2012 to 2016).
T15809 152486-152625 Sentence denotes Only de novo cases that fulfilled the 2008 World Health Organization (WHO) classification criteria for the diagnosis of MPAL were selected.
T71509 152626-152656 Sentence denotes Molecular data were collected.
T50596 152657-152735 Sentence denotes Clinical, laboratory, cytogenetic and bone marrow findings were also reviewed.
T49217 152736-152834 Sentence denotes Statistical analysis was performed using GraphPad Prism with significance set at a p-value < 0.05.
T26726 152835-152843 Sentence denotes Results:
T42159 152844-152909 Sentence denotes We identified 11 cases of MPAL that were examined by a NGS panel.
T6409 152910-153005 Sentence denotes There were 6 (55%) men and 5 (45%) women with a median age of 64 years (range, 28 to 89 years).
T73603 153006-153092 Sentence denotes Two patients had BCR-ABL1 rearrangement and one patient had KMTA2 (MLL) rearrangement.
T16500 153093-153217 Sentence denotes A B-cell/myeloid (B/My) immunophenotype was more common (6/11, 55%) than a Tcell/myeloid (T/My) immunophenotype (4/11, 36%).
T40965 153218-153279 Sentence denotes There was one case with a Bcell/T-cell (B/T) immunophenotype.
T77047 153280-153348 Sentence denotes Mutations in one or more genes were detected in 6/11 (55%) patients.
T3393 153349-153481 Sentence denotes A total of 13 distinct mutations were found in ASXL1, DNMT3A, FLT3, IDH1, IDH2, JAK2, MLH1, NOTCH1, NRAS, RUNX1, TET2, TP53 and WT1.
T72840 153482-153537 Sentence denotes FLT3-ITD was the only recurrent mutation in 2 patients.
T34853 153538-153589 Sentence denotes Mutations were not detected in five patients (45%).
T98676 153590-153736 Sentence denotes Whereas all 4 patients with a T/My immunophenotype harbored at least one mutation, only 2 of six patients with B/My immunophenotype had mutations.
T85812 153737-153812 Sentence denotes Conventional cytogenetics showed a normal karyotype in 3/11 (27%) patients.
T87032 153813-153930 Sentence denotes One (9%) had one chromosomal abnormality, one (9%) had two abnormalities, and 6/11 (55%) had 3 or more abnormalities.
T18017 153931-154082 Sentence denotes Five of 6 patients (83.3%) with a B/My immunophenotype had a complex karyotype, but none of patients with T/My immunophenotype had a complex karyotype.
T8095 154083-154213 Sentence denotes Combining cytogenetics and gene mutational analysis, 10 of 11 (90.9%) patients had either cytogenetic aberration or gene mutation.
T79622 154214-154279 Sentence denotes Only one (9.1%) patient had a normal karyotype without mutations.
T88550 154280-154292 Sentence denotes Conclusions:
T99026 154293-154354 Sentence denotes Our study shows that genomic aberrations in MPAL are complex.
T51958 154355-154579 Sentence denotes Cases of MPAL with a B/My immunophenotype appear to be more cytogenetically complex with less gene mutations, whereas cases with a T/My immunophenotype are the converse, less cytogenetically complex with more gene mutations.
T22745 154580-154681 Sentence denotes Our data suggest that underlying mechanisms of leukemogenesis differ between B/My MPLA and T/My MPAL.
T11351 154682-154684 Sentence denotes R.
T67567 154685-154698 Sentence denotes Faryabi, G.W.
T78109 154699-154711 Sentence denotes Schwartz, Y.
T58130 154712-154722 Sentence denotes Zhou, A.W.
T155 154723-154735 Sentence denotes Lehman, J.J.
T2089 154736-154753 Sentence denotes Morrissette, K.S.
T57363 154754-154775 Sentence denotes Elenitoba-Johnson, M.
T56426 154776-154829 Sentence denotes Carroll University of Pennsylvania, Philadelphia, PA.
T21374 154830-154970 Sentence denotes Introduction: FLT3 internal tandem duplication (FLT3/ITD) is a common somatic mutation and oncogenic driver in acute myeloid leukemia (AML).
T7888 154971-155065 Sentence denotes Hence targeting mutated FLT3 serves as an important therapeutic strategy in FLT3-mutated AMLs.
T71921 155066-155171 Sentence denotes To this end, several inhibitors of FLT3 (FLT3i) have been developed and are in phase III clinical trials.
T93448 155172-155252 Sentence denotes Despite the effectiveness of FLT3i, the overall response rate is lower than 60%.
T48142 155253-155486 Sentence denotes Although the tandem-duplicated sequences of FLT3/ITD is always in-frame resulting in an elongated protein product, it varies in position, length, number of tandem duplications, and inserted spacer nucleotides in between duplications.
T76362 155487-155649 Sentence denotes We hypothesize that the various forms of tandem duplication and spacer sequences defining the elongated FLT3 protein could modify the response to FLT3i treatment.
T14812 155650-155814 Sentence denotes However, a detailed study of the effect of specific FLT3/ITD class on the clinical outcome is hampered by the complexity of classifying various FLT3/ITD structures.
T494 155815-155823 Sentence denotes Methods:
T6131 155824-155924 Sentence denotes Here, we present a computational framework, ITDprofiler to identify and classify FLT3/ITD structure.
T6133 155925-156083 Sentence denotes Utilizing efficient implementation of Suffix Tree algorithm, ITDprofiler automatically identifies tandem duplications and the interspersed inserted sequences.
T33016 156084-156190 Sentence denotes ITDprofiler identified two major categories of FLT3/ITDs which we have designated as; typical or atypical.
T41249 156191-156416 Sentence denotes Typical-ITD is defined as tandem duplication without or with insertion of only native exon 14 sequences of the FLT3 genes; whereas an insertion of sequences exogenous to exon 14 of the FLT3 gene is designated as atypical-ITD.
T25940 156417-156612 Sentence denotes Moreover, ITDprofiler automatically defines the duplicated amino acid residues and provides visual representation of the placement of ITD sequences and highlights duplicated and spacer sequences.
T60133 156613-156621 Sentence denotes Results:
T84453 156622-156802 Sentence denotes Using the ITDprofiler, we have analyzed over 200 AML patients sequenced at the Center for Personalized Diagnostics of the University of Pennsylvania and 107 were FLT3/ITD-positive.
T24960 156803-156926 Sentence denotes Our method segregates typicalfrom atypical-ITD patients with more than 99% accuracy compared to manually annotated samples.
T56412 156927-157002 Sentence denotes We observed atypical-ITD in more than 34% of the FLT3/ITDpositive patients.
T65071 157003-157059 Sentence denotes Atypical-ITD patients were more frequently multi-clonal.
T94840 157060-157231 Sentence denotes Our preliminary analysis suggests that the residues of the JM zipper region important for maintenance of the autoinhibitory conformation of FLT3 are frequently duplicated.
T51414 157232-157301 Sentence denotes This observation is consistent with earlier studies in pediatric AML.
T82843 157302-157314 Sentence denotes Conclusions:
T76387 157315-157587 Sentence denotes Our standalone and web-based ITDprofile program enables accurate and efficient characterization of FLT3/ITD structure and provides a clinically validated platform to study the effect of variations in FLT3/ITD structure on the outcome of the treatment with FLT3 inhibitors.
T47994 157588-157592 Sentence denotes H43.
T46490 157593-157696 Sentence denotes Acute Myeloid Leukemia with Concurrent Biallelic CEBPA Mutation and FLT3 Internal Tandem Duplication C.
T17914 157697-157711 Sentence denotes Soderquist, K.
T49499 157712-157733 Sentence denotes Elenitoba-Johnson, A.
T5836 157734-157742 Sentence denotes Bagg, S.
T5712 157743-157752 Sentence denotes Luger, A.
T9605 157753-157761 Sentence denotes Perl, M.
T88969 157762-157773 Sentence denotes Carroll, J.
T24354 157774-157831 Sentence denotes Morrissette University of Pennsylvania, Philadelphia, PA.
T64223 157832-157845 Sentence denotes Introduction:
T26627 157846-157964 Sentence denotes Acute myeloid leukemia (AML) is a neoplasm of hematopoietic precursor cells with altered proliferation and maturation.
T32082 157965-158158 Sentence denotes The current WHO classification of AML is based on various pathologic features including, amongst others, the presence of recurrent chromosomal rearrangements as well as CEBPA and NPM1mutations.
T4291 158159-158286 Sentence denotes Recurrent mutations in a myriad of additional genes, including FLT3, are known to have prognostic and therapeutic implications.
T90462 158287-158401 Sentence denotes Biallelic CEBPA mutations are associated with a good prognosis whereas FLT3-ITD mutations confer a poor prognosis.
T57300 158402-158476 Sentence denotes Concurrent biallelic CEBPA mutations and FLT3-ITD are relatively uncommon.
T85912 158477-158582 Sentence denotes Some studies suggest that AML with biallelic CEBPA mutations are less genetically complex than other AML.
T90953 158583-158591 Sentence denotes Methods:
T40215 158592-158768 Sentence denotes We conducted a retrospective analysis of AML patient samples with concurrent biallelic CEBPA and FLT3-ITD mutations that were identified using a search of electronic databases.
T65789 158769-158934 Sentence denotes Demographic and clinical variables (age, therapy received, disease response), longitudinal molecular cytogenetic data, and bone marrow histomorphology were analyzed.
T1673 158935-159027 Sentence denotes Total numbers of patients concurrently tested for CEBPA and FLT3 alterations were tabulated.
T19858 159028-159036 Sentence denotes Results:
T26585 159037-159207 Sentence denotes One hundred eighty six AML patients from May 1, 2015 to May 15, 2016 were tested with a panel of 68 genes commonly altered in myeloid neoplasms, including CEBPA and FLT3.
T5529 159208-159260 Sentence denotes Of these, 9 (4.8%) showed biallelic CEBPA mutations.
T12355 159261-159355 Sentence denotes Unexpectedly, 6 of the 9 (66%) patients with biallelic CEBPA mutations had FLT3-ITD mutations.
T2789 159356-159546 Sentence denotes In 5 of 6 cases with concurrent mutations, CEBPA mutations were identified at higher allele frequency than FLT3; in 2 of 6 cases, the FLT3-ITD were identified at allele frequencies below 1%.
T15600 159547-159624 Sentence denotes All FLT3-ITD were confirmed by DNA PCR followed by capillary electrophoresis.
T33030 159625-159720 Sentence denotes The nine cases with biallelic CEBPAmutations showed an average of 4.4 additional mutated genes.
T73699 159721-159796 Sentence denotes Of 8 cases with available cytogenetic data, 4 (50%) had a normal karyotype.
T82232 159797-159888 Sentence denotes Of the 6 patients with concurrent CEBPA and FLT3 alterations, AML subclassification varied.
T58576 159889-159957 Sentence denotes Five of 5 patients achieved a remission with induction chemotherapy.
T651 159958-159969 Sentence denotes Conclusion:
T652 159970-160232 Sentence denotes Though the sample size is small, and our observations based on a single tertiary institutional cohort may be confounded by a referral bias, our data suggests that co-occurrence of biallelic CEBPA mutation and FLT3-ITD may be more common than previously reported.
T27002 160233-160387 Sentence denotes In our cohort, most CEBPA mutations occurred at higher allele frequency than the FLT3-ITDs, suggesting they may have preceded the acquisition of FLT3-ITD.
T52056 160388-160491 Sentence denotes Additionally, AML with biallelic CEBPA mutations show more genetic complexity than previously reported.
T53304 160492-160639 Sentence denotes A larger cohort with long term follow-up is needed to determine if response to treatment and outcome vary from other AMLs that lack these features.
T52721 160640-160705 Sentence denotes Neoplasms Using a Next-Generation Sequencing (NGS)-Based Assay C.
T49405 160706-160714 Sentence denotes Ho, J.C.
T11960 160715-160733 Sentence denotes Gomez-Gelvez, M.H.
T75930 160734-160742 Sentence denotes Syed, A.
T28724 160743-160752 Sentence denotes Zehir, W.
T33070 160753-160759 Sentence denotes Yu, T.
T98275 160760-160769 Sentence denotes Baldi, M.
T16827 160770-160781 Sentence denotes Ladanyi, A.
T79882 160782-160791 Sentence denotes Dogan, J.
T31100 160792-160799 Sentence denotes Yao, K.
T9130 160800-160810 Sentence denotes Nafa, M.E.
T12384 160811-160871 Sentence denotes Arcila Memorial Sloan Kettering Cancer Center, New York, NY.
T46147 160872-160885 Sentence denotes Introduction:
T43965 160886-161021 Sentence denotes Lymphoid and plasma cell neoplasms are characterized by clonallyrestricted T-cell receptor (TCR) or immunoglobulin (Ig) rearrangements.
T40296 161022-161246 Sentence denotes Across clinical laboratories, this is generally demonstrated with standardized multiplex polymerase chain reaction (PCR) assays, in which V-J or D-J products are separated by fragment sizes on capillary electrophoresis (CE).
T62038 161247-161451 Sentence denotes However, this approach has relatively low sensitivity and does not provide the specific clonal sequence information required for tracking a clone at low level or in minimal residual disease (MRD) setting.
T51785 161452-161636 Sentence denotes In this study, we assessed the performance of a NGS-based assay, LymphoTrack (LT) (Invivoscribe), for detection of low level and MRD in comparison to CE and flow cytometry (FC) assays.
T98250 161637-161796 Sentence denotes Methods: DNA was extracted from bone marrow, blood, and formalin-fixed paraffin-embedded tissue from 30 patients with diagnostic and post-therapy (PT) samples.
T65949 161797-161930 Sentence denotes For clonal Ig rearrangement, PCR primers flanking the IGH conserved framework region 1 (FR1) in VH and conserved JH region were used.
T40009 161931-162022 Sentence denotes For clonal TCR rearrangement, primers flanking the TRG conserved V and J regions were used.
T55352 162023-162241 Sentence denotes The amplified products were sequenced on the Illumina MiSeq platform, and analyzed with the proprietary LymphoTrack analysis software, which provided the quantitation and V-J gene family usages of all unique sequences.
T93861 162242-162440 Sentence denotes The patient-specific diagnostic clonal sequences were used to detect MRD in subsequent samples, and compared to concurrent CE jmd.amjpathol.org ■ The Journal of Molecular Diagnostics and FC results.
T3529 162441-162449 Sentence denotes Results:
T69765 162450-162658 Sentence denotes The study included 30 diagnostic and 37 PT samples, from patients with plasma cell neoplasms (PCN) (n=9), acute lymphoblastic leukemias (n=7), mature B-cell neoplasms (n=8), and mature T-cell neoplasms (n=6).
T46815 162659-162795 Sentence denotes Median number of sequencing reads for PT samples was 440,600, with the expected clonal sequences detected in as low as 0.0045% of reads.
T98405 162796-162917 Sentence denotes In 20/37 (54.1%) PT samples, MRD was detected by CE and/or FC; LT detected MRD in 19/20 of these cases (sensitivity=95%).
T98060 162918-163018 Sentence denotes LT did not detect MRD in a case of PCN, but FC detected 0.0028% plasma cells suspicious for disease.
T28371 163019-163116 Sentence denotes In 8/37 (21.6%) PT samples, only LT detected MRD, whereas FC and CE failed to detect any disease.
T67683 163117-163194 Sentence denotes In 11/37 (29.7%) PT samples, both LT and FC detected MRD, whereas CE did not.
T93241 163195-163330 Sentence denotes In 2/8 cases (25%) with MRD detected by LT only, subsequent patient samples showed overt disease with a median follow-up of 2.5 months.
T33219 163331-163485 Sentence denotes 7/37 (18.9%) PT samples were negative for MRD by all assays, and 6/7 (85.7%) of these patients remained diseasefree with a median follow-up of 3.5 months.
T59422 163486-163498 Sentence denotes Conclusions:
T33293 163499-163718 Sentence denotes Compared to CE and FC, LymphoTrack provides comparable or better MRD detection sensitivity of lymphoid neoplasms, and with increased diagnostic certainty by utilizing patient-specific clonal sequences for MRD detection.
T22526 163719-163732 Sentence denotes Introduction:
T12074 163733-163916 Sentence denotes Congenital neutropenia encompasses a group of disorders broadly defined by a persistent or recurrently low absolute neutrophil count (ANC) caused by a constitutional genetic mutation.
T97701 163917-164076 Sentence denotes A subset of these patients have a form of severe congenital neutropenia (SCN), with ANC levels below 200, and are at increased risk of leukemic transformation.
T3507 164077-164236 Sentence denotes The clinical diagnosis of SCN is challenging given its rarity (10 cases per million people) and overlap with other inherited syndromes that result in low ANCs.
T78979 164237-164330 Sentence denotes Here we describe a novel exome sequencing-based panel for the clinical identification of SCN.
T24231 164331-164339 Sentence denotes Methods:
T48969 164340-164417 Sentence denotes Blood samples were obtained from 10 patients with low ANCs and suspected SCN.
T69020 164418-164531 Sentence denotes DNA was extracted and enriched using hybrid-capture based exome sequencing probes targeting a 54 megabase region.
T19678 164532-164602 Sentence denotes The resulting libraries were sequenced using 2x101bp paired-end reads.
T26329 164603-164681 Sentence denotes Data was aligned and variants were called with a variety of open source tools.
T28534 164682-165002 Sentence denotes Variants were compared to a custom database containing ~1,000 previously reported mutations in 24 genes associated with SCN or related diseases including AP3B1, CSF3R, CXCR2, CXCR4, ELANE, G6PC3, GATA2, GFI1, HAX1, JAGN1, KRAS, LAMTOR2, LYST, NRAS, RAB27A, RUNX1, SBDS, SLC37A4, TAZ, TCIRG1, USB1, VPS13B, VPS45 and WAS.
T96622 165003-165011 Sentence denotes Results:
T9263 165012-165182 Sentence denotes An average of 10 gigabases of sequencing data per case was obtained with an average on target efficiency of 62%, resulting in 97% of the exome covered to a depth of >20x.
T29712 165183-165281 Sentence denotes Of the 10 sequenced cases, 4 (40%) carried a previously described neutropenia-associated mutation.
T63730 165282-165418 Sentence denotes The most frequently mutated gene (2 cases) was SBDS; mutations in this gene have been associated with Shwachman-Bodian-Diamond Syndrome.
T12921 165419-165551 Sentence denotes One case (10%) carried a mutation in ELANE, which encodes the neutrophil elastase gene and is the most commonly mutated gene in SCN.
T9312 165552-165687 Sentence denotes One case carried a mutation in CXCR4 that encodes the C-X-C chemokine receptor type 4 gene that has been associated with WHIM syndrome.
T65488 165688-165808 Sentence denotes Four patients (40%) had variants in genes associated with SCN that have not been previoulsy described in the literature.
T86178 165809-165923 Sentence denotes In 2 patients (20%), no variants with an obvious physiologic relationship to congenital neutropenia were detected.
T96344 165924-165935 Sentence denotes Conclusion:
T10613 165936-166084 Sentence denotes Here we show that clinical exome sequencing can identify canonical SCN-associated mutations in approximately 40% of patients with suspected disease.
T50041 166085-166273 Sentence denotes Given the breadth of gene mutations associated with SCN and the difficulty of establishing a clinical diagnosis of SCN, exome based sequencing is a useful assay for confirming a diagnosis.
T40996 166274-166416 Sentence denotes Further, by cataloging novel mutations, clinical exome sequencing has the potential to discover new genes involved in the pathogenesis of SCN.
T72825 166417-166523 Sentence denotes FusionPlex, a Targeted RNA-Sequencing (RNA-Seq) Assay Using Next-Generation Sequencing (NGS) Technology C.
T19712 166524-166530 Sentence denotes Ho, R.
T99730 166531-166542 Sentence denotes Benayed, P.
T82385 166543-166557 Sentence denotes Sukhadia, K.A.
T5500 166558-166572 Sentence denotes Mullaney, K.M.
T71218 166573-166581 Sentence denotes Rios, L.
T20731 166582-166592 Sentence denotes Wang, M.F.
T96788 166593-166603 Sentence denotes Berger, M.
T32238 166604-166617 Sentence denotes Ladanyi, M.E.
T55796 166618-166678 Sentence denotes Arcila Memorial Sloan Kettering Cancer Center, New York, NY.
T42828 166679-166692 Sentence denotes Introduction:
T9360 166693-166918 Sentence denotes Fluorescence in-situ hybridization (FISH) and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) are common detection methods for gene fusions, but both are laborious, low-throughput, and lack scalability.
T93259 166919-167051 Sentence denotes Here we describe the performance of a clinically validated, targeted RNA-Seq assay for fusion detection in hematologic malignancies.
T96565 167052-167212 Sentence denotes The assay allows the simultaneous assessment of fusions involving 42 genes in a single assay without prior knowledge or need for targeting of the partner genes.
T12726 167213-167397 Sentence denotes Methods: RNA was extracted and reverse-transcribed from 44 blood or bone marrow samples submitted for targeted fusion assessment by qRT-PCR or FISH, based on clinicopathologic history.
T78907 167398-167574 Sentence denotes The cDNA fragments were amplified using Archer's Anchored Multiplex PCR with a custom primer panel targeting 42 genes involved in recurrent fusions in hematologic malignancies.
T34437 167575-167681 Sentence denotes The products were sequenced on the Illumina MiSeq platform and analyzed with the Archer analysis pipeline.
T90525 167682-167794 Sentence denotes Reproducibility and sensitivity studies were performed on replicates and serially diluted samples, respectively.
T81073 167795-167850 Sentence denotes The findings were compared to FISH and qRT-PCR results.
T14833 167851-167859 Sentence denotes Results:
T54593 167860-168120 Sentence denotes A total of 44 samples were analyzed, including B-lymphoblastic leukemia (n=23), chronic myelogenous leukemia (n=8), acute promyelocytic leukemia (n=7), acute myeloid leukemia (n=4), mixed phenotype acute leukemia (n=1), and diffuse large B-cell lymphoma (n=1).
T99089 168121-168365 Sentence denotes The Archer assay detected all common BCR-ABL1 fusions, including e1a2 (p190) (n=10), e13a2/b2a2 (p210) (n=4), and e14a2/b3a2 (p210) (n=6), as well as uncommon fusions not detected by our standard qRT-PCR assays: e13a3/b2a3 (n=1) and e1a3 (n=1).
T20954 168366-168422 Sentence denotes The t(9;22)(q34;q11) was confirmed by FISH in all cases.
T50707 168423-168692 Sentence denotes Other fusions detected included ETV6-RUNX1 (n=10), PML-RARA (n=7), RUNX1-RUNX1T1 (n=1), CBFB-MYH11 (n=1), EIF4A2-BCL6 (n=1), ETV6-ABL1 (n=1), and KMT2A-AFF1 (n=1), which were all fully characterized by this NGS assay in concordance with the expected fusion breakpoints.
T68741 168693-168771 Sentence denotes Reproducibility studies showed excellent interand intra-assay reproducibility.
T87568 168772-169048 Sentence denotes Sensitivity studies showed lower limits of fusion detection at 1% for BCR-ABL1 (quantitated by qRT-PCR and adjusted to the international scale (IS)), and 1% for PML-RARA, ETV6-RUNX1, and RUNX1-RUNX1T1 (quantitated by qRT-PCR and normalized to control cell lines with fusions).
T50976 169049-169061 Sentence denotes Conclusions:
T69129 169062-169241 Sentence denotes Our targeted RNA-Seq assay using Archer FusionPlex technology provides a more comprehensive analysis for gene fusions in hematologic malignancies at the time of initial diagnoses.
T19932 169242-169470 Sentence denotes It provides several advantages, including high multiplexing capability and scalability, and detection of uncommon variant fusions, as well as fusions involving alternative partners, providing further insights into tumor biology.
T94744 169471-169475 Sentence denotes K.R.
T98905 169476-169488 Sentence denotes Bessonen, M.
T9293 169489-169498 Sentence denotes Mai, L.A.
T97686 169499-169512 Sentence denotes Frederick, R.
T61535 169513-169521 Sentence denotes He, D.S.
T60758 169522-169560 Sentence denotes Viswanatha Mayo Clinic, Rochester, MN.
T39455 169561-169574 Sentence denotes Introduction:
T31124 169575-169738 Sentence denotes Somatic hypermutation (SHM) of the immunoglobulin variable heavy chain (IGVH) genes is an important prognostic marker in B-cell chronic lymphocytic leukemia (CLL).
T74960 169739-169853 Sentence denotes SHM is commonly detected by PCR amplification of the clonal IGVH-JH region followed by Sanger sequencing (PCR-SS).
T84358 169854-169945 Sentence denotes Next-generation sequencing (NGS) technology has recently been used to determine SHM in CLL.
T22928 169946-170026 Sentence denotes We present our experience using NGS for the detection of IGVH SHM status in CLL.
T81907 170027-170124 Sentence denotes Methods: RNA was isolated from peripheral or bone marrow samples and reverse-transcribed to cDNA.
T55894 170125-170292 Sentence denotes PCR-SS of cDNA was performed with individual leader (LDR) VH1-VH7 family primers in separate reactions, followed by sequencing (Applied Biosystems Inc., Carlsbad, CA).
T16991 170293-170395 Sentence denotes If a LDR PCR product was absent, a second PCR was performed with individual FR1 family and JH primers.
T62035 170396-170555 Sentence denotes Sequencher version 6.0 (Gene Codes Corporation, Ann Arbor, MI) and the IMGT human IGH reference database (http://www.imgt.org) were used for sequence analysis.
T65893 170556-170651 Sentence denotes NGS was performed using the LymphoTrack IGH somatic mutation kit (Invivoscribe, San Diego, CA).
T46396 170652-170714 Sentence denotes Multiplex LDR and JH region primers were used for initial PCR.
T33699 170715-170905 Sentence denotes In the absence of a clonal band by LDR PCR, the FR1 and JH family primer mix was used and both LDR and FR1 products were pooled and sequenced on the MiSeq platform (Illumina, San Diego, CA).
T79953 170906-171068 Sentence denotes NGS analysis was performed using the manufacturersupplied LymphoTrack software and NCBI IgBLAST reference, as well as additional lab-specific analytic assessment.
T21386 171069-171077 Sentence denotes Results:
T50857 171078-171129 Sentence denotes 45 patient samples were analyzed by PCR-SS and NGS.
T30889 171130-171205 Sentence denotes A minimum 5% clonal B-cells were required for adequacy of the NGS platform.
T61570 171206-171344 Sentence denotes A quality threshold of 10 5 minimum total sequence reads and a dominant clonal population of >10% of the total reads per sample were used.
T10575 171345-171423 Sentence denotes Comparison of PCR-SS with NGS revealed that NGS had an accuracy rate of 95.6%.
T36203 171424-171571 Sentence denotes Two of 45 cases (4.4%) were considered truly discordant likely resulting from differences in single reaction in PCR-SS versus multiplex PCR in NGS.
T16399 171572-171718 Sentence denotes Minor differences in data analysis were also observed in VH gene annotation, likely reflecting variations between IMGT and IgBLAST reference data.
T23779 171719-171779 Sentence denotes We identified 11 distinct data patterns in the NGS platform.
T29025 171780-171906 Sentence denotes These patterns were subsequently employed for improved analysis of the NGS data in the clinical molecular diagnostic scenario.
T26475 171907-171919 Sentence denotes Conclusions:
T48743 171920-172035 Sentence denotes This study demonstrates the NGS technique is acceptable for clinical use in the determination of SHM status in CLL.
T92233 172036-172121 Sentence denotes Results obtained are highly accurate in comparison to the current standard of PCR-SS.
T12428 172122-172211 Sentence denotes Advantages of NGS include direct determination of the IGVH rearrangement and percent SHM.
T29898 172212-172338 Sentence denotes In conclusion, NGS for IGVH SHM analysis in CLL is feasible to implement in the routine clinical molecular diagnostic setting.
T30744 172339-172341 Sentence denotes V.
T73137 172342-172356 Sentence denotes Pattanayak, S.
T11785 172357-172372 Sentence denotes Duraisamy, J.K.
T91592 172373-172384 Sentence denotes Lennerz, D.
T85428 172385-172405 Sentence denotes Dias-Santagata, L.P.
T45050 172406-172414 Sentence denotes Le, A.J.
T39173 172415-172426 Sentence denotes Iafrate, V.
T36102 172427-172476 Sentence denotes Nardi Massachusetts General Hospital, Boston, MA.
T57826 172477-172490 Sentence denotes Introduction:
T35685 172491-172624 Sentence denotes Activating hotspot mutations in RAS family members (primarily NRAS and KRAS) are common in many cancers, including myeloid neoplasms.
T79439 172625-172703 Sentence denotes The majority of RAS hotspot mutations occur at codons Gly12, Gly13, and Gln61.
T92885 172704-172945 Sentence denotes For example, whereas mutations to Asp are most common among NRAS Gly12 hotspot mutations in hematopoietic malignancies, mutations to Ser, Ala, Val, Cys, and Arg are also observed, suggesting functional comparability among different variants.
T85420 172946-173066 Sentence denotes However, concomitant variants are not thought to be common either at the level of the RAS isoform or variant amino acid.
T39266 173067-173171 Sentence denotes Here, we describe the occurrence and dynamics of concomitant RAS mutations in a clinical testing cohort.
T13557 173172-173180 Sentence denotes Methods:
T31389 173181-173421 Sentence denotes Targeted next-generation sequencing data from an assay testing exons and hotspots in 39 genes (SNaPshot NGS), including Gly12, Gly13, Gln61, and Ala146 for both NRAS and KRAS, were evaluated for 391 patients (440 samples) with hematological
T9261 173422-173639 Sentence denotes The Journal of Molecular Diagnostics ■ jmd.amjpathol.org malignancies (predominantly AML, MDS, and multiple myeloma) over a period of clinical testing from October 2014 to March 2016 at Massachusetts General Hospital.
T25891 173640-173735 Sentence denotes Results: NRAS or KRAS mutations were identified in 97/440 (22%) samples (from 76/391 patients).
T12286 173736-173922 Sentence denotes Of the 97 samples with NRAS/KRAS mutations, 74 had a single detectable mutation in NRAS or KRAS and 23 samples had multiple mutations in NRAS/KRAS, often at low variant allele fractions.
T61266 173923-174016 Sentence denotes 16 samples had two, four had three, two had four, and one had seven co-occurring mutation(s).
T20430 174017-174106 Sentence denotes Ten patients who had concomitant mutations had testing performed at multiple time points.
T29094 174107-174380 Sentence denotes Of those patients, only one had no changes in the number and identities of concomitant variants, whereas four had a decreased number of variants, three had an increased number, and two had different variants (but the same number of variants) between subsequent time points.
T85119 174381-174575 Sentence denotes At the variant level, of the patients with concomitant mutations, 16/20 (75%) had at least one NRAS G12 or G13 variant, compared to 16/56 patients (29%) with a single mutation only (p = 0.0013).
T54578 174576-174734 Sentence denotes Of the 20 patients with concomitant mutations, 6/20 (30%) had NRAS Gly12Ser mutations, compared to 3/56 (5%) patients with a single RAS mutation (p = 0.0084).
T33954 174735-174747 Sentence denotes Conclusions:
T14426 174748-174827 Sentence denotes Concomitant NRAS/KRAS mutations are not uncommon in hematopoietic malignancies.
T40861 174828-174996 Sentence denotes NRAS G12/G13 mutations are enriched among samples with concomitant mutations, suggesting that various NRAS hotspot mutations may confer different activation properties.
T44947 174997-175288 Sentence denotes In addition, the variation in NRAS/KRAS mutations among multiple time points in the same patients suggests that the mutations are occurring in different clones, consistent with intratumoral heterogeneity and arguing against a role for NRAS/KRAS mutations as driver mutations in these tumors.
T20791 175289-175302 Sentence denotes Introduction:
T42156 175303-175542 Sentence denotes First recognized as a distinct entity in 1938, Diamond Blackfan anemia (DBA) is a genetically and clinically heterogeneous disorder characterized by pure red blood cell aplasia, variable congenital anomalies and a predisposition to cancer.
T23602 175543-175676 Sentence denotes The genes identified to date that are mutated in DBA all encode ribosomal proteins associated with either the small or large subunit.
T51316 175677-175790 Sentence denotes DBA is typically treated with steroids, red blood cell transfusions, and hematopoietic stem cell transplantation.
T55149 175791-175957 Sentence denotes Herein we report an Asian male presenting with macrocytic anemia, neutropenia, genitourinary malformations and growth retardation due to a novel RPL35A gene mutation.
T21879 175958-175971 Sentence denotes Presentation:
T68804 175972-176060 Sentence denotes This patient was born premature at 33 weeks and was one of the non-identical male twins.
T72695 176061-176221 Sentence denotes At 4 months of age, he was found small for his age (2% WHO growth percentiles) with multiple genitourinary malformations, HGB 7.2 g/dl, MCV 102.1 and HCT 20.6%.
T95531 176222-176299 Sentence denotes He had no significant family history and his twin brother was normal in size.
T48830 176300-176351 Sentence denotes The anemia didn't response to iron supplementation.
T98716 176352-176505 Sentence denotes Bone marrow evaluations revealed relative lymphocytic and megakaryocytic hyperplasia, relative granulocytic hypoplasia, and decreased marrow iron stores.
T45235 176506-176514 Sentence denotes Methods:
T41833 176515-176600 Sentence denotes Genomic DNA was isolated from blood specimen using a standardized kit and quantified.
T34857 176601-176845 Sentence denotes Sequence enrichment of the targeted coding exons and adjacent intronic nucleotides was performed by a bait-capture methodology using long biotinylated oligonucleotide probes, followed by polymerase chain reaction and next-generation sequencing.
T98036 176846-176952 Sentence denotes Ten genes were sequenced, including SBDS, RPL11, RPL35a, RPL5, RPS10, RPS17, RPS19, RPS24, RPS26 and RPS7.
T51344 176953-177028 Sentence denotes Any mutations were confirmed by conventional dideoxy DNA sequence analysis.
T12199 177029-177037 Sentence denotes Results:
T72101 177038-177198 Sentence denotes Sequence analysis revealed a novel p.R76P variant, located in coding exon 3 of the RPL35a gene, resulting from a G to C substitution at nucleotide position 227.
T98578 177199-177283 Sentence denotes The highly conserved arginine residue at codon 76 was replaced by a proline residue.
T25154 177284-177369 Sentence denotes This variant was not reported in population based cohorts in several major databases.
T37009 177370-177506 Sentence denotes In addition, this alteration is predicted to be possibly damaging and deleterious by PolyPhen and SIFT in silico analyses, respectively.
T75095 177507-177658 Sentence denotes A steroid therapy started and this patient's anemia gradually improved; one year later, laboratory tests showed HGB 11.5 g/dl, MCV 103.1 and HCT 33.0%.
T6529 177659-177671 Sentence denotes Conclusions:
T79850 177672-177796 Sentence denotes We report a novel variant of unknown significance in the RPL35a gene (p.R76P) in a patient with DBA characteristic findings.
T20125 177797-178035 Sentence denotes This variant is likely the pathogenic mutation of this patient's DBA, because genetically this variant changed a highly conserved amino acid in available vertebrate species, and clinically this patient responded well to steroid treatment.
T50085 178036-178053 Sentence denotes Leukemia (CLL) I.
T80370 178054-178065 Sentence denotes Hubbard, A.
T82857 178066-178076 Sentence denotes Layton, B.
T90451 178077-178144 Sentence denotes Tandon Molecular Pathology Laboratory Network, Inc., Maryville, TN.
T53407 178145-178242 Sentence denotes Introduction: CLL is the most common leukemia affecting adults in western and European countries.
T8215 178243-178449 Sentence denotes IGHV somatic hypermutation (SHM) status is a baseline prognostic indicator routinely assessed at initial diagnosis and is associated with indolent disease course and favorable overall survival when present.
T92607 178450-178539 Sentence denotes SHM is defined by < 98% sequence homology to most closely matched germline IGHV segments.
T52871 178540-178727 Sentence denotes The current standard for evaluation of SHM in CLL involves RNA extraction from patient peripheral blood or bone marrow samples followed by reverse transcription-PCR and Sanger sequencing.
T776 178728-178960 Sentence denotes Limitations of the traditional Sanger sequencing-based methodology include the necessity of prompt specimen receipt due to RNA lability and insensitivity in detecting SHM for clonal CLL populations comprising < 50% of total B-cells.
T94351 178961-179196 Sentence denotes We investigated the potential utility of an available next-generation sequencing (NGS)-based assay to determine SHM status using DNA from known CLL samples previously tested at an outside reference laboratory by IGHV Sanger sequencing.
T52265 179197-179205 Sentence denotes Methods:
T28981 179206-179254 Sentence denotes Twenty-three CLL patient specimens were studied.
T46864 179255-179395 Sentence denotes NGS testing consisted of target enrichment PCR using LymphoTrack primers (Invivoscribe) with adapters for sequencing on the Ion Torrent PGM.
T40864 179396-179477 Sentence denotes The LymphoTrack PGM v2.0 software was used to process and analyze sample results.
T92028 179478-179622 Sentence denotes Mutation rates for the most abundant sequence reads were evaluated based on comparison to partial V genes by the re considered positive for SHM.
T51781 179623-179631 Sentence denotes Results:
T78741 179632-179809 Sentence denotes Of the 23 specimens analyzed by IGHV Sanger sequencing, SHM status could not be determined in 5 cases (22%) due to inability of the assay to detect a dominant clonal population.
T99420 179810-179899 Sentence denotes In contrast, the NGS assay successfully yielded informative results for all 23 specimens.
T4620 179900-180095 Sentence denotes For the 18 samples with reported Sanger sequencing results, 6 (33%) were hypermutated and 12 (67%) were non-hypermutated, with 100% concordance noted between the Sanger Sequencing and NGS assays.
T75335 180096-180309 Sentence denotes The NGS assay also demonstrated complete concordance with expected results based on correlation with other CLL metrics including flow cytometry demonstration of clonal B-cell subsets and ZAP70 expression patterns.
TextSentencer_T472 180310-180391 Sentence denotes Conclusions: NGS may provide an effective replacement for IGHV Sanger sequencing.
TextSentencer_T473 180392-180603 Sentence denotes The utilization of DNA instead of RNA as a starting material, and greater sensitivity of detection of clonal populations, may also represent improvements over the traditional Sanger Sequencing based methodology.
TextSentencer_T474 180604-180775 Sentence denotes The technical feasibility of utilizing patient DNA, as opposed to RNA, also raises the possibility of obtaining clinically valid results from FFPE tissue biopsy specimens.
TextSentencer_T475 180776-180789 Sentence denotes Introduction:
TextSentencer_T476 180790-180954 Sentence denotes Chronic myelomonocytic leukemia (CMML) is characterized by persistent absolute monocytosis (>1 x 10 9 /L) in the peripheral blood (PB) and dysplasia in >1 lineages.
TextSentencer_T477 180955-181104 Sentence denotes In the absence of dysplasia, an acquired clonal cytogenetic/molecular abnormality is required or causes for reactive monocytosis have to be excluded.
TextSentencer_T478 181105-181253 Sentence denotes Oligomonocytic CMML (OM-CMML) showing increased monocytes in BM (>10% of the cellularity) but no absolute monocytosis in the PB occurs occasionally.
TextSentencer_T479 181254-181434 Sentence denotes These cases often show relative monocytosis and are likely classified as myelodysplastic syndrome (MDS) or myelodysplastic/myeloproliferative neoplasm, unclassifiable (MDS/MPN, U).
TextSentencer_T480 181435-181489 Sentence denotes A subset of these cases eventually develop overt CMML.
TextSentencer_T481 181490-181604 Sentence denotes Better characterization of OM-CMML is essential since the distinction between CMML and MDS is clinically relevant.
TextSentencer_T482 181605-181613 Sentence denotes Methods:
TextSentencer_T483 181614-181869 Sentence denotes Thirty OM-CMMLs (>10% monocytes in BM and >10% PB monocytes with absolute monocyte count of 0.5-1x10 9 /L) and 20 overt CMMLs (11 CMML0, 6 CMML1, 3 CMML2/AML) were compared, of which 21/30 and 20/20 cases were subjected to 21-or 45-gene mutation analysis.
TextSentencer_T484 181870-181924 Sentence denotes Results: OM-CMML included 21 men and 9 women (med age:
TextSentencer_T485 181925-181946 Sentence denotes 69 y/o, range 19-87).
TextSentencer_T486 181947-182065 Sentence denotes OM-CMML had lower WBC, lower absolute PB monocyte count and lower percentage of BM monocytes than overt CMML (p<0.05).
TextSentencer_T487 182066-182182 Sentence denotes The demographic distribution, the remaining CBC values and the degree of dysplasia were not significantly different.
TextSentencer_T488 182183-182250 Sentence denotes 14/28 OM-CMML and 1/20 overt CMML had abnormal karyotypes (p<0.01).
TextSentencer_T489 182251-182329 Sentence denotes 7/30 OM-CMML had prior chemotherapy (CT) whereas 1/20 overt CMML had prior CT.
TextSentencer_T490 182330-182384 Sentence denotes 6/7 OM-CMMLs with prior CT showed abnormal karyotypes.
TextSentencer_T491 182385-182435 Sentence denotes 9/31 (29%) OM-CMMLs progressed to overt CMML (med:
TextSentencer_T492 182436-182475 Sentence denotes 3 months, range 1 months to 58 months).
TextSentencer_T493 182476-182589 Sentence denotes Mutations in ASXL1, TET2 and SRSF2 were frequently seen in OM-CMMLs and overt CMMLs (13/21 versus 17/20; p=0.16).
TextSentencer_T494 182590-182722 Sentence denotes Overt CMML contained more cases with >2 concurrent mutations in the 3 genes when compared with OM-CMML (11/20 versus 5/21, p=0.058).
TextSentencer_T495 182723-182801 Sentence denotes 2/5 OM-CMMLs with >2 concurrent mutations in the 3 genes developed overt CMML.
TextSentencer_T496 182802-182892 Sentence denotes The remaining 3 cases showed persistent relative but no absolute monocytosis at follow-up.
TextSentencer_T497 182893-182961 Sentence denotes Mutations in CBL and RUNX1 were found more frequently in overt CMML.
TextSentencer_T498 182962-182974 Sentence denotes Conclusions:
TextSentencer_T499 182975-183111 Sentence denotes Mutations in ASXL1, TET2 and SRSF2 were frequently found in OM-CMML, indicating the genetic similarities between overt CMML and OM-CMML.
TextSentencer_T500 183112-183286 Sentence denotes The findings suggest at least a subset of OM-CMMLs likely represent early phase CMMLs, which was further substantiated by the development of overt CMML in some OM-CMML cases.
T7281 183287-183332 Sentence denotes Rearrangements in Hematopoietic Malignancies.
T46075 183333-183337 Sentence denotes D.P.
T12133 183338-183351 Sentence denotes Simmons, F.C.
T60517 183352-183361 Sentence denotes Kuo, E.P.
T56014 183362-183374 Sentence denotes Garcia, N.I.
T85819 183375-183389 Sentence denotes Lindeman, L.E.
T35601 183390-183406 Sentence denotes Macconaill, M.D.
T42578 183407-183457 Sentence denotes Stachler Brigham and Women's Hospital, Boston, MA.
T60292 183458-183471 Sentence denotes Introduction:
T86030 183472-183661 Sentence denotes Next-generation sequencing provides a breadth of knowledge about the alterations that drive cancer, including single nucleotide variants, copy number changes and chromosomal rearrangements.
T75322 183662-183881 Sentence denotes The latter is accomplished by selective inclusion of intragenic regions known to be involved in rearrangements to allow detection of structural alterations, including novel translocation partners not detectable by FISH.
T72043 183882-184069 Sentence denotes Our panel targets exons of genes (N=300) known to be important in many types of cancer and intragenic regions known to be involved in selected rearrangements (e.g., IGH, ABL, N=36 genes).
T74491 184070-184253 Sentence denotes We report the findings from jmd.amjpathol.org ■ The Journal of Molecular Diagnostics this targeted panel to identify translocations in 243 patients with various hematopoietic cancers.
T42996 184254-184262 Sentence denotes Methods:
T12963 184263-184415 Sentence denotes Samples were selected from patients with hematopoietic malignancies consented under the Dana Farber institutional review board approved protocol 11-104.
T8477 184416-184538 Sentence denotes Genomic regions of interest were targeted by hybrid capture for next-generation sequencing on the Illumina HiSeq platform.
T31436 184539-184709 Sentence denotes Single nucleotide variants, copy number variants, and structural variants were processed through a bioinformatics pipeline and interpreted by a pathologist or geneticist.
T61714 184710-184771 Sentence denotes The results from these interpretations were further analyzed.
T4185 184772-184780 Sentence denotes Results:
T78095 184781-184935 Sentence denotes Two hundred forty three (243) patients with hematopoietic malignancies were evaluated, including 75% with lymphoid neoplasms and 25% with myeloid disease.
T44304 184936-185073 Sentence denotes Structural variants were reported in 33% of patients, and potentially actionable SNV or CNV findings were reported in 61% of individuals.
T34404 185074-185218 Sentence denotes Structural variants were reported in 36% of individuals with other actionable SNV or CNV findings and 27% of individuals without other findings.
T35111 185219-185346 Sentence denotes BCR-ABL translocations were identified in chronic myelogenous leukemia (N=2, 100%) and acute lymphoblastic leukemia (N=2, 50%).
T37019 185347-185466 Sentence denotes In acute myeloid leukemia, MLL rearrangements (N=2, 8%) and FLT3 internal tandem duplications (N=4, 17%) were reported.
T61490 185467-185661 Sentence denotes A variety of findings were reported in B cell non-Hodgkin lymphomas including IGH-BCL2 (N=25, 17%), IGH-CCND1 (N=3, 2%) and IGH-MYC (N=2, 2%) rearrangements as well as SOCS1 deletions (N=3, 2%).
T35876 185662-185867 Sentence denotes Novel translocations identified included a RAF1-MTAP rearrangement in a histiocytic dendritic cell lymphoma, as well as three different IGH rearrangements in a single case of diffuse large B cell lymphoma.
T46798 185868-185880 Sentence denotes Conclusions:
T31885 185881-186035 Sentence denotes Structural variants were identified in patients with a broad spectrum of hematopoietic malignancies using a targeted next-generation DNA sequencing panel.
T90319 186036-186291 Sentence denotes Targeted capture of introns that are frequently involved in rearrangements in human cancer allows for identification of previously reported and novel structural variants within a sequencing panel and can provide additional valuable diagnostic information.
T34991 186292-186305 Sentence denotes Introduction:
T58897 186306-186424 Sentence denotes A commercial (HemaVision) multiplex RT-PCR assay is used widely for detecting 28 fusion transcripts in acute leukemia.
T68409 186425-186554 Sentence denotes However, it is difficult to find other recurrent fusion transcripts, especially with novel or cryptic chromosomal rearrangements.
T70928 186555-186691 Sentence denotes The purpose of this study was to identify significant leukemia fusion genes using targeted or untargeted next-generation RNA sequencing.
T71368 186692-186700 Sentence denotes Methods:
T21182 186701-187070 Sentence denotes We selected 10 acute leukemia patients with novel translocations by G-banding who were investigated by untargeted RNA sequencing and 10 acute leukemia patients with normal karyotype and negative HemaVision result who were investigated by targeted RNA sequencing and one ALL patient with BCR-ABL1/t(9;22) and one AML patient with CBFB-MYH11/inv(16) as positive controls.
T78484 187071-187202 Sentence denotes For untargeted RNA sequencing, total RNA was extracted from leukemia cells and cDNA libraries were constructed with TruSeq RNA kit.
T54804 187203-187252 Sentence denotes Paired-end sequencing was performed on HiSeq2500.
T99452 187253-187377 Sentence denotes For targeted RNA sequencing, total RNA from each bone marrow sample was converted to cDNA using Ovation cDNA Module (NuGEN).
T21772 187378-187577 Sentence denotes Multiplexed enriched libraries were produced by Ovation Fusion Panel Target Enrichment System V2 (NuGEN) which includes all exons of 502 known cancer fusion genes, and NGS was performed on HiSeq2500.
T84174 187578-187708 Sentence denotes Reads were aligned with TopHat/BowTie, and two algorithms such as deFuse and FusionCatcher were used to detect fusion transcripts.
T21110 187709-187799 Sentence denotes The candidate fusion transcripts were confirmed with RT-PCR followed by Sanger sequencing.
T54552 187800-187808 Sentence denotes Results:
T61335 187809-188156 Sentence denotes From the 10 acute leukemia patients with novel translocations by G-banding, we found 5 in-frame fusion genes exactly matched on translocation breakpoints from 3 AML patients and 1 B-ALL patient: USP34-ASAP3/t(1;2)(p36.1;p11.2), MAZ-MKL1/t(16;22)(p11.2;q13), MLL-SEPT6 and SEPT6-CDCA5/t(X;11)(q24;q13), and RCSD1-ABL1/t(1;9)(q24;q34), respectively.
T24488 188157-188328 Sentence denotes In the targeted RNA sequencing, as positive controls, we were able to identify the predicted BCR-ABL1 and CBFB-MYH11 fusions with both deFuse and FusionCatcher algorithms.
T41937 188329-188383 Sentence denotes The patient with BCR-ABL1 had also PAX5-ZCCHC7 fusion.
T30464 188384-188584 Sentence denotes Six of 10 patients with normal karyotype were found to have leukemiaassociated fusion genes; NUP98-NSD1 in two AML, PAX5-ZCCHC7 in B-ALL, MLLT10-C5 in AML, TAL1-PDZK1IP1 in AML, and MTA1-EPS15 in AML.
T34440 188585-188597 Sentence denotes Conclusions:
T95763 188598-188905 Sentence denotes Using targeted or untargeted next-generation RNA sequencing, we have discovered 5 candidate fusion genes from 4 patients of 10 acute leukemia patients with novel translocations (40%) and 2 known fusion genes and 3 novel fusion genes from 6 patients of 10 acute leukemia patients with normal karyotype (60%).
T42921 188906-189095 Sentence denotes Targeted cancer gene panel should be useful for the detection of recurrent fusion genes in acute leukemia patients rather than multiplex RT-PCR. values and long-term outcome was determined.
T2140 189096-189104 Sentence denotes Methods:
T25089 189105-189241 Sentence denotes The QuantideX qPCR BCR-ABL IS Kit from Asuragen uses standard TaqMan chemistry to quantitate BCR-ABL1 and the ABL1 reference gene RNA's.
T36504 189242-189423 Sentence denotes Associated software reports an international scale BCR-ABL1 value and a log-transformed MR value, with a 3 logreduction from pre-treatment baseline represented as 0.1% IS and MR3.0.
T31657 189424-189607 Sentence denotes Three laboratories performed BCR-ABL1 testing on 96 chronic phase CML patient's banked RNA specimens collected from two hospitals and drawn 12 to 18 months after starting TKI therapy.
T94943 189608-189833 Sentence denotes Clinical events (TKI therapy change, loss of complete hematologic or cytogenetic response, progression to accelerated phase/blast crisis, kinase domain mutation, or death) were recorded through 36±4 months after starting TKI.
T55576 189834-189968 Sentence denotes Two operators per site also tested serially-diluted reproducibility samples (range MR1.0 to MR4.0) in multiple replicates over 5 days.
T56864 189969-190106 Sentence denotes The 95% limit of detection (LOD) for the assay was defined as the median measured %IS value of four analogous serially-diluted specimens.
T36734 190107-190115 Sentence denotes Results:
T42660 190116-190174 Sentence denotes Fifty one patients had MR<3.0 at 12 to 18 months post-TKI.
T75407 190175-190339 Sentence denotes Of these 51 patients who did not achieve a major molecular response (MMR), 20 had a subsequent clinical event, 17 had no event, and 14 were lost to follow-up (LFU).
T43805 190340-190370 Sentence denotes Forty-five 18 months post-TKI.
T49267 190371-190461 Sentence denotes Of these 45 patients who did achieve MMR, 8 had an event, 28 had no event, and 9 were LFU.
T67492 190462-190742 Sentence denotes Kaplan-Meier survival curves demonstrated a 22% prolongation of eventfree survival (95% CI 2% to 42%) at 3 years in the MMR (versus non-MMR) group [p=0.028; 58% (95% CI 44% to 75%) for MR<3 versus 80% (95% CI 68% to 93%) reproducibility %CV (log-transformed) between 1.9 and 4.2%.
T52448 190743-190969 Sentence denotes The 95% LOD for both breakpoint transcripts (e13a2 & e14a2) was MR4.7 (0.002%IS), allowing sensitive detection of the MR4.5 cutoff that defines "complete molecular response" in ongoing treatment-free remission clinical trials.
T42390 190970-190982 Sentence denotes Conclusions:
T67608 190983-191211 Sentence denotes The QuantideX qPCR BCR-ABL IS Kit has excellent reproducibility and analytical sensitivity, and the achievement of MR>3 (major molecular response) by this assay predicts prolonged event-free survival in TKI-treated CML patients.
T59572 191212-191266 Sentence denotes Sequencing Method in Acute B-Lymphoblastic Leukemia S.
T60433 191267-191276 Sentence denotes Cheng, G.
T69112 191277-191290 Sentence denotes Inghirami, W.
T47597 191291-191339 Sentence denotes Tam Weill Cornell Medical College, New York, NY.
T63738 191340-191353 Sentence denotes Introduction:
T43083 191354-191544 Sentence denotes Minimal residual disease (MRD) is a powerful predictor for relapse of acute B-lymphoblastic leukemia (B-ALL), and its detection has been integrated as part of modern B-ALL therapy protocols.
T36103 191545-191672 Sentence denotes Traditional MRD assays rely on flow cytometry (FACS) or PCR, and recently NGS has emerged as a novel and more sensitive method.
T82891 191673-191806 Sentence denotes However, the current NGS assay is proprietary and its protocol is difficult to interpret and follow by a routine clinical laboratory.
T48426 191807-191871 Sentence denotes A simpler but highly sensitive and reliable NGS assay is needed.
T63668 191872-191880 Sentence denotes Methods:
T34201 191881-192163 Sentence denotes Here we applied LymphoTrack platform (Invivoscrible) with minor modification to capture all immunoglobulin heavy chain VDJ rearrangements from evaluable pre-treatment and post-treatment B-ALL samples, and developed a novel computer algorithm to identify tumor-associated clonotypes.
T41207 192164-192256 Sentence denotes Briefly, B-ALL genomic DNA was amplified with barcoded primer sets in a single PCR reaction.
T54012 192257-192336 Sentence denotes Resulting libraries were purified and quantified, followed by Miseq sequencing.
T80686 192337-192451 Sentence denotes LymphoTrack software (Invivoscribe) and our custom algorithm were used to analyze NGS data to generate MRD values.
T98077 192452-192654 Sentence denotes Assay performance characteristic, including analytical sensitivity, intra-run and interrun reproducibility, and linearity, were assessed by using B-ALL DNA serially diluted with normal PBMC genomic DNA.
T91422 192655-192768 Sentence denotes For concordance with FACS, we tested >50 B-ALL clinical samples, and compared the NGS results with those of FACS.
T2759 192769-192777 Sentence denotes Results:
T74595 192778-192859 Sentence denotes The assay can detect as few as 5 tumor cells among one million normal leukocytes.
T62891 192860-193113 Sentence denotes At a dilution of 1 in 250,000, intra-run analysis showed a mean (± SE) patientspecific tumor clone frequency of 0.00074% (± 0.00017%); at a dilution of 1 in 100,000, inter-run analysis showed a mean (± SE) tumor clone frequency of 0.00093% (± 0.00027%).
T63975 193114-193236 Sentence denotes Linearity studies demonstrated a high correlation (R 2 =0.999, a slope of 0.92) between reference and observed MRD levels.
T51564 193237-193328 Sentence denotes FASC versus NGS results showed concordance in 25 (78.1%) of 32 MRD + or MRD -B-ALL samples.
T40843 193329-193435 Sentence denotes In 6 of 26 (23.1%) samples that were MRDor inconclusive by FACS, MRD was positive by the sequencing assay.
T48366 193436-193526 Sentence denotes The median MRD level of these Miseq-MRD + samples was 0.0424% with a range of 0.006-0.35%.
T83465 193527-193664 Sentence denotes Our results indicate ultrasensitivity and excellent quantitative accuracy of the assay with high intra-run and inter-run reproducibility.
T6675 193665-193677 Sentence denotes Conclusions:
T30009 193678-193782 Sentence denotes We described here a simple, reliable, highly sensitive and accurate approach for MRD detection in B-ALL.
T63814 193783-193899 Sentence denotes It is advantageous over FACS because of its increased sensitivity as well as ease and consistency of interpretation.
T16036 193900-194039 Sentence denotes Above all, this method can be adopted by any clinical laboratories equipped with the basic physical and human resources for performing NGS.
T80799 194040-194192 Sentence denotes This method will be a useful adjunct in clinical management of patients with B-ALL. samples from patients with known/suspected hematologic malignancies.
T76306 194193-194266 Sentence denotes NGS was performed using a 35 gene panel targeting commonly mutated genes.
T7541 194267-194444 Sentence denotes 200ng sheared genomic DNA was prepared using a SureSelectXT capture reagent (Agilent, Santa Clara, CA) and the library sequenced on the MiSeq platform (Illumina, San Diego, CA).
T89891 194445-194605 Sentence denotes NGS data was processed through a proprietary bioinformatics pipeline (Mayo NGS Workbench) for alignment, base calling, and insertion/deletion (indel) detection.
T38061 194606-194700 Sentence denotes The processed data was annotated for final classification and clinical significance reporting.
T58155 194701-194709 Sentence denotes Results:
T91646 194710-194857 Sentence denotes One acute myeloid leukemia (AML) patient had a CEBPA c.318del involving 100% of the sequence reads (SR), with no coverage depth loss at this locus.
T30778 194858-195009 Sentence denotes Chromosomal microarray demonstrated a 26 megabase (Mb) copy-neutral loss of heterozygosity (cnLOH) event encompassing CEBPA from 19q13.11 to 19q13.43 .
T9449 195010-195158 Sentence denotes A second patient with B-lymphoblastic leukemia (B-ALL) harbored an IGH/BCL2 fusion and trisomy 8 (92%) and a low level BCR/ABL1 fusion (7%) by FISH.
T94372 195159-195312 Sentence denotes Manual review of the NGS data revealed a concurrent FLT3-ITD in c.1817_1818ins87 in 30% of the SR, providing additional information for targeted therapy.
T23813 195313-195421 Sentence denotes The last patient with AML had decreased coverage in a region of ASXL1 associated with clipped (unmapped) SR.
T21033 195422-195533 Sentence denotes Analysis of the clipped SR showed 100% homology to the TSHZ2 gene 20.5 Mb telomeric to ASXL1 on chromosome 20q.
T82306 195534-195614 Sentence denotes These findings were consistent with FISH data showing del(20q) in 46% of nuclei.
T29723 195615-195710 Sentence denotes The genomic break disrupts exon 14 of the ASXL1 gene and joins to the 3' UTR of the TSHZ2 gene.
T4510 195711-195723 Sentence denotes Conclusions:
T20959 195724-195831 Sentence denotes Standard NGS pipeline analysis identifies single base and small indel variants in a straightforward manner.
T34689 195832-196011 Sentence denotes Although wider genomic analysis (e.g. whole exome) is desirable for comprehensive detection, it imposes practical limitations of sample throughput, sensitivity and large datasets.
T7785 196012-196252 Sentence denotes Our experience with a targeted NGS platform suggests that even with small-scale targeted panels, recognition of novel pathogenic associations or analytic anomalies can enhance interpretation of data and may help guide clinical intervention.
T45548 196253-196255 Sentence denotes N.
T87002 196256-196266 Sentence denotes Patkar, R.
T27401 196267-196278 Sentence denotes Kodgule, G.
T42275 196279-196288 Sentence denotes Raval, C.
T93333 196289-196300 Sentence denotes Kakirde, S.
T95886 196301-196310 Sentence denotes Joshi, S.
T72285 196311-196324 Sentence denotes Chaudhary, R.
T8161 196325-196340 Sentence denotes Mascerhenas, K.
T2907 196341-196353 Sentence denotes Kulkarni, P.
T18495 196354-196366 Sentence denotes Tembhare, P.
T53222 196367-196428 Sentence denotes Subramanian Tata Memorial Centre, Mumbai, Maharashtra, India.
T89488 196429-196442 Sentence denotes Introduction:
T70994 196443-196572 Sentence denotes Recent whole genome and exome sequencing studies have identified key driver mutations that are harboured by myeloid malignancies.
T43909 196573-196757 Sentence denotes Amongst these, acute myeloid leukemia (AML) is perhaps the most biologically heterogeneous entity comprising of chromosomal abnormalities and somatic mutations at a DNA sequence level.
T82994 196758-196859 Sentence denotes It is important to recognize these mutations as some of them are of prognostic and therapeutic value.
T58777 196860-196974 Sentence denotes Here, we identify somatic mutations in AML using a 54 gene panel using targeted NGS on an Illumina MiSeq platform.
T20132 196975-196983 Sentence denotes Methods:
T17904 196984-197060 Sentence denotes Ten patients of AML (diagnosed as per the WHO 2008 criteria) were sequenced.
T54328 197061-197134 Sentence denotes FISH and conventional karyotyping was done to risk stratify the patients.
T18795 197135-197193 Sentence denotes Approximately 50 ng of genomic DNA was used as a template.
T57440 197194-197264 Sentence denotes Oligonucleotides were hybridized to target regions of the genomic DNA.
T22168 197265-197309 Sentence denotes This was followed by extension and ligation.
T32188 197310-197388 Sentence denotes Barcode indices and sequencing adapters were added by PCR based amplification.
T32009 197389-197464 Sentence denotes Once the libraries were prepared they were purified, normalized and pooled.
T23084 197465-197526 Sentence denotes Sequencing was done on a MiSeq system using the V3 chemistry.
T79340 197527-197580 Sentence denotes Base calling was done by the MiSeq Reporter software.
T49709 197581-197693 Sentence denotes Sequence alignment and generation of VCF files was done using Basespace and analyzed using VariantStudio v2.2.1.
T35160 197694-197702 Sentence denotes Results:
T51907 197703-197831 Sentence denotes Four patients could be classified as intermediate cytogenetic risk whereas the rest were favourable cytogenetic risk, (mean age:
T89417 197832-197853 Sentence denotes 31.4 years; M:F=3:2).
T78768 197854-197972 Sentence denotes Analysis of data quality indicated that all of the samples met the predefined acceptance criteria (mean Q30 of >91.2).
T17360 197973-198024 Sentence denotes The average amplicon mean coverage depth was 7463x.
T2450 198025-198147 Sentence denotes Mutations were detected in 9/10 samples (90%), which included base substitutions as well as indels at a sensitivity of 6%.
T11695 198148-198201 Sentence denotes An average of 2.2 mutations were detected per sample.
T50152 198202-198378 Sentence denotes Mutations affected diverse biological pathways such as signalling pathways, genes regulating epigenetic pathways, transcription pathways and genes encoding the RNA spliseosome.
T9345 198379-198650 Sentence denotes Mutations were detected in KIT (4 samples) followed by GATA2 (3 samples), ASXL1 (2 samples), DNMT3A (2 samples), RUNX1 (2 samples), TET2 (2 samples), FLT3 (1 sample), IDH2 (1 sample), NPM1 (1 sample), NRAS (1 sample), SMC3 (1 sample), U2AF1 (1 sample) and WT1 (1 sample).
T35232 198651-198741 Sentence denotes Patients with core binding factor leukemia predominantly harbored KIT and ASXL1 mutations.
T68358 198742-198825 Sentence denotes A single intermediate risk patient harboured a coexisting NPM1 and DNMT3A mutation.
T67358 198826-198838 Sentence denotes Conclusions:
T38365 198839-198951 Sentence denotes The TruSight myeloid sequencing panel is a comprehensive test that identifies mutations in myeloid malignancies.
T87058 198952-199075 Sentence denotes The assay has a relatively simple and straight forward workflow and is very suitable for application in a clinical setting.
T10380 199076-199188 Sentence denotes Our results indicate importance of comprehensive molecular profiling of AML in the era of personalised medicine.
T65266 199189-199191 Sentence denotes A.
T67464 199192-199200 Sentence denotes Hill, J.
T43651 199201-199211 Sentence denotes Peters, G.
T59641 199212-199222 Sentence denotes Hosler, K.
T22012 199223-199259 Sentence denotes Murphy ProPath Services, Dallas, TX.
T18544 199260-199273 Sentence denotes Introduction:
T33306 199274-199421 Sentence denotes There are 3 main subtypes of myeloproliferative neoplasms: polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF).
T63639 199422-199566 Sentence denotes Somatic mutations in the calreticulin (CALR) gene have recently been identified in the majority of ET and PMF patients that lack a JAK2mutation.
T38105 199567-199634 Sentence denotes There have been over 50 different CALR mutations described to date.
T97720 199635-199784 Sentence denotes These genetic alterations consist of insertions, deletions, or a complex combination of insertions/deletions (indels) within exon 9 of the CALR gene.
T58815 199785-199900 Sentence denotes The vast majority of the currently reported mutations leads to a +1 base pair (bp) shift in the open reading frame.
T97871 199901-200080 Sentence denotes This frameshift results in a common novel amino acid sequence of the Cterminal domain and elimination of the KDEL amino acid sequence required for endoplasmic reticulum retention.
T42826 200081-200244 Sentence denotes Given that most known mutations result in the same downstream reading frame, it is likely that the sequence plays a role in the oncogenicity of the mutant protein.
T5156 200245-200389 Sentence denotes Methods: CALR mutational analysis was performed by PCR amplification of exon 9 followed by fragment analysis via capillary electrophoresis (CE).
T57276 200390-200456 Sentence denotes The same region of exon 9 was then sequenced by the Sanger method.
T97178 200457-200524 Sentence denotes Results: CE results were consistent with a 1 bp insertion mutation.
T89030 200525-200691 Sentence denotes Sanger sequencing of the same region revealed 2 separate mutations within exon 9, a 2 bp insertion and a 1 bp deletion occurring 76 bases downstream of the insertion.
T40137 200692-200772 Sentence denotes The sequencing data was in agreement with the CE result of an overall 1 bp gain.
T80131 200773-200904 Sentence denotes Taken together, it can be concluded that this patient has a 2 base insertion and 1 base deletion on the same allele of CALR exon 9.
T6926 200905-201060 Sentence denotes The 2 bp insertion found in the patient's sample produces a +1 reading frame shift, resulting in the common mutation sequence for the first 13 amino acids.
T2670 201061-201187 Sentence denotes However, the downstream deletion changes the reading frame to +2 compared to wildtype, generating a novel amino acid sequence.
T75166 201188-201352 Sentence denotes This novel mutant sequence extends 10 amino acids longer and results in a greater addition of positively charged amino acids compared to the common mutant sequence.
T89462 201353-201365 Sentence denotes Conclusions:
T6899 201366-201490 Sentence denotes To our knowledge, a CALR mutation with an initial +1 and subsequent +2 open reading frame has not been previously described.
T10812 201491-201590 Sentence denotes The unique nature of this mutation may provide some insight into critical sequence for oncogenesis.
T20165 201591-201755 Sentence denotes This mutant does contain the first 13 amino acids of the common mutant sequence; therefore, sequence downstream of this core may be less important for oncogenicity.
T46587 201756-201894 Sentence denotes Alternatively, it is possible that the additional positively charged amino acids may enhance the oncogenic effect of this unique mutation.
T41286 201895-201988 Sentence denotes Hypermutation Status in Splenic Marginal Zone Lymphomas Using Next-Generation Sequencing J.C.
T59210 201989-202005 Sentence denotes Gomez-Gelvez, C.
T72941 202006-202012 Sentence denotes Ho, W.
T39651 202013-202021 Sentence denotes Yu, M.H.
T11228 202022-202030 Sentence denotes Syed, A.
T3013 202031-202040 Sentence denotes Zehir, T.
T4773 202041-202050 Sentence denotes Baldi, A.
T95981 202051-202060 Sentence denotes Dogan, M.
T63423 202061-202072 Sentence denotes Ladanyi, J.
T18127 202073-202080 Sentence denotes Yao, K.
T46 202081-202089 Sentence denotes Nafa, M.
T24022 202090-202150 Sentence denotes Arcila Memorial Sloan-Kettering Cancer Center, New York, NY.
T96296 202151-202164 Sentence denotes Introduction:
T22441 202165-202296 Sentence denotes Splenic marginal zone lymphoma (SMZL) is a rare indolent B-cell neoplasm involving spleen, bone marrow (BM) and, frequently, blood.
T28957 202297-202488 Sentence denotes Its distinction from similar indolent B-cell malignancies may be often challenging, particularly when diagnosis must be based on the BM findings alone without the support of spleen histology.
T84494 202489-202751 Sentence denotes Prior studies have shown that SMZL exhibit specific immunoglobulin heavy variable gene (IGHV) gene biases which are distinct from other entities and thus ancillary testing could be potentially utilized to aid in the diagnosis or further stratifying this disease.
T51228 202752-202896 Sentence denotes This assessment is, however, often not feasible in the clinical setting as current methods are laborious and not performed in most laboratories.
T89035 202897-203126 Sentence denotes In this study, we explore the utility of next-generation sequencing (NGS) for the clinical characterization of IGHV in a cohort of SMZL and compare it to other subtypes of marginal zone lymphomas (MZL) reported in the literature.
T89830 203127-203274 Sentence denotes Methods: BM samples from patients with an established diagnosis of SMZL and submitted for routine clonality assessment were selected for the study.
T48172 203275-203477 Sentence denotes After establishing the presence of IGH clonality by capillary electrophoresis, the samples were analyzed using an NGS assay targeting IGH-FR1 (Lymphotrack, Invivoscribe) and sequenced by Illumina MiSeq.
T14469 203478-203567 Sentence denotes Data was analyzed using the LymphoTrack IGH-FR1 and Somatic Hypermutation (SHM) software.
T84179 203568-203664 Sentence denotes Clinical and other ancillary laboratory data were collected from the electronic medical records.
T69582 203665-203673 Sentence denotes Results:
T13464 203674-203713 Sentence denotes A total of 20 BM samples were analyzed.
T67543 203714-203801 Sentence denotes Patients included 8 women and 12 men with a median age at diagnosis of 68 years (range:
T95131 203802-203812 Sentence denotes 47 to 87).
T37026 203813-203900 Sentence denotes IGHV families most frequently rearranged were IGHV3 (11/20, 55%) and IGHV4 (6/20, 30%).
T18099 203901-204092 Sentence denotes The IGHV genes most frequently rearranged were IGHV4-34 (4/20, 20%) followed by IGHV3-23 (2/20, 10%), IGHV3-30 (2/20, 10%), IGHV3-33 (2/20, 10%), IGHV3-73 (2/20, 10%) and IGHV1-3 (2/20, 10%).
T84952 204093-204158 Sentence denotes Using a 98% identity cut-off value, 12/20 cases (60%) showed SHM.
T25480 204159-204246 Sentence denotes Review of the literature showed similar pattern of IGHV usage to other subtypes of MZL.
T47429 204247-204259 Sentence denotes Conclusions:
T64481 204260-204354 Sentence denotes We confirm that SMZL have a biased IGHV gene usage, which is in keeping with prior literature.
T50965 204355-204549 Sentence denotes This usage, however, has significant overlap with other subtypes of MZL considered in the differential diagnosis and therefore does not provide a means of discrimination for diagnostic purposes.
T76972 204550-204691 Sentence denotes This finding however, suggests that the pathogenesis of SMZL may involve epitopes or an antigenic trigger common to other indolent lymphomas.
T71147 204692-204870 Sentence denotes Whether particular molecular characteristics of the IG receptors might be associated with clinical outcome, genetic or phenotypic features is an area that deserves further study.
T59370 204871-204884 Sentence denotes Introduction:
T61022 204885-205041 Sentence denotes Incidence of non-Hodgkin lymphoma (NHL) is increasing in sub-Saharan Africa (SSA), primarily due to human immunodeficiency virus (HIV) and population aging.
T81539 205042-205125 Sentence denotes Many aggressive NHL subtypes are causally associated with Epstein-Barr virus (EBV).
T99572 205126-205351 Sentence denotes We hypothesized that plasma EBV DNA is a potential tumor marker associated with clinical outcome and risk of recurrence in patients with jmd.amjpathol.org ■ The Journal of Molecular Diagnostics aggressive NHL in this setting.
T54931 205352-205360 Sentence denotes Methods:
T78793 205361-205585 Sentence denotes The Kamuzu Central Hospital Lymphoma Study is a prospective cohort of patients with pathologically confirmed lymphoproliferative disorders receiving treatment under local conditions at a national teaching hospital in Malawi.
T163 205586-205692 Sentence denotes Plasma EBV DNA was measured at enrollment, midtreatment, and treatment completion, using a real-time qPCR.
T23335 205693-205881 Sentence denotes Overall survival (OS) and progression-free survival (PFS) were evaluated by Kaplan-Meier methods, and survival differences by baseline plasma EBV DNA were assessed using the log-rank test.
T44417 205882-206008 Sentence denotes Results: EBV viral loads were available from 70/81 (86%) adults with aggressive NHL enrolled between May 2013 to October 2015.
T74442 206009-206202 Sentence denotes Diagnoses included diffuse large B-cell lymphoma (DLBCL, N=43), Burkitt lymphoma (N=6), NK and Tcell lymphomas (N=6), plasmablastic lymphoma (N=4), and other high grade B-cell lymphomas (N=11).
T11076 206203-206258 Sentence denotes The majority of patients (59%, N=41) were HIV-positive.
T91350 206259-206351 Sentence denotes Baseline plasma EBV DNA was detected in 37/70 (53%) at a median level of 3.9 log10copies/mL.
T70672 206352-206672 Sentence denotes Baseline plasma EBV DNA >3.0 log10copies/mL was associated with inferior OS (P = 0.007) and PFS (P = 0.009), and these associations were similar when analyses were restricted to patients with aggressive B-cell NHL (N=64, P = 0.039 and 0.048 for PFS) or diffuse large B-cell lymphoma (P = 0.007 for OS and 0.023 for PFS).
T85304 206673-206837 Sentence denotes In addition, serial assessments during treatment demonstrated that clinically relapsed or progressed NHL was typically associated with rising plasma EBV DNA levels.
T14581 206838-206996 Sentence denotes Conclusions: EBV viral load monitoring is an implementable tool which may have utility as a prognostic and response assessment tool for aggressive NHL in SSA.
T19563 206997-207010 Sentence denotes Introduction:
T51819 207011-207090 Sentence denotes Acute myeloid leukemia (AML) carries a high mortality rate and economic burden.
T74739 207091-207221 Sentence denotes Elucidating the heterogeneity of AML will aid in understanding the hematopoietic stem cell (HSC) self-renewal and differentiation.
T62104 207222-207461 Sentence denotes Though AML is classified as a myeloid neoplasm, we were interested in determining the prevalence of clonal rearrangements within the immunoglobulin heavy (IGH) and light chain (IGK), T-cell receptor gamma (TRG) loci in AML patient samples.
T63304 207462-207603 Sentence denotes Methods: DNA was extracted from a random sampling of 200 AML anonymized patient residual peripheral blood (PB) or bone marrow (BM) specimens.
T53075 207604-207664 Sentence denotes Each DNA sample was tested for 6 different PCR master mixes:
T29994 207665-207799 Sentence denotes IdentiClone IGH Tubes A, B, C, which target the framework (FR) 1, 2, and 3 regions, respectively; IGK Tube A, IGK Tube B, and TRG 2.0.
T36164 207800-207865 Sentence denotes Amplicon products were analyzed using the ABI 3500 XL instrument.
T91960 207866-207956 Sentence denotes Based on the florescent signals, clonal (positive) or polyclonal (negative) were assessed.
T91747 207957-207965 Sentence denotes Results:
T29139 207966-208089 Sentence denotes The IdentiClone IGH assay identified 23 (12%), 14 (7%) and 16 (8%) clonal positive samples for FR 1, 2 and 3, respectively.
T79054 208090-208213 Sentence denotes Combining all 3 IGH tubes increased the clonal detection rate to 28 (14%) with 172 (86%) samples determined to be negative.
T9427 208214-208332 Sentence denotes The IdentiClone IGK assays identified 17 (9%) and 11 (6%) clonal positive samples for Tube A and Tube B, respectively.
T95571 208333-208442 Sentence denotes Combining the two IGK tubes increased clonal detection to 23 (12%), with 175 (88%) determined to be negative.
T25152 208443-208563 Sentence denotes Combining all 5 IGH + IGK tubes, 35 (18%) clonal positive samples and 165 (83%) clonal negative samples were identified.
T33746 208564-208662 Sentence denotes The TRG 2.0 assay detected 85 (43%) clonal positive samples and 114 (57%) clonal negative samples.
T75972 208663-208837 Sentence denotes Overall, using 6 tubes of PCR MM across IGH, IGK and TRG assays, 99 (50%) samples were identified as clonal positive and 101 (50%) samples were identified as clonal negative.
T28575 208838-208850 Sentence denotes Conclusions:
T50024 208851-208944 Sentence denotes Approximate 50% of AML samples demonstrated at least one clonal IG or TCR gene rearrangement.
T65032 208945-209169 Sentence denotes Although it is unclear if it is the malignant myeloid cells or companion lymphoid cells that harbor these somatic gene rearrangements, the relatively high percentage in AML makes this an area worthy of further investigation.
T25031 209170-209183 Sentence denotes Introduction:
T13908 209184-209497 Sentence denotes Next-generation sequencing (NGS) of hematopoietic and lymphoid neoplasm genomes promises to revolutionize oncology, with the ability to design and use targeted drugs, to predict outcome and response, and to classify patients' responses to treatment more thoroughly using more predictive combinations of mutations.
T94217 209498-209687 Sentence denotes It is of critical importance that the NGS platform chosen, the chemistry utilized and the well vetted bioinformatics are then applied consistently for future adoption in clinical decisions.
T89083 209688-209955 Sentence denotes To illustrate the increased capacity and resolution of NGS for the comprehensive characterization of patients with hematologic cancers, we sequenced both clinical patient samples and contrived cell lines using a novel specific targeted strategy involving DNA and RNA.
T14884 209956-210232 Sentence denotes Using contrived cell lines, we utilize maximum gene coverage, long read lengths, and higher sequencing depth to accurately detect variants, indels and breakpoints critical in both hematologic development and for tracking minimal residual disease (MRD) and clonal architecture.
T96804 210233-210418 Sentence denotes We demonstrate limit of detection that allows for complete characterization of the molecular changes, with high sensitivity and specificity, thus allowing clonal architecture discovery.
T91874 210419-210427 Sentence denotes Methods:
T20906 210428-210611 Sentence denotes To examine these cancers, we targeted coding exons (571 genes) and potential genomic breakpoint regions within known somatic gene fusions (360 genes) comprising the MyHeme gene panel.
T81136 210612-210741 Sentence denotes We sequenced target loci on the Illumina MiSeq platform to an average depth of coverage 1000x for cell lines and patient samples.
T92717 210742-210917 Sentence denotes Using a custom bioinformatics pipeline, we performed thorough mutation detection analyses to identify single nucleotide variants (SNVs), indels, inversions and translocations.
T3472 210918-211020 Sentence denotes In addition, we calculated allelic frequencies to investigate potential aneuploidy, LOH and clonality.
T67876 211021-211126 Sentence denotes Using contrived samples, we were able to determine limit of detection rates, sensitivity and specificity.
T69519 211127-211135 Sentence denotes Results:
T9390 211136-211267 Sentence denotes Our analyses of targeted sequencing results from cell lines identified the published genomic variants within MyHeme targeted genes.
T41193 211268-211333 Sentence denotes Critically, our assay enabled detection of variants as low as 5%.
T37222 211334-211462 Sentence denotes In many cases, these variants were more fully characterized for their precise genomic breakpoints and inserted sequence content.
T68603 211463-211475 Sentence denotes Conclusions:
T29774 211476-211714 Sentence denotes We demonstrate that by specifically targeting driver genes using the MyHeme gene panel, we can comprehensively characterize mutations for AML, ALL, Non-Hodgkin's, Multiple Myeloma cell lines and patients with these hematologic conditions.
T81325 211715-211921 Sentence denotes Our results show this assay can comprehensively characterize the cancer genome of patients, identifying not only primary clones, but secondary clones that are present in at least 5% of the patient's sample.
T39850 211922-211935 Sentence denotes Introduction:
T77841 211936-212096 Sentence denotes By implementing novel technologies on the Ion S5 sequencing platform, read lengths of 600 bp were achieved, equivalent to Sanger capillary sequencing platforms.
T26620 212097-212105 Sentence denotes Methods:
T32799 212106-212295 Sentence denotes Leveraging innovations in isothermal amplification, Ion Sphere particles templating, and sequencing chemistry, we demonstrate efficient, high quality sequencing with a 600 to 700 base mode.
T72560 212296-212449 Sentence denotes These advancements were used to sequence human leukocyte antigen (HLA) libraries, a haplotyping application requiring long reads for phasing information.
T59156 212450-212458 Sentence denotes Results:
T30641 212459-212583 Sentence denotes Using TypeStream auto-analysis, concordance rates of 99.7% were achieved for HLA-A, B, C, DRB1, DQB1, DPB1, and DRB345 loci.
T63949 212584-212719 Sentence denotes The higher output of the Ion S5 system allowed us to type 96 samples on one Ion 530 chip with substantially improved typing resolution.
T74 212720-212732 Sentence denotes Conclusions:
T4752 212733-212907 Sentence denotes We believe that these longer reads have the ability to span the 500bp intron, linking exon 2 and 3 of the Class 1 genes, leading to less ambiguity and proper gametic phasing.
T25989 212908-213104 Sentence denotes With long contiguous, accurate reads, we demonstrate the potential to dramatically reduce sample cost and time by multiplexing up to 192 samples on one chip and delivering results in a single day.
T86265 213105-213391 Sentence denotes Introduction: MYD88 L265P, a diagnostic marker for Waldenström macroglobulinemia (WM) /lymphoplasmacytic lymphoma (LPL), has also been described in IgM-monoclonal gammopathy of undetermined significance and in other types of lymphomas, therefore abating initially predicted specificity.
T58195 213392-213489 Sentence denotes We recently demonstrated a novel approach to assess MYD88 L265P mutation status (Burnworth et al.
T68106 213490-213524 Sentence denotes AMP 2015; platform presentation) .
T78129 213525-213633 Sentence denotes Combining flow cytometric cell sorting (FACS) with molecular analysis increased the specificity for WM/ LPL.
T76516 213634-213791 Sentence denotes Mutation analysis supports a diagnosis of WM/LPL with high confidence only if the MYD88 L265P is detected in both the plasma-cell and B-lymphocyte fractions.
T75679 213792-213917 Sentence denotes Cases with MYD88 L265P present only in either the plasma-cell or B-lymphocyte clones may represent two independent neoplasms.
T3293 213918-214075 Sentence denotes The current study compares analysis for MYD88 L265P in plasma-cell and B-lymphocytes populations sorted by FACS to plasma cells enriched with magnetic beads.
T46516 214076-214084 Sentence denotes Methods:
T55135 214085-214167 Sentence denotes Four specimens with known WM/LPL and 4 specimens with suspected LPL were analyzed.
T70996 214168-214314 Sentence denotes By flow cytometry, all specimens had restriction for the same immunoglobulin light chain in both the plasma-cell clone and the B-lymphocyte clone.
T74622 214315-214515 Sentence denotes FACS isolated clonal plasma and B lymphoid cell fractions, whole bone marrows as well as magnetic bead enriched CD138+ collections were tested for the presence of MYD88 mutations by Sanger sequencing.
T62575 214516-214697 Sentence denotes Results: MYD88 L265P was detected in the FACS isolated plasma and B lymphoid cell fractions as well as in the CD138+ magnetic bead collection of all specimens with confirmed WM/LPL.
T88208 214698-214865 Sentence denotes Magnetic CD138+ collections tested positive for MYD88 L265P in all specimens revealing positive MYD88 mutation status in both the FACS plasma and FACS B-cell fraction.
T25852 214866-215098 Sentence denotes In contrast, enriched CD138+ cell fractions collected by magnetic beads tested positive for bone marrow aspirate specimens harboring MYD88 L265P in only the FACS isolated CD19+ B lymphoid cells but not in FACS isolated plasma cells.
T95683 215099-215111 Sentence denotes Conclusions:
T65751 215112-215278 Sentence denotes Confirming the presence of MYD88 L265P in both the plasma-cell and B-lymphocyte populations is an important prerequisite to distinguish LPL/WM from related disorders.
T70902 215279-215425 Sentence denotes In this study we demonstrate that flow cytometric cell sorting (FACS) is required for specific MYD88 mutation status of the plasma cell component.
T59660 215426-215535 Sentence denotes Magnetic CD138+ bead enrichment will collect cross-contaminations of other cell types, such as B lymphocytes.
T7885 215536-215687 Sentence denotes Therefore, lymphoid malignancies carrying MYD88 L265P will result in false-positive test results in plasma cell collections enriched by magnetic beads.
T70998 215688-215889 Sentence denotes FACS rather than enrichment with magnetic beads is required for separate analysis of the plasma-cell and B-lymphocyte populations for MYD88 L265P, which is necessary for a specific diagnosis of WM/LPL.
T19466 215890-215892 Sentence denotes C.
T56842 215893-215905 Sentence denotes Moung 1 , Y.
T11143 215906-215916 Sentence denotes Liu 2 , J.
T98305 215917-215932 Sentence denotes Intrieri 1 , L.
T72386 215933-215945 Sentence denotes Borsu 1 , K.
T81753 215946-215959 Sentence denotes Nafa 1 , M.E.
T68178 215960-216071 Sentence denotes Arcila 1 1 Memorial Sloan Kettering Cancer Center, New York, NY; 2 Weill Cornell Medical College, New York, NY.
T2774 216072-216085 Sentence denotes Introduction:
T30356 216086-216205 Sentence denotes The identification of the BRAF V600E mutation in hairy cell leukemia (HCL) has both diagnostic and therapeutic utility.
T12717 216206-216361 Sentence denotes The presence of increased reticulin fibrosis in all bone marrow (BM) samples of patients with HCL constitutes a distinct challenge for mutation assessment.
T27336 216362-216503 Sentence denotes In the vast majority of cases, BM aspirates are scant or result in a "dry tap." In a proportion of patients, the BM may also be hypocellular.
T46702 216504-216592 Sentence denotes Testing methods should therefore be highly sensitive to allow detection of the mutation.
T13414 216593-216753 Sentence denotes In this study, we compare two techniques which incorporate locked nucleic acid (LNA) probes to increase the sensitivity of the V600E mutation detection in HCLs.
T97803 216754-216819 Sentence denotes Methods: HCL cases with archived DNA were selected for the study.
T29469 216820-216912 Sentence denotes All concurrent biopsy sections, flow cytometry and IHCs were reviewed to ensure correlation.
T9781 216913-217043 Sentence denotes Molecular testing was performed by Sanger sequencing with and without an LNA probe to suppress the amplification of wild-type DNA.
T89132 217044-217149 Sentence denotes Digital PCR (dPCR) was performed using LNA probes for both wild-type and mutant DNA on a RainDrop system.
T62043 217150-217281 Sentence denotes Results were compared to standard Sanger sequencing and IHC (when available) using the BRAF V600E mutation specific antibody (VE1).
T81334 217282-217290 Sentence denotes Results:
T1935 217291-217407 Sentence denotes A total of 21 HCL cases were tested by dPCR and the LNA-PCR sequencing assays (18 BMs and 3 peripheral bloods (PB)).
T21476 217408-217492 Sentence denotes Seventeen cases (81%) were positive for the BRAF V600E mutation and 4 were negative.
T28476 217493-217560 Sentence denotes There was 100% concordance between the two high sensitivity assays.
T54310 217561-217633 Sentence denotes Both methods proved superior to both standard Sanger sequencing and IHC.
T64090 217634-217773 Sentence denotes Five of 17 positive cases were not detected using standard Sanger sequencing whereas only 1 of 9 positive cases tested by IHC was negative.
T66989 217774-217830 Sentence denotes The sensitivity of both high sensitivity assays is 0.1%.
T94028 217831-217843 Sentence denotes Conclusions:
T39363 217844-218032 Sentence denotes The detection of the BRAF V600E in BM and PB samples is often challenging due to intrinsic characteristics of the leukemia which result in low tumor yield in the specimens that are tested.
T55657 218033-218174 Sentence denotes LNA probes used in LNA-PCR Sanger sequencing and digital PCR are extremely valuable tools for increasing the sensitivity of a standard assay.
T76227 218175-218674 Sentence denotes Both high sensitivity assays showed equal performance. dPCR has multiple advantages compared to LNA-PCR Sanger sequencing as it requires significantly less DNA and allows for a much rapid turnaround time of 1 day post extraction versus 3 days with LNA-PCR. dPCR is also quantitative, providing valuable information for monitoring of patients. gene are the most common mutations found in acute myeloid leukemia (AML) and are characterized by an aggressive phenotype with a high prevalence of relapse.
T61236 218675-218798 Sentence denotes Internal tandem duplication (ITD) mutations within the juxtamembrane domain are the most common mutations in the FLT3 gene.
T95061 218799-218940 Sentence denotes The development of a sensitive and specific assay for FLT3/ITD mutations represents a significant advancement in guiding treatment decisions.
T29710 218941-218949 Sentence denotes Methods:
T43551 218950-219085 Sentence denotes The next-generation sequencing (NGS) MRD assay was designed to target exons 14 and 15 of the FLT3 gene with a single PCR amplification.
T59090 219086-219229 Sentence denotes Amplicons from up to 24 samples were purified, pooled and sequenced before being analyzed using proprietary software developed by Invivoscribe.
T54186 219230-219417 Sentence denotes Validation was performed by spiking in fixed amounts of mutant DNA into wild-type DNA to establish a sensitivity equivalent to detection of at least one ITD-containing cell out of 10,000.
T57913 219418-219483 Sentence denotes The DNA input of the assay was 700 ng (>100,000 cell equivalent).
T26231 219484-219557 Sentence denotes The assay was applied to bone marrow DNA from patients with FLT3/ITD AML.
T4335 219558-219566 Sentence denotes Results:
T21242 219567-219808 Sentence denotes The FLT3/ITD MRD assay can detect mutations with a mutant cell sensitivity of 10 -4 (1 mutant cell in a background of ten thousand normal cells) which is equivalent to an allelic sensitivity of 5x10 -5 when a single mutant allele is present.
T30932 219809-219902 Sentence denotes The linearity of the assay is excellent in the mutation/total reads range of 10 -1 to 10 -5 .
T18065 219903-220061 Sentence denotes Excellent precision and reproducibility of the assay was demonstrated in the range of 10 -3 to 10 -5 by testing DNA from two cell lines and a clinical sample.
T37767 220062-220219 Sentence denotes Fifteen clinical follow-up samples determined to be negative for the FLT3/ITD mutation by the standard CLIA-certified assay were tested by the NGS MRD assay.
T6936 220220-220385 Sentence denotes There was no detectable FLT3/ITD mutation in six of these samples by the MRD assay, which are concordant with clinical outcomes that these patients are disease free.
T49025 220386-220514 Sentence denotes TheFLT3/ITD mutations were detected with read frequencies in the range of 1.38x10 -6 to 3.67x10 -3 in the rest of the 9 samples.
T25540 220515-220641 Sentence denotes The ITD lengths detected by NGS were the same as these detected by the CLIA-certified assay in the original diagnosis samples.
T43834 220642-220707 Sentence denotes These nine patients either relapsed or are still under treatment.
T62477 220708-220852 Sentence denotes The NGS FLT3/ITD MRD assay is highly specific and is at least two orders of magnitude more sensitive than current commercially available assays.
T55326 220853-220959 Sentence denotes The results of clinical sample tested by the NGS MRD assay showed 100% concordance with clinical outcomes.
T68074 220960-221031 Sentence denotes This assay provides a reliable tool to assess MRD in leukemia patients.
T72099 221032-221036 Sentence denotes A.R.
T45920 221037-221047 Sentence denotes Carson, Z.
T97955 221048-221055 Sentence denotes Xie, V.
T21477 221056-221069 Sentence denotes McClain, J.E.
T42482 221070-221080 Sentence denotes Miller, T.
T39912 221081-221135 Sentence denotes Stenzel Invivoscribe Technologies, Inc, San Diego, CA.
T83849 221136-221149 Sentence denotes Introduction:
T87598 221150-221251 Sentence denotes The nucleophosmin (NPM1) gene is an important marker for acute myeloid leukemia (AML) stratification.
T31458 221252-221438 Sentence denotes NPM1 is one of the most commonly mutated genes in AML, with mutations seen in roughly 35% of patients at diagnosis, and in approximately 60% of adult cytogenetically normal AML patients.
T99248 221439-221629 Sentence denotes Importantly, NPM1 mutations generally confer a slightly more favorable outcome in AML patients and can mitigate poor prognoses when they occur concurrently with activating mutations in FLT3.
T7570 221630-221766 Sentence denotes As a commonly mutated gene with prognostic value in AML, NPM1 is an appropriate biomarker for minimal residual disease (MRD) monitoring.
T52000 221767-221855 Sentence denotes MRD detection has proven to be valuable in the clinical management of patients with AML.
T94837 221856-222014 Sentence denotes NPM1 is of particular importance, as it was recently shown that the presence of NPM1 mutants after chemotherapy was associated with a greater risk of relapse.
T43971 222015-222190 Sentence denotes Thus, the development of a sensitive and reliable assay to detect NPM1 mutations at low frequencies represents a significant advancement in guiding treatment for AML patients.
T88244 222191-222199 Sentence denotes Methods:
T40898 222200-222385 Sentence denotes The assay targets exon 12 of the NPM1 gene using a single optimized PCR amplification that was developed to overcome inherent challenges caused by repetitive sequence across this locus.
T8410 222386-222480 Sentence denotes Using DNA input of 700 ng (>100,000 cell equivalents), up to 24 samples are amplified per run.
T82869 222481-222621 Sentence denotes The PCR libraries are then purified, pooled and sequenced using a next-generation sequencing (NGS) platform to a depth of at least 100,000x.
T66783 222622-222709 Sentence denotes Sequenced reads are then analyzed using proprietary software developed by Invivoscribe.
T71105 222710-222862 Sentence denotes The specificity and sensitivity of the NPM1 mutation detection were established and validated by spiking fixed amounts of mutant DNA into wild-type DNA.
T55907 222863-222871 Sentence denotes Results:
T63370 222872-223018 Sentence denotes We have developed an amplicon-based NGS assay to detect NPM1 mutations with a detection sensitivity that can be used to track MRD in AML patients.
T29724 223019-223258 Sentence denotes This assay can detect mutations with a mutant cell sensitivity of at least 10 -4 (1 mutant cell in a background of ten thousand normal cells), which is equivalent to an allelic sensitivity of 5x10 -5 when a single mutant allele is present.
T28420 223259-223389 Sentence denotes We performed linearity and show that the assay's performance is excellent in the in the allele frequency range of 10 -1 to 10 -5 .
T32737 223390-223637 Sentence denotes In addition, we assayed cell lines and clinical samples multiple times (>20 replicates each) to establish high precision and reproducibility for the detection of different NPM1 mutations, including the most commonly seen 4bp insertion (COSM17559).
T27949 223638-223650 Sentence denotes Conclusions:
T23172 223651-223866 Sentence denotes The NPM1 MRD NGS assay is a highly specific test that can detect NPM1 mutations with a sensitivity at least two orders of magnitude greater than current commercially available assays without sacrificing specificity.
T34503 223867-223933 Sentence denotes This assay provides a reliable tool to assess MRD in AML patients.
T34073 223934-223947 Sentence denotes Introduction:
T53210 223948-224041 Sentence denotes Burkitt lymphoma is one of the most common non-Hodgkin lymphomas in children and adolescents.
T49594 224042-224140 Sentence denotes The genetic hallmark of Burkitt lymphoma is chromosomal translocations involving the MYC oncogene.
T12641 224141-224257 Sentence denotes Up to 90% of the cases have a translocation between MYC and one of the three immunoglobulin loci: IGH, IGK, and IGL.
T75598 224258-224419 Sentence denotes A small group of MYC-negative lymphoma cases sharing similar pathomorphology, immunophenotype, and clinical presentation of Burkitt Lymphoma have been described.
T95030 224420-224515 Sentence denotes However, the genomic alteration of these Burkitt-like lymphomas have not been well illustrated.
T10579 224516-224719 Sentence denotes We present the comprehensive genomic analyses of a MYC-negative Burkitt lymphoma case using conventional cytogenetics fluorescence in situ hybridization (FISH) and high resolution SNP array technologies.
T74136 224720-224728 Sentence denotes Methods:
T58879 224729-224816 Sentence denotes A 13 year-old boy presented with right side neck swelling and an enlarged right tonsil.
T83443 224817-224886 Sentence denotes Tonsillectomy specimen showed malignancy resembling Burkitt lymphoma.
T16536 224887-225079 Sentence denotes Chromosomal analysis, FISH with CEP8/MYC/IGH tricolor dual fusion probe and KMT2A dual color break apart probe (Abbott Molecular), and high resolution SNP array were performed on the specimen.
T46783 225080-225151 Sentence denotes A literature review of MYCnegative Burkitt lymphoma was also conducted.
T99994 225152-225257 Sentence denotes Results: FISH analysis found no evidence of IGH/MYC gene rearrangement but three copies of KMT2A signals.
T56450 225258-225587 Sentence denotes Chromosomal analysis revealed two related abnormal clones: the stemline showed a derivative chromosome 11 consisting of an inverted duplication of 11q12.3-11q24.1 and a translocation between the derivative chromosome 11 at band 11q24.1 and the chromosome 12 at band 12p11.2; the subclone displayed an extra copy of chromosome 19.
T88596 225588-225808 Sentence denotes High resolution SNP array studies demonstrated a 59.5Mb mosaic duplication at chromosome 11q12.3-q24.1 followed by a 12.7Mb terminal deletion at 11q24.1; and two discontinuous duplications at 12p13.33 and 12p13.32p11.22.
T90380 225809-225943 Sentence denotes Array analysis also identified a copy number neutral loss of heterozygosity (cnLOH) involving almost entire long arm of chromosome 18.
T95731 225944-225956 Sentence denotes Conclusions:
T87389 225957-226143 Sentence denotes We present a case of MYC-negative Burkitt lymphoma with partial duplication and partial deletion of chromosome 11q, unbalanced translocation between chromosomes 11 and 12, and cnLOH 18q.
T50587 226144-226328 Sentence denotes Literature review found that duplication of proximal 11q region with or without telomeric deletion have been described in patients with Burkitt-like lymphoma without MYC rearrangement.
T74267 226329-226453 Sentence denotes Our case adds additional evidence supporting the association of chromosome 11q aberration and MYC-negative Burkitt lymphoma.
T23553 226454-226551 Sentence denotes The clinical significance of additional genomic findings in this patient remains to be explained.
T58328 226552-226554 Sentence denotes B.
T60338 226555-226569 Sentence denotes Van Deusen, M.
T17472 226570-226582 Sentence denotes Bessette, L.
T42621 226583-226594 Sentence denotes Johnson, A.
T85568 226595-226605 Sentence denotes Berlin, M.
T18104 226606-226617 Sentence denotes Banos, L.M.
T16953 226618-226629 Sentence denotes Griffin, E.
T86513 226630-226643 Sentence denotes Reckase, J.A.
T34917 226644-226653 Sentence denotes Stahl, A.
T82734 226654-226663 Sentence denotes Licon, B.
T29172 226664-226699 Sentence denotes Kudlow ArcherDX, Inc., Boulder, CO.
T66418 226700-226713 Sentence denotes Introduction:
T92950 226714-226899 Sentence denotes Acute myeloid leukemia (AML) oncogenesis is thought to require multiple somatic mutations in a "two-hit" process to 1) increase proliferation and 2) prevent maturation of myeloid cells.
T71356 226900-227061 Sentence denotes Whereas FLT3 and KIT mutations are associated with increased proliferation, NPM1, CEBPA and several other mutations can be associated with maturation inhibition.
T70784 227062-227261 Sentence denotes The most common mutations in AML are internal tandem duplications (ITDs) in FLT3, which are detected in more than 20% of pediatric and adult AML cases and are associated with an aggressive phenotype.
T52396 227262-227415 Sentence denotes As FLT3-ITD expressed kinases are sensitive to tyrosine kinase inhibitors, they are of considerable interest for the development of novel AML treatments.
T95699 227416-227548 Sentence denotes Capillary gel electrophoresis can detect ITDs but cannot be easily coupled with assays to detect other mutation types common in AML.
T11396 227549-227654 Sentence denotes Next-generation sequencing (NGS)-based methods enable comprehensive detection of multiple mutation types.
T63711 227655-227842 Sentence denotes However, detection of ITDs by NGS is particularly challenging, in part because of their highly variable nature and the difficulties of mapping repeated sequences to a wild-type reference.
T6653 227843-228041 Sentence denotes Anchored Multiplex PCR (AMP) is a target enrichment strategy for NGS that uses molecular barcoded adaptors and genespecific primers, permitting open-ended capture of DNA fragments from a single end.
T13523 228042-228195 Sentence denotes We present an approach based on AMP technology and a novel bioinformatics algorithm to detect ITDs in FLT3 as well as other mutation types common in AML.
T73813 228196-228204 Sentence denotes Methods:
T50682 228205-228353 Sentence denotes We developed the Archer VariantPlex Core AML library preparation assay for NGS to detect FLT3-ITDs from genomic DNA extracted from clinical samples.
T52347 228354-228457 Sentence denotes We designed AMP probes to cover the commonly mutated juxtamembrane domain and tyrosine kinase domain 1.
T22493 228458-228602 Sentence denotes We further developed a novel de novo sequence assembly algorithm based on over 2000 in silico datasets representing a large range of known ITDs.
T33724 228603-228611 Sentence denotes Results:
T81916 228612-228781 Sentence denotes In silico datasets enabled optimization of the VariantPlex Core AML analysis algorithm, resulting in the detection of over 98% of in silico ITDs with no false positives.
T24114 228782-228964 Sentence denotes The VariantPlex Core AML library preparation assay in conjunction with the optimized analysis algorithm enabled sensitive NGSbased detection of ITDs in 16 AML-positive blood samples.
T62692 228965-229061 Sentence denotes These results were consistent with results obtained from standard capillary gel electrophoresis.
T79469 229062-229202 Sentence denotes In addition, point mutations in the TKD of FLT3 and insertions in NPM1 exon 11 were detected in 2/7 and 5/7 FLT3-ITD positive blood samples.
T77264 229203-229215 Sentence denotes Conclusions:
T9309 229216-229315 Sentence denotes Our data show that AMP enables accurate NGS-based detection of FLT3-ITDs from clinical DNA samples.
T23747 229316-229488 Sentence denotes As this approach can detect multiple mutation types from a single sample, our VariantPlex Core AML kit enables simultaneous detection of multiple mutations relevant in AML.
T45044 229489-229493 Sentence denotes J.T.
T56734 229494-229503 Sentence denotes Kim, K.J.
T34091 229504-229516 Sentence denotes Newsom, H.Y.
T51478 229517-229525 Sentence denotes Wang, P.
T84035 229526-229582 Sentence denotes Starostik University of Florida Health, Gainesville, FL.
T94749 229583-229596 Sentence denotes Introduction:
T1703 229597-229745 Sentence denotes Enteropathy-associated T-cell lymphoma (EATL) is a rare type of malignant peripheral T-cell lymphoma (PTCL) typically affecting the small intestine.
T96320 229746-229860 Sentence denotes Clinically, EATL has generally shown a poor response to chemotherapy and no personalized targeted-therapies exist.
T72620 229861-229976 Sentence denotes EATL is known to be highly associated with celiac disease, harboring the same genetic background as celiac disease.
T93804 229977-230063 Sentence denotes About 90% of PTCLs carry cytogenetic abnormalities although none is specific for PTCL.
T91471 230064-230237 Sentence denotes In one study, EATL was characterized by frequent complex gains of 9q31.3-qter (70% of cases), or by an almost mutually exclusive 2.5-megabase loss of 16q12.1 (23% of cases).
T48582 230238-230359 Sentence denotes Amplification of NOTCH1 and ABL1 gene loci has been observed in EATL, but the gene mutational profile is largely unknown.
T19772 230360-230474 Sentence denotes The aim of this study was to analyze EATL for SNVs and short indels with a next-generation sequencing (NGS) assay.
T26486 230475-230483 Sentence denotes Methods:
T60203 230484-230587 Sentence denotes Genomic DNA from FFPE tissue was extracted using phenol/chloroform and sequenced using a LDT, GatorSeq.
T7630 230588-230771 Sentence denotes The assay is a hybridization capture-based next-generation targeted resequencing assay for deep sequencing of 1207 exons of 76 key cancer genes encompassing 242,205 bp of genomic DNA.
T65609 230772-230836 Sentence denotes 24 EATL tumors were analyzed on the Illumina NextSeq instrument.
T19588 230837-230988 Sentence denotes Sequence data were then processed using a customized analysis pipeline designed to accurately detect base substitutions and short insertions/deletions.
T91127 230989-230997 Sentence denotes Results:
T44354 230998-231113 Sentence denotes The patient population consisted of 24 cases with even split between males and females with average age 59.3 years.
T30136 231114-231264 Sentence denotes The most frequently detected alterations were seen in the following genes: BCOR, BRAF, EZH2, FAM5C, IDH1, MLL, MYH11, NOTCH1, NUP214, RUNX1, and TET2.
T56736 231265-231375 Sentence denotes Conclusions: EATLs display multiple genomic alterations known to be present in other hematologic malignancies.
T87339 231376-231485 Sentence denotes GatorSeq allows for a rapid and cost-effective mutational profiling of hematologic malignancies such as EATL.
T7092 231486-231775 Sentence denotes The information generated by the NGS technologies enables clinicians to make improved diagnostic and treatment decisions. (CLL) have been shown to bear mutations which contribute to oncogenic signaling and therapy resistance, or associated with biologically relevant subsets and prognosis.
T67386 231776-231952 Sentence denotes Therefore, we developed and analytically validated a next-generation sequencing (NGS) panel capturing 220 genes relevant to these diseases for use in a clinical diagnostic lab.
T90786 231953-231961 Sentence denotes Methods:
T85705 231962-232062 Sentence denotes Target enrichment for the NGS assay (FOCUS::Lymphoid) was developed using a hybrid-capture approach.
T83562 232063-232166 Sentence denotes Probes (N=4086) were designed (Nimbledesign, Roche, Inc.) to capture all coding exons of the 220 genes.
T97314 232167-232242 Sentence denotes Sequencing was performed on either a Miseq or Nextseq500 sequencing system.
T2078 232243-232425 Sentence denotes Library preparation and sequencing methods were established using a set of 210 formalin-fixed, paraffin-embedded (FFPE) or freshfrozen (FF) DNAs from DLBCL, FL, MCL, or CLL patients.
T47499 232426-232500 Sentence denotes Target performance metrics were set using 95 samples from within this set.
T39893 232501-232594 Sentence denotes Analytical validation of the assay used an additional 60 FF or FFPE DNAs and normal controls.
T6851 232595-232665 Sentence denotes A diluted Jurkat cell line was used as a positive control in all runs.
T6783 232666-232891 Sentence denotes After sequencing, alignment and variant calling were performed usingCLC Genomics Workbench v2.1 (CLCbio) according to build hg19/GRCh37 reference genome with variant annotation using Cartegenia Bench Lab NGS v4.2.2 (Agilent).
T66713 232892-232900 Sentence denotes Results:
T5989 232901-232966 Sentence denotes The average read depth was found to be 590X (FF) and 540X (FFPE).
T56669 232967-233071 Sentence denotes Target performance metrics were with >10% targets not achieving this metric were excluded from analysis.
T16433 233072-233168 Sentence denotes Two genes (CTBP2, PDPK1) and 7 targets failed to consistently achieve this and are not analyzed.
T68865 233169-233282 Sentence denotes The sensitivity of the assay is 5% allele variant frequency (AVF) at coverage >200X, and 10% between 60X to 200X.
T96982 233283-233398 Sentence denotes Comparison of DNAs (N=39) run on the NGS panel versus other CLIA-approved sequencing tests showed 100% concordance.
T90006 233399-233575 Sentence denotes Precision of the assay was tested using 7 FF and 4 FFPE DNAs where 99% concordance was achieved between 104 variants SNVs, 14 indels, 1 MNV) from samples run on different days.
T26801 233576-233699 Sentence denotes Intra-run assessment (N=3) showed 29 variants (24 SNVs and 5 indels) were consistently detected, yielding 100% concordance.
T96930 233700-233786 Sentence denotes Hapmap and normal DNAs (5 FF and 3 FFPE) were found to harbor no pathogenic mutations.
T35988 233787-233874 Sentence denotes Variants are reproducible with starting material as low as 50 ng (FFPE) and 25 ng (FF).
T95437 233875-233887 Sentence denotes Conclusions:
T25047 233888-233992 Sentence denotes The Focus::Lymphoid NGS panel enables detection of somatic variants relevant to mature B-cell neoplasms.
T11232 233993-234109 Sentence denotes Analytical validation demonstrates the robustness of the assay and can be used for the management of these diseases.
T9446 234110-234123 Sentence denotes Introduction:
T49507 234124-234289 Sentence denotes Bordetella pertussis (B. pertussis), the etiologic agent for whooping cough is still a public health issue despite widespread vaccination of most children in the US.
T50120 234290-234428 Sentence denotes This is due to the failure of some parents to vaccinate their children, and waning immunity, typically between the ages of 11 to 18 years.
T68731 234429-234606 Sentence denotes The very contagious nature of B. pertussis is responsible for localized community outbreaks of whooping cough requiring the need for a rapid, highly specific and sensitive test.
T1659 234607-234746 Sentence denotes Luminex has developed the ARIES, a sample to answer automated instrument, which is designed for their proprietary MultiCode PCR technology.
T15193 234747-234808 Sentence denotes The ARIES uses test cassettes into which the sample is added.
T10678 234809-234895 Sentence denotes The MultiCode PCR primers are manually added to a tube that clips on to the cassettes.
T1979 234896-234996 Sentence denotes Once placed into the ARIES instrument, nucleic acid extraction and PCR analysis are fully automated.
T30335 234997-235211 Sentence denotes The purpose of this study was to determine the suitability of the ARIES platform for detection of B. pertussis and B. parapertussis DNA directly from patient samples compared to our current Light Cycler-based test.
T92801 235212-235342 Sentence denotes Methods: B. pertussis and B. parapertussis MultiCode PCR primers and ARIES test cassettes were obtained from Luminex (Austin, TX).
T81683 235343-235511 Sentence denotes Testing was performed, according to standard instrument settings using Luminex SYNCT software, on 200 μL of patient sample (nasopharyngeal swabs in M4 transport media).
T22406 235512-235631 Sentence denotes Our current method uses the same MultiCode primers and reagents with analysis performed on the Roche Light Cycler (LC).
T34903 235632-235765 Sentence denotes For the latter DNA was extracted from 200 μL of patient sample using the bioMerieux EasyMag, and eluted into 50 μL of EasyMag Buffer.
T69931 235766-235841 Sentence denotes Five (5) μL of DNA was added to the master-mix for a final volume of 25 μL.
T23123 235842-235950 Sentence denotes PCR amplification and melting point analysis were performed according to the manufacturer's recommendations.
T22844 235951-235959 Sentence denotes Results:
T15390 235960-236087 Sentence denotes Forty patient samples collected as above were tested for B. pertussis and B. parapertussis DNA on the ARIES and LC instruments.
T55232 236088-236140 Sentence denotes Agreement between the two PCR methods was very good.
T63973 236141-236197 Sentence denotes One sample negative on the LC was positive by the ARIES.
T68040 236198-236265 Sentence denotes Three samples previously positive by the LC were negative by ARIES.
T40458 236266-236360 Sentence denotes These samples had a very high Ct value on the LC (>40) and had been stored in the freezer long
T21763 236361-236423 Sentence denotes The Journal of Molecular Diagnostics ■ jmd.amjpathol.org term.
T68302 236424-236543 Sentence denotes Otherwise the ARIES Ct values for nearly all the samples tested were lower than for the LC by an average of 3.2 cycles.
T65797 236544-236638 Sentence denotes This is expected since the ARIES utilizes a higher input of the sample DNA than the LC method.
T73387 236639-236651 Sentence denotes Conclusions:
T11471 236652-236727 Sentence denotes The ARIES instrument is a robust, simple to use, sample to result platform.
T23397 236728-236838 Sentence denotes The performance of the ARIES B. pertussis assay compared very well to results obtained using the Light Cycler.
T84171 236839-236929 Sentence denotes The major advantage of the ARIES method is the elimination of all sample processing steps.
T8080 236930-236932 Sentence denotes A.
T30032 236933-236946 Sentence denotes Rezaei 1 , N.
T17797 236947-236968 Sentence denotes Guaring-Angulo 1 , J.
T91854 236969-236982 Sentence denotes Walker 2 , J.
T60243 236983-236996 Sentence denotes Graham 1 , N.
T91847 236997-237074 Sentence denotes Dhiman 1 1 med fusion, Lewisville, TX; 2 Roche Diagnostics, Indianapolis, IN.
T94483 237075-237088 Sentence denotes Introduction:
T2525 237089-237307 Sentence denotes As one of the largest regional laboratories and an integrated Roche Molecular Center of Excellence for molecular diagnostics in Texas, our objective was to consolidate and fully automate our routine viral load testing.
T43579 237308-237485 Sentence denotes We performed a comparative workflow analysis of the Cobas p630 Cobas AmpliPrep/Cobas TaqMan (CAP/CTM) with the Cobas 6800 System as a high throughput molecular testing solution.
T38664 237486-237642 Sentence denotes Our aim was to integrate best practices with lean workflow principles and automation to maximize efficiency, improve turnaround time (TAT) and reduce waste.
T76893 237643-237651 Sentence denotes Methods:
T41862 237652-237844 Sentence denotes Current workflow was evaluated in terms of total volumes, batch sizes, TAT, waste generated and hands-on and walkaway time during pre-analytical, analytical and post-analytical testing phases.
T65971 237845-237984 Sentence denotes These data were analyzed in a performance matrix to identify opportunities for error, overproduction, wait times, waste and spaghetti maps.
T94194 237985-238083 Sentence denotes The outcomes were used to drive recommendations for improved workflow using the Cobas 6800 System.
T87269 238084-238092 Sentence denotes Results:
T75222 238093-238344 Sentence denotes As a leading reference laboratory for transplant centers and AIDS clinics, we consolidated Cytomegalovirus (CMV), Human Immunodeficiency Virus (HIV), Hepatitis B and C (HBV and HCV) viral load assays on CAP/CTM platform in 2013 on International Scale.
T79684 238345-238501 Sentence denotes Current CAP/CTM usage for one shift operation is at a median of 235 (range 171 to 491) samples and 5 (range 2 to 8) runs per day, posing a challenge to TAT.
T83460 238502-238689 Sentence denotes The current workflow had 56 process steps and the performance matrix showed inefficiencies at batched rack preparation, work list review and manual pipetting workaround for short volumes.
T40223 238690-238798 Sentence denotes There were a few efficiency advantages, including complete chain of custody and directionally LIS interface.
T51456 238799-238955 Sentence denotes We observed a ~63% reduction in process steps (35 out of 56) in transitioning to the COBAS 6800, including elimination of 70% of the potential error points.
T47380 238956-239076 Sentence denotes This had a projected time savings of ~1.7 hours per day, improved total TAT by ~4 hours and reduced solid wastes by 80%.
T34641 239077-239089 Sentence denotes Conclusions:
T60353 239090-239307 Sentence denotes The Cobas 6800 System dramatically streamlined and standardized the process for viral load testing by reducing the number of process steps, eliminating potential error points and reducing TAT, hands-on time and waste.
T45188 239308-239448 Sentence denotes Other efficiencies gained were the consolidation of seven instruments into a single instrument, which lead to space and maintenance savings.
T2260 239449-239584 Sentence denotes In conclusion, the Cobas 6800 System was identified as a lean option for molecular viral load testing for a large reference laboratory.
T45814 239585-239587 Sentence denotes T.
T37568 239588-239601 Sentence denotes Sundin 1 , D.
T72217 239602-239616 Sentence denotes Seidman 1 , K.
T11124 239617-239633 Sentence denotes Slaughter 1 , M.
T27854 239634-239649 Sentence denotes Aunchman 1 , L.
T6113 239650-239664 Sentence denotes Grissom 1 , J.
T33971 239665-239677 Sentence denotes Inman 1 , R.
T39519 239678-239689 Sentence denotes Post 1 , A.
T19526 239690-239702 Sentence denotes Shean 1 , K.
T87082 239703-239718 Sentence denotes Gonzalez 2 , N.
T36939 239719-239806 Sentence denotes Suhail 1 1 Sentara Healthcare, Norfolk VA 2 Focus Diagnostics, Cypress CA Introduction:
T85031 239807-240017 Sentence denotes Herpes simplex virus 1 and 2 (HSV-1 and HSV-2) are relatively large double-stranded DNA viruses associated with a wide range of clinical infections including genital, dermal, ocular, and central nervous system.
T52829 240018-240115 Sentence denotes Prompt detection and diagnosis of HSV illnesses are critical for infection control and treatment.
T95688 240116-240276 Sentence denotes Due to numerous biological areas known to harbor HSV, a vast array of clinical specimen sources may be obtained to test for accurate diagnosis of HSV infection.
T16688 240277-240516 Sentence denotes In this study, performance of the Focus Diagnostics HSV real-time PCR assay for quantitative detection of HSV-1 and -2 in whole blood and CSF and qualitative detection of HSV-1 and -2 in APTIMA GEN-PROBE and ThinPrep samples was evaluated.
T76148 240517-240525 Sentence denotes Methods:
T72721 240526-240710 Sentence denotes A total of 441 samples (blood/plasma [n=180], CSF [n=72], ThinPrep [n=92], APTIMA swabs [n=97]) were tested utilizing a combination of retrospective samples and control-spiked samples.
T13980 240711-240962 Sentence denotes Samples were extracted using Roche MagNA Pure 96 system with the DNA Isolation Kit DNA/Viral NA SV 2.0 and subsequently tested by the Focus 3M TM Integrated Cycler using Focus HSV-1 or HSV-1 GEN II and HSV-2 Primer Pairs and 2.5X Universal Master Mix.
T6348 240963-241090 Sentence denotes Linear ranges, accuracy, analytical sensitivity, analytical specificity, precision, and limit of detection (LOD) were assessed.
T40722 241091-241099 Sentence denotes Results:
T75495 241100-241346 Sentence denotes Real-time PCR testing of 441 specimens, revealed the Focus HSV assay has excellent analytical sensitivity and analytical specificity [HSV-1 100% positive predictive value (PPV)/ 100% negative predictive value (NPV); HSV-2 99.32% PPV/ 99.03% NPV].
T67204 241347-241404 Sentence denotes Out of 441 samples, only 2 discrepant results were found.
T39994 241405-241526 Sentence denotes Referee testing on discrepant samples with a third party also using the Focus HSV assay were concordant with our results.
T30360 241527-241724 Sentence denotes Inter-assay variability was measured using log values from AcroMetrix HSV-1 and HSV-2 low and high positive CSF controls, with average coefficients of variation (CV) of 1.8% and 1.1%, respectively.
T49110 241725-241938 Sentence denotes Quantified AcroMetrix HSV-1 and HSV-2 Plasma Panels were analyzed with 5 replicates of each dilution to determine intra-assay variability [HSV-1 2.9% (CV); HSV-2 4.4% (CV)] and linearity (250-1,000,000 copies/mL).
T60977 241939-242033 Sentence denotes Additional serial dilutions of the panels were evaluated to determine an LOD of 250 copies/mL.
T61905 242034-242046 Sentence denotes Conclusions:
T10696 242047-242286 Sentence denotes These results demonstrate the benefit of the Focus Diagnostics HSV assay for quantitative results on whole blood and CSF samples, and qualitative results for ThinPrep and APTIMA GEN-PROBE samples from patients with suspected HSV infection.
T50162 242287-242435 Sentence denotes Furthermore, qualitative and quantitative assays can be run simultaneously in a clinical laboratory improving workflow and decreasing control costs.
T52239 242436-242572 Sentence denotes Use of this molecular assay will ensure efficient, reliable and reproducible analysis of HSV infections in a single real-time PCR assay.
T98002 242573-242690 Sentence denotes Kidneys from hepatitis C virus-positive (HCV+) organ donors are almost exclusively transplanted into HCV+ recipients.
T17804 242691-242861 Sentence denotes Because the number of HCV+ kidneys far exceeds the number of patients willing and able to accept such organs, nearly two-thirds of these kidneys are discarded every year.
T12695 242862-243100 Sentence denotes The development of new therapies for HCV that cure more than 95% of patients, coupled with the vast shortage of available kidneys, have led some transplant professionals to consider transplanting HCV+ kidneys into HCV-negative recipients.
T60300 243101-243340 Sentence denotes One potential barrier to doing so is the ability to rapidly genotype organ donors for HCV, since viral genotype guides therapy and no pan-genotypic antiviral regimens exist that can safely be used in patients with impaired kidney function.
T36259 243341-243537 Sentence denotes Also, it is not known whether donor stabilization procedures, such as fluid or drug infusions, affect genotyping ability (e.g., by reducing viremia or introducing PCR inhibitors into circulation).
T57733 243538-243656 Sentence denotes Our aim was to determine whether archived plasma samples from past organ donors could be accurately genotyped for HCV.
T57214 243657-243665 Sentence denotes Methods:
T25880 243666-243842 Sentence denotes A local organ procurement organization performed a retrospective review of their database to identify previous organ donors that were HCV+ on a qualitative nucleic acid screen.
T6103 243843-243938 Sentence denotes Archived frozen plasma samples were obtained for 17 such donors, along with donor demographics.
T99372 243939-244054 Sentence denotes RNA was extracted from the thawed samples using the QIAamp DSP Viral RNA kit on the QIAcube (Qiagen, Valencia, CA).
T87190 244055-244176 Sentence denotes HCV genotypes were detected using the eSensor HCVg Direct Test (RUO) and XT-8 System (GenMark Diagnostics, Carlsbad, CA).
T10854 244177-244378 Sentence denotes All samples were also genotyped by Sanger sequencing (Retrogen, San Diego, CA) and subjected to quantitative HCV viral load testing (COBAS AmpliPrep/TaqMan system, Roche Diagnostics, Indianapolis, IN).
T6903 244379-244387 Sentence denotes Results:
T48974 244388-244530 Sentence denotes The samples obtained were collected over a 10 month period primarily from Caucasian donors, both male and female, between 21 and 57 years old.
T94424 244531-244585 Sentence denotes All samples were successfully extracted and genotyped.
T88871 244586-244701 Sentence denotes The majority of samples (n=14) were HCV Genotype 1a with the remainder being Genotype 2b (n=1) or Genotype 3 (n=2).
T72450 244702-244783 Sentence denotes All genotyping results were concordant with those obtained via Sanger sequencing.
T36641 244784-244896 Sentence denotes The average HCV viral load across the samples was ~ 1.6 million IU/mL (range: ~16,000 IU/mL to 7 million IU/mL).
T39906 244897-244909 Sentence denotes Conclusions:
T92118 244910-245044 Sentence denotes Albeit a limited sample size, we found that viral RNA from plasma of organ donors can be successfully extracted and genotyped for HCV.
T50341 245045-245189 Sentence denotes Treatment provided to stabilize donors prior to organ procurement does not appear to interfere with the ability to extract or amplify viral RNA.
T53033 245190-245352 Sentence denotes The capacity to provide an accurate HCV genotype suggests that clinical trials designed to transplant HCV+ kidneys into HCV-negative recipients could be feasible.
T77545 245353-245366 Sentence denotes Introduction:
T36313 245367-245523 Sentence denotes Mortality rates for women with cervical cancer in low-and middleincome countries (LMICs) are approximately three times higher than in high income countries.
T99244 245524-245639 Sentence denotes Limited access to high-risk HPV (hrHPV) screening and adequate followup are major contributors to these statistics.
T59868 245640-245896 Sentence denotes Successful use of molecular analysis for hrHPV testing has been limited to established laboratories experienced with highly complex testing techniques, effectively creating a significant barrier to increased hrHPV screening of at-risk populations in LMICs.
T98415 245897-246062 Sentence denotes Here we report the development of a simplified point-of-care (POC) field protocol and results from field-testing of an hrHPV molecular assay in the LMIC of Honduras.
T49759 246063-246071 Sentence denotes Methods:
T3969 246072-246287 Sentence denotes A total of 662 women from outreach clinics in a textile factory in San Pedro Sula (402), and a rural village clinic in Locomapa (260), were screened for 14 hrHPV types in portable rudimentary POC field laboratories.
T77478 246288-246379 Sentence denotes Three cervical specimens from each women were collected using standard sampling techniques.
T61687 246380-246673 Sentence denotes One sample was immediately preserved for follow-up Pap smear testing at La Liga Contra el Cancer in San Pedro Sula and one sample was inactivated and shipped to the Laboratory of Clinical Genomics and Advanced Technologies (CGAT) at Dartmouth-Hitchcock Medical Center for confirmation studies.
T28436 246674-247016 Sentence denotes DNA from the remaining sample was extracted into a crude jmd.amjpathol.org ■ The Journal of Molecular Diagnostics lysate and hrHPV testing was performed onsite using the QuanDx High-Risk HPV Genotyping Assay on the ZeeSan SLAN-96S RT-PCR System following a modified protocol, including applying crude extract directly to lyophilized reagents.
T75083 247017-247102 Sentence denotes The presence of any of the 14 hrHPV types was determined through melt curve analysis.
T80388 247103-247111 Sentence denotes Results:
T91269 247112-247267 Sentence denotes Of the 662 samples tested, the total validity rate was 76.2% (505/662), with 73.1% (294/402) and 81.4% (210/260) from the factory and village respectively.
T46320 247268-247411 Sentence denotes Swabs tested by a similar method in the CGAT laboratory had a validity rate of 97.1% (136/140) in a controlled clinical laboratory environment.
T8883 247412-247526 Sentence denotes 13.0% (86/662) of samples tested in Honduras and 14.3% (20/140) of samples tested at CGAT were positive for hrHPV.
T72063 247527-247539 Sentence denotes Conclusions:
T84812 247540-247725 Sentence denotes The successful establishment of two portable field laboratories and implementation of high throughput molecular testing has shown the potential for same-day diagnosis of hrHPV in LMICs.
T54290 247726-247898 Sentence denotes Rapid diagnosis would allow for immediate health counseling and services for patients who can be challenging to track due to poor communications infrastructure and poverty.
T54807 247899-248176 Sentence denotes Increasing the access for large numbers of women to molecular testing for hrHPV could dramatically impact mortality rates of cervical cancer in LMICs by correctly identifying women at highest risk for cervical cancer and immediately directing clinical follow-up as appropriate.
T49925 248177-248190 Sentence denotes Introduction:
T26175 248191-248374 Sentence denotes Persistent infection with high risk (HR) human papillomavirus (HPV) is a principal cause of cervical cancer and is associated with greater than 99% of cervical cancer cases worldwide.
T33844 248375-248498 Sentence denotes Numerous FDA approved or cleared laboratory testing platforms exist for the detection of HR HPV in gynecological specimens.
T27012 248499-248699 Sentence denotes The goal of this study was to verify and implement the Cobas HPV Test (Roche Diagnostics, Inc., Indianapolis, IN) in our laboratory and to evaluate the effects on various aspects of specimen workflow.
T53284 248700-248708 Sentence denotes Methods:
T27787 248709-248925 Sentence denotes Verification studies, using 421 patient specimens, were performed to switch clinical HR HPV testing from the Cervista HR HPV and the HPV 16/18 genotyping assays (Hologic, Inc., Marlborough, MA) to the Cobas HPV Test.
T6603 248926-248996 Sentence denotes The Cobas assay was always performed in the HPV 16/18 genotyping mode.
T26511 248997-249131 Sentence denotes Discrepant results were resolved by repeat testing as well as by using the Linear Array HPV Genotyping Test (Roche Diagnostics, Inc.).
T82378 249132-249214 Sentence denotes Data were compared for various workflow metrics between the two testing platforms.
T53361 249215-249223 Sentence denotes Results:
T54001 249224-249358 Sentence denotes The overall, positive, and negative agreements between the Cervista and Cobas HR HPV assays were 92.9%, 87.9%, and 94.3% respectively.
T19786 249359-249483 Sentence denotes Notably, our high positive agreement was due in part to our laboratory's data analysis algorithm of Cervista HR HPV results.
T61119 249484-249656 Sentence denotes The majority of discrepant results were HPV positive on the Cobas and HPV negative on the Cervista assay, suggestive of increased analytical sensitivity of the Cobas assay.
T27624 249657-249931 Sentence denotes The percent positivity by cytologic diagnosis was evaluated after Cobas implementation and ranged from approximately 3% positive for negative for intraepithelial lesion or malignancy (NILM) to approximately 85% positive for high-grade squamous intraepithelial lesion (HSIL).
T68802 249932-250009 Sentence denotes Upon switching testing platforms various workflow improvements were observed.
T41104 250010-250267 Sentence denotes For a subset of samples, the Cobas assay eliminated the need for a reflex HPV 16/18 genotyping assay which reduced TAT by approximately three days and the physical distance traveled across our Cytopathology and Molecular Pathology laboratories by 1.5 miles.
T38732 250268-250424 Sentence denotes The Cobas assay also eliminated secondary review of results and reduced the number of indeterminate/inconclusive results as well as the repeat testing rate.
T29109 250425-250490 Sentence denotes All of these improvements resulted in decreased laboratory costs.
T34601 250491-250503 Sentence denotes Conclusions:
T15603 250504-250668 Sentence denotes Improvements following this laboratory testing methodology change were observed in specimen workflow, analytical testing metrics, and overall laboratory operations.
T85225 250669-250859 Sentence denotes Taken together, these improvements translate into an earlier HR HPV laboratory result for patients and clinicians within our health system with a potential for overall improved patient care.
T45867 250860-251061 Sentence denotes Recently, different classes of promising new anti-TB agents, as the Benzothiazinones (BTZs), that results active against M. tuberculosis drugsusceptible, MDR and XDR clinical isolates, were discovered.
T6738 251062-251169 Sentence denotes Many of these drugs hit the same target, DprE1, an enzyme involved in arabinogalactan biosynthetic pathway.
T25068 251170-251402 Sentence denotes Based on the analysis of dprE1 gene, two ready-to-use assays were developed, one for TB diagnosis and identification of MTBC or MAC infections and one to identify single point mutations associated with resistance in M. tuberculosis.
T35345 251403-251411 Sentence denotes Methods:
T3930 251412-251462 Sentence denotes The 2 assays were based on Real-Time PCR analysis.
T87243 251463-251638 Sentence denotes Each test tube contains all the required PCR components in a freeze-dried form, exploiting STAT-NAT technology that allows the room-temperature storage of the mix for 2 years.
T48559 251639-251837 Sentence denotes The MTBC/MAC screening kit include specific sets of primers and probes, one to amplify a fragment of M. tuberculosis IS6110 direct repeat region and one to amplify a fragment of M. avium dprE1 gene.
T36740 251838-251944 Sentence denotes For drug resistant TB assay, the STAT-NAT technology was associated with a novel quenching probe (Qprobe).
T76436 251945-252059 Sentence denotes The assay allows the discrimination between the DprE1 inhibitors and Rifampicin (RIF) sensitive/resistant strains.
T73370 252060-252176 Sentence denotes Specific sets of primers and Qprobes were designed to amplify fragments of M. tuberculosis dprE1 gene and rpoB gene.
T83836 252177-252275 Sentence denotes In each assay, another set of primers and probe was included as an internal amplification control.
T99619 252276-252284 Sentence denotes Results:
T13201 252285-252389 Sentence denotes The LOD of MTBC/MAC screening assay was 1 and 10 copies/reaction for MTBC and MAC species, respectively.
T54025 252390-252472 Sentence denotes The assays did not cross-react with any of the other mycobacterial species tested.
T27263 252473-252665 Sentence denotes Regarding the drug resistant TB assay, the genomic DNA obtained from M. tuberculosis DprE1 inhibitors-resistant mutants, M. tuberculosis MDR and wild-type strains, was tested using this assay.
T9736 252666-252801 Sentence denotes The results showed unambiguous discrimination between mycobacterial strains that are resistant or sensitive to RIF or DprE1 inhibitors.
T77753 252802-252814 Sentence denotes Conclusions:
T31647 252815-253051 Sentence denotes The high-sensitivity and specificity of these two assays, associated with the ready-to-use and room temperature storage of the STAT-NAT technology, would have a direct impact on the early and correct management of the affected patients.
T55720 253052-253166 Sentence denotes Overall, these technologies would be a valuable tool in the rapid diagnosis of other tuberculosis drug-resistance.
T31269 253167-253180 Sentence denotes Introduction:
T53529 253181-253330 Sentence denotes Escherichia coli is a common cause of septicemia and urinary tract infection, and the increasing rate of quinolone resistance has become problematic.
T29828 253331-253469 Sentence denotes The global spread of quinolone-resistant E. coli sequence type (ST) over the past decade has been assessed using DNA sequencing technique.
T42423 253470-253575 Sentence denotes However, they have limitations in discriminating genetically similar microbes and requires time and cost.
T55282 253576-253780 Sentence denotes Therefore, we investigated the applicability of Raman spectroscopy which measures the spectral phase of molecular vibration as a typing tool to discriminating ciprofloxacin (CIP)-nonsusceptible E.coliSTs.
T65772 253781-253995 Sentence denotes Methods: CIP-nonsusceptible E. coli isolates, collected from blood cultures, were pre-characterized regarding the minimum inhibitory concentration (MIC) of CIP and extended-spectrum betalactamase (ESBL) production.
T10508 253996-254173 Sentence denotes E. coli ATCC 25922 was used as susceptible control, and the resistance E. coliSTs between ST131 and ST1193 isolates were analyzed and compared with Raman spectroscopy technique.
T1084 254174-254332 Sentence denotes All Raman spectra were obtained through gold-coated surface-enhanced Raman scattering (SERS) technique using a SENTERRA confocal Raman system (Bruker Optics).
T22087 254333-254422 Sentence denotes The enhancement factor of this gold-deposited substrate was roughly calculated 2.4×10 7 .
T68653 254423-254646 Sentence denotes The principal component analysis (PCA) algorithm and Multi-support vector machine (SVM) classifiers with a one-against-one approach onto this two-dimensional plot were used to discriminate between ST131 and ST1193 isolates.
T68343 254647-254655 Sentence denotes Results:
T48177 254656-254788 Sentence denotes The optimal separating lines could be obtained from the SVM classification algorithm using the training data (n=20) from each group.
T11734 254789-254920 Sentence denotes A linear kernel on the score plot of PC1-PC3 clearly showed a difference between the control and two CIPnonsusceptible E. coli STs.
T70165 254921-255125 Sentence denotes The score plot of PC1-PC2 showed individual characteristics of three groups, which yielded a sensitivity of 100% and a specificity of 100% for discriminating between strains of ST131, ST1193 and controls.
T73202 255126-255138 Sentence denotes Conclusions:
T15843 255139-255321 Sentence denotes This study suggested the high sensitivity of Raman spectroscopybased identification method for discrimination of different CIP-nonsusceptible E. coli STs without additional labeling.
T97261 255322-255324 Sentence denotes H.
T71655 255325-255332 Sentence denotes Lee, Y.
T60129 255333-255340 Sentence denotes Kim, J.
T43139 255341-255349 Sentence denotes Choi, H.
T70020 255350-255359 Sentence denotes Kanga, S.
T76919 255360-255367 Sentence denotes Cho, T.
T22191 255368-255440 Sentence denotes Park Kyung Hee University, School of Medicine, Seoul, Republic of Korea.
T16862 255441-255454 Sentence denotes Introduction:
T39604 255455-255576 Sentence denotes The microbiome study using NGS-metagenomics on the human body was widely performed on the gut, skin, vagina, oral cavity.
T77228 255577-255757 Sentence denotes However, respiratory tract has not been actively studied, and the microbiome of healthy lungs is very poorly understood due to difficulties in obtaining proper respiratory samples.
T89400 255758-255890 Sentence denotes Few studies on lung microbiome showed diverse results owing to various specimen types, different sampling methods and study designs.
T30647 255891-255975 Sentence denotes We investigated the microbiome on healthy trachea with a controlled sampling method.
T95751 255976-255984 Sentence denotes Methods:
T79714 255985-256085 Sentence denotes The subjects were adult patients who needed general anesthesia for operation no longer than 2 hours.
T31113 256086-256232 Sentence denotes We excluded the patients with any conditions related with respiratory tract (infection, smoker, allergy, cancer and decreased pulmonary function).
T98644 256233-256332 Sentence denotes Patients with infections on other sites, metabolic or nutritional abnormalities were also excluded.
T95461 256333-256399 Sentence denotes We collected tips of intubation tubes used for general anesthesia.
T95271 256400-256463 Sentence denotes The depth of intubation were 21cm for female and 23cm for male.
T98878 256464-256603 Sentence denotes The collected tips were washed with DEPC water and the spin-down pellets were used for NGS-metagenomics with Mi-Seq system (Illumina Inc.).
T34763 256604-256656 Sentence denotes Paired-ends fastq data were merged using FLASH (ver.
T73893 256657-256665 Sentence denotes 1.2.11).
T12949 256666-256740 Sentence denotes Pre-processing and clustering of reads were done with CD-HIT-OTU software.
T20010 256741-256809 Sentence denotes Taxonomic assign and diversity analysis were done with QIIME-UCLUST.
T97128 256810-257025 Sentence denotes Cultures were performed in both aerobic and anaerobic conditions with media routinely used in clinical microbiologic laboratory, and obtained colonies were identified with MALDI-TOF MS (Bruker) to the species level.
T89193 257026-257034 Sentence denotes Results:
T932 257035-257138 Sentence denotes The included patients were eight females with breast cancer stage under T1 and one male with hydrocele.
T71881 257139-257231 Sentence denotes All of the samples showed plateau in rare fraction curve and sufficient reads were obtained.
T37377 257232-257455 Sentence denotes The median (range) number of identified species and genus by MALDI-TOF MS were 11 (8 to 17) and 5 (3 to 8) whereas the operational taxonomic unit (OTU) by NGS-metagenomics were 64 (53 to 71) and 53 (45 to 52), respectively.
T36290 257456-257526 Sentence denotes The top 5 frequent OTU in genus level (% total, range) were Prevotella
T47409 257527-257747 Sentence denotes The Journal of Molecular Diagnostics ■ jmd.amjpathol.org (23.1, 31.9 to 9.6), Streptococcus (12.2, 25.2 to 4.6), Veillonella (8.9, 10.8 to 5.7), Neisseria (8.3, 27.7 to 1) and Alloprevotella (8, 15 to 2.4), respectively.
T15605 257748-257841 Sentence denotes The samples showed relatively consistent patterns of OTU composition among enrolled subjects.
T37883 257842-257854 Sentence denotes Conclusions:
T28892 257855-257998 Sentence denotes Our controlled sampling method using the endotracheal tube was useful for collecting healthy respiratory tract specimens without ethical issue.
T48056 257999-258125 Sentence denotes The OTU and their compositions shown in our study were consistent with previously reported studies on healthy lung microbiota.
T1943 258126-258234 Sentence denotes To establish the diagnosis of acute HIV infection is extremely important from a public health point of view.
T56465 258235-258590 Sentence denotes This study aims to detect acute HIV infection in patients seen in emergency cares with signs and symptoms of mononucleosis or aseptic meningitis syndrome, but who have negative results to serological tests for mononucleosis, CMV and toxoplasmosis; as well as for the detection of enterovirus and HSV I / II by nucleic acids amplification techniques (NAT).
T46138 258591-258599 Sentence denotes Methods:
T56815 258600-258849 Sentence denotes This study was conducted from February 2014 to February 2015 in 5 different hospital settings of the Hospital Israelita Albert Einstein, four exclusively private patient intake sites and one site for admitting patients from the public health system.
T69061 258850-259327 Sentence denotes It was included patients over 18 years with signs and symptoms suggestive of mononucleosis syndrome (fever, sore throat, cervical nodes) in which the doctor asked for mononucleosis, cytomegalovirus and toxoplasmosis serologies whose results were negative; as well as patients with aseptic meningitis (fever, headache, nausea or vomiting and neck stiffness) who underwent lumbar puncture with Enterovirus and Herpes virus I and II detection request by NAT with negative results.
T22629 259328-259525 Sentence denotes The patients were analyzed for the presence of anti-HIV antibodies as well as the presence of viral RNA (with a sensitivity of 20 copies / ml and linearity ranging from 47 to 10,000,000 copies/ml).
T76823 259526-259534 Sentence denotes Results:
T48808 259535-259658 Sentence denotes In a collected sample of 30 patients during the years 2013-2015, two of them were positive for HIV serology and viral load.
T14503 259659-259820 Sentence denotes The sample population consisted of 16 females and 12 males aged between 18-49 years and the two positive cases were males with 32 and 45 years old, respectively.
T63660 259821-260038 Sentence denotes Acute HIV infection incidence was 6.67% in the target population of the study.In the study, the incorporation of the following laboratory tests were necessary: HIV viral load ($ 73.00) and anti-HIV serology ($ 17.26).
T16498 260039-260051 Sentence denotes Conclusions:
T32742 260052-260263 Sentence denotes Our results demonstrate that it is important to detect acute HIV infection to prevent HIV spreading to other patients and to help acute HIV patients to receive an appropriate antiretroviral therapy Introduction:
T36534 260264-260388 Sentence denotes Increasing prevalence of antibiotic resistance (AbX) bacteria in the last decade is of great concern in healthcare settings.
T7397 260389-260422 Sentence denotes The most common type of blaCTX-M.
T45256 260423-260532 Sentence denotes The CTX-M genes encode for a complex and non-homogenous group of enzymes that are seen in Enterobacteriaceae.
T56781 260533-260637 Sentence denotes Klebsiella pneumoniae carbapenemase (KPC) is the most common carbapenemase gene type reported in the US.
T5790 260638-260782 Sentence denotes KPC is produced by the blaKPC gene in Enterobacteriaceae, Acinetobacter and Pseudomonas species and confers resistance to all (ß) lactam agents.
T9422 260783-260958 Sentence denotes Of greater clinical significance worldwide are the carbapenemase metallo-(ß)-lactamases (MBL) variants: blaNDM, blaVIM and blaIMP seen in Enterobacteriaceae and P. aeruginosa.
T51118 260959-261096 Sentence denotes We have designed a multiplex PCR assay that detects most variants for these five genes that confer AbX in various gram negative bacteria.
T41528 261097-261105 Sentence denotes Methods:
T75866 261106-261167 Sentence denotes Organisms were ordered from American Type Culture Collection.
T26441 261168-261234 Sentence denotes Samples were extracted on the MagNA Pure LC or MagNA Pure Compact.
T19754 261235-261391 Sentence denotes Nested PCR and detection was done with either dual labeled hydrolysis probe or LC Green signal and melting curve analysis on the Bio-Rad CFX96 Touch System.
T26766 261392-261515 Sentence denotes Nested PCR primers for outer PCR1 and inner PCR2 were designed for; IMP, KPC, NDM, VIM and CTX-M groups; 1, 2, 8, 9 and 25.
T75785 261516-261604 Sentence denotes All 5 assays were multiplexed using the outer PCR primers for 25 cycles comprising PCR1.
T92147 261605-261737 Sentence denotes The amplicon was diluted 1:100 and added to 6 subsequent PCR reactions with inner primers and either a hydrolysis probe or LC Green.
T88201 261738-261852 Sentence denotes The CTX-M types 1 and 9 were multiplexed together for PCR2 and CTX-M groups 2, 8 and 25 were multiplexed together.
T80319 261853-261924 Sentence denotes Eighty two bacterial and viral isolates were run for all 5 AbX markers.
T58036 261925-262032 Sentence denotes We designed 9 positive control, 500 base pair gBlocks that were synthesized by Integrated DNA Technologies.
T74326 262033-262093 Sentence denotes All positive detections were confirmed by Sanger sequencing.
T93589 262094-262102 Sentence denotes Results:
T6435 262103-262163 Sentence denotes The assay was performed for 82 bacterial and viral isolates.
T44692 262164-262259 Sentence denotes Eighteen isolates had previous ESBL, carbapenemase or MBL detection by other molecular methods.
T80717 262260-262303 Sentence denotes Some samples had multiple resistance genes.
T36009 262304-262458 Sentence denotes All eighteen expected positive samples (12 CTX-M, 2 IMP, 1 KPC, 1 NDM, and 4 VIM) were detected by the multiplex assay and confirmed by Sanger sequencing.
T43160 262459-262513 Sentence denotes No unexpected negatives or positives were encountered.
T89525 262514-262540 Sentence denotes All assay controls passed.
T68854 262541-262629 Sentence denotes Limit of detection for each gene was determined to be at least 1000 copies per reaction.
T81540 262630-262642 Sentence denotes Conclusions:
T22773 262643-262755 Sentence denotes We have designed a real-time PCR assay that is sensitive and specific for 5 antibiotic resistance gene families.
T67740 262756-262788 Sentence denotes The assay can be run in 3 hours.
T59759 262789-262894 Sentence denotes It is sensitive and specific for the target genes with no observable interference from human genomic DNA.
T83666 262895-262926 Sentence denotes This assay is not FDA approved.
T95489 262927-262931 Sentence denotes J.R.
T11221 262932-262956 Sentence denotes Rebello Pinho 1,3 , A.R.
T98234 262957-262973 Sentence denotes Marra 2,3 , D.T.
T24538 262974-262991 Sentence denotes Malheiro 2 , S.C.
T57155 262992-263006 Sentence denotes Oller 2 , R.A.
T36453 263007-263021 Sentence denotes Santana 2 , R.
T79582 263022-263037 Sentence denotes Sitnik 2 , R.C.
T78301 263038-263054 Sentence denotes Petroni 2 , G.F.
T42280 263055-263071 Sentence denotes Dastoli 2 , E.A.
T69777 263072-263089 Sentence denotes Rossetto 2 , T.Z.
T66560 263090-263106 Sentence denotes Camargo 2 , P.D.
T77295 263107-263125 Sentence denotes Gonçalves 2 , M.D.
T94325 263126-263140 Sentence denotes Martino 2 , J.
T26920 263141-263159 Sentence denotes Pasternak 2 , C.L.
T32761 263160-263194 Sentence denotes Using the Cepheid SmartCycler S.J.
T63558 263195-263214 Sentence denotes Deharvengt 1 , S.A.
T19639 263215-263230 Sentence denotes Turner 1 , L.S.
T1855 263231-263247 Sentence denotes Kennedy 2 , G.J.
T81111 263248-263443 Sentence denotes Tsongalis 1 1 Dartmouth Hitchcock Medical Center, Norris Cotton Cancer Center and Geisel School of Medicine, Lebanon, NH; 2 Norris Cotton Cancer Center and Geisel School of Medicine, Lebanon, NH.
T80428 263444-263457 Sentence denotes Introduction:
T53713 263458-263539 Sentence denotes Cancer of the uterine cervix (CC) is the most common cancer in women in Honduras.
T13609 263540-263636 Sentence denotes The main risk factor is infection with one or more high-risk human papillomavirus (hrHPV) types.
T97518 263637-263766 Sentence denotes The aim of this study was to develop an affordable assay to detect the genotype-specific prevalence of the 14 hrHPV types for CC.
T23484 263767-263775 Sentence denotes Methods:
T38503 263776-263872 Sentence denotes Primer3 Input and pave Papilloma virus genome database websites were used to design primer sets.
T12546 263873-263965 Sentence denotes The uMelt SM Software was used to predict the melt curve shape and melting temperature (Tm).
T12748 263966-264131 Sentence denotes DNA samples included human genomic DNA (gDNA) (Promega), gBlocks gene fragments of the 14 hrHPV types (IDT) and DNA extracted from previously tested patient samples.
T77624 264132-264227 Sentence denotes The enzyme used for PCR amplification was SsoFast EvaGreen (Biorad) on the Cepheid SmartCycler.
T40363 264228-264236 Sentence denotes Results:
T60279 264237-264362 Sentence denotes We selected one primer pair for each of the 14 hrHPV types producing a specific amplicon and giving a single peak melt curve.
T36648 264363-264478 Sentence denotes Primer sets were combined and tested with only gDNA to confirm that no cross-reaction occurred between the primers.
T19578 264479-264541 Sentence denotes This reaction was included in every run as a negative control.
T95801 264542-264651 Sentence denotes The primer combinations without a high background were used with up to 5 gBlocks as template in one reaction.
T21898 264652-264735 Sentence denotes Melt curves were obtained with Tm differences that could differentiate hrHPV types.
T56818 264736-264798 Sentence denotes This reaction was included in every run as a positive control.
T68176 264799-264957 Sentence denotes The multiplex PCR reactions were then run using DNA extracted from cervical swab specimens collected in Honduras, previously tested with the QuanDx HPV assay.
T86898 264958-265017 Sentence denotes The results were highly consistent between the two methods.
T82412 265018-265030 Sentence denotes Conclusions:
T88127 265031-265165 Sentence denotes The SmartCycler can be used for multiplex Real-Time PCR melting curve analysis for identifying up to five hrHPV types in one reaction.
T33537 265166-265242 Sentence denotes The main advantages of this technique are its affordability and flexibility.
T12024 265243-265360 Sentence denotes 1 1 Louisiana State University Health Sciences Center, New Orleans, LA; 2 University Medical Center, New Orleans, LA.
T55443 265361-265374 Sentence denotes Introduction:
T67687 265375-265514 Sentence denotes Chlamydia trachomatis and Neisseria gonorrhoeae (CT/NG) are the most common sexually transmitted bacterial infections in the United States.
T74427 265515-265661 Sentence denotes Infection with either organism can be asymptomatic and is associated with significant urogenital and reproductive tract sequelae in men and women.
T56462 265662-265816 Sentence denotes CT/NG infections among men who have sex with men (MSM) often involve in the rectum and pharynx, and commonly occur without concomitant urethral infection.
T47721 265817-266002 Sentence denotes Currently, CT/NG infections are most commonly detected using nucleic acid amplification tests (NAATs) due to enhanced analytical sensitivity, specificity and ease of specimen transport.
T98898 266003-266146 Sentence denotes The Roche Cobas CT/NG v2.0 test is an automated, in vitro NAAT for the qualitative detection of CT/NG DNA in several urogenital specimen types.
T27822 266147-266290 Sentence denotes Using this test on the Cobas 4800 system, the objective of this study is to validate CT/NG detection from rectal and pharyngeal swab specimens.
T61202 266291-266299 Sentence denotes Methods:
T71977 266300-266521 Sentence denotes Using the CT/NG v2.0 and the Aptima Combo 2 CT/NG assays, CT/NG detection was performed on 20 dual rectal swab samples and 18 dual pharyngeal swab samples collected from the HIV Outpatient Clinic (HOP) in New Orleans, LA.
T13677 266522-266702 Sentence denotes An additional comparative assessment using the CT/NG v2.0 was performed using a panel of known positive/negative rectal (n=15) and pharyngeal (n=19) specimens from a secondary lab.
T13992 266703-266814 Sentence denotes Lastly, CT spiking experiments were performed to further assess assay performance in pharyngeal swab specimens.
T12665 266815-266868 Sentence denotes Results were analyzed using Cohen's kappa statistics.
T98843 266869-266877 Sentence denotes Results:
T86066 266878-267019 Sentence denotes Sensitivity and specificity values for both rectal and pharyngeal CT/NG were 100% between the CT/NG v2.0 and the Aptima Combo 2 CT/NG assays.
T47768 267020-267200 Sentence denotes In the secondary comparison of known positive/negative specimens on the Cobas CT/NG v2.0 test, sensitivity and specificity values for pharyngeal CT/NG and rectal CT were both 100%.
T9628 267201-267363 Sentence denotes One rectal specimen deemed NG-positive at the reference lab was not detected in our testing, yielding a sensitivity and specificity of 89% and 100%, respectively.
T34598 267364-267485 Sentence denotes CT spiking experiments showed excellent interassay reproducibility with coefficients of variation that were less than 5%.
T89795 267486-267566 Sentence denotes Kappa coefficients we're classified as very good or perfect for all comparisons.
T44527 267567-267569 Sentence denotes J.
T44114 267570-267579 Sentence denotes Li 1 , S.
T40231 267580-267595 Sentence denotes Favaloro 2 , C.
T25575 267596-267610 Sentence denotes McGowin 1 , C.
T25774 267611-267619 Sentence denotes Kletecka
T89228 267620-267756 Sentence denotes The results confirm that CT/NG detection from rectal and pharyngeal swab samples can be performed successfully on the Cobas 4800 system.
T3697 267757-267770 Sentence denotes Introduction:
T45822 267771-267868 Sentence denotes Cryptococcus is a human pathogenic fungus and is implicated in pneumonia and meningoencephalitis.
T85270 267869-267945 Sentence denotes Cryptococcus spp. have been reported to cause 15% of AIDS-related mortality.
T83141 267946-268100 Sentence denotes C. neoformans is found worldwide and C. gattii has been associated with tropical as well as temperate locations such as British Columbia and Northwest US.
T12389 268101-268300 Sentence denotes Molecular methodologies including PCR in its many forms are essential for fast, sensitive detection jmd.amjpathol.org ■ The Journal of Molecular Diagnostics of C. gattii and C. neoformans infections.
T73511 268301-268309 Sentence denotes Methods:
T97271 268310-268415 Sentence denotes Two independent nested realtime PCR assays were each designed to detect both C. gattii and C. neoformans.
T97379 268416-268593 Sentence denotes The cox2 and atp6 genes were chosen because they are located in the mitochondrial genome of Cryptococcus and are present in multiple copies, increasing sensitivity of detection.
T54974 268594-268728 Sentence denotes Each PCR assay was designed with an outer primer pair, an inner primer pair, and a hydrolysis probe for fluorescent signal generation.
T81225 268729-268799 Sentence denotes The inner primers were tailed with M13 tails to facilitate sequencing.
T57272 268800-268914 Sentence denotes The outer primer PCR product was diluted 1:10 and added to the inner PCR reaction for an effective 1:100 dilution.
T97258 268915-269104 Sentence denotes Both assays were evaluated for C. gattii and C. neoformans identification using a panel of reference strains representing all 4 genogroups of C. gattii and various strains of C. neoformans.
T44292 269105-269204 Sentence denotes Further testing of 25 culture-confirmed clinical cerebrospinal fluid (CSF) specimens was performed.
T83153 269205-269341 Sentence denotes CSF quantitative culture, cross-sectionally collected during antifungal therapy, ranged in burden from 10 CFU/mL to >100,000 CFU/mL CSF.
T64454 269342-269413 Sentence denotes Nucleic acid was extracted from 200 μL CSF and 10 μL were used for PCR.
T44800 269414-269422 Sentence denotes Results:
T62155 269423-269571 Sentence denotes The COX2 and the ATP6 realtime nested PCR assays detected all C. gattii and C. neoformans reference strains including the 4 genogroups of C. gattii.
T5802 269572-269703 Sentence denotes The limit of detection of both assays was between 100 copies/reaction to 1,000 copies/reaction when compared to synthetic template.
T79811 269704-269789 Sentence denotes The COX2 and ATP6 assays did not detect any of the non-cryptococcal organisms tested.
T2305 269790-269877 Sentence denotes Compared with culture, the COX2 and ATP6 assays each showed 80% (20 of 25) concordance.
T87322 269878-270021 Sentence denotes Of 18 culture-positive CSF specimens from patients with confirmed Cryptococcus infection, both assays resulted in 13 (72%) positive detections.
T6924 270022-270198 Sentence denotes Bi-directional sequencing revealed that the COX2 assay did not amplify a C. gattii strain at 660 CFU/mL and the ATP6 assay did not amplify a C. neoformans strain at 120 CFU/mL.
T49041 270199-270268 Sentence denotes The COX2 and ATP6 assays tested negative in all culture negative CSF.
T64231 270269-270281 Sentence denotes Conclusions:
T19467 270282-270447 Sentence denotes The two PCR assays are able to detect C. gattii and C. neoformans species faster than culture, suggesting reduction in mortality and morbidity with prompt treatment.
T60312 270448-270572 Sentence denotes A comparison with quantitative culture shows the described COX2 and ATP6 assays each perform well for testing CSF specimens.
T63055 270573-270606 Sentence denotes These assays are not FDA-cleared.
T97033 270607-270620 Sentence denotes Introduction:
T72110 270621-270721 Sentence denotes Fever is a common medical problem that accounts for a large proportion of pediatric hospital visits.
T74194 270722-270893 Sentence denotes Symptoms displayed are often non-specific and it is difficult to distinguish between viral syndromes, serious bacterial infection (SBI), or non-infectious causes of fever.
T36791 270894-271132 Sentence denotes Even though the majority of cases are thought to be viral in origin, many patients receive extensive evaluation including blood, urine, and cerebrospinal fluid (CSF) cultures and then require hospitalization until an SBI can be ruled out.
T57956 271133-271305 Sentence denotes A rapid, easy-to-use, comprehensive diagnostic test could benefit patient care by potentially reducing antibiotic use or influencing hospital admission/discharge decisions.
T7668 271306-271475 Sentence denotes To aid in viral identification associated with pediatric fevers, BioFire Diagnostics is developing the FilmArray (FA) Febrile Infant (FI) Panel for use on the FA System.
T95606 271476-271552 Sentence denotes The FA FI Panel simultaneously tests for ten viruses using 200 μL of plasma.
T38589 271553-271651 Sentence denotes Two minutes of hands-on time are required and comprehensive results are returned in about an hour.
T42409 271652-271660 Sentence denotes Methods:
T86189 271661-271808 Sentence denotes One hundred ninety six (196) fever (reported temperature > 38.0°C) or a blood/CSF culture where an SBI was suspected, were evaluated in this study.
T39120 271809-271878 Sentence denotes This study was approved by participating institution's review boards.
T41418 271879-272039 Sentence denotes Residual standard of care (SOC) specimens as well as prospectively collected samples were evaluated by the FA FI Panel and results were compared to SOC testing.
T12752 272040-272048 Sentence denotes Results:
T2883 272049-272119 Sentence denotes Eighty viruses were observed in 196 plasma specimens (41% positivity).
T49396 272120-272260 Sentence denotes The most frequently detected viruses were human herpesvirus 6 (19), adenovirus (14), human herpes virus 7 (13), and Epstein-Barr virus (13).
T38953 272261-272383 Sentence denotes The FA FI Panel provided a positive virus identification in 34% (15/44) of cases where all SOC test results were negative.
T5347 272384-272572 Sentence denotes Many viruses included in the FA FI Panel are not part of SOC testing, however the FA FI Panel was concordant with positive SOC results for adenovirus and enterovirus in 60% (4/7) of cases.
T83567 272573-272648 Sentence denotes Discordance may be due to different sample matrices (nasal swab v. plasma).
T14839 272649-272661 Sentence denotes Conclusions:
T19852 272662-272829 Sentence denotes Because febrile pediatric patients often exhibit non-specific symptoms, discriminating between viral, bacterial, or non-infectious causes of high fever is challenging.
T50958 272830-272916 Sentence denotes Testing with the FA FI Panel resulted in a virus detection in 41% (80/196) of samples.
T23999 272917-273025 Sentence denotes Detection of some viruses, such as enterovirus, is known to be associated with a reduced probability of SBI.
T50887 273026-273159 Sentence denotes These results suggest that the FA FI Panel could be a useful system to rapidly aid in identifying pathogens causing fever in infants.
T93249 273160-273165 Sentence denotes ID17.
T69317 273166-273322 Sentence denotes Evaluation of the ARIES HSV-1/2 Assay for the Detection and Differentiation of Herpes Simplex Virus 1 and 2 from Blood, Respiratory, and Ocular Specimens M.
T78679 273323-273331 Sentence denotes Espy, C.
T90138 273332-273341 Sentence denotes Irish, M.
T37023 273342-273379 Sentence denotes Binnicker Mayo Clinic, Rochester, MN.
T83022 273380-273393 Sentence denotes Introduction:
T7671 273394-273574 Sentence denotes Herpes simplex virus types 1 and 2 (HSV-1/HSV-2) can be found in a wide range of clinical specimens, including genital, dermal, cerebrospinal fluid, respiratory, ocular, and blood.
T96609 273575-273703 Sentence denotes The use of real-time PCR is a sensitive and specific method for the detection and differentiation of HSV-1/2 in these specimens.
T9434 273704-273852 Sentence denotes Historically, specimens submitted for real-time PCR are processed to extract nucleic acids, followed by amplification and analysis by real-time PCR.
T68264 273853-273999 Sentence denotes Recently, Luminex Corporation (Austin, TX) developed an automated platform (ARIES) that can perform all of these functions on a single instrument.
T30122 274000-274062 Sentence denotes The ARIES is FDA-cleared for testing genital/dermal specimens.
T74727 274063-274244 Sentence denotes In this study, we sought to evaluate the performance of the ARIES HSV-1/2 system using a variety of non-FDA cleared sample types, including blood, respiratory, and ocular specimens.
T81624 274245-274253 Sentence denotes Methods:
T95415 274254-274527 Sentence denotes One hundredseventeen retrospective clinical specimens (respiratory [n=66], ocular [n=28], and whole blood [n=23] that were determined to be positive for HSV-1 (n=72), HSV-2 (n=9), HSV non-typeable (n=6) or HSV negative (n=30) by routine testing were included in this study.
T3574 274528-274754 Sentence denotes Routine testing consisted of extraction of 200 μl of sample on the MagNA Pure (Roche Diagnostics), followed by analysis of 5 μl of extract using the Roche HSV-1/2 analyte specific reagents (ASR) on the LightCycler 2.0 (Roche).
T88692 274755-274856 Sentence denotes An aliquot of each sample (200μl) was also tested by the ARIES assay per manufacturer's instructions.
T85818 274857-275013 Sentence denotes Results were analyzed by comparing the ARIES results to the results obtained by routine testing, which was considered the reference standard for this study.
T72379 275014-275022 Sentence denotes Results:
T73702 275023-275214 Sentence denotes Following testing of the one hundred-seventeen specimens, the ARIES HSV-1/2 system demonstrated a sensitivity of 100% for HSV-1 (72/72) and HSV-2 (9/9) when compared to the Roche HSV-1/2 ASR.
T5473 275215-275352 Sentence denotes The six samples that were resulted as "HSV non-typeable" by routine testing were determined to be positive for HSV-1 by the ARIES system.
T19107 275353-275425 Sentence denotes The specificity of the ARIES HSV-1/2 assay was found to be 100% (30/30).
T39537 275426-275438 Sentence denotes Conclusions:
T12012 275439-275590 Sentence denotes Testing of blood, respiratory and ocular specimens by the ARIES HSV-1/2 assay showed comparable performance to routine specimen processing and testing.
T98631 275591-275798 Sentence denotes The results of this study suggest that the ARIES system may serve as an option for clinical laboratories that are seeking an automated platform that performs all current PCR functions on a single instrument.
T11479 275799-275813 Sentence denotes Kubasek 2 , R.
T94361 275814-275961 Sentence denotes Widen 2 1 Federal University of São Paulo, Sao Paulo, Brazil; 2 Tampa General Hospital, Tampa, FL; 3 Children's Healthcare of Atlanta, Atlanta, GA.
T33826 275962-275975 Sentence denotes Introduction:
T32852 275976-276223 Sentence denotes Achromobacter xylosoxidans (AX), Burkholderia cepacia (BC), Pseudomonas aeruginosa (PSA) and Stenotrophomonas maltophilia (SM) are non-fermentative Gram negative rods frequently isolated from the respiratory tract of Cystic Fibrosis (CF) patients.
T6041 276224-276414 Sentence denotes The aim of this study was to develop a multiplex PCR test to detect AX, BC, PSA and SM directly from CF patient's respiratory samples using the open mode system of the BD MAX platform (BDM).
T5547 276415-276423 Sentence denotes Methods:
T69025 276424-276527 Sentence denotes A total of 402 CF respiratory samples, with concurrent culture, were tested by the new CF BDM PCR test.
T80778 276528-276657 Sentence denotes A specific set of forward and reverse primers, as well as a probe for each one of the four targets tested, were designed inhouse.
T28702 276658-276768 Sentence denotes In addition, a set of primers and a probe to detect the beta globin (BG) gene was used as an internal control.
T43089 276769-276936 Sentence denotes Before testing, samples were transferred into a 500μL tube of SL solution (Copan Diagnostics, California) and held for 15 minutes at room temperature for liquefaction.
T13252 276937-277035 Sentence denotes Liquefied samples were treated with Proteinase K at 60°C for 30 minutes and at 95°C for 5 minutes.
T11417 277036-277138 Sentence denotes After treatment, 250μL of each sample was inoculated into the BD MAX Sample Preparation Reagent Tubes.
T32160 277139-277315 Sentence denotes Extraction and multiplex PCR were performed by the BD MAX system, using the BD MAX ExK TNA-2 extraction kits, the BD MAX TNA MMK master mix and the specific primers and probes.
T25264 277316-277351 Sentence denotes Cycling conditions were as follows:
T82854 277352-277414 Sentence denotes 80°C for 10min and 40 cycles of 95°C for 15s and 60°C for 60s.
T61473 277415-277515 Sentence denotes The Limit of Detection (LoD) of each target was evaluated by testing seven 10-fold serial dilutions.
T8125 277516-277692 Sentence denotes PCR performance, interference and inhibition were evaluated by spiking eight negative clinical samples with more than one ATCC strain, each one representing a different target.
T67323 277693-277860 Sentence denotes Specificity was performed by testing different species of mycobacteria (n=26), aerobic bacteria (n=15) and candida (n=7) commonly identified in respiratory infections.
T87996 277861-277869 Sentence denotes Results:
T9124 277870-278007 Sentence denotes Out of 402 samples tested, 227 were identified as negative and 175 as positive by culture, for at least one of the targets tested by PCR.
T33046 278008-278168 Sentence denotes Among culture positive samples, the new PCR was able to identify 21 out of 27 (78%) AX, 4 out of 5 (80%) BC, 138 out of 140 (99%) PSA and 29 out of 34 (85%) SM.
T36406 278169-278317 Sentence denotes In addition, the new CF BDM test was able to identify 35 samples as positive that were initially negative by culture (8 AX, 2 BC, 12 PSA and 13 SM).
T56403 278318-278396 Sentence denotes The LoD was 1.5 x 10 2 CFU/mL for AX, BC and PSA and 1.5 x 10 3 CFU/mL for SM.
T89921 278397-278544 Sentence denotes The new multiplex test demonstrated no cross reactivity to related organisms and correctly identified the species correspondent to each PCR target.
T44196 278545-278556 Sentence denotes Conclusion:
T5837 278557-278747 Sentence denotes The new multiplex test performed on the BD MAX System proved to be a specific and sensitive test to detect AX, BC, PSA and SM by real-time PCR on an automated sample-in results-out platform.
T3760 278748-278761 Sentence denotes Introduction:
T75925 278762-278951 Sentence denotes Diagnosis of lower respiratory bacterial infections largely rely on timeconsuming culture-based identification and quantification of bacteria from heterogeneous lower respiratory specimens.
T6038 278952-279013 Sentence denotes Background levels of colonizing flora vary between specimens.
T71971 279014-279176 Sentence denotes PCR assays that provide quantitative information of bacterial loads can potentially be useful to distinguish lower respiratory tract infections from colonization.
T93283 279177-279259 Sentence denotes We designed, evaluated, and compared two multiplex quantitative PCR (qPCR) assays.
T31123 279260-279460 Sentence denotes Each multiplex qPCR assay was designed to detect 16 pathogens. qPCR assay results from 82 clinical specimens (23 tracheal aspirate, 23 BAL, and 36 sputum) were compared to clinical laboratory results.
T35281 279461-279469 Sentence denotes Methods:
T1694 279470-279861 Sentence denotes Organisms evaluated include Acinetobacter baumanniicalcoaceticus complex (ABC complex), Escherichia coli, Enterobacter cloacae, E. aerogenes, Klebsiella pneumoniae, K. oxytoca,Haemophilus influenzae, Proteus sp., Moraxella catarrhalis, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Serratia marcescens, Streptococcus pyogenes, S. agalactiae, S. pneumoniae, and Staphylococcus aureus.
T54462 279862-279976 Sentence denotes Two individual multiplex qPCR assays were designed to detect these using 2 separate gene targets for each analyte.
T28442 279977-280074 Sentence denotes Extracted nucleic acid from 82 clinical specimens were tested and quantified by both qPCR assays.
T28933 280075-280213 Sentence denotes Data from each assay were compared for number of positive and negative detections, quantitative titer, and agreement with culture results.
T69707 280214-280222 Sentence denotes Results:
T73878 280223-280321 Sentence denotes The limit of detection for the majority of analytes was estimated to genomic equivalents/reaction.
T83584 280322-280434 Sentence denotes Testing the same clinical specimens by both qPCR assays showed good correlation between each quantified analyte.
T65015 280435-280641 Sentence denotes Compared to culture, percent positive agreement (PPA) was 100% for all analytes except K. pneumoniae (70%, both qPCR assays), S. maltophilia (75%, one qPCR assay), and P. aeruginosa (90%, both qPCR assays).
T85303 280642-280751 Sentence denotes Concordant analytes included 19 P. aeruginosa, 7 K. pneumoniae, 7 S. marcescens, 6 Proteus sp., 5 E. coli, 5.
T29686 280752-280899 Sentence denotes E. aerogenes, 4 S. agalactiae, 4 S. maltophilia, 4 ABC complex, 3 K. oxytoca, 3 E. cloacae, 2 H. influenzae, 2 S. pneumoniae, and 1 M. catarrhalis.
T77752 280900-281013 Sentence denotes Percent negative agreement was greater than 80% for all analytes except ABC complex, S. maltophilia, and E. coli.
T70543 281014-281026 Sentence denotes Conclusions:
T814 281027-281154 Sentence denotes Both multiplex qPCR assays have near perfect PPA with culture when testing lower respiratory specimens for bacterial detection.
T35305 281155-281307 Sentence denotes A higher number of detections was observed using multiplex qPCR than culture, however PCR quantification suggests sub-clinical levels in some specimens.
T53097 281308-281444 Sentence denotes Quantification can be incorporated to study clinically relevant limits for organisms that commonly colonize the lower respiratory tract.
T57796 281445-281478 Sentence denotes These assays are not FDAapproved.
T93307 281479-281481 Sentence denotes M.
T69580 281482-281496 Sentence denotes Alikhan 1 , V.
T47001 281497-281509 Sentence denotes Tesic 2 , K.
T40819 281510-281521 Sentence denotes Kaul 3 , S.
T65644 281522-281692 Sentence denotes Das 3 1 NorthShore University Health System/University of Chicago, Chicago, IL; 2 University of Chicago, Chicago, IL; 3 NorthShore University Health System, Evanston, IL.
T4739 281693-281706 Sentence denotes Introduction:
T13194 281707-281911 Sentence denotes Invasive fungal infections (IFIs) can be life-threatening, especially in immunocompromised patients, making rapid and reliable identification of causative species essential for early and adequate therapy.
T35956 281912-282087 Sentence denotes Microscopic identification of fungi from formalin-fixed, paraffin-embedded (FFPE) specimens may provide presumptive identification, and routine culture can take days to weeks.
T57002 282088-282189 Sentence denotes Rapid identification of fungi directly from FFPE tissues could be of immense benefit in patient care.
T35844 282190-282362 Sentence denotes Adequate and efficient extraction of DNA from FFPE tissue is paramount to ensuring purity for subsequent molecular genetic studies which can provide more robust speciation.
T35306 282363-282624 Sentence denotes Herein, we describe the use of the PinPoint Slide DNA Isolation System (Zymo Research) as a convenient and effective method of DNA isolation and extraction from FFPE tissues for definitive identification of fungal microorganisms by subsequent Sanger sequencing.
T57911 282625-282781 Sentence denotes Methods: IFIs from 23 patients were selected from 2 institutions, of which 21 showed fungal organisms by Gomori methanamine silver (GMS) stain on histology.
T46631 282782-282861 Sentence denotes The sources of infections included sinus contents, liver, bone, lung, and skin.
T79888 282862-283058 Sentence denotes Areas with the highest organism burden were selected from each slide, and DNA was isolated and extracted using the PinPoint Slide DNA Isolation System according to the manufacturer's instructions.
T22005 283059-283233 Sentence denotes The recovered DNA was subjected to polymerase chain reaction (PCR) analysis using specific primers against the internal transcribed spacer-2 region of the ribosomal RNA gene.
T87497 283234-283316 Sentence denotes The resultant sequencing data was compared against a homology search using BLASTn.
T20974 283317-283382 Sentence denotes Concurrent fungal culture results were compared to sequence data.
T4363 283383-283391 Sentence denotes Results:
T58187 283392-283490 Sentence denotes Fungal DNA was successfully amplified in 18 of 23 specimens with histologically-proven IFIs (78%).
T86366 283491-283636 Sentence denotes Sequencing showed a variety of pathogens including Aspergillis spp., Pseudoallescheria boydii, Fusarium, Curvularia, Alternaria, and Candida spp.
T30094 283637-283847 Sentence denotes Of the 23 cases, 9 had no growth on fungal culture; 6 of these had positive identification of a fungal pathogen on PCR analysis, and, in an additional 2 cases, identification was made by a reference laboratory.
T19097 283848-283901 Sentence denotes Two culture-positive cases were negative by PCR (9%).
T9109 283902-283973 Sentence denotes Cultures and PCR were concordant in 11 of the 12 remaining cases (92%).
T99685 283974-283986 Sentence denotes Conclusions:
T53605 283987-284138 Sentence denotes Direct sequencing from FFPE specimens provides a rapid method of fungal species identification in IFIs and is superior to conventional culture methods.
T74653 284139-284239 Sentence denotes The extraction system evaluated in this study provides adequate and good quality DNA for sequencing.
T71037 284240-284344 Sentence denotes The method can provide rapid identification of the pathogen at the species level and help guide therapy.
T64900 284345-284486 Sentence denotes Additionally, FFPE tissues with IFI can be stored and DNA retrieved for future studies years later, providing a valuable source for research.
T98990 284487-284500 Sentence denotes Introduction:
T9727 284501-284588 Sentence denotes Respiratory viral infections are the most common worldwide cause of infectious disease.
T63631 284589-284697 Sentence denotes We investigated the incidences and age-related/seasonal variations of respiratory virus infections in Korea.
T62671 284698-284706 Sentence denotes Methods:
T5763 284707-284913 Sentence denotes A total of 3,467 respiratory specimens from patients with acute respiratory infection symptoms in Hwaseong, Korea were tested for respiratory viruses using a multiplex real-time PCR kit during 2013 to 2015.
T79914 284914-284922 Sentence denotes Results:
T25975 284923-285058 Sentence denotes At least one virus was detected in 2,561 of the 3,467 specimens (73.9%), and 706 specimens (20.4%) were sitive for two or more viruses.
T28406 285059-285195 Sentence denotes Two viruses were simultaneously detected in 604 specimens (17.4%), 96 specimens (2.8%) had three viruses, and 6 (0.2%) had four viruses.
T87795 285196-285320 Sentence denotes The most frequently detected viruses were rhinovirus (23.9%), respiratory syncytial virus B (15.5%), and adenovirus (12.5%).
T45118 285321-285530 Sentence denotes Most of the patients (with and without a detected virus) were children, and young children (<5 years old) were significantly more likely to have two or more viruses, compared to older individuals (P < 0.0001).
T42812 285531-285574 Sentence denotes Most viruses exhibited seasonal variations.
T39726 285575-285832 Sentence denotes Coronavirus and respiratory syncytial virus were prevalent during the late autumn and winter, metapneumovirus was common during the spring, bocavirus was common during late spring and early summer, and parainfluenza was common during late spring and summer.
T241 285833-285845 Sentence denotes Conclusions:
T14288 285846-286025 Sentence denotes This study revealed that the incidence of respiratory virus infections and co-infection rates were high, especially among children, and most viruses exhibited seasonal variations.
T12321 286026-286151 Sentence denotes These findings can enhance our understanding of the distributions of respiratory viruses according to patient age and season.
T33158 286152-286154 Sentence denotes A.
T84415 286155-286167 Sentence denotes Ricketts, C.
T60670 286168-286177 Sentence denotes Netti, S.
T86060 286178-286227 Sentence denotes Kazi Qnostics, Glasgow, Scotland, United Kingdom.
T36472 286228-286241 Sentence denotes Introduction:
T53112 286242-286422 Sentence denotes Accurate and Reproducible viral load determination plays a critical role in clinical diagnostics particularly in monitoring patients' response to treatment and disease progression.
T19352 286423-286613 Sentence denotes In this study, 4 transplant associated viral targets were analysed over multiple sites and molecular platforms to assess whether the presence of a recognised standard helped improve results.
T34176 286614-286729 Sentence denotes Assay reproducibility and accuracy were examined by comparing how different platforms quantified identical samples.
T42008 286730-286938 Sentence denotes Two of the targets; Human Cytomegalovirus (CMV) and Epstein Barr Virus s), the other targets, John Cunningham Virus (JCV) and BK Virus (BKV) had no international standards available at the time of this study.
T27600 286939-286947 Sentence denotes Methods:
T5479 286948-287061 Sentence denotes Control materials were prepared in plasma at a titre that falls within the linear range for most clinical assays.
T57232 287062-287149 Sentence denotes The controls were characterised internally using in house PCR and digital PCR (BioRad).
T80541 287150-287283 Sentence denotes Labs were asked to treat the materials as a clinical sample and to return quantitative data along with information on the assay used.
T47045 287284-287373 Sentence denotes The data were separated by assay, anonymised, collated and non-compliant results removed.
T96703 287374-287452 Sentence denotes Datasets were included if more than five laboratories reported compliant data.
T58746 287453-287461 Sentence denotes Results:
T66335 287462-287597 Sentence denotes Five hundred and seventy-eight CMV results were collected across 7 assay groups, 67% were commercial kits, 37% were expressed in IU/ml.
T36474 287598-287672 Sentence denotes 260 EBV results were collected across 5 groups, 55% commercial, 30% in IU.
T69905 287673-287783 Sentence denotes The consensus values reported in c/ml and IU/ml were closely aligned (CMV 3.86 c/3.92 IU, EBV 4.09 c/3.97 IU).
T62027 287784-287857 Sentence denotes 190 JCV results were collected across 3 groups, 33% were commercial kits.
T23648 287858-287931 Sentence denotes 445 BKV results were collected across 5 groups, 47% were commercial kits.
T76622 287932-288048 Sentence denotes Standard deviation of the consensus mean was comparable for all 4 targets, (CMV 0.36, EBV 0.40, BKV 0.47, JCV 0.46).
T71006 288049-288183 Sentence denotes JCV and BKV were higher, although this may be due to fact that there are currently more commercial kits used in CMV and EBV detection.
T63876 288184-288241 Sentence denotes Values for dPCR were in line with the observed consensus.
T34938 288242-288290 Sentence denotes CMV, JCV and BKV t 10 of the observed consensus.
T92042 288291-288329 Sentence denotes For EBV dPCR was 0.4 Log10 c/ml lower.
T71170 288330-288401 Sentence denotes All dPCR values were within 1 standard deviation of the consensus mean.
T43630 288402-288414 Sentence denotes Conclusions:
T89042 288415-288640 Sentence denotes The use of recognised international standards is essential in supporting and improving the traceability and comparison of results across laboratories which in turn can help improve the accuracy and reproducibility of results.
T15804 288641-288945 Sentence denotes However in the absence of International standards well characterised control materials and good QC practice are essential in helping the laboratory to monitor and improve the accuracy and reproducibility of their assays on a daily basis which also helps define future International Standard requirements.
T70195 288946-288948 Sentence denotes A.
T92643 288949-288964 Sentence denotes Hindupur 1 , J.
T29721 288965-288977 Sentence denotes Evans 2 , C.
T38493 288978-288990 Sentence denotes Maity 3 , S.
T48352 288991-289004 Sentence denotes Raines 4 , B.
T35714 289005-289020 Sentence denotes Loeffler 4 , S.
T13127 289021-289034 Sentence denotes Elagin 4 , V.
T31573 289035-289081 Sentence denotes Slepnev 1 Meridian Bioscience, Cincinnati, OH.
T56191 289082-289095 Sentence denotes Introduction:
T23741 289096-289334 Sentence denotes Current molecular methods for testing Zika virus face several challenges such as 1) simplification of sample preparation from blood, 2) reagent stability under ambient conditions, 3) ease-of-use for the end-user and 4) affordable pricing.
T31253 289335-289503 Sentence denotes Reverse transcription coupled Loop mediated isothermal amplification (RT-LAMP) is a highly sensitive, rapid molecular method which can be used to detect Zika virus RNA.
T52488 289504-289592 Sentence denotes We report on the performance of a simplified Zika assay in an easy to use LAMP platform.
T45063 289593-289601 Sentence denotes Methods:
T39496 289602-289765 Sentence denotes The Meridian illumigene Zika (Research Use Only, RUO) reverse transcription coupled DNA Amplification Assay, uses one step RT-LAMP to detect the RNA of Zika virus.
T96944 289766-289877 Sentence denotes During LAMP amplification, magnesiumpyrophosphate accumulates, changing the absorbance of the reaction mixture.
T58630 289878-289952 Sentence denotes The absorbance change is measured by the Meridian illumipro-10 instrument.
T23965 289953-290076 Sentence denotes A one step purification based on chemical lysis and gel filtration was designed to extract Zika virus RNA from whole blood.
T20656 290077-290211 Sentence denotes Purification procedure uses gravity-driven gel filtration column, Meridian M-prep that produces amplifiable RNA within 5 to 7 minutes.
T98391 290212-290309 Sentence denotes Simulated blood samples were prepared by mixing particles of Zika virus with donor blood samples.
T34105 290310-290395 Sentence denotes Viral DNA was purified using M-prep and directly amplified in one step RT-LAMP assay.
T69313 290396-290460 Sentence denotes Amplification of target was detected using illumipro-10 readers.
T95592 290461-290469 Sentence denotes Results:
T30941 290470-290541 Sentence denotes The Limit of Detection (LoD) of illumigene Zika assay is 840 Copies/mL.
T12251 290542-290650 Sentence denotes The assay also detected the synthetic templates representing highly polymorphic seven strains of Zika virus.
T38802 290651-290744 Sentence denotes In addition, the assay also detected the RNA of commercially available strains of Zika virus.
T35598 290745-290842 Sentence denotes No cross-reactivity was observed with synthetic templates of closely related Dengue type 1 and 2.
T33740 290843-290855 Sentence denotes Conclusions:
T84875 290856-291051 Sentence denotes The RT-LAMP based illumigene Zika assay was demonstrated to detect Zika virus from whole blood samples with high analytical sensitivity using an extremely simple procedure, in less than one hour.
T87030 291052-291145 Sentence denotes No blood fractionation or additional lab equipment (centrifuge or heat blocks) was necessary.
T97724 291146-291242 Sentence denotes The described RT-LAMP may be particularly suitable for deployment in resources limited settings.
T9994 291243-291337 Sentence denotes This provides a much needed alternative to the more complex molecular test for Zika diagnosis.
T63840 291338-291343 Sentence denotes NOTE:
T96587 291344-291431 Sentence denotes The illumipro-10 and M-prep are not cleared for use with the illumigene Zika RUO assay.
T55271 291432-291534 Sentence denotes M-prep is CE Marked, not cleared for use in the United States of America. (68) samples were evaluated.
T5890 291535-291639 Sentence denotes All samples were nasopharyngeal swabs (NPSW) collected in BD viral transport media (VTM), stored at 4°C.
T17903 291640-291771 Sentence denotes Primary patient testing was performed on either the BioFire (BF) FilmArray Respiratory Panel or the Cepheid Xpert FLU/AB/RSV assay.
T25102 291772-291827 Sentence denotes The maximum time from collection to evaluation was 72h.
T69924 291828-291889 Sentence denotes Testing on all three platforms was performed on the same day.
T69142 291890-291966 Sentence denotes Alere is CLIA waived for direct nasal swabs, CLIA moderate for swabs in VTM.
T49599 291967-292046 Sentence denotes Sofia is CLIA waived for nasal swabs and NPSW tested directly, and NPSW in VTM.
T62806 292047-292085 Sentence denotes Xpress is CLIA waived for NPSW in VTM.
T10322 292086-292153 Sentence denotes All three assays were run according to manufacturers' instructions.
T27181 292154-292174 Sentence denotes Evaluation Criteria:
T77344 292175-292282 Sentence denotes Samples had to be positive with at least two systems (BF, Alere, Xpert, Sofia) to be included in the study.
T28639 292283-292362 Sentence denotes Several operators performed testing and evaluated hands-on time for each assay.
T4553 292363-292371 Sentence denotes Results:
T10590 292372-292510 Sentence denotes Ten samples were negative for Flu A,B,RSV; 4 samples were Flu A-, B-, RSV+; and 6 samples were Flu A,B,RSV-and positive for other targets.
T63963 292511-292568 Sentence denotes Twenty-seven samples were Flu A+; 21 samples were Flu B+.
T85586 292569-292640 Sentence denotes Of the 68 samples, 22 were discrepant on at least one of the 3 systems.
T32314 292641-292781 Sentence denotes Four of the discrepant samples positive for Flu A in BF and negative with all three comparison assays were excluded from further evaluation.
T77513 292782-292826 Sentence denotes Forty-six samples agreed across all systems:
T96262 292827-292873 Sentence denotes 20 Flu A/B-, 16 Flu A+/ B-, and 10 Flu B+/ A-.
T94736 292874-292886 Sentence denotes Sensitivity:
T98995 292887-292957 Sentence denotes Alere 82.6% A+, 81% B+; Xpress 100% Flu A,B; Sofia 69.6% A+, 47.6% B+.
T6136 292958-292970 Sentence denotes Specificity:
T47920 292971-292994 Sentence denotes 100% for all platforms.
T40436 292995-293040 Sentence denotes Hands-on/testing times for each platform are:
T27008 293041-293110 Sentence denotes Sofia 2.5 min set up time, 15 min incubation and read totaling18 min.
T9785 293111-293208 Sentence denotes Alere 15 minutes directly from swab and 18 min if testing swab in VTM; about 2 min hands-on time.
T48097 293209-293258 Sentence denotes Xpress 1 to 2 min set-up time, 60 min incubation.
T56671 293259-293271 Sentence denotes Conclusions:
T94643 293272-293378 Sentence denotes Timely testing at the POC with a rapid accurate influenza test will directly impact appropriate treatment.
T46104 293379-293461 Sentence denotes The Xpert Xpress platform had the highest sensitivity (100%) with results in 1 hr.
T20676 293462-293553 Sentence denotes The sensitivity of Alere was 82.6% and 81% for Flu A and B respectively, results in 18 min.
T42299 293554-293662 Sentence denotes Sofia had the lowest sensitivity with 69.6% and 47.6% for Flu A and B respectively; results in about 18 min.
T52471 293663-293818 Sentence denotes When considering rapid systems, accuracy, hands-on time, incubation time, and interface capability should all be considered for the final system selection.
T32444 293819-293821 Sentence denotes Y.
T51522 293822-293828 Sentence denotes Lu, S.
T88006 293829-293841 Sentence denotes Adhikari, L.
T31893 293842-293849 Sentence denotes Liu, K.
T20108 293850-293895 Sentence denotes Norman Thermo Fisher Scientific, Fremont, CA.
T48534 293896-294081 Sentence denotes Introduction: EBV is the pathogenic agent for infectious mononucleosis, as well as a variety of lymphoid and epithelial malignancies in immunocompetent and immunosuppressed individuals.
T18507 294082-294191 Sentence denotes EBV DNA load measurement by quantitative PCR has been shown to be a potential diagnostic and monitoring tool.
T62950 294192-294394 Sentence denotes Despite the availability of the WHO standard for EBV since 2011, variability of assay amplification target sites, particularly between different laboratories, still leads to variability in test results.
T88324 294395-294506 Sentence denotes In this study, we evaluated the potential contribution of reference material diversity to EBV test variability.
T66036 294507-294515 Sentence denotes Methods:
T99162 294516-294774 Sentence denotes The performance of EBV strain B95-8 obtained from different commercial sources was compared across different platforms (including ABI, Roche, and EliTech), different laboratories, and different EBV target genes (including IR1, LMP2, BNRF1, EBNA1, and BKRF1).
T49024 294775-294783 Sentence denotes Results:
T76171 294784-294908 Sentence denotes Results were normalized to the WHO EBV standard to evaluate which source materials exhibited similar performance to the WHO.
T39048 294909-295007 Sentence denotes Surprisingly, up to a 6fold difference in titers was observed across assays for the same material.
T81141 295008-295098 Sentence denotes Differences in titers were also observed from different EBV sources run on the same assay.
T40693 295099-295111 Sentence denotes Conclusions:
T60924 295112-295256 Sentence denotes Our data indicate that gene copy number varies across B95-8 source materials, which affects traceability to the WHO depending on the assay used.
T32484 295257-295473 Sentence denotes This was especially apparent when value assignment was based on a qPCR assay targeting variable regions of the EBV genome, which caused either under-or over-estimation of titer relative to the WHO standard by 2 fold.
T74778 295474-295631 Sentence denotes One EBV B95-8 source performed consistently across assay platforms when compared to the WHO standard (CV at 24.4% compared to the 44.9% from another source).
T49795 295632-295643 Sentence denotes Conclusion:
T1969 295644-295804 Sentence denotes Significant test variability can be artificially introduced through the use of secondary standards that are not genetically similar to the WHO B95-8 EBV strain.
T54023 295805-295994 Sentence denotes EBV reference material testing against multiple targets and laboratories has allowed for the determination of the most consistent EBV B95-8 source for use as a secondary reference material.
T36584 295995-296008 Sentence denotes Introduction:
T45973 296009-296114 Sentence denotes Hepatitis C virus (HCV) is a common chronic viral infection worldwide and a major cause of liver disease.
T37037 296115-296213 Sentence denotes Direct-acting antiviral therapies used to treat HCV infection are typically based on HCV genotype.
T62201 296214-296470 Sentence denotes Here, we describe our validation of the research use only (RUO) eSensor HCVg DirectTest (HCVD) (GenMark Diagnostics, Carlsbad, CA) to change from our laboratory's previous method, the Versant HCV Genotype 2.0 Assay (LiPA) (Siemens Healthcare, Malvern, PA).
T7387 296471-296639 Sentence denotes The main advantages of HCVD (objective resulting, shorter turnaround time), as well as its drawbacks (difficulty with mixed infections), have previously been described.
T52128 296640-296808 Sentence denotes During our validation, we uncovered an additional limitation of the assay not previously reported, as well as a potential way to improve genotyping of mixed infections.
T77823 296809-296817 Sentence denotes Methods:
T65517 296818-297076 Sentence denotes Archived plasma samples (n=81) and commercially available HCV genotyping panels (n=2) extracted using the QIAamp DSP Viral RNA kit and QIAcube (Qiagen, Valencia, CA) were used to evaluate the performance characteristics of HCVD and the XT-8 System (GenMark).
T62875 297077-297166 Sentence denotes HCVD was assessed for analytical sensitivity and specificity, concordance, and precision.
T96758 297167-297314 Sentence denotes Discrepant samples were resolved through repeat testing, altering the extraction method (GenMark), and Sanger sequencing (Retrogen, San Diego, CA).
T58925 297315-297414 Sentence denotes Results: HCVD was able to accurately genotype all commercial control samples (n=15, genotypes 1-6).
T81994 297415-297491 Sentence denotes The approximate analytical sensitivity was an HCV viral load of 1,000 IU/mL.
T37068 297492-297585 Sentence denotes Precision studies showed concordance of results for multiple samples over several replicates.
T9688 297586-297756 Sentence denotes HCVD demonstrated 100% analytical specificity (n=20) and an overall concordance rate of 90% (55 of 61 clinical samples previously genotyped by LiPA or Sanger sequencing).
T29842 297757-297968 Sentence denotes The 6 discrepant results were from samples with mixed infections (n=3), a low viral load (218 IU/mL) (n=1), a genotype 4f infection (n=1), and a prior indeterminate result by LiPA (accurately genotyped by HCVD).
T76590 297969-298087 Sentence denotes We found that altering the extraction method allowed HCVD to detect 2 of the 3 mixed infections previously not called.
T45119 298088-298213 Sentence denotes Additionally, we discovered that viral nucleic acid from genotype 4f cross-reacts with reagent signal probes for genotype 1b.
T977 298214-298343 Sentence denotes This led a genotype 4f sample to be genotyped as a Type 1 plus Type 4 co-infection and then as a Type 1b in subsequent reactions.
T80342 298344-298356 Sentence denotes Conclusions:
T40214 298357-298540 Sentence denotes Overall, the RUO eSensor HCVg Direct Test performed favorably relative to LiPA; after discrepant resolution, the overall concordance rate between the two methods was 93% (n=57 of 61).
T40358 298541-298790 Sentence denotes Whereas HCVD had difficulty with type 4f and mixed infections in our hands, modifying the extraction method allowed for genotyping of previously missed coinfected samples, suggesting a potential solution for laboratories encountering similar issues.
T87445 298791-298859 Sentence denotes Introduction: HPV is the most common sexually transmitted infection.
T37631 298860-298970 Sentence denotes Persistent infection with oncogenic high risk HPV (hrHPV) types causes virtually all cases of cervical cancer.
T30721 298971-299082 Sentence denotes HPV detection and prevention algorithms are moving targets, with evolving guidelines and diagnostic techniques.
T24842 299083-299191 Sentence denotes Recent studies have demonstrated regional and population differences in the distribution of hrHPV genotypes.
T74923 299192-299300 Sentence denotes Bronx, NY is ethnically diverse with 52% Hispanic, 32% African-American, 12% White, 2% Asian, and 2% others.
T92254 299301-299425 Sentence denotes Almost one-third (31.8%) of its residents are foreign-born and a majority (50.5%) of its births are to foreign-born mothers.
T59986 299426-299434 Sentence denotes Methods:
T55630 299435-299585 Sentence denotes Utilizing a retrospective query of LIS database we studied SurePath cervicovaginal cytology and Cobas HPV results reported between 10-5-15 and 5-6-16.
T84238 299586-299659 Sentence denotes Patients aged 16-95 (average age 43), with racial distribution including:
T20839 299660-299762 Sentence denotes African-American 32.4%, Other mixed, Hispanic35%, Caucasian 14.4%, Asian 0.7%, Declined/Unknown 17.5%.
T86633 299763-299894 Sentence denotes A total of 18,333 or 73.2% underwent testing for hrHPV, which separately reports HPV 16, HPV 18 and a pool of 12 other hrHPV types.
T52335 299895-299903 Sentence denotes Results:
T71135 299904-299964 Sentence denotes Among all samples tested, 3901 or 21.4% were hrHPV positive.
T103 299965-300019 Sentence denotes All 27 of COBAS-tested HSIL cases were hrHPV positive.
T44046 300020-300208 Sentence denotes In keeping with the increased risk of progression reported for HPV16, the percentage that were positive for HPV 16 alone or in combination was 37% in HSIL versus 14.8% in patients overall.
T80692 300209-300300 Sentence denotes However, only one of the HSIL cases was HPV 18 positive; majority (66.7%) was OHR positive.
T15551 300301-300322 Sentence denotes Further evaluation is
T97340 300323-300531 Sentence denotes The Journal of Molecular Diagnostics ■ jmd.amjpathol.org needed to determine if this OHR pool includes individual genotypes that in our population carry a higher risk of persistence and progression to cancer.
T44010 300532-300544 Sentence denotes Conclusions:
T7782 300545-300617 Sentence denotes In this diverse population, the 21.4% hrHPV is higher than Athena study.
T19870 300618-300756 Sentence denotes Percentage of hrHPV positive results, both overall and for individual genotypes, increases with increasing level of cytologic abnormality.
T36036 300757-300866 Sentence denotes HPV 16 only, is present in 1.6% of cases diagnosed as NSIL and increases to 22.2% in cases diagnosed as HSIL.
T85670 300867-300930 Sentence denotes OHR types only, are present in 13.6% of NSIL and 59.3% of HSIL.
T23187 300931-301031 Sentence denotes Moreover OHR was the only type identified in one case that proved to show invasive cancer on biopsy.
T95872 301032-301131 Sentence denotes This seems contrary to prior reports that HPV 16 and 18 account for a majority of invasive cancers.
T97260 301132-301346 Sentence denotes Given the ethnic diversity of population, and in light of prior studies indicative of regional differences in prevalent genotypes, further genotyping with the most abnormal cytology may reveal additional genotypes.
T34760 301347-301562 Sentence denotes This study suggests that there is a need for more studies in ethnically diverse populations to identify other high risk genotypes, and plan risk and ethnic specific prevention as well as tests for earlier diagnosis.
T22708 301563-301685 Sentence denotes Secondly to compare the cost, TAT and diagnostic yield of different algorithms for the detection of respiratory pathogens.
T96595 301686-301805 Sentence denotes Finally, to test the Simplexa assay on BAL samples, which has only been validated on nasopharyngeal swabs (NTS) so far.
T61982 301806-301814 Sentence denotes Methods:
T10137 301815-301899 Sentence denotes We collected 125 NTS and 25 BAL samples from symptomatic immunocompromised patients.
T40244 301900-302064 Sentence denotes Samples for which Simplexa and TAC results were discordant underwent verification testing using the multiplex real-time FTD Flu/HRSV assay (Fast-track Diagnostics).
T44491 302065-302170 Sentence denotes The TAC assay is based on singleplex RT-PCR, targeting 24 viruses, 8 bacteria and 2 fungi simultaneously.
T25669 302171-302179 Sentence denotes Results:
T25084 302180-302301 Sentence denotes As expected, the overall sensitivity was significantly lower for DFA testing than for the two molecular methods (p<0.05).
T94955 302302-302441 Sentence denotes However, when considering results for each pathogen separately, the difference in performance was only statistically significant for Flu A.
T61006 302442-302576 Sentence denotes The Simplexa direct test missed one RSV, one Flu A and two Flu B positive samples in comparison to the TAC assay and verification PCR.
T52306 302577-302693 Sentence denotes One sample was found strongly positive for Flu A by Simplexa, but was negative by DFA, TAC and verification testing.
T21786 302694-302851 Sentence denotes Nevertheless, the differences in individual and overall sensitivity and specificity of Simplexa testing were not significant compared to TAC testing (p>0.1).
T61511 302852-302944 Sentence denotes For BAL samples only (n=25), the sensitivity and specificity of the Simplexa assay was 100%.
T31501 302945-303064 Sentence denotes In total, DFA identified 14 samples (9.3%) and Simplexa testing found 24 (16%) samples positive with one pathogen each.
T42708 303065-303146 Sentence denotes The TAC assay identified 93 samples with one or more respiratory pathogens (62%).
T69571 303147-303260 Sentence denotes More than half (54%) of Simplexa negative samples were positive by TAC for other pathogens than RSV, Flu A and B.
T9080 303261-303307 Sentence denotes A co-infection rate of 15.3% was found by TAC.
T52490 303308-303436 Sentence denotes The estimated costs and TAT were 8.2€ and 2 hours for DFA, 31.8€ and 1.5 hours for Simplexa and 56€ and 6 hours for TAC testing.
T68614 303437-303449 Sentence denotes Conclusions:
T77101 303450-303672 Sentence denotes Based on these results, performing a first line molecular method such as the Simplexa test instead of DFA would be necessary to obtain an acceptable overall sensitivity, albeit at a higher cost generated in the laboratory.
T9461 303673-303840 Sentence denotes Performing the TAC as a second line test for patients with a negative Simplexa result would increase the diagnostic yield significantly, albeit at an even higher cost.
T50828 303841-303854 Sentence denotes Introduction:
T96151 303855-303990 Sentence denotes Human monocytic ehrlichiosis and human granulocytic anaplasmosis are tick borne bacterial diseases, found in several states in the U.S.
T83185 303991-304227 Sentence denotes We developed and evaluated a multiplexed, real-time PCR assay to detect A. phagocytophillum (ANA) and several Ehrlichia species (E. chaffeensis, E. ewingii and E. muris) (EHR) in whole blood specimens using the Luminex ARIES instrument.
T17182 304228-304451 Sentence denotes Selective primers, labeled with several different fluorescent dyes, facilitate the detection of ANA, EHR and an internal control in whole blood specimens in a single-step, multiplexed, qPCR assay using the ARIES instrument.
T3500 304452-304528 Sentence denotes This will allow for rapid diagnosis with increased sensitivity in less time.
T14968 304529-304537 Sentence denotes Methods:
T76470 304538-304548 Sentence denotes Specimens:
T43258 304549-304690 Sentence denotes Multiplexed, real-time PCR methods, based on the unique MultiCode base pair (isoC:isoG), were developed for diagnosis of tick borne diseases.
T32487 304691-304841 Sentence denotes Analytical specificity studies were assessed by testing whole blood, collected from separate volunteers, spiked with 10 fold dilutions of ANA and EHR.
T36762 304842-304963 Sentence denotes Primers were added to the ready mix tubes, attached them to the cassettes and then loaded the samples on to the cassette.
T40400 304964-304973 Sentence denotes Reagents:
T70330 304974-305121 Sentence denotes Primer pairs are designed to include a fluorescent reporter labeled primer with an isoC on the 5'end and an unlabeled primer and obtained from IDT.
T70478 305122-305345 Sentence denotes The primer sequences for ANA are: forward 5'-CAG TCG TGA ATG TAG AGG GAA AAA C-3'; reverse 5'-GGA ATC CCC CTT CAG GAA CTT G-3' and for EHR are: forward 5'-AAT GCT TCT ACT GCT ACT GT-3'; reverse 5'-GCT CCA CCA TGA GCT GG-3'.
T70999 305346-305398 Sentence denotes Ready mix and cassettes were purchased from Luminex.
T54506 305399-305599 Sentence denotes The cassette contains all reagents needed to run PCR and all the steps including extraction, purification, amplification, detection reagent and sample processing control are contained in the cassette.
T63837 305600-305716 Sentence denotes Instrument: ARIES is an in vitro diagnostic medical device for detection of nucleic acids by fluorescence based PCR.
T61943 305717-305725 Sentence denotes Results:
T38542 305726-305902 Sentence denotes The limit of detection (LOD) was determined for both targets and was defined as the lowest concentration of each organism in the 10 spiked samples that produced a CT value <40.
T17196 305903-306061 Sentence denotes Thus, the overall LOD for each ANA and EHR in whole blood was shown to be 36.7±1.5 and 36.06±0.7, respectively (n=5), at a concentration of 100 copies per ml.
T86744 306062-306189 Sentence denotes In a limited study, 17 specimens (2 EHR positives and 15 negatives) correlated with an in-house PCR assay for the same targets.
T82989 306190-306202 Sentence denotes Conclusions:
T41610 306203-306381 Sentence denotes The availability of an ARIES detection system involves minimal technologist time and increases the ability to rapidly diagnose with improved sensitivity of tick borne infections.
T96976 306382-306384 Sentence denotes A.
T10877 306385-306401 Sentence denotes Sanchez 1,3 , B.
T43181 306402-306420 Sentence denotes Rodic-Polic 2 , K.
T19310 306421-306567 Sentence denotes Culbreath 3 1 University of New Mexico, Albuquerque, NM; 2 DiaSorin Molecular LLC, Cypress, CA; 3 Tricore Reference Laboratories, Albuquerque, NM.
T91376 306568-306581 Sentence denotes Introduction:
T41739 306582-306772 Sentence denotes Norovirus, the most common cause of acute gastroenteritis in the United States, causes epidemic outbreaks among children and adults, leading to 19 million to 21 million new cases every year.
T7000 306773-306892 Sentence denotes Though 90% of adults are seropositive for Norovirus antibodies, immunity is not long-lasting and reinfection can occur.
T22712 306893-307004 Sentence denotes The illness tends to be self-limited, comprising of nausea, vomiting, non-bloody diarrhea and abdominal cramps.
T13196 307005-307142 Sentence denotes The symptoms, caused by transient malabsorption, can last up to 6 weeks following a viral challenge, but usually resolve within 72 hours.
T53370 307143-307314 Sentence denotes Currently, there are 6 recognized Norovirus genogroups, all belonging to the Caliciviridae family-the GI, GII, and GIV being the genogroups most commonly affecting humans.
T15593 307315-307454 Sentence denotes Reliable detection of Norovirus has the potential to improve patient management decisions and facilitate prompt infection control measures.
T7123 307455-307632 Sentence denotes This study evaluates the performance characteristics of the FOCUS Diagnostics RT-PCR molecular assay using analyte-specific reagents (ASR) for the detection of Norovirus GI/GII.
T97532 307633-307641 Sentence denotes Methods:
T11774 307642-307751 Sentence denotes To evaluate the accuracy of the assay, we compared our results with those obtained with the reference method.
T88924 307752-307841 Sentence denotes One hundred and twelve formed and unformed stool samples were analyzed during this study:
T57308 307842-308058 Sentence denotes 30 previously tested positive samples and 25 previously tested negative samples as well as 57 samples which were collected for the detection of other gastrointestinal pathogens, and comprised the random sample group.
T13428 308059-308157 Sentence denotes Nucleic acid extraction was performed using the NucliSENS easyMAG system (bioMerieux, Durham, NC).
T95029 308158-308243 Sentence denotes Extracted samples were subjected to RT-PCR qualitative analysis on Integrated Cycler.
T78626 308244-308355 Sentence denotes Analytical specificity of the assay was evaluated by testing a panel of most common gastrointestinal pathogens.
T70694 308356-308466 Sentence denotes Analytical sensitivity was determined by performing serial dilutions of Norovirus GI/GII quantitated controls.
T9655 308467-308475 Sentence denotes Results:
T60351 308476-308585 Sentence denotes Among 30 known positive samples previously tested, 26 were confirmed positive by the FOCUS Diagnostics assay.
T29343 308586-308752 Sentence denotes The remaining 4 discrepant samples were subsequently repeated by our method and then confirmed negative by the reference method, resulting in 100% positive agreement.
T13658 308753-308824 Sentence denotes Of the 57 randomly selected samples, 4 were positive for Norovirus GII.
T33174 308825-308896 Sentence denotes No cross reactivity with members of the specificity panel was observed.
T38410 308897-308909 Sentence denotes Conclusions:
T99946 308910-309095 Sentence denotes The preliminary results of our evaluation study indicate that Focus Diagnostics RT-PCR assay for qualitative detection of Norovirus I/II is a sensitive and reproducible molecular assay.
T86322 309096-309243 Sentence denotes This assay proved to be robust and easy to perform which makes it suitable for the reliable detection of Norovirus I/II in the clinical laboratory.
T92053 309244-309246 Sentence denotes C.
T81520 309247-309256 Sentence denotes Cheng, Y.
T32037 309257-309268 Sentence denotes Parocua, B.
T47031 309269-309279 Sentence denotes Torres, M.
T939 309280-309321 Sentence denotes Tabb DiaSorin Molecular LLC, Cypress, CA.
T27313 309322-309335 Sentence denotes Introduction:
T9136 309336-309450 Sentence denotes Enteroviruses (EV) and human parechoviruses (HPeV) are single stranded RNA viruses from the Picornaviridae family.
T82185 309451-309527 Sentence denotes EV are the most common pathogens associated with aseptic (viral) meningitis.
T54139 309528-309622 Sentence denotes However, HPeV have been reported to cause up to 5% of these cases in the pediatric population.
T36292 309623-309811 Sentence denotes The Simplexa EV & HPeV Direct assay (Simplexa assay) is a sample-to-answer assay that detects EV and HPeV from 50 μl of CSF without any additional specimen preparation or extraction steps.
T33891 309812-309861 Sentence denotes The test result is available in about 90 minutes.
T54575 309862-310038 Sentence denotes The goal of this study was to evaluate Simplexa assay performance for analytical limit of detection (LoD), microbial inhibition, method comparison and competitive interference.
T93577 310039-310047 Sentence denotes Methods:
T40204 310048-310101 Sentence denotes A LoD study was performed on 5 EV and 2 HPeV strains.
T4633 310102-310221 Sentence denotes For the microbial inhibition study, contrived samples containing EV and HPeV were spiked with 24 interfering pathogens.
T76186 310222-310292 Sentence denotes A panel of 101 CSF specimens was used for the method comparison study.
T66862 310293-310383 Sentence denotes Due to lack of HPeV positive specimens, 29 contrived and 1 HPeV clinical sample were used.
T9456 310384-310528 Sentence denotes Each specimen was tested in singlicate using the Simplexa assay and results were compared to previously reported PCR results (reference method).
T43327 310529-310761 Sentence denotes The competitive inhibition panel consisted of near LoD EV and HPeV samples contrived using synthetic spinal fluid (SF), challenged with jmd.amjpathol.org ■ The Journal of Molecular Diagnostics high concentrations of competing virus.
T15059 310762-310770 Sentence denotes Results:
T68778 310771-310954 Sentence denotes LoD concentratons for 5 EV strains (Coxsackievirus A3, A17, Echovirus 21, Enterovirus 68 and Poliovirus 2) were confirmed at 0.0325, 0.0125, 0.0325, 6.5 and 26 TCID50/mL respectively.
T81457 310955-311050 Sentence denotes LoD for HPeV strains (HPeV-1 and HPeV-3) were confirmed at 1.25 and 4.7 TCID50/mL respectively.
T9553 311051-311154 Sentence denotes No inhibition was observed with 24 potentially interfering organisms in the microbial inhibition study.
T9295 311155-311305 Sentence denotes Positive and negative agreements in method comparison between Simplexa and the reference method were 98% (50/51) and 100% (50/50) respectively for EV.
T12040 311306-311384 Sentence denotes All 30 HPeV samples were detected as positive (30/30) with the Simplexa assay.
T14647 311385-311577 Sentence denotes In competitive inhibition, all replicates containing low concentrations of one target virus (EV or HPeV) were detected in the presence of high concentrations of the second virus (EV or HPeV) .
T41957 311578-311646 Sentence denotes No inhibition to detection of low virus concentrations was observed.
T11891 311647-311659 Sentence denotes Conclusions:
T68686 311660-311807 Sentence denotes The Simplexa Direct assay was able to detect EV strains at LoDs ranging from 0.0125 to 26 TCID50/mL and two HPeV strains at 1.25 and 4.7 TCID50/mL.
T92453 311808-311927 Sentence denotes The assay demonstrated 98% positive and 100% negative agreements for EV detection and 100% positive detection for HPeV.
T56445 311928-312024 Sentence denotes No cross reactivity or competitive inhibition was observed using the bacteria or viruses tested.
T40281 312025-312122 Sentence denotes The Simplexa assay is currently in development and is not currently for sale and not FDA cleared.
T107 312123-312316 Sentence denotes Introduction: PCR primers used to determine fungal species identification cited from phylogenetic studies often need to undergo optimization and validation for use in the clinical laboratories.
T3653 312317-312589 Sentence denotes Except for the "universal primers" which target the internal transcribed spacer (ITS) as well as domains 1 and 2 of the large ribosomal subunit -TUB) and calmodulin (CAL) genes are also frequently used in phylogenetic studies and have been applied in clinical diagnostics.
T66113 312590-312719 Sentence denotes We retrospectively reviewed 593 clinical and -TUB and CAL primers for identification of Aspergillus species by Sanger sequencing.
T55146 312720-312961 Sentence denotes Methods: DNA was extracted from 593 isolates of various Aspergillus spp. representing clinically significant species by bead-beating -TUB and CAL regions were then PCR amplified using M13 tagged BT2a/BT2b, and cmd5/cmd6 primers respectively.
T31943 312962-313067 Sentence denotes PCR products were sequenced bi-directionally with M13F/M13R primers using the BigDye sequencing reagents.
T65965 313068-313150 Sentence denotes Raw sequences were aligned and edited using the Lasergene software (DNASTAR, Inc).
T60656 313151-313215 Sentence denotes Finished sequences were queried through the GenBank nr database.
T57772 313216-313320 Sentence denotes The top 50 scoring hits were combined with phenotype data to identify each isolate to the species level.
T730 313321-313437 Sentence denotes Results: -TUB (Bt2a/Bt2b) and CAL (cmd5/cmd6) primers were selected to test 94% and 84% of 593 Aspergillus isolates.
T1440 313438-313560 Sentence denotes Sequences were successfully obtained in 94% (527/560) and 72% (355/496) of isolates -TUB and CAL primer set, respectively.
T2280 313561-313638 Sentence denotes Among these 593 isolates, 33 species and 4 species complexes were identified.
T15764 313639-313862 Sentence denotes The major pathogenic Aspergillus species found were A. fumigatus (44%; 260/593), species in the Aspergillus section Nigri, (25%; 146/593), A. flavus (8%; 49/593), as well as Aspergillus terreus species complex (6%; 35/593).
T83351 313863-314020 Sentence denotes Failure of PCR amplification -TUB) and 28% (CAL) isolates, which was mostly caused by biological issues (lack of primer binding sequence in a given species).
T58104 314021-314184 Sentence denotes However, species and species complex level identification using both -TUB and CAL sequence data in combination with phenotype data was successful in all 593 cases.
T86644 314185-314197 Sentence denotes Conclusions:
T79544 314198-314249 Sentence denotes In the molecular identification of Aspergillus spp.
T73470 314250-314515 Sentence denotes -TUB (Bt2a/Bt2b) primer had the highest success rate in producing sequence data, but did not produce a species level identification for some aspergilli (e.g., Section Nigri) due to lack of phylogenetic differences and/or sufficient coverage in the GenBank database.
T70334 314516-314694 Sentence denotes The CAL (cmd5/cmd6) primer alone was able to identify isolates to the species level in 72% of the cases, thus making this primer useful when used in conjunction with -TUB primer.
T62753 314695-314730 Sentence denotes Simplexa Bordetella Direct Assay Y.
T4162 314731-314738 Sentence denotes Xie, H.
T15222 314739-314746 Sentence denotes Mai, J.
T60228 314747-314755 Sentence denotes Chen, M.
T10780 314756-314797 Sentence denotes Tabb Diasorin Molecular LLC, Cypress, CA.
T70952 314798-314811 Sentence denotes Introduction:
T83854 314812-314989 Sentence denotes Bordetella pertussis is the main cause of whooping cough, however other Bordetella species, such as Bordetella parapertussis and Bordetella holmesii, can cause similar symptoms.
T97073 314990-315119 Sentence denotes The Simplexa Bordetella Direct assay is in development as a sample-to-answer assay performed on the Integrated Cycler instrument.
T37048 315120-315281 Sentence denotes Nasopharyngeal swab specimens collected in transport media are loaded directly onto a Direct Amplification Disc without extraction or other specimen preparation.
T35437 315282-315408 Sentence denotes The Simplexa Bordetella Direct assay was developed to detect and differentiate B. pertussis, B. parapertussis and B. holmesii.
T1094 315409-315517 Sentence denotes The goal of this verification study was to evaluate the performance of the Simplexa Bordetella Direct assay.
T62798 315518-315526 Sentence denotes Methods:
T26635 315527-315693 Sentence denotes Limit of detection (LoD), analytical reactivity, reproducibility and substance interference studies were performed to evaluate Simplexa Bordetella Direct performance.
T32953 315694-315795 Sentence denotes Limit of detection (LoD) studies were performed to determine the analytical sensitivity of the assay.
T11932 315796-315873 Sentence denotes Ten additional B. pertussis strains were evaluated for analytical reactivity.
T70361 315874-315948 Sentence denotes A reproducibility study was performed with medium and low positive panels.
T96833 315949-316055 Sentence denotes A panel of potentially interfering substances was tested to determine whether any inhibition was observed.
T42705 316056-316064 Sentence denotes Results:
T93235 316065-316190 Sentence denotes LoD studies showed that the Simplexa Bordetella Direct assay detected B. pertussis B. parapertussis B. holmesii at 60 CFU/ml.
T75384 316191-316295 Sentence denotes All 10 B. pertussis Inter-and intra-assay reproducibility assays yielded <4.0% coefficient of variation.
T20595 316296-316373 Sentence denotes No inhibition or interference was observed from any of the substances tested.
T2244 316374-316385 Sentence denotes Conclusion:
T72661 316386-316600 Sentence denotes The Simplexa Bordetella Direct assay was capable of directly detecting and differentiating B. pertussis, B. parapertussis and B. holmesii without up-front nucleic acid extraction from nasopharyngeal swab specimens.
T98354 316601-316723 Sentence denotes The assay and instrumentation provide a compact system for rapid (~80 minutes) detection directly from nasal swab samples.
T89364 316724-317031 Sentence denotes This assay can be performed simultaneously with Simplexa Flu A/B & RSV Direct and Simplexa Respiratory Virus Direct to detect a panel of 9 respiratory pathogens (Flu A, Flu B, RSV, adenovirus, parainfluenza virus (1, 2, 3 & 4) , hMPV, B. pertussis, B. parapertussis and B. holmesii) from one patient sample.
T21910 317032-317190 Sentence denotes Simplexa Bordetella Direct and Simplexa Respiratory Virus Direct assays are in development; they are not currently available for sale and are not FDA cleared.
T52116 317191-317318 Sentence denotes There are estimated to be at least 150,000 cases a year in the USA, with Arizona accounting for at least 90,000 (60%) of these.
T7145 317319-317505 Sentence denotes The PathoGene Coccidioides Assay is a qualitative real-time PCR-based assay that detects Coccidioides spp. target DNA from bronchial alveolar lavage (BAL) or bronchial wash (BW) samples.
T82572 317506-317669 Sentence denotes Extracted DNA is added to an assay cartridge that contains all the reagents for amplification and detection of the Coccidioides target DNA and an internal control.
T44581 317670-317851 Sentence denotes The cartridge is placed in the GeneSTAT instrument and the assay is initiated by the user; all subsequent steps of the assay are performed by the GeneSTAT without user intervention.
T17814 317852-317933 Sentence denotes Assay turn-around time from cartridge loading to results generation is 1.5 hours.
T90681 317934-317942 Sentence denotes Methods:
T57251 317943-318133 Sentence denotes The Coccidioidesspecific PCR assay targets a 106-bp sequence that is present in multiple copies within the C. posadasii and C. immitis genomes (assay patent licensed from TGen, Phoenix, AZ).
T726 318134-318199 Sentence denotes The human RNase P gene acts as an internal control for the assay.
T77076 318200-318306 Sentence denotes Amplified PCR products are detected with FAM-(Coccidioides) and AP593-labeled (RNase P) hydrolysis probes.
T21671 318307-318424 Sentence denotes C. posadasiispherules and extracted DNA from both Coccidioides strains were obtained from TGen North (Flagstaff, AZ).
T39061 318425-318433 Sentence denotes Results:
T92210 318434-318506 Sentence denotes Limit of Detection (LoD) for C. posadasii spherules was 50 spherules/mL.
T82402 318507-318588 Sentence denotes The LoD for extracted DNA from both Coccidioides strains was 10 genome copies/mL.
T11184 318589-318689 Sentence denotes The assay was 100% specific when screened against 47 different bacterial, viral, and fungal species.
T72399 318690-318910 Sentence denotes Clinical performance was determined vs. current gold-standard culture testing at three external sites, and the overall clinical sensitivity and specificity of the assay was 100% (55/55) and 98.9% (275/278), respectively.
T62588 318911-318923 Sentence denotes Conclusions:
T20249 318924-319053 Sentence denotes The PathoGene Coccidioides Assay is a sensitive and specific method for direct detection of Coccidioides DNA from BAL/BW samples.
T65478 319054-319163 Sentence denotes The assay may offer a significant improvement over current serology and culture-based testing methods for VF:
T67718 319164-319311 Sentence denotes Serological testing is rapid but is problematic in that the immune response is delayed after infection and this can lead to false negative results.
T50307 319312-319403 Sentence denotes Fungal culture is specific but it can take 3 weeks or longer to confirm a negative culture.
T70840 319404-319579 Sentence denotes The PathoGene assay is able to generate accurate test results the same day as sample collection, and this should result in improved patient outcomes and reduced medical costs.
T27371 319580-319602 Sentence denotes For research use only.
T90291 319603-319641 Sentence denotes Not for use in diagnostics procedures.
T80306 319642-319655 Sentence denotes Introduction:
T2866 319656-319769 Sentence denotes Accurate and rapid identification of Aspergillus species is often complicated by overlapping phenotypic features.
T98528 319770-319888 Sentence denotes Species of the Aspergillus section Nigri are significant pathogens causing allergic reactions and invasive infections.
T69546 319889-320022 Sentence denotes Molecular methods are often used in combination with phenotype data to finalize a species level identification in a clinical setting.
T65175 320023-320190 Sentence denotes We retrospectively reviewed 146 Aspergillus section Nigri -TUB) and 2 sets of commonly used calmodulin (CAL) primers for molecular identification by Sanger sequencing.
T6016 320191-320314 Sentence denotes Methods: DNA was extracted from 146 Aspergillus section Nigri isolates by bead-beating and the EZ1 DNA Tissue Kit (Qiagen).
T22372 320315-320409 Sentence denotes Regions of the --TUB (BT2a/BT2b), as well as CAL (CL1F/2AR & cmd5/cmd6), primers respectively.
T66412 320410-320475 Sentence denotes PCR products were sequenced using the BigDye sequencing reagents.
T32821 320476-320558 Sentence denotes Raw sequences were aligned and edited using the Lasergene software (DNASTAR, Inc).
T80345 320559-320623 Sentence denotes Finished sequences were queried through the GenBank nr database.
T24824 320624-320728 Sentence denotes The top 50 scoring hits were combined with phenotype data to identify each isolate to the species level.
T64904 320729-320901 Sentence denotes Results: -TUB (Bt2a/Bt2b) and CAL (CL1F/2AR) primers were used when Section Nigri was suspected, whereas the CAL (cmd5/cmd6) primer set was used when this was not the case.
T73397 320902-321098 Sentence denotes Sequences were successfully obtained for all isolates using the Bt2a/Bt2b primers (118/118) and CL1F/2AR primers (84/84), whereas sequences were successful in only 77% (51/66) cases when cmd5/cmd6
T4302 321099-321174 Sentence denotes The Journal of Molecular Diagnostics ■ jmd.amjpathol.org primers were used.
T48344 321175-321663 Sentence denotes Sequence alignment of the calmodulin amplicons indicate that differences in the consensus sites used to generate CAL primers and the actual calmodulin sequence of these species account for the disparate success rates for -TUB, CL1F/2AR primer sets and phenotype data, species in Aspergillus section Nigri were able to be identified in 86/146 cases as A. brasiliensis (2), A. japonicas (2), A. luchuensis (1), A. neoniger (1), A. niger (24), A. tubingensis (37), and A. welwitschiae (19) .
T27947 321664-321718 Sentence denotes Sixty isolates could not be differentiated to species.
T19465 321719-321731 Sentence denotes Conclusions:
T49590 321732-321997 Sentence denotes Whereas -TUB had a high success rate in producing sequence data, -TUB alone lacks sufficient phylogenetic information to fully resolve section Nigri, highlighting the importance of using multiple genetic regions to make species level identification for these fungi.
T92214 321998-322144 Sentence denotes The CAL cmd5/6 primer was shown to be a useful marker to identify many Aspergillus spp. in other sections but with a relative higher failure rate.
T84722 322145-322413 Sentence denotes In contrast, the CAL CL1F/2AR was found to be the superior marker for generating sequence data used to resolve the isolates to the species level in Aspergillus section Nigri. , also known as yeast infection, accounts for about a quarter of reported cases of vaginitis.
T79614 322414-322584 Sentence denotes Whereas CV is typically treated with standard antifungal azole treatments, some C. glabrata infections are resistant to azoles and are instead treated with polyene drugs.
T52406 322585-322659 Sentence denotes Accurate diagnosis of CV infections therefore improves treatment outcomes.
T66556 322660-322887 Sentence denotes This study evaluated the clinical and analytical performance of a research use real-time transcription-mediated amplification (TMA) test for the qualitative detection of 5 common Candida species on the automated Panther System.
T96035 322888-322896 Sentence denotes Methods:
T14781 322897-323031 Sentence denotes Vaginal swab specimens were collected from 362 women symptomatic for vaginitis or vaginosis at 11 clinical sites in the United States.
T5002 323032-323213 Sentence denotes The samples were tested with a research use real-time TMA assay for 5 Candida species (Candida group: C. albicans, C. parapsilosis, C. tropicalis, C. dubliniensis; and C. glabrata).
T12112 323214-323277 Sentence denotes Test results were compared to blood agar and Chromagar results.
T57079 323278-323393 Sentence denotes Subjects from whom specimens yielded a blood agar score of 3+ or 4+ were considered positive for Candida vaginitis.
T92238 323394-323441 Sentence denotes Speciation was determined by Chromagar results.
T95910 323442-323515 Sentence denotes The results were also compared to BD Affirm assay results when available.
T14048 323516-323524 Sentence denotes Results:
T28363 323525-323703 Sentence denotes Compared to the culture reference standard, the CV TMA assay had 97.5% sensitivity and 91.9% specificity, whereas the BD Affirm assay had 71.4% sensitivity and 97.5% specificity.
T21674 323704-323884 Sentence denotes The CV assay had 77% positive predicted value and 99.2% negative predicted value, whereas the BD Affirm assay had 88.7% positive predicted value and 92.4% negative predicted value.
T80182 323885-323950 Sentence denotes Analytical sensitivity of the CV TMA assay (in CFU/mL) was C.alb:
T30106 323951-323963 Sentence denotes 1521, C.dub:
T79462 323964-323976 Sentence denotes 2085, C.par:
T53102 323977-323989 Sentence denotes 1786, C.tro:
T45278 323990-324001 Sentence denotes 387, C.gla:
T24627 324002-324005 Sentence denotes 56.
T5622 324006-324090 Sentence denotes Testing with 44 non-candida microorganisms showed no cross-reactivity or inhibition.
T21291 324091-324103 Sentence denotes Conclusions:
T28824 324104-324317 Sentence denotes The Candida real-time RUO TMA assay showed good agreement with traditional culture methods in clinical sample testing and has been demonstrated to be both sensitive and specific for detection of Candida vaginitis.
T34085 324318-324378 Sentence denotes To address this, reagents have been developed and evaluated.
T30203 324379-324509 Sentence denotes DiaSorin Molecular's ZIKV Primer Pairs are designed for real-time PCR amplification and detection of conserved NS-1 and ENV genes.
T65122 324510-324659 Sentence denotes This study was done to determine the crossreactivity, analytical reactivity and ZIKV primer pair's ability to multiplex with Chikungunya Primer Pair.
T20086 324660-324668 Sentence denotes Methods:
T92748 324669-324759 Sentence denotes Each ZIKV Primer Pair contains forward primer, reverse primer, and a CFR610 labeled probe.
T20938 324760-324847 Sentence denotes The CHIKV Primer Pair contains forward primer, reverse primer, and a FAM labeled probe.
T1107 324848-324921 Sentence denotes A panel of 46 bacteriae/ viruses was tested to evaluate cross reactivity.
T89256 324922-325007 Sentence denotes Five ZIKV strains (Asian/ African lineages) were evaluated for analytical reactivity.
T21991 325008-325084 Sentence denotes A set of 40 ZIKV contrived and blinded plasma and urine samples were tested.
T47085 325085-325215 Sentence denotes Samples were extracted on the Roche MagNA Pure LC, using the Total Nucleic Acid Isolation Kit and tested in a real-time PCR assay.
T8925 325216-325224 Sentence denotes Results:
T80362 325225-325323 Sentence denotes No cross-reactivity was detected when closely related flaviviruses or other pathogens were tested.
T93937 325324-325445 Sentence denotes Analytical reactivity studies demonstrated that the ZIKV primer pairs detect both Asian and African lineages efficiently.
T81031 325446-325548 Sentence denotes The primer pairs correctly identified 40 /40 contrived samples including Martinique and MR766 strains.
T68288 325549-325648 Sentence denotes The ZIKV/CHIKV multiplex demonstrated 100% correlation compared to CHIKV primers run as singleplex.
T54079 325649-325661 Sentence denotes Conclusions:
T25312 325662-325798 Sentence denotes DiaSorin Molecular has developed two primer pairs for real-time PCR amplification and detection of the conserved ZIKV NS1 and ENV genes.
T8776 325799-325921 Sentence denotes Specificity studies demonstrate that this primer pair does not cross-react with other pathogens found in plasma and urine.
T32955 325922-326000 Sentence denotes Analytical reactivity studies demonstrate detection of known lineages of ZIKV.
T11216 326001-326080 Sentence denotes Both ZIKV Primer Pairs are compatible for multiplexing with CHIKV primer pairs.
T28409 326081-326164 Sentence denotes These analyte specific reagents should be compatible with all modern thermocyclers.
T50500 326165-326178 Sentence denotes Introduction:
T99104 326179-326270 Sentence denotes Treatment and management of HIV-1 infection has improved dramatically in the recent decade.
T6927 326271-326431 Sentence denotes With more people utilizing antiretroviral treatment options, and with markedly longer life spans, the importance of infection monitoring has risen dramatically.
T48563 326432-326607 Sentence denotes Options for the quantitative monitoring of HIV-1 infection must be sensitive, inclusive of many genotypes, and easy to introduce in the work flow of the diagnostic laboratory.
T77244 326608-326789 Sentence denotes The Hologic Aptima HIV-1 Quant Dx assay (Aptima), was compared to the Abbott RealTime HIV-1 assay (RealTime) for the ability to sensitively detect HIV-1 in human clinical specimens.
T7680 326790-326798 Sentence denotes Methods:
T20761 326799-326911 Sentence denotes Clinical specimens from HIV-1 infected individuals were collected in EDTA-plasma collection tubes and processed.
T68674 326912-327101 Sentence denotes Plasma specimens representing a broad range of HIV-1 RNA copy numbers (10e1-10e6), both frozen and non-frozen, were tested by RealTime and Aptima according to the manufacturer's procedures.
T93558 327102-327302 Sentence denotes Commercial panels, including the Acrometrix HIV-1 copies/ml, the Seracare Worldwide HIV and the WHO 3 rd HIV-1 IS were also analyzed on both platforms to assess accuracy and inclusivity, respectively.
T63142 327303-327438 Sentence denotes Acute HIV-1 specimens, found to contain HIV-1 RNA in the absence of HIV-1 antibody were also assessed quantitatively on both platforms.
T68894 327439-327447 Sentence denotes Results:
T71014 327448-327632 Sentence denotes One hundred and fifty-five (155) samples were tested by both methods, showing an overall agreement of 80.0% (124/155) with a Kappa statistic of 0.628 (SE 0.052; 95% CI 0.527 to 0.730).
T89047 327633-327667 Sentence denotes With regard to discordant samples:
T81579 327668-327733 Sentence denotes 12 Samples <30 copies/mL by Aptima were not detected by RealTime.
T32962 327734-327825 Sentence denotes Eight (8) samples that were detected <40 copies/mL by RealTime were not detected by Aptima.
T19112 327826-327899 Sentence denotes Ten (10) samples quantified by RealTime that were Detected <30 by Aptima.
T84274 327900-328072 Sentence denotes In an analysis of 92 clinical samples with quantitative results in both assays, the slope 95% CI was equal to 0.9921 to 1.076 with an intercept 95% CI of -0.1700 to 0.2023.
T98056 328073-328229 Sentence denotes All subtypes tested (A, B, C, D, F, G, H, CRF01-AE and CRF01-AG) from a panel of the most prevalent subtypes worldwide were accurately quantified by Aptima.
T45213 328230-328442 Sentence denotes Workflow evaluations comparing the Aptima assay on Panther to the RealTime assay on m2000 revealed that Aptima was measurably easier to use, and required substantially less hands-on time than the compared system.
T21764 328443-328455 Sentence denotes Conclusions:
T59197 328456-328554 Sentence denotes The Aptima assay is capable of accurately detecting and quantifying HIV-1 RNA in clinical samples.
T95908 328555-328635 Sentence denotes The test generates results highly comparable to an existing FDA approved system.
T35320 328636-328788 Sentence denotes Aptima is capable of accurately detecting and quantifying HIV-1 RNA from a wide variety of subtypes, and is easier to use than existing cleared methods.
T36188 328789-328949 Sentence denotes The system appears to be highly effective for use in the assessment of viral loads in HIV-1 patients, as it establishes more facile workflows for laboratorians.
T32973 328950-328954 Sentence denotes K.B.
T38481 328955-328968 Sentence denotes Pierce 1 , A.
T45078 328969-328982 Sentence denotes Hopper 2 , S.
T54274 328983-328994 Sentence denotes Holt 2 , A.
T93482 328995-329010 Sentence denotes Blaschke 1 , K.
T83806 329011-329024 Sentence denotes Ampofo 1 , K.
T77900 329025-329041 Sentence denotes Korgenski 2 , A.
T15459 329042-329057 Sentence denotes Phillips 2 , M.
T99463 329058-329071 Sentence denotes Dickey 2 , R.
T71755 329072-329087 Sentence denotes GrandPre 2 , J.
T35888 329088-329187 Sentence denotes Daly 2 1 University of Utah, Salt Lake City, UT; 2 Primary Children's Hospital, Salt Lake City, UT.
T66383 329188-329201 Sentence denotes Introduction:
T13739 329202-329284 Sentence denotes Pharyngitis is one of the most common reasons for visits to health care providers.
T29296 329285-329541 Sentence denotes Rapid, accurate identification of the presence or absence of Group A Streptococcus (GAS) as a cause of pharyngitis is important for both appropriate treatment when it is present, and to reduce inappropriate use of antibiotics in cases of viral pharyngitis.
T463 329542-329800 Sentence denotes Groups G (GGS) and C Strep (GCS), though part of normal human flora, are also know to cause pharyngitis and invasive disease, particularly in the immune compromised, and the presence of either entity warrants antibiotic treatment in the presence of symptoms.
T99858 329801-329951 Sentence denotes Microbiologic culture remains the gold standard for diagnosis of strep pharyngitis, but takes 24 hours to 48 hours to yield definitive identification.
T87517 329952-330020 Sentence denotes New molecular tests are challenging culture's efficacy in this role.
T66699 330021-330359 Sentence denotes The Solana Strep Complete Assay (Quidel Corporation, San Diego, CA) is a rapid molecular test designed to identify the presence or absence of GAS and GCS/GGS in throat swabs from symptomatic patients via qualitative isothermal helicase-dependent amplification (HDA) endpoint detection by fluorescent probe within one hour of swab receipt:
T90839 330360-330368 Sentence denotes Methods:
T38701 330369-330607 Sentence denotes Throat swabs obtained for GAS and GGS/GCS testing at Primary Children's Hospital (PCH) in Salt Lake City, Utah per standard of care and at physician discretion over the time period of April through June of 2016 were included in the study.
T23631 330608-330642 Sentence denotes Data collection is still underway.
T92516 330643-330760 Sentence denotes Each throat swab was first plated using PCH standard procedures and then tested with the Solana Strep Complete Assay.
T21583 330761-330935 Sentence denotes Pledgets from culture swab transport tubes were forwarded to Quidel for culture-based testing for GAS and GGS/GCS and for testing with an alternative nucleic acid test (PCR).
T84320 330936-331008 Sentence denotes PCH and Quidel culture outcomes determined the consensus culture result.
T16627 331009-331094 Sentence denotes PCR testing was used for discrepancy resolution between consensus culture and Solana.
T97634 331095-331103 Sentence denotes Results:
T31608 331104-331183 Sentence denotes To date, a total of 176 swabs were tested by both Solana and consensus culture.
T54021 331184-331243 Sentence denotes Forty-eight (27%) were culture positive for GAS or GGS/GCS.
T28239 331244-331295 Sentence denotes Of these, 42 (24%) were positive on Solana testing.
T26419 331296-331367 Sentence denotes Of 128 (73%) culture-negative swabs, 119 (68%) were negative by Solana.
T84307 331368-331491 Sentence denotes In 4 cases where culture was negative and Solana positive, PCR showed the presence of GAS or GGS/GCS DNA within the sample.
T7568 331492-331598 Sentence denotes In 6 culture positive cases where Solana was negative, PCR identified no GAS or GGS/GCS DNA in the sample.
T27858 331599-331761 Sentence denotes After discrepancy resolution, sensitivity, specificity, accuracy, PPV and NPV of the Solana Strep Complete Assay were, respectively, 100%, 96%, 97%, 90% and 100%.
T55863 331762-331774 Sentence denotes Conclusions:
T68514 331775-331937 Sentence denotes Use of this rapid method for identification of GAS and GGS/GCS in clinical samples has the potential to refine current methods of diagnosis for strep pharyngitis.
T38124 331938-331940 Sentence denotes S.
T31174 331941-331950 Sentence denotes Das, K.A.
T61392 331951-331962 Sentence denotes Mangold, J.
T60487 331963-331975 Sentence denotes Behles, K.L.
T17080 331976-331986 Sentence denotes Kaul, R.B.
T50109 331987-332044 Sentence denotes Thomson NorthShore University HealthSystem, Evanston, IL.
T8213 332045-332058 Sentence denotes Introduction:
T94928 332059-332170 Sentence denotes Identification of mycobacteria by traditional biochemical and phenotypic characteristics is slow and laborious.
T17457 332171-332297 Sentence denotes Additionally, with over 150 species of atypical mycobacteria, accurate phenotypic identification is also difficult to achieve.
T12392 332298-332434 Sentence denotes Molecular methods are often sought as they are rapid, can impact patient care, and provide reliable identification to the species level.
T84983 332435-332576 Sentence denotes We instituted a rapid molecular method for identification of mycobacteria directly from Mycobacterial Growth Indicator Tube (MGIT) specimens.
T65761 332577-332711 Sentence denotes We analyzed the performance of this assay in comparison to other techniques available for the identification of mycobacterial species.
T26753 332712-332720 Sentence denotes Methods:
T78759 332721-332839 Sentence denotes A PCR-melt curve analysis (melt) method has been validated in our laboratory for rapid identification of mycobacteria.
T61641 332840-332970 Sentence denotes This realtime assay uses FRET probes targeting the hsp65 sequence region and can be performed from both MGIT tube and solid media.
T7670 332971-333085 Sentence denotes The assay is currently used as an initial identification method as soon as growth becomes evident in a MGIT broth.
T6610 333086-333199 Sentence denotes Confirmation of melt results if warranted is usually achieved by additional tests either alone or in combination.
T45874 333200-333383 Sentence denotes These include phenotypic characteristics such as growth rate, DNA probes, 16S rRNA or rpoB gene sequencing, and matrix assisted laser desorption and ionization-time of flight (MALDI).
T25172 333384-333531 Sentence denotes Herein, we review identification results of positive cultures for mycobacteria over a 2-year period to determine the performance of the melt assay.
T82606 333532-333644 Sentence denotes In a subset of isolates, we also compare results of species identification obtained using the different methods.
T15861 333645-333653 Sentence denotes Results:
T43437 333654-333703 Sentence denotes A total of 395 positive cultures were identified.
T17810 333704-333961 Sentence denotes In 89.6% of the isolates (including M. gordonae, Mycobacterium avium complex, M. chelonae, M. fortuitum and M. abscessus), melt curve results provided an initial identification that matched the phenotypic characteristics or results from DNA probes or MALDI.
T99003 333962-334080 Sentence denotes We compared identification in a subset of 30 positive cultures where an alternate identification method was performed.
T4977 334081-334404 Sentence denotes The melt results agreed with the alternate method in 15 cultures, but failed (n=1), or was indeterminate (n=1) in two isolates, Indeterminate melt curves obtained in 11 isolates were identified as new species not included in the analysis scheme (e.g., M. neoaurum, M. parafinicum, M. elephantis, M. yongonense, M. celatum).
T62826 334405-334417 Sentence denotes Conclusions:
T79745 334418-334605 Sentence denotes The real-time PCR-melt curve analysis can be performed from MGIT broth immediately after growth becomes evident as opposed to the MALDI or sequencing which requires growth on solid media.
T29855 334606-334814 Sentence denotes Although our study includes limited number of isolates with alternate identification, the melt assay appears to be rapid and reliable for the initial identification of commonly isolated mycobacterial species.
T31804 334815-334817 Sentence denotes Y.
T26049 334818-334826 Sentence denotes Chen, D.
T61124 334827-334865 Sentence denotes Hsia Danner Laboratory, San Diego, CA.
T1680 334866-334879 Sentence denotes Introduction:
T62939 334880-334933 Sentence denotes M genitalium was first identified in the early 1980s.
T27971 334934-335158 Sentence denotes According to 2010 study by University of Washington, the prevalence of M genitalium for combined men and women was 1.0% compared with 0.4%, 4.2%, and 2.3% for gonococcal, chlamydial, and trichomonal infections, respectively.
T26519 335159-335345 Sentence denotes For men, epidemiology studies indicated M genitalium responsible for up to 45% of nongonococcal and nonchlamydial urethritis, and approximately 30% of cases were persistent or recurrent.
T89616 335346-335422 Sentence denotes For women, the organism can be found in the vagina, cervix, and endometrium.
T80397 335423-335532 Sentence denotes It could be attributable for 10%-30% cases of women with clinical cervicitis and 2%-22% cases of PID (median:
T42594 335533-335563 Sentence denotes 10%) depending on the setting.
T38230 335564-335684 Sentence denotes M genitalium diagnostic specimens can be obtained from both male and female urine as well as cervical and vaginal swabs.
T45424 335685-335845 Sentence denotes Genetic testing can speed up diagnosis and improve treatment decisions since it is a slow-growing organism which can take up to 6 months to grow in the culture.
T71578 335846-336024 Sentence denotes Danner Laboratory has validated a Transcription-Mediated Amplification (TMA) based assay using the automated Panther System enabling a fast turnaround for M genitalium diagnosis.
T5742 336025-336033 Sentence denotes Methods:
T43626 336034-336220 Sentence denotes A batch of 92 urine and vaginal swab samples, 62 positive and 30 negative of M genitalium, was kindly provided by Springfield-Greene County Health Department and used for the validation.
T98474 336221-336341 Sentence denotes The samples were collected in tubes with a solution which released RNA and protected it from degradation during storage.
T49860 336342-336571 Sentence denotes The assay was performed with the Panther System in three major steps which all take place in one single tube: capturing the targeted rRNA, target amplification by TMA, and RNA amplicon detection by hybridization protection assay.
T59598 336572-336692 Sentence denotes In addition, analytical performance of the assay including sensitivity, specificity, and reproducibility were evaluated.
T59871 336693-336701 Sentence denotes Results:
T85357 336702-336781 Sentence denotes It has been determined that the assay has 94% sensitivity and 100% specificity.
T40542 336782-336875 Sentence denotes The inter-assay and intra-assay variability were determined as 1% and 1.2 % CV, respectively.
T11492 336876-336970 Sentence denotes The assay was specific and did not cross react with any of the common genital tract pathogens.
T3136 336971-337030 Sentence denotes Limit of detection (LOD) of the assay was at 278 copies/mL.
T2767 337031-337043 Sentence denotes Conclusions:
T8014 337044-337132 Sentence denotes The assay was validated using a panel of characterized urine and vaginal swab specimens.
T68557 337133-337222 Sentence denotes It shows excellent clinical and analytical sensitivity, specificity, and reproducibility.
T8221 337223-337342 Sentence denotes In addition, the automated Panther System was capable of accomplishing the tests in 3 hours with minimal hands on time.
T65891 337343-337449 Sentence denotes Therefore, it is an ideal assay to replace the traditional M genitalium detection by culturing techniques.
T68868 337450-337452 Sentence denotes A.
T24213 337453-337466 Sentence denotes Greninger, C.
T67366 337467-337479 Sentence denotes Johnston, D.
T64012 337480-337490 Sentence denotes Koelle, M.
T90987 337491-337500 Sentence denotes Huang, K.
T75904 337501-337546 Sentence denotes Jerome University of Washington, Seattle, WA.
T46236 337547-337560 Sentence denotes Introduction:
T72710 337561-337719 Sentence denotes Herpes simplex virus 2 (HSV2) is the most common cause of genital herpes, causing 417 million infections worldwide among adults aged 15 years to 49 years old.
T96303 337720-337929 Sentence denotes Phylogenomic analysis of viral infections is a powerful technique for understanding transmission of the virus and detecting variants that may be associated with clinical phenotypes, i.e., antiviral resistance.
T39528 337930-338109 Sentence denotes HSV2 genomics is challenging due to its incredibly high GC% (70%), presence of multiple repeats, and presence at low concentration in a high concentration of human DNA background.
T84315 338110-338118 Sentence denotes Methods:
T1955 338119-338172 Sentence denotes We developed a tiling IDT xGen capture panel to HSV2.
T19417 338173-338364 Sentence denotes DNA libraries were prepared using NEB fragmentase, end-repair/dA tailing, barcoded ligation protocol and pooled 8-ways prior to capture based on a relative HSV2 to human beta-globin DNA qPCR.
T45761 338365-338456 Sentence denotes Libraries were sequenced on an Illumina MiSeq to achieve ~100X coverage of the HSV2 genome.
T91249 338457-338505 Sentence denotes Reads were aligned using Geneious v9.1 software.
T82783 338506-338514 Sentence denotes Results:
T87416 338515-338621 Sentence denotes Approximately 130 genomes from HSV2 strains from clinical swabs and cultured isolates have been recovered.
T29627 338622-338824 Sentence denotes Capture sequencing allowed for the recovery of whole genomes from clinical swabs with concentrations below 10^3 copies per mL, an increased level of detection of 4 to 5 logs beyond shotgun metagenomics.
T52809 338825-339022 Sentence denotes Comparison of sequences between genomes recovered from clinical swabs versus 1X cultured passage virus often revealed no mutational changes, consistent with the slow molecular clock of herpesvirus.
T76592 339023-339103 Sentence denotes The HSV2 panel recovered for approximately 30% of the genome from HSV1 isolates.
T43273 339104-339270 Sentence denotes Strikingly, multiple clinical swabs demonstrated recombination between HSV1 and HSV2 sequence in drug targets such as the ribonucleotide reductase and DNA polymerase.
T82147 339271-339283 Sentence denotes Conclusions:
T88130 339284-339384 Sentence denotes We demonstrate rapid and cost-effective recovery of HSV2 whole genomes direct from clinical samples.
T34341 339385-339526 Sentence denotes These sequences demonstrate a complex genomic relationship between HSV1 and HSV2 sequences that require further biochemical characterization.
T51233 339527-339529 Sentence denotes E.
T99111 339530-339549 Sentence denotes Rakhmanaliev 1 , P.
T73621 339550-339567 Sentence denotes Ariyaratne 1 , A.
T1265 339568-339578 Sentence denotes Yeo 1 , C.
T71492 339579-339589 Sentence denotes Lee 1 , P.
T43138 339590-339611 Sentence denotes Nimitsuntiwong 2 , C.
T25049 339612-339628 Sentence denotes Wathtphan 2 , Z.
T37595 339629-339639 Sentence denotes Rui 1 , E.
T29333 339640-339656 Sentence denotes Passomsub 2 , W.
T60467 339657-339675 Sentence denotes Chantratita 2 , G.
T75867 339676-339715 Sentence denotes Michel 1 1 Vela Research Singapore Pte.
T2090 339716-339794 Sentence denotes Ltd., Singapore; 2 Ramathibodi Hospital Mahidol University, Bangkok, Thailand.
T74711 339795-339808 Sentence denotes Introduction:
T59316 339809-339959 Sentence denotes Resistance of HIV to antiretroviral drugs is the most common cause for therapeutic failure in people infected with Human Immunodeficiency Virus (HIV).
T1786 339960-340203 Sentence denotes Objective of this study was to compare 2 sequencing-based HIV-1 drug resistance monitoring systems: a CLIP-based system (TruGene HIV-1 Genotyping Kit) and a novel next-generation sequencing (NGS)-based test (Sentosa SQ HIV-1 Genotyping Assay).
T68646 340204-340212 Sentence denotes Methods:
T86638 340213-340544 Sentence denotes We used an automated NGS-based integrated workflow, comprised of 1) a robotic liquid handling system for nucleic acid extraction and NGS library preparation (Sentosa SX101); 2) instruments for deep sequencing; 3) kits for RNA extraction, HIV NGS library preparation and deep sequencing, and 4) data analysis and reporting software.
T93511 340545-340673 Sentence denotes Reporting includes 86 Drug Resistance Mutations (DRMs) across the Reverse Transcriptase (RT), Protease (PR) and Integrase genes.
T69591 340674-340765 Sentence denotes 111 prospective plasma samples from patents infected with HIV-1 were tested for this study.
T91219 340766-340774 Sentence denotes Results:
T66195 340775-340815 Sentence denotes All samples were tested on both systems.
T11053 340816-340866 Sentence denotes 97.3% (108/111) samples were subtyped as CRF01_AE.
T71007 340867-340961 Sentence denotes In total, 647 DRMs were detected (435 in the RT, 199 in the PR and 13 in the Integrase genes).
T89873 340962-341031 Sentence denotes The Sentosa HIV Assay detected 100% (199/199) and L10I 20.7%(23/111).
T28588 341032-341173 Sentence denotes Although DRMs are reported by the system the software does not provide interpretation of the data with regard to usage in patient management.
T42337 341174-341186 Sentence denotes Conclusions:
T88621 341187-341315 Sentence denotes Detection and reporting of DRMs is critical for drug regiment and can minimize the development of resistance to antiviral drugs.
T55957 341316-341586 Sentence denotes High sensitivity (up to 5% mutation frequency) and the comparatively The Journal of Molecular Diagnostics ■ jmd.amjpathol.org short turnaround time of 2.5 days make this NGS-based workflow a promising new tool for detecting relevant mutations in HIV-1 treatment targets.
T55792 341587-341600 Sentence denotes Introduction:
T68306 341601-341820 Sentence denotes Metagenomic next-generation sequencing (mNGS) for pan-pathogen detection has been successfully tested in proof of concept studies to aid in difficult diagnostic cases, but is not available in a clinical setting to date.
T10963 341821-342003 Sentence denotes The UCSF Clinical Microbiology Lab has developed and validated a clinical mNGS assay for diagnosis of infectious causes of meningitis and encephalitis from cerebrospinal fluid (CSF).
T28779 342004-342101 Sentence denotes We evaluated performance characteristics of the assay using contrived and excess patient samples.
T37692 342102-342110 Sentence denotes Methods:
T96135 342111-342271 Sentence denotes Two libraries per sample were constructed from DNA and RNA portions of extract and prepared using Nextera XT, with 5-20 million sequences generated per library.
T68573 342272-342367 Sentence denotes Internal controls consisting of DNA and RNA phage particles were spiked in prior to extraction.
T52753 342368-342537 Sentence denotes An external positive control made of 7 representative organisms (DNA virus, RNA virus, Gram-negative rod, Gram-positive coccus, yeast, mold, and parasite) was developed.
T55789 342538-342625 Sentence denotes Positive control dilutions in synthetic CSF were used to determine limits of detection.
T42050 342626-342770 Sentence denotes To document the effects of inhibition, hemolyzed RBC and human DNA and RNA were added prior to extraction to the positive control and sequenced.
T56947 342771-342832 Sentence denotes The accuracy study included 105 CSF samples (known positives:
T45513 342833-342902 Sentence denotes 28 DNA viral, 11 RNA viral, 5 bacterial, 14 fungal, and 1 parasitic).
T73676 342903-343086 Sentence denotes For bioinformatics analysis, the SURPI pipeline for pan-pathogen detection from mNGS data was optimized to report positives within established cutoffs after normalization (SURPIclin).
T70483 343087-343211 Sentence denotes An algorithm for automatic reporting of detected pathogens was developed with final interpretation by laboratory physicians.
T67369 343212-343451 Sentence denotes Discrepancy testing was conducted when possible using additional orthogonal molecular tests when mNGS detected organisms not routinely tested for by reference methods, and to establish the presence or absence of microorganism nucleic acid.
T7143 343452-343460 Sentence denotes Results:
T73216 343461-343601 Sentence denotes We established quality metrics, threshold values, and limits of detection of between 0.01-130 genomic copies or colony forming units per mL.
T97938 343602-343766 Sentence denotes Gross hemolysis and high white blood cell counts reduced assay sensitivity; however, the phage-spiked internal control was a reliable indicator of sensitivity loss.
T6229 343767-343861 Sentence denotes The assay exhibited 76.4% sensitivity and 98.3% specificity compared to reference lab results.
T23584 343862-344029 Sentence denotes 27/87 (31%) of organisms undetected by mNGS were rare isolates by culture or Ct >37 by qPCR; when removed from the accuracy study, assay sensitivity improved to 90.9%.
T32471 344030-344042 Sentence denotes Conclusions:
T48599 344043-344194 Sentence denotes A mNGS assay for pan-pathogen detection in the context of samples from patients with encephalitis or meningitis was developed and clinically validated.
T31791 344195-344244 Sentence denotes Performance characteristics are highly promising.
T50671 344245-344385 Sentence denotes Clinical utility is being evaluated in a 300-patient prospective study supported by the California Initiative to Advance Precision Medicine.
T63348 344386-344399 Sentence denotes Introduction:
T39524 344400-344502 Sentence denotes Viral respiratory infections are a significant cause of morbidity and mortality during the flu season.
T18137 344503-344692 Sentence denotes Rapid virus identification provides critical information for the treatment, management and isolation of infected patients, and can offer substantial economic savings to health care systems.
T75037 344693-344891 Sentence denotes Previously we modeled various strategies of implementing rapid viral detection in our health care system and calculated the predicted effects on laboratory workflow, turn-around time (TAT) and cost.
T67111 344892-345092 Sentence denotes Here we review the observed impact on these laboratory metrics following the implementation of a rapid molecular viral detection assay as the initial testing modality for selected patient populations.
T65745 345093-345101 Sentence denotes Methods:
T75015 345102-345385 Sentence denotes During the 2015 to 2016 flu season, rapid virus identification for influenza A/B and respiratory syncytial virus (RSV) was performed using the Cepheid Xpert Flu/RSV XC assay for patient samples originating from the emergency department (ED) and the perinatal evaluation center (PEC).
T15473 345386-345558 Sentence denotes Rapid test negative/inconclusive samples were reflexed to our laboratory developed, real-time PCR based nine analyte respiratory virus panel (RVP) for confirmatory testing.
T5264 345559-345647 Sentence denotes For RVP reflexed specimens, concordance rates between the two platforms were calculated.
T68143 345648-345742 Sentence denotes The effects of rapid viral identification on laboratory workflow, TAT and cost were evaluated.
T89160 345743-345751 Sentence denotes Results:
T62091 345752-345924 Sentence denotes Over 700 respiratory specimens were analyzed using the rapid molecular assay, accounting for one fifth of all specimens requiring viral testing during this past flu season.
T4958 345925-346035 Sentence denotes One fourth (26%) of samples tested by the rapid molecular assay were positive for either influenza A/B or RSV.
T76217 346036-346155 Sentence denotes Compared with the RVP assay, the Xpert had a sensitivity of 99% for influenza A, 100% for influenza B, and 90% for RSV.
T36441 346156-346264 Sentence denotes Discordant samples displayed a high Ct value on the RVP assay, suggesting a low viral load in these samples.
T50942 346265-346369 Sentence denotes Laboratory costs increased by 1.5 or 2.5 fold for rapid test positive and negative samples respectively.
T11487 346370-346453 Sentence denotes A median reduction in TAT of 20 hours was observed for rapid test positive samples.
T80464 346454-346661 Sentence denotes For rapid test negative samples that underwent additional RVP testing, the final TAT was not statistically different compared to samples that did not undergo rapid testing (unequal variances t-test, P>0.05).
T89965 346662-346674 Sentence denotes Conclusions:
T50980 346675-346769 Sentence denotes The Xpert assay results were highly concordant with our RVP results for influenza A/B and RSV.
T47102 346770-347004 Sentence denotes Furthermore, initial rapid molecular testing resulted in a drastic reduction in TAT for influenza or RSV positive samples, without significantly altering the TAT for rapid testing negative samples that required additional RVP testing.
T99705 347005-347189 Sentence denotes These findings suggest that the implementation of rapid testing improved the management of ED and PEC patients during flu season and may significantly reduce overall health care costs.
T90122 347190-347203 Sentence denotes Introduction:
T55223 347204-347333 Sentence denotes Viral respiratory tract infections are some of the most common illnesses and cause significant morbidity and mortality worldwide.
T40000 347334-347508 Sentence denotes Among noninfluenza respiratory viruses, Human Bocavirus (BVS), Human Metapneumovirus (hMPV), Parainfluenza (PIV), and Respiratory Syncytial Virus (RSV) are the most frequent.
T47438 347509-347654 Sentence denotes Molecular methods have significantly improved the diagnosis of acute respiratory tract infections as they offer high sensitivity and specificity.
T23330 347655-347909 Sentence denotes VIASURE Real-Time Detection kits (CerTest) are novel assays which contain all the necessary components for the PCR in a stabilized format, which allows its shipment and jmd.amjpathol.org ■ The Journal of Molecular Diagnostics storage at room temperature.
T81446 347910-348033 Sentence denotes The aim of this study is to compare Real-time PCR assays and multiplex PCR followed by visualization in low-density arrays.
T65676 348034-348042 Sentence denotes Methods:
T82380 348043-348279 Sentence denotes A prospective comparative study was performed in 320 throat swab samples from patients with clinical suspicion of viral respiratory infections collected at Hospital Clínico Universitario Lozano Blesa (Spain) during three winter seasons:
T47604 348280-348300 Sentence denotes 2014, 2015 and 2016.
T64429 348301-348423 Sentence denotes These samples were tested with VIASURE monoplex assays for the detection of BVS, hMPV, PIV 1, 2, 3 and 4, and RSV A and B.
T39662 348424-348755 Sentence denotes The results were compared with the data obtained by a microarray method, CLART PneumoVir (Genomica), and confirmed by liquid-format commercial Real-Time PCR kits: "RealStar RSV RT-PCR Kit" and "RealStar hMPV RT-PCR Kit" (Altona Diagnostics), "RIDA GENE Parainfluenza" (R-biopharm) and "FTD HAdV/HMPV/HBoV" (Fast Track Diagnostics).
T23675 348756-348764 Sentence denotes Results:
T13427 348765-348848 Sentence denotes Two hundred and eighty samples were positive for some of these viruses by Genomica:
T86291 348849-349019 Sentence denotes 36 for BVS, 50 for hMPV (A and B), 8 for PIV1, 3 for PIV2, 41 for PIV3, 19 for PIV4, 61 for RSVA and 62 for RSVB; and 40 samples were negative for all of these pathogens.
T21100 349020-349099 Sentence denotes These samples were tested using the pertinent VIASURE monoplex assay obtaining:
T11068 349100-349305 Sentence denotes 28 positive samples for BVS by VIASURE out of 35 positive samples for BVS by Genomica (28/36), 45/50 for hMPV, 3/8 for PIV1, 3/3 for PIV2, 34/41 for PIV3, 13/19 for PIV4, 59/61 for RSVA and 59/62 for RSVB.
T26914 349306-349476 Sentence denotes Therefore, the sensitivity reached for VIASURE may be estimated 70%-100% for BVS, hMPV, PIV3, RSVA, and RSVB and <70% for PIV1 and PIV4 (probably due to low sample size).
T54630 349477-349603 Sentence denotes However, the commercial qPCR kits used leading to the similar results and then, showing a 98.2% concordance rate with VIASURE.
T60900 349604-349785 Sentence denotes Conclusions: "VIASURE Real-Time PCR Detection Kits" represent an efficient diagnostic tool for the detection of noninfluenza viruses as they show a high sensitivity and specificity.
T90856 349786-349876 Sentence denotes Moreover, the results obtained by VIASURE were supported by commercial Real-time PCR kits.
T23757 349877-350046 Sentence denotes These data suggest that visualization of PCR in arrays platforms may result in a higher sensibility than fluorescent signal or leads to putative false positives results.
T40160 350047-350060 Sentence denotes Introduction:
T9713 350061-350182 Sentence denotes Genotypic and phenotypic resistance assays are used to assess viral strains and inform selection of treatment strategies.
T22110 350183-350335 Sentence denotes Standard genotypic drugresistance testing in ARV-naive persons involves testing for mutations in the reverse transcriptase (RT) and protease (PR) genes.
T97412 350336-350580 Sentence denotes If transmitted Integrase strand transfer inhibitor (INSTI) resistance is a concern, providers should supplement standard genotypic resistance testing with an INSTI genotype test the same approach is used in persons failing INSTI-based regimens.
T49832 350581-350689 Sentence denotes In this study we evaluate performance of ACL LDT HIV Integrase genotype assay for drug-resistance detection.
T29687 350690-350698 Sentence denotes Methods:
T88565 350699-350851 Sentence denotes Forty HIV positive plasma specimens were tested using ACL LDT HIV Integrase genotype (HIVING) assay and compared to results provided by ARUP Laboratory.
T49193 350852-350882 Sentence denotes Method 1: ACL LDT HIVGT assay:
T2876 350883-351135 Sentence denotes Abbott ViroSeq HIV-1 Integrase Sample Preparation Kit extraction, amplification on ABI 9700, detection by (36 cm) CE Sequencing on ABI 3130xl instrument, data assemble and analysis by ViroSeq analysis software provided by Abbott (last updated in 2011).
T10128 351136-351175 Sentence denotes Method 2: ARUP Laboratories/comparator.
T79742 351176-351498 Sentence denotes HIV Integrase sequencing data of forty (40) clinical samples belonging to a cohort of treatment-experienced patients was generated using three software; RUO ViroSeq HIV Integrase assay (Abbott Molecular, US 2011), RUO ViroScore-HIV system from Advanced Biological Laboratories 2013 and Stanford HIVdb v7.0.1 February 2014.
T57955 351499-351507 Sentence denotes Results:
T14062 351508-351776 Sentence denotes The ACL HIV Integrase assay performance was evaluated by assessing analytical specificity, sensitivity, interfering substances, and accuracy (clinical sensitivity and specificty) based on HIV positive specimens (in plasma, with or without drug resistance mutation(s)).
T84063 351777-352204 Sentence denotes ACL LDT HIVING produced 90% clinical sensitivity (18/19) and specificity (19/21) all samples were genotype M, subtype B (40) with viral loads ranging from (1000 to 150,000 cp/mL), analytical sensitivity 100% at VL=1000 cp/mL for HIV subtypes (B, G, C, CRF01_AE) and per in silico analysis HIV genotypes (D, F, A, CRF02_AG), analytical specificity 100% (11/11) in comparison to potentially cross-reactive microorganisms/viruses.
T69398 352205-352443 Sentence denotes Reproducibility was assessed by testing 20 times positive and negative control (expected result for positive QC were B genotype, amplicon length 864bp and (G123S, A124T, R127K, N232D, A265V) variants detected) all replicates matched 100%.
T77713 352444-352511 Sentence denotes Precision of the assay is 100% (in range 1000 cp/mL of viral load).
T42880 352512-352524 Sentence denotes Conclusions:
T4776 352525-352739 Sentence denotes This study demonstrates that the ACL LDT HIV Integrase sequencing assay is a sensitive and reproducible method for detecting HIV Integrase genotype and determination of drug-resistance status in clinical specimens.
T18300 352740-352969 Sentence denotes Introduction: HPV genotyping is relevant for studying the natural history of HPV infections and the diseases associated therewith, for monitoring vaccine efficacy, and for identifying novel HPV candidates for vaccine development.
T63918 352970-353099 Sentence denotes There is currently a need for a sensitive and specific HPV genotyping assay that is cost effective for testing large sample sets.
T73203 353100-353187 Sentence denotes The HPV prevalence at oral, penile, and anal sites is not well documented in Greek men.
T23171 353188-353316 Sentence denotes We developed a next-generation sequencing (NGS) assay for HPV genotyping, and applied it to test a cohort of Greek male samples.
T55896 353317-353325 Sentence denotes Methods:
T81029 353326-353464 Sentence denotes The assay uses PGMY09/11 primers modified by lllumina adapter and patient-specific index sequences to amplify the HPV L1 consensus region.
T37484 353465-353496 Sentence denotes Input DNA requirement is 10 ng.
T53724 353497-353568 Sentence denotes Up to 200 samples can be sequenced per flow cell on the Illumina MiSeq.
T69211 353569-353851 Sentence denotes HPV genotyping is obtained via a customized bioinformatics pipeline, which removes ambiguous and off-target sequences, aligns the filtered data to 179 HPV genomes from the Papillomavirus Episteme (PaVe) database, and scores each read based on its alignment to a specific HPV genome.
T15108 353852-354186 Sentence denotes A test cohort of 29 samples with previously characterized HPV status by a CLIA-certified multiplex qPCR and linear array, including HPV 16 (n=2), HPV 18 (n=2), HPV 33 (n=1), HPV 39 (n=1), HPV 45 (n=2), and 22 negative high-risk HPV specimens was used to assess the assay performance and to establish HPV positive and negative cutoffs.
T76407 354187-354412 Sentence denotes We then applied this methodology to test 882 samples of oral mucosa, glans penis, and anal mucosa from 294 Greek men with uncharacterized HPV status and genotype that were collected during their routine STD clinic visit at A.
T80434 354413-354448 Sentence denotes Syggros Hospital in Athens, Greece.
T25437 354449-354516 Sentence denotes Forty-three of these samples were genotyped by qPCR for comparison.
T3822 354517-354525 Sentence denotes Results:
T58621 354526-354596 Sentence denotes Concordance between NGS and qPCR was 96.5% (28/29) in the test cohort.
T74151 354597-354686 Sentence denotes One sample was positive only by NGS for HPV 59, which is not targeted by the qPCR method.
T90682 354687-354733 Sentence denotes The discordant sample was p16 positive by IHC.
T31784 354734-354833 Sentence denotes In 49% of Greek subjects, at least one HPV subtype was detected in one or more of the tested sites.
T45929 354834-354896 Sentence denotes HPV 6 was the most commonly found HPV type in this population.
T15737 354897-354987 Sentence denotes Genital warts were reportedly present in 42/53 (79%) of men with an HPV 6 or 11 infection.
T16285 354988-355054 Sentence denotes Correlation of NGS and qPCR for the Greek samples was 91% (40/44).
T88002 355055-355170 Sentence denotes Phylogenetic analysis showed that all HPV types found were found in the Alphapapillomavirus branch of the HPV tree.
T58898 355171-355183 Sentence denotes Conclusions:
T15610 355184-355321 Sentence denotes This NGS assay may be a viable platform for population studies and for routine diagnosis of HPV-associated cancers in a clinical setting.
T41664 355322-355500 Sentence denotes Results of this study could be used to create various intervention programs for Greek men, and to provide justification for expanding the current HPV vaccination schedule to men.
T13969 355501-355719 Sentence denotes Introduction: PCR tests for influenza and respiratory syncytial virus (RSV) must be sensitive and accurate to effectively guide patient care, ideally with the capability to be performed in multiple healthcare settings.
T93514 355720-356034 Sentence denotes We sought to 1) compare the overall sensitivity and specificity of the Cepheid Xpert Flu+RSV Xpress Assay to that of the Prodesse ProFlu+ test, and 2) evaluate concordance of results with those of ProFlu+ when performed by individuals with no formal laboratory training in point of care (CLIA-waived) environments.
T19732 356035-356043 Sentence denotes Methods:
T2596 356044-356439 Sentence denotes Nasopharyngeal (NP) swabs consisting of fresh-prospectively collected samples and pre-selected frozen specimens were obtained and tested from patients with flu-like symptoms presenting to emergency departments (EDs), outpatient (OP) and urgent care clinics (UC), and from hospital inpatients (IP) at 12 clinical sites (10 categorized as CLIA waived) during the 2014 -2015 respiratory flu season.
T11022 356440-356477 Sentence denotes Failed assays were retested one time.
T14668 356478-356612 Sentence denotes Performance of the Xpert Flu+RSV Xpress test was compared to the PCR-based ProFlu+ Assay and percentages of agreement were determined.
T66050 356613-356621 Sentence denotes Results:
T2192 356622-356790 Sentence denotes 2435 NP swabs (2176 fresh, 259 frozen) were obtained from a patient population consisting of 422 (17.3%) < 5 years, 421 (17.3%) 6-21 years, 1302 (53.5%) 22years of age.
T43497 356791-356892 Sentence denotes Samples were collected from 1120 (46%) ED; 565 (23.2%) OP; 688 (28.3%) UC patients and 62 (2.5%) IPs.
T19822 356893-357010 Sentence denotes Xpert Flu+RSV Xpress Assay tests for 94.9% (2329/2453) of specimens were successfully completed on the first attempt.
T61512 357011-357126 Sentence denotes Following a single retesting of 124 failed specimens, an overall 99.3% (2435/2453) assay success rate was achieved.
T21556 357127-357385 Sentence denotes Compared to the ProFlu+ Assay, the Xpert Flu+RSV Xpress Assay demonstrated a high positive, negative, and overall percent agreement, for detection of flu A, flu B and RSV for all samples tested in moderately complex and CLIA-waived settings (Tables 1 and 2).
T1170 357386-357505 Sentence denotes Introduction: K. kingae is a fastidious Gram-negative bacillus that causes bone and joint infections in young children.
T43842 357506-357575 Sentence denotes It can also cause bacteremia and endocarditis in adults and children.
T53387 357576-357680 Sentence denotes The primary method for diagnosing K. kingae infections is culture, which is, unfortunately, insensitive.
T61131 357681-357868 Sentence denotes We designed a LightCycler real-time PCR assay to detect K. kingae in blood and synovial fluid which can potentially be used to improve the diagnosis of infections caused by this organism.
T90931 357869-357877 Sentence denotes Methods:
T26384 357878-357984 Sentence denotes The K. kingae PCR assay amplifies a 156 bp portion of rtxB, a single-copy gene within the RTX toxin locus.
T94755 357985-358211 Sentence denotes The assay consists of a single forward primer, two internal FRET hybridization probes, one labeled with fluorescein and the other with LC Red 610, and a single reverse primer and is performed on the LightCycler 2.0 instrument.
T63327 358212-358321 Sentence denotes A plasmid containing the amplified sequence was created and used to determine the assay's limit of detection.
T40716 358322-358458 Sentence denotes Genomic material from a library of bacterial organisms, including non-kingae, Kingella species, was used to assess for cross reactivity.
T14991 358459-358632 Sentence denotes Thirty EDTA blood specimens and 30 synovial fluid specimens were spiked with a reference strain of K. kingae (DSM 7536) to a final concentration of approximately 130 CFU/μL.
T24211 358633-358751 Sentence denotes The spiked specimens were extracted and assayed along with 10 negative blood and 10 negative synovial fluid specimens.
T72552 358752-358760 Sentence denotes Results:
T34449 358761-358975 Sentence denotes Gel analysis of the DNA amplified from genomic DNA of K. kingae produced a single band at the appropriate size, indicating the desired target had been amplified; sequencing confirmed amplification of the rtxB gene.
T20756 358976-359088 Sentence denotes The limit of detection was 1 target copy/μl, as confirmed in 6 out of 6 replicates tested at this concentration.
T99146 359089-359246 Sentence denotes Cross reactivity studies were negative, which included Moraxella bovis which contains an RTX toxin locus with homology to the RTX locus present in K. kingae.
T29133 359247-359413 Sentence denotes The molecular assay detected K. kingae in all spiked blood and synovial fluids and yielded no false positive results in the negative blood and synovial fluids tested.
T89935 359414-359560 Sentence denotes We developed a highly sensitive and specific assay with the potential to improve the detection of K. kingae in blood and synovial fluid specimens.
T71482 359561-359812 Sentence denotes Future studies are needed on clinical specimens containing K. kingae. -lactams are the antibiotics most widely used all over the world, have also given rise to a continuous increase of resistance and driven diversification of the resistance --lactams.
T75692 359813-360027 Sentence denotes The extended--lactamases, such as CTX-M and SHV, hydrolyze cephalosporins and monobactams; whereas K. pneumoniae carbapenemases -lactamase genes reside in plasmids, transferring between different stains or species.
T70630 360028-360036 Sentence denotes Methods:
T45336 360037-360235 Sentence denotes Routine clinical MicroScan and Etest methods were used to profile the susceptibility of antimicrobial resistance in K. pneumoniae clinical isolates recovered from patient specimens during 2005-2014.
T60788 360236-360346 Sentence denotes Additional PCR amplification followed by Sanger sequencing was used for gene detection of blaCTX-M and blaKPC.
T49644 360347-360473 Sentence denotes A combination of short-and long-read whole genome sequencing was used for three isolates positive to both blaCTX-M and blaKPC.
T95078 360474-360599 Sentence denotes Comparative genomic analyses were further conducted to characterize detailed resistance gene structures and genomic features.
T89916 360600-360608 Sentence denotes Results:
T90162 360609-360706 Sentence denotes We identified three K. pneumoniae isolates, CN1, CR14 and NY9, carrying both blaCTX-M and blaKPC.
T81326 360707-360768 Sentence denotes Through de novo assembly, we obtained three complete genomes.
T44440 360769-360876 Sentence denotes In CR14 and NY9, blaCTX-M and blaKPC were carried in distinctive plasmids with distinctive incompatibility.
T65613 360877-360987 Sentence denotes In contrast, CN1 harbors in chromosome one blaKPC-2 and three blaCTX-M-15 genes, in addition to one blaSHV-11.
T38404 360988-361217 Sentence denotes Whereas blaCTX-M-15 genes were transferred via the same insertion sequence ISEcp1, blaKPC-2 gene was mobilized in the context of insertion sequence Tn4401a, which was found conjugative with a PsP3-like prophage in CN1 chromosome.
T42055 361218-361434 Sentence denotes Intriguingly, downstream of the newly generated Tn4401a-prophage genomic island, CN1 carried a CRISPR-Cas array with four spacers targeting a variety of K. pneumoniae plasmids carrying antimicrobial resistance genes.
T32056 361435-361659 Sentence denotes It suggests the evolving CRISPR-Cas, a bacterial immune system preventing phage infection and plasmid transfer, might have induced the mobilization of resistance genes from plasmids into chromosome under the selective force.
T24806 361660-361671 Sentence denotes Conclusion:
T86163 361672-361741 Sentence denotes We observed the emergence of K. pneumoniae isolates -lactamase genes.
T15114 361742-362004 Sentence denotes Our comparative genomic analyses revealed their shared genomic features and dynamic evolutionary events occurring in chr -lactam resistance from plasmids to chromosome depicts the currently complex pandemic scenario of multidrug resistant K. pneumoniae isolates.
T10496 362005-362018 Sentence denotes Introduction:
T59122 362019-362153 Sentence denotes Acute respiratory infections are a significant public health issue; therefore rapid diagnosis is crucial for appropriate patient care.
T95479 362154-362259 Sentence denotes The accuracy of these rapid multiple pathogen detection panels is critical to start and guide treatments.
T96206 362260-362378 Sentence denotes Best practice and regulations require laboratories to establish quality control programs for every assay they perform.
T11900 362379-362607 Sentence denotes The routine use of quality controls that are consistent lot to lot assists the laboratory in identifying shifts, trends, and increased frequency of random errors caused by variations in the test system, such as failing reagents.
T31421 362608-362794 Sentence denotes Maine Molecular Quality Controls Inc (MMQCI) has developed a synthetic, multiplexed control to simultaneously monitor all 12 common pathogens detected by BioFire's FilmArray RP EZ Assay.
T21096 362795-362803 Sentence denotes Methods:
T19883 362804-363060 Sentence denotes Synthetic, multiplex molecular constructs containing indidual genome segments of all respiratory pathogens were designed in silico to create a singular piece of synthetic DNA, ligated into MMQCI vectors and transformed to create stable frozen clone stocks.
T88996 363061-363171 Sentence denotes RNA transcripts were generated and quantified by 260/280 UV spec and formulated in MMQCI's proprietary matrix.
T56690 363172-363415 Sentence denotes The control was titrated to an optimal level for positive detection on the FilmArray RP EZ Assay to ensure reliable simultaneous detection of all targets included in the panel, at a concentration close enough to LOD to monitor the test system.
T16043 363416-363424 Sentence denotes Results:
T76839 363425-363612 Sentence denotes Three different manufactured lots of RP EZ control were tested across three FilmArray RP EZ assay pouch lots (n=35), with 100% detection of all targets with a standard deviation of 2 Cps.
T93544 363613-363696 Sentence denotes RP EZ Negative control panel gave accurate results across three BioFire pouch lots.
T32721 363697-363873 Sentence denotes Clinical data supplied by BioFire, consisted of 10 days of testing at three sites of three positive and three negative controls run per day (total of 60 control runs per site).
T69602 363874-363948 Sentence denotes Multiple operators participated in testing at each of the three locations.
T53898 363949-364046 Sentence denotes Three lots of external control material and 3 lots of RP EZ pouches were tested across all sites.
T13044 364047-364181 Sentence denotes 100% concordance was seen for all three positive controls and 97.8% concordance for all negative controls from 182 RP EZ test results.
T54011 364182-364425 Sentence denotes Conclusions: MMQCI's proprietary matrix and stabilization buffers allow for the synthetic RNA to be maintained as a stable and reliable control that can be carried through the entire molecular diagnostic assay including the extraction process.
T49941 364426-364723 Sentence denotes The RP EZ Control Panel offers a ready-to use, non-infectious control material designed to monitor multiplex assays with just two controls.This qualtitative, synthetic molecular control material provides a wellcharacterized reference to be used for verification, validation or as routine controls.
T16783 364724-364780 Sentence denotes Panel with High-Throughput BioCode MDx3000 Instrument J.
T83659 364781-364793 Sentence denotes Kirchner, C.
T31677 364794-364803 Sentence denotes Knoth, M.
T81605 364804-364814 Sentence denotes Henrie, B.
T57162 364815-364823 Sentence denotes Shah, T.
T29246 364824-364836 Sentence denotes Ul-Hasan, D.
T3666 364837-364848 Sentence denotes Mantzke, A.
T49632 364849-364857 Sentence denotes Pham, M.
T80616 364858-364906 Sentence denotes Aye Applied BioCode, Inc., Santa Fe Springs, CA.
T76903 364907-364920 Sentence denotes Introduction:
T69465 364921-364993 Sentence denotes Gastroenteritis is the second most common illness after the common cold.
T10746 364994-365169 Sentence denotes Globally, diarrhea accounts for approximately 2 million annual deaths in children under 5 years old, or about 2,000 child deaths per day (Boschi-Pinto C, Velebit L, Shibuya K.
T54726 365170-365175 Sentence denotes Bull.
T89928 365176-365195 Sentence denotes World Health Organ.
T82091 365196-365215 Sentence denotes 2008; 86(9):710-7).
T69425 365216-365369 Sentence denotes High-throughput multiplex assays allow for both rapid identification of diarrhea-causing pathogens and enhanced infection control in healthcare settings.
T62303 365370-365532 Sentence denotes Using proprietary barcoded magnetic bead (BMB) technology, Applied BioCode has developed a molecular diagnostic assay for detection of gastrointestinal pathogens.
T82342 365533-365648 Sentence denotes Concurrently, we have developed the BioCode MDx3000, an automated high-throughput instrument with a 96-well format.
T4170 365649-365657 Sentence denotes Methods:
T90185 365658-365784 Sentence denotes The BioCode MDx3000 instrument integrates and automates PCR, post-PCR sample handling and detection steps in a 96-well format.
T79138 365785-365897 Sentence denotes Unpreserved stool or Cary-Blair samples were subjected to bead lysis and extracted with an automated instrument.
T86413 365898-366119 Sentence denotes Extracted samples were combined with RT-PCR chemistry and loaded onto the BioCode MDx3000 instrument where they were amplified by PCR, transferred to a 96-well plate and captured by target-specific probes coupled to BMBs.
T84516 366120-366316 Sentence denotes The presence of captured target sequence(s) was detected by a fluorescence conjugate and qualitative results were determined by median fluorescence intensity (MFI) values relative to assay cutoff.
T50257 366317-366325 Sentence denotes Results:
T29571 366326-366615 Sentence denotes The BioCode GI Pathogen Panel is a high multiplex assay with preliminary limit of detection (LoD) comparable to currently available molecular assays (STEC 2.4E+03 CFU/mL, Rotavirus 6E+02 TCID50/mL, Cryptosporidium (hominis and parvum) 2.5E+04 oocytes/mL, C. difficile A+B+ 7.7E+02 CFU/mL).
T36789 366616-366889 Sentence denotes The panel is highly specific and gave robust MFI for 60 targeted GI pathogens, including synthetic jmd.amjpathol.org ■ The Journal of Molecular Diagnostics mimics of norovirus GI 1-8 and GII 1-14, while not cross-reacting with the off-target organisms tested in this study.
T71291 366890-366933 Sentence denotes No well-to-well contamination was observed.
T31897 366934-367037 Sentence denotes Equivalent detection was observed between unpreserved stool and samples in Cary-Blair transport medium.
T85720 367038-367185 Sentence denotes Evaluation of performance with a set of clinical specimens showed 91% positive agreement and 94% negative agreement compared to a FDA cleared test.
T77573 367186-367198 Sentence denotes Conclusions:
T63243 367199-367474 Sentence denotes The BioCode MDx3000, automated for integration of RT-PCR, post-PCR sample handling and detection steps, enables multiplex molecular detection in 96-well format whereas BioCode GI Pathogen Panel specifically detects and discriminates 18 bacteria/toxins, viruses and parasites.
T97306 367475-367727 Sentence denotes In combination, this platform and assay will allow users to perform multiplex molecular detection in a high throughput format, thereby simplifying the workflow, reducing hands-on time and minimizing contamination risk. (>80%) between the three methods.
T28134 367728-367929 Sentence denotes The ART assay detected 12 %and 14% additional patient samples that CAP/CTM and altona missed, respectively; whereas CAP/CTM detected 3% and 10% patient samples that ART and altona missed, respectively.
T86275 367930-368088 Sentence denotes Method bias ranged from -1.2 log IU/mL between Roche and altona, -0.2 between Abbott and altona and finally 0.8 log IU/mL between Abbott and Roche (Table 1 ).
T27208 368089-368256 Sentence denotes High degree of agreement was observed between methods at the CAP/CTM and altona LOD (>79%) whereas relatively low level of agreement was observed at the ART LOD (>7%).
T6434 368257-368268 Sentence denotes Conclusion:
T34442 368269-368422 Sentence denotes Method comparison demonstrated the importance of further understanding differences which contribute to assay variability in light of the 1 st CMV WHO IS.
T50668 368423-368487 Sentence denotes Overall method bias ranged from 0.8 log IU/mL to -1.2 log IU/mL.
T38511 368488-368709 Sentence denotes Additionally, this study demonstrated that the ART assay detected CMV residual viremia more readily than the other two commercial assays and in turn would be able to identify early therapy failures or disease progression.
T45935 368710-368860 Sentence denotes Dilutions of viral material were tested and probit analysis was subsequently performed to determine the limit of detection (LOD) for each sample type.
T93311 368861-369002 Sentence denotes Patient samples positive for other viruses commonly identified in plasma and urine specimens were tested to determine analytical specificity.
T27100 369003-369161 Sentence denotes Inter-assay and intra-assay precision studies involved a panel of 6 specimens tested independently by 3 technologists and tested in triplicate within the run.
T72311 369162-369269 Sentence denotes Additionally, method comparison analyses were done to determine the degree of agreement between the assays.
T58372 369270-369278 Sentence denotes Results:
T70534 369279-369449 Sentence denotes Compared to the ViraCor and Scott & White assays, the clinical sensitivity using the Focus ASR assay was 98.3% (59/60); 100% (40/40) for plasma and 95% (19/20) for urine.
T59692 369450-369540 Sentence denotes Clinical specificity was 97.1% (68/70); 96% (48/50) for plasma and 100% (20/20) for urine.
T19924 369541-369657 Sentence denotes Regression analysis of plasma and urine results revealed a high correlation (R 2 = 0.9804 and 0.9467, respectively).
T53745 369658-369763 Sentence denotes A strong correlation was also seen using Pearson correlation; r = 0.9901 for plasma and 0.9730 for urine.
T91657 369764-369879 Sentence denotes Bland-Altman plots for plasma and urine determined the mean bias to be -0.227 log10 and -0.616 log10, respectively.
T17213 369880-369984 Sentence denotes The LOD with a 95% confidence interval was determined to be 54 cp/mL for plasma and 119 cp/mL for urine.
T79089 369985-370180 Sentence denotes Furthermore, no cross-reactivity was observed for any analytical specificity samples tested and all precision studies produced equivalent results, yielding coefficients of variation less than 6%.
T33545 370181-370193 Sentence denotes Conclusions:
T58803 370194-370564 Sentence denotes The Focus ASR assay performed comparably to the assays performed by ViraCor and Scott & White and provides accurate and reproducible results for the detection and quantification of BKV viral load in plasma and urine specimens. (BKV) replication is recommended to identify post-transplant patients at risk of BKV-associated diseases and to guide therapeutic intervention.
T77861 370565-370747 Sentence denotes BKV quantification in urine by real-time PCR is noninvasive and can be informative, as a high level of BK viruria usually precedes viremia and potential nephropathy by 4 to 12 weeks.
T74915 370748-370956 Sentence denotes However, the heterogeneity of qPCR methods among different laboratories limits interlaboratory comparison and hampers the establishment of thresholds to distinguish asymptomatic viruria with clinical disease.
T65686 370957-371098 Sentence denotes This study aimed to evaluate the performance of the QIAsymphony RGQ system for BKV DNA quantification (BK-Q) using artus BKV QS-RGQ reagents.
T18250 371099-371190 Sentence denotes Results were compared to our Laboratory-Developed Test (LDT) for correlation and agreement.
T26434 371191-371199 Sentence denotes Methods:
T90432 371200-371332 Sentence denotes Total nucleic acid extraction and qPCR reaction set-up were performed using an integrated mode on the QIAsymphony SP/AS instruments.
T61970 371333-371435 Sentence denotes The QIAsymphony DSP Virus/Pathogen Mini kit in combination with the Complex200 protocol (input volume:
T73972 371436-371459 Sentence denotes 200 μL, elution volume:
T32409 371460-371492 Sentence denotes 60 μL) were used for extraction.
T72513 371493-371589 Sentence denotes The artus BKV QS-RGQ reagents (Qiagen) were used to run the qPCR on the Rotor Gene Q instrument.
T80525 371590-371698 Sentence denotes Calibration was determined using serial dilutions of a commercially available BKV (ExactDx, Fort Worth, TX).
T72949 371699-371900 Sentence denotes Linearity, precision and lower limit of quantification (LoQ) were evaluated using a serial dilution panel from a patient sample nominally quantified in-house and then diluted in urine negative for BKV.
T11911 371901-372052 Sentence denotes Correlation and bias were assessed using 31 urine specimens (21 positive, 10 undetectable) from transplant recipients previously quantified by the LDT.
T8902 372053-372061 Sentence denotes Results:
T23825 372062-372134 Sentence denotes Linearity was observed from 3.18 to 7.0 Log10 BKV copies/mL (R 2 =0.99).
T50008 372135-372215 Sentence denotes The LoQ was 1,510 copies/mL which is well below the clinical significant values.
T48959 372216-372301 Sentence denotes The assay showed high precision with standard deviations from 0.05 to 0.20 Log10 (CV:
T20152 372302-372312 Sentence denotes 1% to 8%).
T64382 372313-372389 Sentence denotes Quantitative data obtained with BK-Q correlated (Pearson's r = 0.95) but was
T26411 372390-372501 Sentence denotes The Journal of Molecular Diagnostics ■ jmd.amjpathol.org not identical to LDT, with BK-Q values trending lower.
T14700 372502-372558 Sentence denotes All undetectable samples by LDT were below the BK-Q LoQ.
T46392 372559-372571 Sentence denotes Conclusions:
T68025 372572-372615 Sentence denotes The BK-Q demonstrated reliable performance.
T49333 372616-372772 Sentence denotes Monitoring of BKV replication with standardized system may enable clinical laboratories to assess quantification with greater reproducibility and precision.
T60895 372773-372884 Sentence denotes The liquid Amies appears to be a useful culture medium to improve stability and viability of certain organisms.
T56597 372885-373013 Sentence denotes Our data shows that ES 480C could be used as a universal collection medium for molecular testing in a clinical microbiology lab.
T69469 373014-373054 Sentence denotes Step Algorithm for the Diagnosis of C. .
T45406 373055-373282 Sentence denotes In addition to the FDA cleared HSV1&2 assay, the ARIES system also has the ability to run ASRs and LDTs by using either ASR primers or primers purchased from a third-party vendor, ARIES extraction cassettes and ARIES Ready Mix.
T93902 373283-373505 Sentence denotes Objective of this study was to verify the performance of HSV1&2 assay, and validate ASR primers for Candida albicans (CA), Candida glabrata (CG), Gardnerella vaginalis (GV) and Trichomonas vaginalis (TV) on ARIES platform.
T84025 373506-373514 Sentence denotes Methods:
T55268 373515-373631 Sentence denotes The vaginosis panel (VP) comprised of CA/CG run in one ARIES extraction cassette and GV/TV run in a second cassette.
T1592 373632-373737 Sentence denotes Primers were loaded into ARIES Ready Mix tubes prior to snapping them onto the ARIES extraction cassette.
T64416 373738-373802 Sentence denotes ARIES HSV IVD cassettes were used per the manufacturer protocol.
T85278 373803-373840 Sentence denotes All 3 assays used 200μL input volume.
T47264 373841-374047 Sentence denotes Results were reviewed using cycle threshold (Ct) and melt curve (TM) analysis on ARIES for the HSV IVD tests per manufacturer protocol and in the SYNCT software using the UDP (User Defined Protocol) for VP.
T45888 374048-374143 Sentence denotes The limit of detection, clinical accuracy, precision, specificity and stability were evaluated.
T14039 374144-374387 Sentence denotes For clinical accuracy, the VP panel on ARIES was compared to either BD Affirm VPIII (for GV, CA and TV) or Aptima TV assay on Hologic Panther platform (for TV) whereas HSV1&2 assays were compared to ROCHE LightCycler HSV 1/2 Qualitative assay.
T17860 374388-374507 Sentence denotes Results: ARIES uses Multi-Code real-time PCR technology to identify and discriminate targets using melt curve analysis.
T25345 374508-374630 Sentence denotes Compared to Affirm, ARIES was not only able to accurately identify Candida species but also sub-type all 3 patients as CA.
T65374 374631-374758 Sentence denotes BD Affirm VPIII detected 7 of the 8 GV positives detected on the ARIES but missed a TV sample which was positive on the ARIES .
T78068 374759-374836 Sentence denotes ARIES detected 90.9% (10/11) of the TV positives detected by Aptima TV assay.
T95968 374837-374942 Sentence denotes ROCHE LightCycler HSV 1/2 Qualitative assay missed one of the 8 HSV1 samples that were positive on ARIES.
T89673 374943-375071 Sentence denotes In summary, ARIES was more sensitive than Affirm in accurately identifying TV and equally sensitive compared to Aptima TV assay.
T73994 375072-375122 Sentence denotes Sensitivity for HSV1&2 was similar to ROCHE assay.
T94641 375123-375251 Sentence denotes Conclusions: ARIES offers a sample to answer solution with versatility, flexibility and scalability for a growing molecular lab.
T5531 375252-375370 Sentence denotes It also appears to have good sensitivity as compared to some of the other real-time PCR platforms currently available.
T3103 375371-375653 Sentence denotes The VP assay on ARIES is easy to perform and has better sensitivity compared to currently widely used BD Affirm with ability of also discriminating between Candida species. typically receive empirical treatment with broad-spectrum agents, which are inadequate in up to 30% of cases.
T45990 375654-375752 Sentence denotes Inadequate therapy is associated with increased mortality, morbidity, and length of hospital stay.
T78674 375753-376057 Sentence denotes Rapid identification of BSI pathogens and resistance markers directly from clinical specimens, bypassing the requirement for lengthy culture upstream, would facilitate appropriate therapy to improve survival rates, reduce hospital costs, and diminish the development of antimicrobial resistant organisms.
T15586 376058-376237 Sentence denotes We report the sensitivity of a novel method to detect BSI pathogens and resistance genes directly from whole blood and some validation results of the method on clinical specimens.
T53378 376238-376246 Sentence denotes Methods:
T19845 376247-376402 Sentence denotes The sensitivity of component steps in the Genalysis Blood Stream Infection Identification (G-BSI) assay was established for a large range of BSI pathogens.
T75251 376403-376547 Sentence denotes Sample prep sensitivity was measured by spiking 10 mL samples of healthy donor blood (n=20, per pathogen) at nominal concentrations of 1 CFU/mL.
T636 376548-376642 Sentence denotes Recovered cells were plated on solid medium and quantified after overnight incubation at 37°C.
T90132 376643-376982 Sentence denotes Sensitivity of nested PCR to species-specific ID was measured by spiking genomic copies of C. albicans, S. aureus, E. faecalis, E. coli, and mecA, in the presence of 500 ng human DNA in a titration series into a preamp reaction, with PCR detection by measurement of pH changes by ISFET sensors on a CMOS sensor chip using specific primers.
T54553 376983-377126 Sentence denotes End-to-end functionality was confirmed by testing clinical specimens obtained from consented patients at the University of New Mexico Hospital.
T44492 377127-377135 Sentence denotes Results:
T76238 377136-377269 Sentence denotes Sample prep sensitivity to 1 CFU/mL was demonstrated for nine Gram-positive bacteria, eight Gram-negative bacteria, and three yeasts.
T9201 377270-377385 Sentence denotes Nested PCR showed sensitivity down to a single genomic copy for the four microorganisms and mecA resistance marker.
T41492 377386-377618 Sentence denotes End-to-end functionality was demonstrated on a set of clinical specimens; 7 specimens were correctly identified as negative (concordant with blood culture) and 2 specimens correctly identified as positive (K. pneumoniae, S. aureus).
T73508 377619-377631 Sentence denotes Conclusions:
T96301 377632-377724 Sentence denotes Rapid detection of pathogens in spiked and clinical samples was demonstrated with Genalysis.
T84333 377725-377828 Sentence denotes The current manual process allows pathogen ID by molecular methods, at the 1 CFU/ml level, in <5 hours.
T80933 377829-378006 Sentence denotes Ongoing automation of the process is expected to reduce the total process time to <3 hours, providing therapeutically actionable information in a clinically relevant time frame.
T79884 378007-378263 Sentence denotes Screening for GBS colonization in antepartum women between 35 and 37 weeks' gestation, followed by intrapartum antibiotic treatment for women with positive colonization status has proven to be an effective mechanism for prevention of perinatal GBS disease.
T47520 378264-378501 Sentence denotes Here we describe the implementation of a polymerase chain reaction (PCR) based GBS Assay (Great Basin Scientific), a qualitative in vitro diagnostic test (IVD) for the detection of GBS DNA from vaginal/rectal swabs from antepartum women.
T98175 378502-378510 Sentence denotes Methods:
T69233 378511-378744 Sentence denotes Samples included 20 known GBS, and 20 known E. faecalis QC organisms performed over 20 separate days, and 26 patient specimens run concurrently with the Illumigene Pro GBS Assay (Meridian Bioscience) which was the procedure in place.
T75505 378745-379003 Sentence denotes All samples were initially cultured in Hardy Carrot broth (Hardy Diagnostics #Z140) which uses the Granada medium reaction and contains the necessary components for pigment detection of betahemolytic GBS and produces positive results in as little as 6 hours.
T1390 379004-379411 Sentence denotes Following incubation in enrichment broth (utilizing Hardy Carrot broth as the enrichment broth rather than LIM broth) for 18 -29 hours, samples underwent automated sample preparation and PCR on the PA500 Portrait Analyzer System to amplify a cfb gene sequence specific to the GBS genome which is detected by hybridization probes immobilized on a silica chip surface according to manufacturer's instructions.
T86280 379412-379420 Sentence denotes Results:
T12602 379421-379520 Sentence denotes Of the 20 known positive and 20 negative control specimens tested, all yielded the expected result.
T43468 379521-379606 Sentence denotes Of the 26 patient specimens, 19 were negative by both Illumigene and Great Basin GBS.
T63991 379607-379774 Sentence denotes Of the 7 positive specimens, 3 were positive by pigment change in Carrot broth, 2 were positive by culture, CAMP and/or Vitek GP ID, and 2 were positive by Illumigene.
T63927 379775-379787 Sentence denotes Conclusions:
T40442 379788-379908 Sentence denotes The Great Basin GBS assay demonstrated 100% concordance with expected results over 66 specimens and three methodologies.
T31975 379909-379981 Sentence denotes The Carrot broth has proven an effective enrichment media for the assay.
T84189 379982-380184 Sentence denotes In our current workflow, negative Carrot broth specimens are tested using the Great Basin GBS Assay to detect non-hemolytic strains of GBS. (2008) was conducted on 90 clinical plasma or serum specimens.
T16271 380185-380193 Sentence denotes Results:
T55303 380194-380314 Sentence denotes All 35 of the published ZIKV strain sequences showed 100% homology with at least one of the two amplification reactions.
T14611 380315-380381 Sentence denotes No amplification was observed in ZIKV-negative clinical specimens.
T91981 380382-380543 Sentence denotes No cross-reactivity was observed with any of the pathogens tested and no significant sequence homology was found for any of the 51 organisms evaluated in silico.
T7375 380544-380666 Sentence denotes An assay LoD of at least 0.05 U/mL (TCID50) was established in both plasma and serum on each of the three thermal cyclers.
T73746 380667-380791 Sentence denotes In the method comparison, both assays detected ZIKV in each of 34 specimens and did not detect ZIKV in each of 44 specimens.
T85749 380792-381022 Sentence denotes The VERSANT Zika RNA 1.0 Assay (kPCR) detected ZIKV in an additional 10 specimens that the comparator CDC assay did not, whereas the comparator CDC assay detected ZIKV in 2 specimens that VERSANT Zika RNA 1.0 Assay (kPCR) did not.
T28523 381023-381092 Sentence denotes The VERSANT Zika RNA 1.0 Assay (kPCR) qualitatively detects ZIKV RNA.
T5786 381093-381365 Sentence denotes The assay recognizes a broad spectrum of published ZIKV RNAs in silico, has high analytical sensitivity, is specific to ZIKV among Flaviviridae, and has excellent performance with clinical specimens. (CDI) is a leading cause of hospitalassociated gastrointestinal illness.
T74736 381366-381512 Sentence denotes Many clinical labs use molecular diagnostic assays for C. difficile to screen unformed stool samples from patients with signs and symptoms of CDI.
T8754 381513-381746 Sentence denotes However, a future potential application of such assays is for use in screening asymptomatic patients (either on admission or during a hospital stay) for presence of the C. difficile organism using unformed stool or peri-rectal swabs.
T60946 381747-381892 Sentence denotes This will assist hospitals in isolating asymptomatic carriers, especially from immunocompromised in-patients, to prevent the spread of infection.
T75693 381893-382088 Sentence denotes The goal of this study was to compare the performance of unformed and formed stool samples in the Simplexa C. difficile Direct assay, and compare the Simplexa assay to Cepheid Xpert C. difficile.
T23277 382089-382272 Sentence denotes The reproducibility of the Simplexa Direct assay with formed stool as well as the ability of the Simplexa Direct assay to detect a wide range of C. difficile isolates was also tested.
T63541 382273-382281 Sentence denotes Methods:
T31433 382282-382464 Sentence denotes Limit of detection (LoD) panels were created by serially diluted C. difficile strains ATCC 43255, NAP1A and BAA-1805 and spiking into negative formed stool to mimic clinical samples.
T14019 382465-382571 Sentence denotes The LoD panel was screened with both molecular assays (Simplexa and Xpert) in triplicate to determine LoD.
T75701 382572-382718 Sentence denotes A total of 33 C. difficile isolates ranging in different toxinotypes and ribotypes were evaluated for analytical reactivity in the Simplexa assay.
T38725 382719-382851 Sentence denotes A reproducibility study was performed for the Simplexa assay using medium and low positive contrived samples in formed stool matrix.
T79977 382852-382860 Sentence denotes Results:
T15129 382861-382994 Sentence denotes LoD studies showed that the Simplexa C. difficile Direct assay had similar performance with the three strains tested in formed stool.
T29682 382995-383138 Sentence denotes The approximate LoDs for ATCC 43255, NAP1A and BAA-1805 for Simplexa were 3.1, 400 and 119 CFU/mL and were 1.6, >410 and >121 CFU/mL for Xpert.
T48196 383139-383197 Sentence denotes The Simplexa assay detected all 33 characterized isolates.
T66608 383198-383210 Sentence denotes Conclusions:
T6316 383211-383347 Sentence denotes The Simplexa C. difficile Direct assay was capable of detecting C. difficile strains spanning a wide range of toxinotypes and ribotypes.
T96447 383348-383483 Sentence denotes Comparison between the Simplexa and Xpert assays showed that the assays demonstrated similar sensitivity when tested with formed stool.
T94307 383484-383625 Sentence denotes The Simplexa assay also demonstrated reproducible performance for formed stool samples and may be useful for screening asymptomatic carriers.
T57592 383626-383743 Sentence denotes Simplexa C. difficile Direct is in development; the assay is not currently available for sale and is not FDA cleared.
T64562 383744-383757 Sentence denotes Introduction:
T5640 383758-383862 Sentence denotes The first report of HIV anti-retroviral therapy (ART) resistance occurred in 1989 after the release AZT.
T15174 383863-383990 Sentence denotes Since this initial report, HIV resistance testing, also referred to as HIV genotyping, has become an integral part of HIV care.
T23810 383991-384084 Sentence denotes HIV-ART resistance has significant clinical implications for choosing effective ART regimens.
T71258 384085-384308 Sentence denotes Genotypic resistance assays used by most clinical laboratories detect the presence of specific drug resistance mutations in regions of the HIV genome encoding protease (PRO), reverse transcriptase (RT), and integrase (INT).
T63440 384309-384582 Sentence denotes Results are reported as individual mutations (eg, M184V, the signature mutation for lamivudine resistance) with comments such as "susceptible," "possibly resistant /intermediate," or "resistant" for each antiretroviral agent based on known impact of each mutation reported.
T2070 384583-384670 Sentence denotes Currently, a single FDA cleared assay (Abbott Viroseq) is available for HIV genotyping.
T80811 384671-384855 Sentence denotes We report the validation/comparison of a laboratory developed procedure (LDP) HIV genotype protocol, incorporating the use of SmartGene HIV module, for determination of ART resistance.
T14998 384856-384864 Sentence denotes Methods:
T72158 384865-385041 Sentence denotes A total of n=94 unique HIV cases representing n=15 HIV-1 subtypes were amplified using the Viroseq assay and compared to the HCMC-HIVgt assay spanning PRO, RT, and INT regions.
T13324 385042-385201 Sentence denotes Amplification efficiency was partially determined by concentration of the amplified product(s) prior to Sanger sequencing using the ABI 3500 genetic sequencer.
T85186 385202-385296 Sentence denotes A single extract, using automated EasyMag extraction was used for all amplification reactions.
T5095 385297-385544 Sentence denotes Comparison/correlation of i) amplification efficiencies, ii) failed sequence runs, iii) resistance mutations detected iv) interpretative resistance/susceptible reporting using Viroseq and SmartGene software and v) limit of detection were compared.
T68814 385545-385553 Sentence denotes Results:
T6327 385554-385668 Sentence denotes Overall, 83% correlation in interpretive reporting, including all forms of associated ART resistance was observed.
T45091 385669-385766 Sentence denotes For cases involving "Major HIV Associated Drug Resistance Mutations" correlation improved to 95%.
T94787 385767-385912 Sentence denotes Eight cases (8.5%) failed to produce reliable genotype results using the ViroSeq assay compared to five cases (5.32%) using the HCMC-HIVgt assay.
T31540 385913-385925 Sentence denotes Conclusions:
T95610 385926-386056 Sentence denotes A 95% correlation in detection of clinically significant HIV resistance mutations between the two genotype protocols was observed.
T11574 386057-386273 Sentence denotes Use of the SmartGene HIV-1 module allowed streamlined reporting of all HIV ART drug classes within a single, consolidated patient report and allowed for longitudinal resistance analysis and reports for 100% of cases.
T44299 386274-386524 Sentence denotes The HCMC-HIVgt assay used with the SmartGene HIV-1 module represents an alternative approach for reliable HIV genotyping using LDP protocols for determination and reporting of HIV-1 resistance and a highly cost-effective approach for our institution.
T59923 386525-386538 Sentence denotes Introduction:
T39624 386539-386715 Sentence denotes To comply with CLIA/CAP regulations for NGS data analysis, we developed a bioinformatics pipeline to process raw sequencing data from ampliconbased target enrichment NGS panel.
T47584 386716-386875 Sentence denotes The pipeline has two parts: a Galaxy workflow to generate a processed variant call file (VCF), and variant filtering and annotation using third party software.
T84045 386876-387016 Sentence denotes A modified GRCh37 reference that contained artificially integrated variants was used to generate synthetic sequencing reads in FASTQ format.
T38271 387017-387109 Sentence denotes These FASTQ fileswere processed to assess accuracy, sensitivity, specificity, and precision.
T71194 387110-387175 Sentence denotes These files can be used to re-validate updated pipeline versions.
T94667 387176-387184 Sentence denotes Methods:
T2402 387185-387207 Sentence denotes Synthetic FASTQ files:
T73412 387208-387295 Sentence denotes Wild-type sequences were extracted from hg19/GRCh37 referencing the NGS panel BED file.
T65059 387296-387432 Sentence denotes Spike-in variants were sourced from known mutations in Coriell samples and also contained a number of random variants from dbSNP (v144).
T5429 387433-387611 Sentence denotes Zygosity of Coriell mutations were based on observation in samples and all selected dbSNP variants were randomly assigned zygosity based on a 1:3 homozgous to heterozygous ratio.
T12245 387612-387811 Sentence denotes To mimic the diploid human genome, homozygous variants were integrated into one set of wild-type sequence, heterozygous and homozygous variants were integrated into another set of wild-type sequence.
T11445 387812-387874 Sentence denotes Primers were subsequently added to create synthetic amplicons.
T6958 387875-388026 Sentence denotes In silico sequencing reads in FASTQ format were sampled from these synthetic amplicons by ART using Illumina sequencing error and quality data profile.
T74624 388027-388253 Sentence denotes Ten synthetic samples were generated to assess sensitivity, whereas three wild samples and four duplicate samples simulated by different random seeds were also included for specificity and inter-assay repeatability assessment.
T51507 388254-388378 Sentence denotes Informatics Pipeline: FASTQ files were processed by FASTQ Parallel Groomer tool and Trimmomatic to remove low quality reads.
T43637 388379-388456 Sentence denotes The remaining reads were mapped tohg19/GRCh37 reference genome using BWA-MEM.
T52581 388457-388592 Sentence denotes The alignment files were further intersected by the NGS panel BED file and once the amplicon region was retained, primers were removed.
T50525 388593-388664 Sentence denotes Variant calling for SNVs and short indels was performed with Freebayes.
T59109 388665-388834 Sentence denotes The resulting VCF was uploaded to Bench (Cartagenia, MA) and variants are filtered based on call quality, population frequency, panel artifacts, and known pathogenicity.
T36909 388835-388843 Sentence denotes Results:
T23701 388844-388888 Sentence denotes The following assay metrics were calculated:
T63117 388889-388963 Sentence denotes Accuracy, 99.99%; sensitivity, 98.58%; specificity, 100%; precision, 100%.
T486 388964-388976 Sentence denotes Conclusions:
T40304 388977-389112 Sentence denotes Use of synthetic FASTQ files enables evaluation of the accuracy of an NGS bioinformatic pipeline independent of sequencing run quality.
T37695 389113-389229 Sentence denotes This approach provides a highly efficient, reproducible, cost-effective approach for QC/QA in a clinical laboratory.
T62408 389230-389258 Sentence denotes High-Resolution Melting Z.L.
T57702 389259-389274 Sentence denotes Dwight 1 , C.T.
T5240 389275-389384 Sentence denotes Wittwer 2 1 University of Utah Health Sciences, Salt Lake City, UT; 2 University of Utah, Salt Lake City, UT.
T28412 389385-389398 Sentence denotes Introduction:
T97295 389399-389475 Sentence denotes High-resolution melting is a rapid, low-cost diagnostic tool for genotyping.
T15992 389476-389580 Sentence denotes This technique visualizes the thermodynamic differences in PCR products with fluorescent melting curves.
T94818 389581-389657 Sentence denotes Melting curves can differentiate and identify types and subtypes of viruses.
T86806 389658-389793 Sentence denotes The same virus can exhibit sequence variation between patients and even within a patient due to high rates of replication and mutation.
T88381 389794-389953 Sentence denotes Designing assays that offer specificity and sensitivity can be challenging, forcing inefficient 'shotgun' approaches to primer design and experimental testing.
T32280 389954-390168 Sentence denotes The software presented uses systematic frequency analysis to identify primer pairs that will amplify the intended targets with the highest specificity possible while optionally excluding unwanted types or subtypes.
T98277 390169-390177 Sentence denotes Methods:
T89789 390178-390247 Sentence denotes Multiple sequences for the intended target are input in FASTA format.
T64353 390248-390344 Sentence denotes All sequences are divided into an exhaustive set of possible primers that are within a Tm range.
T98870 390345-390379 Sentence denotes Any redundant primers are removed.
T27277 390380-390440 Sentence denotes Each unique primer is then compared to each target sequence.
T34096 390441-390505 Sentence denotes If a perfect match is found, the consensus score is set to 100%.
T39705 390506-390641 Sentence denotes If a mismatch occurs, both the mismatch Tm and a weighting factor based on mismatch/bulge position and type lowers the consensus score.
T86143 390642-390799 Sentence denotes The average consensus score across all target sequences are ranked and primer sets that match a product size and/or Tm specified by the user are then listed.
T9867 390800-391014 Sentence denotes This consensus portion of the algorithm provides primer pairs that will amplify the intended target (descending order of average consensus rank) that are within selected primer Tms, products sizes, and product Tms.
T42869 391015-391100 Sentence denotes Optionally, users may define unwanted targets by importing a second set of sequences.
T42171 391101-391184 Sentence denotes That is, the desired primers need to avoid amplifying this second set of sequences.
T17748 391185-391324 Sentence denotes Similarly, this segregation score will identify primers with high consensus to the target set and high differentiation of the unwanted set.
T67766 391325-391333 Sentence denotes Results:
T38301 391334-391468 Sentence denotes The software was tested with the goal of amplifying all types of Hepatitis C with sequences obtained from the Los Alamos HCV Database.
T42602 391469-391562 Sentence denotes The software was also tested against Zika sequences obtained from NCBI's Zika Virus Resource.
T38723 391563-391655 Sentence denotes In the case of Zika, NCBI's PrimerBlast was also used to discriminate against other viruses.
T55476 391656-391786 Sentence denotes For HCV and Zika, the top four assays were selected from the software results, and all successfully amplified the intended target.
T89145 391787-391799 Sentence denotes Conclusions:
T41740 391800-391923 Sentence denotes This software provides a straightforward approach to designing assays to amplify and differentiate difficult viral targets.
T49875 391924-392101 Sentence denotes Focusing on assay design rather than primer characteristics in the context of the intended targets greatly reduces the parameters often believed to be required in primer design.
T41995 392102-392115 Sentence denotes Introduction:
T26324 392116-392338 Sentence denotes Whereas laboratory information systems (LISs) have grown increasingly complex to meet the demands of expanding anatomic and clinical pathology laboratories, they continue to neglect the nuances of the molecular laboratory.
T61143 392339-392428 Sentence denotes As a result, basic functionalities necessary for molecular workflow are glaringly absent.
T73684 392429-392676 Sentence denotes This study determined functionality gaps of laboratory information systems (LISs) in molecular laboratories and the associated impact to workflow, efficiency, and security by collecting anonymous survey data from clinical laboratory professionals.
T96927 392677-392685 Sentence denotes Methods:
T64407 392686-392829 Sentence denotes A 34 question survey (30 required + 4 optional questions) was compiled using an online survey tool (Survey Monkey, Palo Alto, California, USA).
T12070 392830-392974 Sentence denotes Participants were recruited through professional molecular society listservs (AMP, ASHI, API, AMIA) and given four weeks to complete the survey.
T30965 392975-393117 Sentence denotes Data collected included participant demographics, scope of testing, software capabilities for the LIS and molecular instruments, and comments.
T9047 393118-393126 Sentence denotes Results:
T33214 393127-393174 Sentence denotes Eighty respondents completed the entire survey.
T17293 393175-393302 Sentence denotes LISs included basic anatomic pathology (26), custom (20), basic clinical pathology (19) and molecularspecific LIS modules (14).
T16514 393303-393381 Sentence denotes 55% of respondents could not record nucleic acid extraction data into the LIS.
T77217 393382-393434 Sentence denotes In contrast, 87.5% listed this ability as important.
T95063 393435-393538 Sentence denotes For sign-out, 65.0% manually type the result into the LIS because no electronic transfer was available.
T11577 393539-393630 Sentence denotes Only 12.5% reported full electronic transmission of results from the instrument to the LIS.
T28509 393631-393683 Sentence denotes Conversely, 96.3% listed this function as important.
T31973 393684-393796 Sentence denotes HIPAA requires that any software with identifiable patient information have audit trails and unique user logons.
T52487 393797-393957 Sentence denotes Only 68.8% of respondents reported ability to access audit trails in the LIS, with the remainder stating that they had to use other mechanisms to get this data.
T11080 393958-394155 Sentence denotes Strikingly, only 16.3% of participants stated that unique user logins were required in all instruments, whereas 41.3% and 42.5% reported unique user logins in some and no instruments, respectively.
T72669 394156-394225 Sentence denotes Conversely, 100% of respondents listed HIPAA compliance as important.
T92106 394226-394238 Sentence denotes Conclusions:
T60522 394239-394333 Sentence denotes The respondents indicated that many basic functionalities are lacking in the majority of LISs.
T90928 394334-394568 Sentence denotes Further, because of the lack of reported access to audit trails and enforcement of unique user logins, these results raise concerns that both molecular laboratory instruments and LISs are not compliant with federal HIPAA security law.
T39195 394569-394779 Sentence denotes Collaboration between molecular professionals and software vendors is necessary to correct these major deficits and is critically important to ensure the continued high quality and safety of molecular practice.
T44723 394780-394793 Sentence denotes Introduction:
T93246 394794-395135 Sentence denotes Current primer trimming approaches in amplicon next-generation sequencing (NGS) data is a double-edged sword, since to minimize variant allele frequency (VAF) dilution effect to mutations at primer binding sites through trimming may compromise the detection of mutations near primer binding sites due to nonalignment at the edge of amplicon.
T90326 395136-395289 Sentence denotes Since the current approaches work in prealignment level, both pre-trimming and post-trimming NGS reads of a given sample need to be analyzed in parallel.
T13627 395290-395416 Sentence denotes The effort in analysis and interpretation is doubled for optimal detection of mutations both at and near primer binding sites.
T76847 395417-395425 Sentence denotes Methods:
T14334 395426-395591 Sentence denotes We developed a post-alignment primer trimming bioinformatics algorithm BAMClipper, which trims primer sequence by soft-clipping BAM alignments of original NGS reads.
T6282 395592-395742 Sentence denotes VAF dilution effect and alignment edge effect are minimized simultaneously to cater for mutations both at and near primer binding sites, respectively.
T42562 395743-395913 Sentence denotes The Perl-based algorithm reads and writes in standard BAM alignment format and needs only primer genomic positions that are commonly provided by commercial panel vendors.
T85045 395914-396055 Sentence denotes It is independent of gene panel or organism and can be readily incorporated into any bioinformatics pipeline without any compilation process.
T17431 396056-396169 Sentence denotes We evaluated BAMClipper with Illumina MiSeq sequencing reads of QIAGEN GeneRead DNAseq Human Breast Cancer Panel.
T1019 396170-396296 Sentence denotes Frozen tissue sample of 6 breast cancer patients with a known germline BRCA1/BRCA2 mutation was analyzed by the 44-gene panel.
T91993 396297-396426 Sentence denotes Original and BAMClipper-processed BAM alignments were processed by the same bioinformatics workflow on a Cray XC30 supercomputer.
T26636 396427-396467 Sentence denotes VAF of the known mutation were compared.
T90260 396468-396476 Sentence denotes Results:
T94391 396477-396636 Sentence denotes Among the 6 known BRCA1/BRCA2 mutations, 5 mutations were overlapped with primer binding sites of nearby amplicons and thus susceptible to VAF dilution effect.
T21295 396637-396697 Sentence denotes VAF was underestimated by 16% to 82% before primer trimming.
T20825 396698-396887 Sentence denotes In an extreme case, VAF was 9% before trimming (51% after trimming) to the extent that common germline mutation callers could miss the heterozygous mutation due to low VAF (false negative).
T94561 396888-396946 Sentence denotes The remaining mutation was not near or at any primer site.
T98189 396947-396985 Sentence denotes Trimming did not affect its VAF (51%).
T15301 396986-396998 Sentence denotes Conclusions:
T19143 396999-397208 Sentence denotes Primer trimming at post-alignment level restored otherwise underestimated VAF of mutations at primer binding sites, without compromising detection of any mutations near the sites due to alignment edge effects.
T80848 397209-397356 Sentence denotes Unnecessarily duplicated computational analysis and human interpretation efforts can be avoided while mutation detection performance is maintained.
T45429 397357-397520 Sentence denotes The algorithm is particularly relevant to clinical molecular pathology laboratories utilizing amplicon-based NGS panel for germline and somatic mutation detection.
T34637 397521-397534 Sentence denotes Introduction:
T15181 397535-397655 Sentence denotes Screening of polymorphisms in PCR primer binding sites is essential during evaluation of any PCR-based diagnostic assay.
T9933 397656-397805 Sentence denotes Otherwise, sequence mismatches between primer and template may result in sub-optimal PCR reaction and potentially false positive or negative results.
T54758 397806-398091 Sentence denotes Although public catalogs of human variation data (e.g. 1000 Genomes Project Phase 3 (1KG) and Exome Aggregation Consortium (ExAC)) and possibly in-house catalogs are rapidly growing, there is a lack of high-throughput tool for laboratories to evaluate hundreds of primer pairs at once.
T90499 398092-398235 Sentence denotes The popular online tool SNPCheck does not support the latest public catalogs and it is also recently announced that the service will wind down.
T93702 398236-398244 Sentence denotes Methods:
T45237 398245-398362 Sentence denotes We developed a user-friendly bioinformatics tool PanelQC for polymorphism screening of multiple primer pairs at once.
T97381 398363-398480 Sentence denotes In addition to the latest 1KG and ExAC catalogs, it also supports any additional catalogs in the standard VCF format.
T7819 398481-398567 Sentence denotes Laboratories can keep the tool on premises to utilize their in-house private catalogs.
T24313 398568-398709 Sentence denotes Allele frequency threshold (e.g. 5%) can be configured to flag any primers potentially affected by polymorphisms within primer binding sites.
T39747 398710-398879 Sentence denotes Various in-house and commercial PCR primer sequences were evaluated by PanelQC with 10% 1KG global minor allele frequency (GMAF) as flagging threshold for demonstration.
T82641 398880-398888 Sentence denotes Results:
T64182 398889-399064 Sentence denotes We tested PanelQC with 296 pairs of laboratory developed assay primers in our ISO15189 accredited molecular pathology laboratory as part of internal regular checking exercise.
T54278 399065-399117 Sentence denotes The automated screening was completed in 15 seconds.
T17283 399118-399307 Sentence denotes We also performed a preliminary screening of a commercial next-generation sequencing (NGS) gene panel (QIAGEN GeneRead DNAseq Human Breast Cancer Panel) and PanelQC completed in 11 minutes.
T85965 399308-399449 Sentence denotes The 44-gene panel comprised 2915 amplicons, of which 121 amplicons (4%) was potentially affected by at least one polymorphism with GMAF >10%.
T85539 399450-399671 Sentence denotes However, whether or how these polymorphisms affect the PCR targeting efficiency remained to be clarified because presence of any countermeasure in the actual reagent was unknown (e.g. degenerate base, additional primers).
T69990 399672-399684 Sentence denotes Conclusions:
T64458 399685-399787 Sentence denotes A new tool PanelQC was developed for efficient screening of polymorphism in multiple PCR primer pairs.
T80236 399788-400063 Sentence denotes As highly multiplexed PCR primer panels (e.g. commercially available NGS gene panels) become increasingly popular, the tool should be particularly useful to facilitate comprehensive polymorphism screening in thousands of primers for high-quality molecular diagnostic service.
T40439 400064-400077 Sentence denotes Introduction:
T16467 400078-400250 Sentence denotes Performance evaluation of next-generation sequencing (NGS)-based clinical molecular diagnostics needs reference materials that are sustainable, comprehensive and realistic.
T17145 400251-400444 Sentence denotes In silico simulation of NGS data with the target mutation profile is a complement (but not replacement) to fully experimental approaches, particularly for the bioinformatics part of evaluation.
T24327 400445-400581 Sentence denotes Full simulation of NGS data from scratch is sustainable and comprehensive but requires panel-specific data modeling to become realistic.
T8690 400582-400777 Sentence denotes Although semi-simulation approach (in silico adding mutations to real NGS reads) looks more flexible and realistic, there are few available and userfriendly tools to perform such semi-simulation.
T56785 400778-400786 Sentence denotes Methods:
T35019 400787-400871 Sentence denotes We developed an easy-touse bioinformatics tool MutationEngineer for semi-simulation.
T11721 400872-401019 Sentence denotes Given a mutation list (in standard VCF format) and BAM alignments of any sample, the tool will return modified BAM alignments with mutations added.
T85396 401020-401146 Sentence denotes Resulting BAM alignments per se or FASTQ reads after back-conversion can be fed into any bioinformatics pipelines of interest.
T43069 401147-401230 Sentence denotes Optionally locus sequencing depth and mutation allele frequency (AF) can be chosen.
T45659 401231-401360 Sentence denotes The tool written in Perl was tested to be compatible with standard Linux system or a Cray XC30 supercomputer without compilation.
T28342 401361-401566 Sentence denotes Semi-simulation of mutations at various depths and AF was performed for a hereditary breast and/or ovarian cancer (HBOC) gene panel (germline mutation) and a myeloid neoplasm gene panel (somatic mutation).
T58381 401567-401575 Sentence denotes Results:
T95624 401576-401755 Sentence denotes We applied semi-simulation on the 4-gene HBOC panel data (Illumina MiSeq) of a breast cancer patient with known mutation status by Sanger sequencing of BRCA1 and BRCA2 full genes.
T78482 401756-401980 Sentence denotes For a panel of 4 mutations (BRCA1, BRCA2, TP53 and PTEN), 24 sets of FASTQ reads are semisimulated corresponding to 4 different depths (20X, 50X, 100X and 200X) and 6 different mutation AF (20%, 25%, 30%, 40%, 50% and 100%).
T54215 401981-402098 Sentence denotes In-house bioinformatics pipeline for clinical reporting detected all 4 mutations in all tested depth-AF combinations.
T73679 402099-402255 Sentence denotes Likewise, JAK2 V617F semi-simulation was performed on the 54-gene myeloid panel data (Illumina MiSeq) of a healthy adult with normal complete blood profile.
T55820 402256-402350 Sentence denotes In-house bioinformatics pipeline detected the mutation in all 18 depth-AF combinations (depth:
T70890 402351-402372 Sentence denotes 100X, 200X, 500X; AF:
T77571 402373-402407 Sentence denotes 10%, 20%, 30%, 40%, 50% and 100%).
T58912 402408-402420 Sentence denotes Conclusions:
T642 402421-402560 Sentence denotes Mutation semi-simulation was demonstrated to be a feasible approach in evaluating bioinformatics aspect of NGS-based molecular diagnostics.
T90645 402561-402701 Sentence denotes MutationEngineer facilitates molecular pathology laboratories to scale up evaluation to a wider mutation spectrum and depth-AF combinations.
T910 402702-402811 Sentence denotes Here we aim to assess the prognostic utility of mutational signatures in ovarian high grade serous carcinoma.
T6154 402812-402820 Sentence denotes Methods:
T87768 402821-403108 Sentence denotes Open access whole exome sequencing data of 15,439 somatic single nucleotide variants of 310 ovarian high grade serous carcinomas from The Cancer Genome Atlas (TCGA) are used to construct a Bayesian model to classify each cancer as either having or lacking a BRCA1/2 mutational signature.
T14818 403109-403324 Sentence denotes We evaluate the association of the BRCA1/2 signature with overall survival on the TCGA dataset and on an independent cohort of 92 ovarian high grade serous carcinomas from the Australian Ovarian Cancer Study (AOCS).
T26089 403325-403656 Sentence denotes Results: BRCA1/2 mutated ovarian carcinomas exhibited a distinctive mutational signature, characterized by a decreased enrichment of C>T transitions at CpG sites compared to BRCA1/2 wildtype carcinomas (11.5% versus 17.8%, p = 0.0004) as well as a relative increase of T>A mutations at GpTpC sites (0.81% versus 0.33%, p = 0.0001).
T24996 403657-403885 Sentence denotes Patients from TCGA with tumors harboring a BRCA1/2 mutational signature have improved survival (57.8 months versus 38.0 months), which is independent of BRCA1/2 gene mutation status, age, stage and grade (HR = 0.52, p = 0.0003).
T19622 403886-404066 Sentence denotes In the AOCS dataset, the BRCA1/2mutational signature is also associated with improved overall survival (46.7 months versus 22.7 months) independent of stage (HR = 0.50, p = 0.004).
T41427 404067-404079 Sentence denotes Conclusions:
T7018 404080-404173 Sentence denotes A BRCA1/2 mutational signature is a prognostic marker in ovarian high grade serous carcinoma.
T60740 404174-404289 Sentence denotes Mutational signature analysis of ovarian cancer genomes may be useful in addition to testing for BRCA1/2 mutations.
T95097 404290-404393 Sentence denotes These analytical methods may expand the clinical indication for the broad sequencing of cancer genomes.
T47272 404394-404407 Sentence denotes Introduction:
T26533 404408-404548 Sentence denotes Germline and somatic mutations in the BRCA1 and BRCA2 genes are highly involved in hereditary and non-hereditary breast and ovarian cancers.
T21148 404549-404707 Sentence denotes A test that detects these mutations from clinically relevant FFPE samples is tremendously valuable for both research and future clinical diagnostics purposes.
T64773 404708-404861 Sentence denotes Large rearrangements (LRs) represent an important portion of BRCA1/2 mutations, in addition to single nucleotide mutations and small insertion/deletions.
T94374 404862-405048 Sentence denotes The sizes of LRs make them difficult to detect using traditional sequencing approaches thereby requiring additional tests such as multiplex ligation dependent probe amplification (MLPA).
T19860 405049-405273 Sentence denotes Recent reports showing feasibility using amplicon-based massively parallel sequencing methods to detect LRs either were not designed for use with FFPE samples, or lack data analysis methods optimized for such an application.
T88909 405274-405402 Sentence denotes We have developed an amplicon-based NGS approach* for FFPE samples that can detect SNVs, small mutations and LRs simultaneously.
T88634 405403-405543 Sentence denotes We have implemented a comprehensive bioinformatics algorithm that detects LRs at high sensitivity, even in the absence of control sample(s).
T83782 405544-405619 Sentence denotes This significantly reduces the cost and labor for BRCA1/2 genetic analyses.
T20673 405620-405628 Sentence denotes Methods:
T86219 405629-405764 Sentence denotes Ion AmpliSeq, a targeted, multiplexed amplification technology was used in combination with the Ion PGM and Ion S5 sequencing platform.
T48001 405765-405871 Sentence denotes An assay was designed to cover 100% of all exons of BRCA1 and BRCA2 with 263 amplicons (targeted regions).
T61952 405872-405962 Sentence denotes Torrent Suite Server and Torrent Variant Caller were used for analysis of small mutations.
T9004 405963-406164 Sentence denotes For LRs, a standalone algorithm was developed that includes custom normalization, detection of whole gene deletions, sample and run quality control metrics as well as exon-level copy number detections.
T66861 406165-406281 Sentence denotes NGS data from libraries made from cell line genomic DNA and FFPE derived DNA was used to train and verify the assay.
T15384 406282-406414 Sentence denotes To test this approach, NGS data was generated from an early access study comprising more than 20 independent research organizations.
T91265 406415-406501 Sentence denotes At least 4 sites included samples with LRs that were previously characterized by MPLA.
T99302 406502-406510 Sentence denotes Results:
T34055 406511-406648 Sentence denotes We demonstrate high sensitivity and specificity for both LR and small mutation detection from cell line, blood and research FFPE samples.
T75981 406649-406848 Sentence denotes We detected a range of LRs, including heterozygous whole gene deletions, single and multiple exon heterozygous deletions, single and multiple exon duplications, and homozygous multiple exon deletion.
T53042 406849-406861 Sentence denotes Conclusions:
T50816 406862-407055 Sentence denotes We have developed an NGS assay and comprehensive data analysis approach capable of detecting both small mutations and LRs simultaneously from FFPE samples with high sensitivity and specificity.
T51670 407056-407078 Sentence denotes For research use only.
T38332 407079-407116 Sentence denotes Not for use in diagnostic procedures.
T97206 407117-407119 Sentence denotes A.
T66286 407120-407140 Sentence denotes Momeni Boroujeni, E.
T54862 407141-407152 Sentence denotes Yousefi, R.
T78499 407153-407166 Sentence denotes Maglantay, R.
T96401 407167-407176 Sentence denotes Gupta, N.
T18419 407177-407250 Sentence denotes Chen State University of New York Downstate Medical Center, Brooklyn, NY.
T46521 407251-407314 Sentence denotes Introduction: FSHD Region Gene 1 is located on chromosome 4q35.
T65717 407315-407388 Sentence denotes This gene is implicated in facioscapulohumeral muscular dystrophy (FSHD).
T936 407389-407497 Sentence denotes The protein product of the gene is believed to play a role in spliceosome formation and pre-mRNA processing.
T21535 407498-407580 Sentence denotes FRG1BP is the pseudogene counterpart of FRG1 which is located on chromosome 20q11.
T6427 407581-407640 Sentence denotes We have evaluated mutations in FRG1BP in different cancers.
T31063 407641-407649 Sentence denotes Methods:
T23820 407650-407756 Sentence denotes Mutational and copy number alteration data from 814 cancer patients were extracted from the TCGA database.
T64624 407757-407850 Sentence denotes The frequency of mutation and copy number alterations of FRG1BP was evaluated in these cases.
T39108 407851-408006 Sentence denotes We used subnetwork analysis, network power analysis to determine the main subnetworks within the mutational profiles of different cancers among our sample.
T56534 408007-408094 Sentence denotes We further performed mutual exclusion analysis between genes implicated in spliceosome.
T91937 408095-408103 Sentence denotes Results:
T82578 408104-408352 Sentence denotes Our results show that FRG1BP is mutated or has copy number alterations in a significant percentage of chromophobe renal cell carcinoma, uterine carcinosarcoma, papillary renal cell carcinoma, adrenocortical carcinoma, pheochromocytoma and melanoma.
T1354 408353-408529 Sentence denotes Subcluster analysis shows FRG1BP is mutated in a significant sublcluster of patients which have unique mutational profiles when compared to other patients with the same cancer.
T34749 408530-408681 Sentence denotes Power centrality analysis showed that mutations in this gene have a power centrality (2.97) comparable to PTEN and PIK3CA (2.38 and 2.31 respectively).
T63184 408682-408791 Sentence denotes We also demonstrated that alterations of FRG1BP are mutually exclusive from FRG1, PRPF19 and CDC40 (p<0.001).
T70338 408792-408803 Sentence denotes Conclusion:
T47709 408804-408885 Sentence denotes To date a comprehensive review of the mutations of FRG1BP has not been performed.
T64755 408886-409028 Sentence denotes Our study shows that FRG1BP is mutated in a considerable subcluster of cancer patients and it appears to be an important gene in some cancers.
T53509 409029-409136 Sentence denotes Our mutual exclusiveness test shows that FRG1BP is likely to play a role in spliceosome formation pathways.
T16495 409137-409324 Sentence denotes Further investigation of FRG1BP's function and role in cancer pathogenesis is needed. has the potential to benefit public health by facilitating the early detection of pathogen outbreaks.
T91996 409325-409486 Sentence denotes Efficient tracking of ID requires 1) broadly-distributed, comprehensive diagnostic testing and 2) rapid electronic collection, analysis and distribution of data.
T32120 409487-409657 Sentence denotes Several FDA-cleared diagnostic platforms are in use in clinical laboratories, which have assays for many of the large groups of infectious agents known to cause diarrhea.
T777 409658-409702 Sentence denotes BioFire's FilmArray (FA) is one such system.
T16200 409703-409740 Sentence denotes The FA GI panel detects 22 pathogens.
T6333 409741-409878 Sentence denotes Whereas the first condition for tracking GI disease has been met, the second condition has not; there is no general, automated electronic
T17760 409879-410020 Sentence denotes The Journal of Molecular Diagnostics ■ jmd.amjpathol.org mechanism for aggregating GI test results from across the United States in realtime.
T76927 410021-410029 Sentence denotes Methods:
T58033 410030-410095 Sentence denotes We have implemented a cloud-based epidemiology network, FA-Trend.
T56344 410096-410254 Sentence denotes The system connects FA Instruments directly to the cloud, automatically exporting electronic de-identified test results to a secure, HIPAA-compliant database.
T81466 410255-410479 Sentence denotes Web-based views of the aggregated data are accessible to various user groups: clinical users can track institutional and local trends, and the public can monitor bacteria, viruses and parasites causing infectious GI disease.
T89563 410480-410621 Sentence denotes This automated approach does not require labor intensive manual processing or data extraction from information systems that vary by hospital.
T90336 410622-410630 Sentence denotes Results:
T96042 410631-410903 Sentence denotes Four US sites are participants in the initial GI FA Trend pilot study, Medical University of South Carolina (SC), Primary Children's Medical Center (UT), UC San Diego Medical Center (CA), and NYU Langone Medical Center (NY), with more than 15 FA Instruments in the cohort.
T53496 410904-411083 Sentence denotes Automated export of the electronic results and archival data began in the fall of 2015 and collectively the group will contribute over 4,000 test results to the project this year.
T50636 411084-411112 Sentence denotes Data presented will include:
T56124 411113-411441 Sentence denotes 1) GI pathogen prevalence, including fluctuations in the prevalence of diarrheagenic E. coli/Shigella among other organisms; 2) Polymicrobial detections, which indicate, at the population level, interactions between pathogens that occur in a patient; 3) Rotavirus prevalence trends compared to CDC NREVSS Rotavirus surveillance.
T24604 411442-411454 Sentence denotes Conclusions:
T69352 411455-411696 Sentence denotes We demonstrate that, when appropriate care is taken to remove protected health information from ID test results, it is possible to address hospital data security concerns and patient privacy issues involving real-time, automated data export.
T95468 411697-411848 Sentence denotes With this infrastructure in place it is straightforward to connect FA Instruments directly to the internet and export deidentified GI pathogen results.
T87990 411849-411963 Sentence denotes The resulting data stream gives us unprecedented visibility of the prevalence and spread of GI infectious disease.
T59957 411964-412191 Sentence denotes The application of DNA sequencing in clinical cancer genomics has proven to be an invaluable tool to characterize acquired tumor mutational load, inform therapeutic routes and to predict treatment response in oncology patients.
T17483 412192-412475 Sentence denotes Despite these powerful benefits, this approach lacks the ability to quantitatively characterize downstream gene expression levels or capture non-genomic transcript variation-two important avenues currently under characterized in next-generation sequencing based clinical diagnostics.
T82300 412476-412702 Sentence denotes RNA sequencing (RNAseq) addresses these issues by quantitatively capturing the entirely of the expressed transcripts in a tissue, while additionally detecting all of the expressed genomic alterations as seen in DNA sequencing.
T839 412703-412930 Sentence denotes One of the barriers to its implementation in a clinical setting is the availability of computational pipelines and bioinformatics tools to comprehensively characterize the broad range of variation present in transcriptome data.
T36531 412931-412939 Sentence denotes Methods:
T19449 412940-413208 Sentence denotes We have developed a cloud-based RNAseq computational workflow to detect expressed variation including SNVs, INDELs, structural variants, gene fusions, differential gene expression, allele specific expression, and novel alternative allele isoforms in a single pipeline.
T34041 413209-413217 Sentence denotes Results:
T92856 413218-413346 Sentence denotes We apply our method to characterize the transcriptomes of pediatric sarcoma and lung adenocarcinoma to identify novel variation.
T46336 413347-413511 Sentence denotes Further, we demonstrate that our approach compares favorably to DNA based sequencing methods when performing variant calling on somatic patient samples.Conclusions:
T30950 413512-413779 Sentence denotes Our cloud-based transcriptome profiling pipeline serves as a proof-of-principle for RNA-guided comprehensive tumor profiling, and may serve as a parallel bioinformatic avenue to analyze, interpret, and apply genomic information for personalized oncology patient care.
T42353 413780-413797 Sentence denotes J. van Riet, N.M.
T82870 413798-413808 Sentence denotes Krol, P.N.
T50088 413809-413826 Sentence denotes Atmodimedjo, J.W.
T97914 413827-413840 Sentence denotes Martens, L.H.
T10715 413841-413854 Sentence denotes Looijenga, G.
T52924 413855-413868 Sentence denotes Jenster, H.J.
T74962 413869-413880 Sentence denotes Dubbink, W.
T71668 413881-413961 Sentence denotes Dinjens, H.W. van de Werken Erasmus MC Cancer Institute, Rotterdam, Netherlands.
T37644 413962-413975 Sentence denotes Introduction:
T45976 413976-414177 Sentence denotes Exploration and visualization of next-generation sequencing (NGS) data originating from targeted multi-gene panels is crucial for analysis of genetic aberrations in both research and clinical settings.
T89026 414178-414355 Sentence denotes However, software for simple, robust and dynamic web-based visualization of single nucleotide polymorphisms (SNPs) in the regions of these targeted multi-gene panels is lacking.
T50059 414356-414544 Sentence denotes Methods: we developed a lightweight Shiny-based web-application, called SNPitty, for interactive visualization and interrogation of single-and multi-sample Variant Call Format (VCF) files.
T77031 414545-414716 Sentence denotes SNPitty is best applicable with data from NGS targeted multi-gene panels to display loss of heterozygosity (LOH) and copy number variation of genetic heterozygous markers.
T50268 414717-414857 Sentence denotes Moreover, SNPitty is capable of generating predefined reports which summarize and highlight the targets-of-interest based on LaTeXtemplates.
T93184 414858-414866 Sentence denotes Results:
T82782 414867-414972 Sentence denotes Here, we apply SNPitty on patient-derived tumor-only glioblastomas (n=3) to assess LOH and amplification.
T30548 414973-415138 Sentence denotes DNA was sequenced on an Ion-Torrent platform using a diagnostic multi-gene panel targeting known genetic aberrations associated with tumor formation and progression.
T22267 415139-415192 Sentence denotes VCFs were subsequently generated using Torrent Suite.
T64653 415193-415332 Sentence denotes Amplification and LOH in regions for known genetic aberrations were clearly indicated for the glioblastoma samples and reported by SNPitty.
T46515 415333-415409 Sentence denotes Conclusion: SNPitty can be used freely at https://ccbc.erasmusmc.nl/SNPitty.
T20250 415410-415573 Sentence denotes Source code and documentation for SNPitty can be obtained under the terms of the GPL-3 open-source license through BitBucket at https://bitbucket.org/ccbc/snpitty.
T52608 415574-415587 Sentence denotes Introduction:
T47775 415588-415779 Sentence denotes Karyotyping, the practice of visually analyzing and recording chromosomal abnormalities, is commonly used to diagnose and determine treatment strategies for patients with malignant neoplasms.
T42260 415780-415907 Sentence denotes Karyotypes are written in the International System for Human Cytogenetic Nomenclature (ISCN), which has a linguistic structure.
T80277 415908-416093 Sentence denotes Thus, assessing karyotypes to identify recurrent cytogenetic abnormalities is performed in a non-computational manner, by visually inspecting long strings of often complex nomenclature.
T51734 416094-416275 Sentence denotes Because ISCN is currently computationally intractable, much of the genomic data stored in tens of thousands of karyotypes remain inaccessible, and potential applications unrealized.
T15419 416276-416448 Sentence denotes To mine these data, we have developed a cytogenetic platform (CytoGPS) that transforms humanreadable ISCN karyotypes into a machine-readable model (Loss-Gain-Fusion [LGF]).
T56973 416449-416663 Sentence denotes As proof of principle, we analyzed karyotype data for chronic lymphocytic leukemia (CLL) in the Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer (http://cgap.nci.nih.gov/Chromosomes/Mitelman).
T64466 416664-416752 Sentence denotes We chose CLL because it contains recurrent, clinically relevant cytogenetic aberrations.
T23414 416753-416863 Sentence denotes This provides us with known aberrations, as well as the opportunity to discover novel cytogenetic aberrations.
T86026 416864-416872 Sentence denotes Methods:
T20457 416873-416996 Sentence denotes Parsing and mapping of ISCN karyotypes was performed in CytoGPS using grammatical rules for writing karyotypes in the ISCN.
T71790 416997-417152 Sentence denotes Applying these rules, we extracted two key pieces of information: the location of the band where an event occurred and the biological result of that event.
T66191 417153-417235 Sentence denotes We classified all events as having three possible outcomes: loss, gain, or fusion.
T87097 417236-417349 Sentence denotes Thus, each ISCN text-based karyotype is represented as a binary vector, to which cluster analysis can be applied.
T78739 417350-417358 Sentence denotes Results:
T80333 417359-417467 Sentence denotes We applied CytoGPS to parse 2051 CLL karyotypes from the Mitelman database into the LGF binary vector model.
T52662 417468-417560 Sentence denotes We then performed cluster analysis on the binary vector karyotypes obtained from these data.
T81199 417561-417698 Sentence denotes We recovered the known recurrent cytogenetic abnormalities in CLL: trisomy 12, deletion 13q, deletion 11q, deletion 17p, and deletion 6q.
T92460 417699-417850 Sentence denotes We also discovered many unusual and novel aberrations, including the co-occurrence of trisomies 18 and 19 and the cooccurrence of monosomies 21 and 22.
T2368 417851-417863 Sentence denotes Conclusions:
T45279 417864-418004 Sentence denotes Karyotype data, which contain clinically important genomic information, are difficult to mine because they are not computationally readable.
T96004 418005-418205 Sentence denotes We describe a method to translate text-based karyotypes into a computationally usable binary vector that retains the biological meaning of the karyotype while facilitating modern informatics analyses.
T32435 418206-418317 Sentence denotes This model will enable researchers to use karyotype data in computational studies in a new and powerful manner.
T2111 418318-418331 Sentence denotes Introduction:
T76712 418332-418505 Sentence denotes Polymerase chain reaction (PCR) followed by high-resolution melting (HRM) of DNA samples is a simple, cost-effective, fast, and powerful, method for genotyping applications.
T61819 418506-418705 Sentence denotes With increased knowledge about DNA sequence variation in genetic targets and the ability to perform high throughput DNA testing, the amount of data is becoming overwhelming for manual interpretation.
T75994 418706-418871 Sentence denotes Automated HRM analysis software has thus emerged as an effective solution toward either assisting a user make the genotyping call or replacing user's interpretation.
T11759 418872-419033 Sentence denotes The goal of this investigation was to develop a novel, scalable software solution for automated genotyping and to assess the genotyping accuracy of the software.
T46001 419034-419042 Sentence denotes Methods:
T84857 419043-419133 Sentence denotes We prototyped a novel software solution to perform automated genotyping based on HRM data.
T27493 419134-419298 Sentence denotes The software is based on nucleic acid thermodynamics theory and apriori information, and was designed so that no user input or manual data manipulation is required.
T26815 419299-419397 Sentence denotes It can operate without any algorithm modification for either small-amplicon or probe-based assays.
T4916 419398-419545 Sentence denotes The software requires a-priori information that is specific to the target being tested as well as melting curve data of a control wild-type sample.
T98793 419546-419722 Sentence denotes A companion software application was developed to allow a user to easily generate the necessary a-priori information for any new HRM assay using a training set of melting data.
T51116 419723-419887 Sentence denotes In this investigation, we performed accuracy testing of the prototype software on two HRM assays: a) small-amplicon MTHFR A1298C and b) probe-based CYP2C9*2 assays.
T15088 419888-420117 Sentence denotes For each assay, a set of melting curves representative of all three genotypes, wild-type (WT), heterozygous (HET), and homogeneous (HOM), as determined by an expert in HRM analysis was collected and used for testing the software.
T17803 420118-420242 Sentence denotes The software genotyping accuracy was derived by comparing the software results with the genotyping calls made by the expert.
T29890 420243-420251 Sentence denotes Results:
T57939 420252-420439 Sentence denotes We collected a) 102 melting curves for MTHFR A1298C consisting of 43 WT, 34 HET, and 25 HOM samples, and b) 50 melting curves for CYP2C9*2 consisting of 25 WT, 15 HET, and 10 HOM samples.
T78580 420440-420525 Sentence denotes All sample melting curve data were acquired on Canon's prototype genotyping platform.
T21810 420526-420593 Sentence denotes For both assays, our software resulted in 100% genotyping accuracy.
T64287 420594-420606 Sentence denotes Conclusions:
T49545 420607-420715 Sentence denotes A novel, scalable software solution to perform automated genotyping based on HRM curves has been prototyped.
T22816 420716-420885 Sentence denotes Results revealed excellent genotyping accuracy of our software and further demonstrated that the underlying methods and mechanics of our software are sound and reliable.
T83908 420886-420890 Sentence denotes J.D.
T75908 420891-420905 Sentence denotes Peterson, C.I.
T87871 420906-420931 Sentence denotes Amos, F.B. de Abreu, W.A.
T86508 420932-420943 Sentence denotes Wells, G.J.
T69309 420944-421061 Sentence denotes Tsongalis Dartmouth Hitchcock Medical Center, Geisel School of Medicine and Norris Cotton Cancer Center, Lebanon, NH.
T94404 421062-421075 Sentence denotes Introduction:
T11177 421076-421276 Sentence denotes In recent years the NGS sample preparation and sequencing processes have become increasingly streamlined, whereas converting raw sequencing data into reportable results remains highly labor intensive.
T85661 421277-421435 Sentence denotes For this reason, automating and integrating the data analysis and variant interpretation processes can be extremely effective in reducing total analysis time.
T96653 421436-421623 Sentence denotes CLC Biomedical Genomics Workbench (BMGW) is a comprehensive NGS data analysis platform designed to create automated bioinformatic workflows for a wide spectrum of sequencing applications.
T74469 421624-421826 Sentence denotes Here we describe a custom BMGW pipeline for analyzing data generated using the Ion Torrent Cancer Hotspot Panel v2, and compare the results with our clinically validated in-house bioinformatic pipeline.
T77937 421827-421986 Sentence denotes Methods: FASTQ files from 30 FFPE samples previously analyzed using our inhouse bioinformatic pipeline were re-processed using a custom BMGW analysis pipeline.
T1093 421987-422128 Sentence denotes Our in-house pipeline utilizes Torrent Suite (v4.0) for alignment and variant calling, and Golden Helix SVS (v.8.3.4) for variant annotation.
T61994 422129-422282 Sentence denotes The BMGW analysis pipeline includes read mapping (hg19), local-realignment and variant calling, and variant annotation from a number of public databases.
T97758 422283-422522 Sentence denotes Identified variants were subjected to a multi-tier filtering process of varying stringency designed to isolate high-quality clinically significant variants passing the minimum validated thresholds (>500x coverage, >5.0% allelic frequency).
T95655 422523-422672 Sentence denotes The BMGW analysis pipeline also incorporates a module to automatically upload filtered VCF files to QCI-interpret for variant curation and reporting.
T43371 422673-422681 Sentence denotes Results:
T57500 422682-422847 Sentence denotes For the 30 FFPE samples, we observed high concordance in total variants called between the custom BMGW and in-house bioinformatics pipeline (>98.0%, 50/51 variants).
T4705 422848-422954 Sentence denotes One point mutation was identified using our in-house analysis pipeline that was not called using the BMGW.
T66441 422955-423070 Sentence denotes In addition, one 3bp deletion was detected using the BMGW pipeline that was not called using our in-house pipeline.
T36775 423071-423197 Sentence denotes Overall, the custom BMGW pipeline showed superior accuracy in annotating complex variants (INDELs and compound substitutions).
T89730 423198-423210 Sentence denotes Conclusions:
T4626 423211-423464 Sentence denotes As laboratories are faced with increasing pressure to both maintain testing turn-around-times, and increase the amount of clinically relevant data that is reported to clinicians, creating highly efficient analytical processes is more critical than ever.
T12109 423465-423598 Sentence denotes The BMGW is a powerful analysis platform for creating automated NGS bioinformatic pipelines for a variety of sequencing applications.
T87022 423599-423818 Sentence denotes The ability to consolidate the data analysis and interpretation processes, while maintaining sensitivity and accuracy in variant calling, suggest it is ideally suited for integration into a clinical testing environment.
T25462 423819-423832 Sentence denotes Introduction:
T31867 423833-423969 Sentence denotes Pathology reports in narrative text format require a primarily manual abstraction of key data elements for entry into cancer registries.
T17379 423970-424156 Sentence denotes Due to delays in the timely receipt of reports and the need for manual abstraction, cancer surveillance reports are often released years after scientific and care standards have changed.
T58402 424157-424505 Sentence denotes This motivated the College of American Pathologists (CAP) and the California Department of Public Health (CDPH) to undertake a pilot project to dramatically reduce the receipt and abstraction time for surgical pathology and biomarker (BMK) data, which currently takes up to 24 months per case, while increasing the quality of the data for analysis.
T307 424506-424703 Sentence denotes The project uses 13 CAP electronic Cancer Checklist (eCC) BMK templates, which follow national standards to provide required data elements (RDEs) required for patient care and accreditation bodies.
T31060 424704-424872 Sentence denotes These templates are used by vendors to standardize the data entry forms used in the pathologist workflow and to standardize the collection and reporting of cancer RDEs.
T56871 424873-425093 Sentence denotes As reporting via cancer BMK templates may soon become standard practice, we describe the experience of the California Cancer Registry (CCR) in using two CAP BMK templates (breast and colon) for structured data reporting.
T99698 425094-425412 Sentence denotes Methods: mTuitive xPert software, using eCC BMK templates, was installed for pathologists at the 10 hospitals within St. Joseph Health (SJH), to create and transmit standardized BMK reports to CCR. mTuitive and Meditech software generated messages containing the eCC BMK RDEs per the NAACCR volume 5 (version 4) guide.
T14270 425413-425529 Sentence denotes Real-time eCC data feeds into the CCR were captured and parsed by the Eureka system (a CCR registry software suite).
T112 425530-425631 Sentence denotes Data presented here were collected from live transmissions to the CCR from February 2015 to May 2016.
T88586 425632-425728 Sentence denotes Results: SJH is now automatically transmitting eCC BMK data daily into Eureka via HL7 messaging.
T69553 425729-425868 Sentence denotes A total of 570 Breast BMK reports from 248 unique patients (range 1 to 4 reports/patient) were received by the CCR during the study period.
T40413 425869-425939 Sentence denotes Analysis of Breast BMK data revealed hormone receptor and HER2 status.
T94770 425940-426072 Sentence denotes 432 of 518 cases tested positive for ER, 376 of 522 cases tested positive for PgR and 38 of 384 cases were positive for HER2 by IHC.
T97716 426073-426127 Sentence denotes A total of 113 colon cancer BMK reports were received.
T53317 426128-426177 Sentence denotes The time course for collection was also analyzed.
T66452 426178-426190 Sentence denotes Conclusions:
T76757 426191-426376 Sentence denotes Automated use of CAP eCC templates for BMK reporting allowed for operational improvement and user satisfaction at SJH and CCR to report and collect standardized cancer case information.
T49417 426377-426823 Sentence denotes The eCC BMK templates facilitate the recording and rapid automated transmission of standardized BMK RDEs, so that inferences from registry data can be drawn in a timely fashion and based upon the most current CAP RDEs. (Lincoln et al., JMD 2015) showing that technically challenging variants for NGS were a substantial fraction of the pathogenic findings in a representative population of 1062 patients receiving a 29-gene hereditary cancer test.
T56804 426824-426982 Sentence denotes We sought to explore this observation in a larger, more diverse data set and to address the challenges these variants pose to test development and validation.
T33494 426983-426991 Sentence denotes Methods:
T15803 426992-427143 Sentence denotes We examined panel test results for over 30,000 patients across 1000 genes associated with cancer, cardiovascular, neurological and pediatric disorders.
T27224 427144-427206 Sentence denotes We examined the Genome in a Bottle (GIAB) dataset (Zook et al.
T366 427207-427330 Sentence denotes Nature Biotech 2014) for the targeted bases of these same genes, and we similarly examined NGS coverage in exome sequences.
T49866 427331-427490 Sentence denotes As samples with "hard" variants are difficult to obtain, we developed large plasmid inserts bearing specific variants engineered into GIAB reference sequences.
T18834 427491-427625 Sentence denotes These were spiked into GIAB genomic DNAs at concentrations that mimic heterozygous changes and sequenced using standard NGS protocols.
T68404 427626-427782 Sentence denotes Multiple assays including linked reads and long-read single molecule sequencing are also being used to improve the GIAB reference data in difficult regions.
T46463 427783-427791 Sentence denotes Results:
T53126 427792-427860 Sentence denotes Consistent with our prior data, challenging variants were prevalent:
T51705 427861-428064 Sentence denotes Of roughly 5000 patients with a pathogenic variant, 2.9% had single-exon CNVs,1.8% a large indel or complex sequence change, and 5.8% a variant in a region of high-GC, low complexity or poor mappability.
T73493 428065-428230 Sentence denotes Even at >250x average depth, 3% to 4% of target bases had low (<20x) coverage in the exome sequences, compared to 0.0% to 0.1% for optimized panels at similar depth.
T63406 428231-428389 Sentence denotes About 20% of targeted bases lie outside the published GIAB highconfidence regions, and, unsurprisingly, very few examples of "hard" GIAB variants are present.
T74291 428390-428509 Sentence denotes However, new draft GIAB data increase the high-confidence regions from 78% to 89% of the genome, improving its utility.
T79956 428510-428726 Sentence denotes Moreover the GIAB samples with engineered fragments generated BAM files resembling those of patients carrying the same events, suggesting these may be a useful tool for test development, optimization, and validation.
T20558 428727-428739 Sentence denotes Conclusions:
T64886 428740-428936 Sentence denotes Most currently published validation studies include few, if any, of the most challenging types of variants, which are indeed diagnostically important and are prevalent across a range of disorders.
T95104 428937-429173 Sentence denotes As standard bioinformatics methods are not designed to detect such variants and standard targeting chemistries provide limited coverage of important genes, laboratories need to do additional work to develop and validate sensitive tests.
T3119 429174-429323 Sentence denotes To help with this, new GIAB data and GIAB samples with engineered variants will be broadly available to the AMP community by the 2016 Annual Meeting.
T633 429324-429337 Sentence denotes Introduction:
T73906 429338-429793 Sentence denotes Minimal residual disease (MRD) monitoring using ultra high sensitive PCR assays for BCR/ABL1 and PML/RARA is critical for the management of patients with chronic myelogenous leukemia, acute promyelocytic leukemia and other hematologic disorders.The laboratory workflows for generation, interpretation, reporting and management of these high-volume assay results are often cumbersome, redundant, and error-prone due to manual transfer of alphanumeric data.
T5182 429794-429932 Sentence denotes Retrospective review of test results is critical for appropriate test selection and clinical interpretation, which adds to the complexity.
T95577 429933-430035 Sentence denotes Using principles of laboratory automation and web technology, we aimed to optimize the assay workflow.
T7238 430036-430044 Sentence denotes Methods:
T18397 430045-430133 Sentence denotes The ABI 7500 real-time PCR module was used for generating the raw quantitative PCR data.
T15675 430134-430274 Sentence denotes A web-based application was developed using ColdFusion and SQL Server 2012 database to automate several components of the existing workflow.
T33396 430275-430419 Sentence denotes Briefly, tab-delimited files, containing the raw data, were exported from the ABI 7500 real-time PCR module and uploaded to the web application.
T97434 430420-430521 Sentence denotes The raw data was processed and presented for review and interpretation via a web user interface (UI).
T97664 430522-430595 Sentence denotes Algorithms for clinical interpretation of test results were incorporated.
T22503 430596-430692 Sentence denotes The application was hosted in our institution's datacenter using appropriate security protocols.
T52340 430693-430701 Sentence denotes Results:
T82527 430702-430838 Sentence denotes This web-application significantly reduced manual transfer of patient and sample information and test results (at least 4 manual steps).
T26950 430839-430993 Sentence denotes Interoperability with our Laboratory Information System (LIS) enabled automated integration of molecular test results with patient and sample information.
T49671 430994-431191 Sentence denotes The interactive UI enabled single view of all historic test results for a given patient in a tabular and graphical format eliminating the need for redundant and time-consuming manual lookup in LIS.
T73538 431192-431341 Sentence denotes Clinical reports in HTML format was generated after review by pathologist and transferred to the LIS using clipboard function for electronic signout.
T85293 431342-431476 Sentence denotes Calculation of clinically reportable information (logarithmic change in transcript levels and International Scale) was also automated.
T55339 431477-431581 Sentence denotes The application was clinically validated using a total of 56 cases and at least 10 data points per case.
T32940 431582-431685 Sentence denotes The validation cohort constituted an appropriate mix of cases with negative, positive and indeterminate
T27511 431686-431756 Sentence denotes The Journal of Molecular Diagnostics ■ jmd.amjpathol.org test results.
T41164 431757-431769 Sentence denotes Conclusions:
T44605 431770-431919 Sentence denotes This web-based application is a user friendly, secure, reliable and efficient tool for management of molecular test results and clinical information.
T76256 431920-432119 Sentence denotes Although significant optimization was achieved, few manual steps could not be eliminated, such as review of printed copy of real-time PCR amplification curves and manual transfer of clinical reports.
T85662 432120-432176 Sentence denotes Further development is underway to address these issues.
T39629 432177-432179 Sentence denotes A.
T810 432180-432200 Sentence denotes Momeni Boroujeni, R.
T79753 432201-432214 Sentence denotes Maglantay, R.
T39920 432215-432289 Sentence denotes Gupta State University of New York Downstate Medical Center, Brooklyn, NY.
T29691 432290-432303 Sentence denotes Introduction:
T29079 432304-432375 Sentence denotes Endometrial carcinomas have two main subtypes: serous and endometrioid.
T40472 432376-432474 Sentence denotes It has been known that serous adenocarcinomas have a worse prognosis than endometrioid carcinomas.
T76352 432475-432555 Sentence denotes They are also believed to be genetically different from endometrioid carcinomas.
T25280 432556-432733 Sentence denotes In this study we aim to cluster the endometrial carcinomas based on their mutational profile and compare the survival statistics of the mutational versus morphologic clustering.
T27004 432734-432742 Sentence denotes Methods:
T87265 432743-432869 Sentence denotes Five hundred and forty-eight cases of endometrial cancers (serous and endometrioid) from The Cancer Genome Atlas were studied.
T37139 432870-432975 Sentence denotes Their survival and mutational data for the most common mutations in endometrial carcinomas was extracted.
T24473 432976-433067 Sentence denotes A network analysis method was used to cluster the cases based on their mutational profiles.
T16581 433068-433260 Sentence denotes The survival of mutational clusters and morphologic clusters were compared using survival statistical tools including Kaplan-Meyer, Log rank and Cox regression tools in R programming language.
T86864 433261-433269 Sentence denotes Results:
T72113 433270-433451 Sentence denotes After adjusting for clinical stage, morphologic clustering of endometrial carcinomas was found to have a better correlation with survival compared to mutational clustering (p value:
T46699 433452-433531 Sentence denotes 0.007) with endometrioid carcinoma having the best prognosis (average survival:
T99801 433532-433618 Sentence denotes 110.55 months) followed by mixed endometrioid and serous carcinomas (average survival:
T35360 433619-433676 Sentence denotes 36.1 months) and serous adenocarcinoma (average survival:
T26477 433677-433691 Sentence denotes 30.09 months).
T44006 433692-433963 Sentence denotes Of note, a subcluster of endometrioid endometrial carcinoma cases with a unique mutational profile (with mutations in PTEN/FGFR2/NRAS/CCND1/CYLC1 and no mutation in PIK3CA and none of type I endometrial carcinoma mutations) have a significantly better prognosis (p value:
T66234 433964-434048 Sentence denotes 0.022) compared to the other endometrioid carcinomas irrespective of clinical stage.
T39922 434049-434115 Sentence denotes Copy number alterations is another predictor of survival (p value:
T22894 434116-434205 Sentence denotes 0.032) whereas overall number of mutations has no meaningful effect on survival (p value:
T573 434206-434213 Sentence denotes 0.102).
T53775 434214-434225 Sentence denotes Conclusion:
T82916 434226-434473 Sentence denotes Our results show that although mutational clustering of endometrial carcinomas is possible, these clusters do not conform to histologic grouping of endometrial carcinomas because of shared mutations between endometrioid and serous adenocarcinomas.
T99768 434474-434559 Sentence denotes Morphologic grouping is a better predictor of outcome compared to genetic clustering.
T39916 434560-434705 Sentence denotes Genetic profiling, however, identified a unique subcluster of endometrioid carcinomas which confer an excellent prognosis, irrespective of stage.
T57662 434706-434719 Sentence denotes Introduction:
T91015 434720-434823 Sentence denotes Proficiency testing (PT) is essential to assess the accuracy and reproducibility of clinical reporting.
T69915 434824-434943 Sentence denotes In 2015, a consortium of 6 academic clinical laboratories was formed to test their respective bioinformatics workflows.
T6885 434944-435101 Sentence denotes Each lab, which performs clinical laboratory developed tests for solid tumors using the same commercial library preparation kit, exchanged 24 FASTQ datasets.
T87419 435102-435290 Sentence denotes As described in a publication earlier this year, 100% concordance in reporting clinically significant single nucleotide variants (SNV) was achieved at all sites who performed the analysis.
T57639 435291-435507 Sentence denotes However, the discordance rate for calling insertion/deletion (indel) mutations was as high as 60% (3/5) for a clinically significant mutation in the EGFR gene and was not uniform among all other submitted challenges.
T1756 435508-435626 Sentence denotes To address this, we chose to re-challenge bioinformatics PT focusing specifically on the detection of indel mutations.
T69380 435627-435635 Sentence denotes Methods:
T33903 435636-435795 Sentence denotes A preliminary series of 10 (8 new, 2 previous) FASTQ datasets produced with the Illumina TruSight Tumor 26 kit and MiSeq sequencers was distributed to 4 sites.
T10254 435796-435924 Sentence denotes The two previous datasets had clinically significant indel mutations that were not detected by 2/4 sites in our published study.
T158 435925-436029 Sentence denotes Data analysis was performed independently by 4 sites and clinically significant mutations were reported.
T64709 436030-436159 Sentence denotes The results were reported back to each site, and an assessment of the performance of the bioinformatics workflows were discussed.
T55236 436160-436277 Sentence denotes An additional 10 FASTQ datasets with complex indel and SNV mutations are in the process of being analyzed by 5 sites.
T31735 436278-436286 Sentence denotes Results:
T48554 436287-436437 Sentence denotes Analysis of our preliminary series of 10 FASTQ datasets demonstrated a dramatic improvement in the identification of clinically significant mutations.
T67056 436438-436484 Sentence denotes We achieved 100% concordance in 9/10 datasets.
T19902 436485-436600 Sentence denotes Only one mutation, a 12 bp insertion in ERBB2, was missed by one site that had previously missed the same mutation.
T16240 436601-436709 Sentence denotes Although the detection rate of indel mutations dramatically improved, nomenclature discrepancies were noted.
T67081 436710-436838 Sentence denotes An additional 10 FASTQ datasets are in the process of being analyzed and the results of all 20 FASTQ datasets will be presented.
T35028 436839-436851 Sentence denotes Conclusions:
T54881 436852-436977 Sentence denotes We observed that despite disparate clinical bioinformatics workflows, consistency in detecting indel mutations is achievable.
T78017 436978-437103 Sentence denotes One site that previously performed poorly in detecting indels upgraded their entire workflow, dramatically improving results.
T53714 437104-437269 Sentence denotes A second site that correctly reported 9/10 datasets, but missed a clinically significant mutation in ERBB2, was able to identify the cause of their software problem.
T1288 437270-437404 Sentence denotes PT via FASTQ exchange provides an essential function in assisting laboratories to achieve robust and reproducible clinical NGS assays.
T42893 437405-437409 Sentence denotes T.M.
T58491 437410-437422 Sentence denotes Pearce, M.N.
T40910 437423-437437 Sentence denotes Nikiforova, S.
T38158 437438-437498 Sentence denotes Roy University of Pittsburgh Medical Center, Pittsburgh, PA.
T33393 437499-437512 Sentence denotes Introduction:
T9301 437513-437928 Sentence denotes Determining the clinical significance of any given sequence variant requires an integrated evaluation of information from multiple genomic data sources, such as variant location mapping, predicted cDNA and amino acid sequence change, minor allele (population) frequency, insilico prediction scores, somatic and germline mutation databases, and comparison with previously identified variants (inhouse knowledgebase).
T14571 437929-438074 Sentence denotes Software tools to visualize the mapping of sequence variant to peptide structure have been developed and used predominantly in research projects.
T82538 438075-438245 Sentence denotes We investigated the scope for applicability of data visualization techniques to assist in the interpretation of sequence variants from NGS-based somatic mutation testing.
T41849 438246-438254 Sentence denotes Methods:
T3632 438255-438392 Sentence denotes We developed a modular, browser-based visualization widget using JavaScript using widely-used, freely-available libraries such as jQuery.
T52018 438393-438563 Sentence denotes The widget can consume publicly-available or custom web services via AJAX (Asynchronous JavaScript and XML) to obtain data for visualization for a variety of annotations.
T44271 438564-438718 Sentence denotes The interactive graphical user interface utilizes HTML, SVG and CSS, and is encapsulated in an HTML element for easy integration with any web application.
T58521 438719-438863 Sentence denotes An application programming interface (API) provides developers with a variety of configuration options for programmatic control over the widget.
T10286 438864-438980 Sentence denotes Intuitive user interface and easy integration into existing clinical workflows were important design considerations.
T90581 438981-438989 Sentence denotes Results:
T86305 438990-439167 Sentence denotes The data visualization widget was successfully incorporated into a clinical variant management and reporting application used at our institution with minimal change in codebase.
T13157 439168-439374 Sentence denotes It rendered an integrated visualization of the variant location, functional domain mapping, and details and prevalence of a variant of interest when compared to in-house knowledgebase or external databases.
T16157 439375-439612 Sentence denotes For example, during review of uncommon or rare somatic variants, the widget provided visual overlay of the existing variant with variants from the COSMIC database while preserving the positional mapping to the functional protein domains.
T22891 439613-439777 Sentence denotes During routine clinical case workup, it minimized the requirement for separate review of multiple data sources such as pFam, uniprot, COSMIC and other data sources.
T24240 439778-439790 Sentence denotes Conclusions:
T24450 439791-439926 Sentence denotes Data visualization is powerful approach for presenting and interpreting complex genomic data, in clinical as well as research settings.
T51989 439927-440059 Sentence denotes We developed a modular, browser-based widget, which was easily configurable and incorporated into our existing clinical application.
T74432 440060-440198 Sentence denotes Integrated data visualization provided a novel and valuable user experience for variant interpretation, especially with high test volumes.
T30268 440199-440212 Sentence denotes Introduction:
T39620 440213-440356 Sentence denotes Somatic copy number alterations (SCNAs) have significant diagnostic and prognostic value in many types of cancer, including glioblastoma (GBM).
T46565 440357-440528 Sentence denotes SCNAs are often identified by fluorescence in situ hybridization (FISH), but massively parallel sequencing (MPS) can also be used to infer copy number based on read depth.
T65076 440529-440771 Sentence denotes It is, however, challenging to predict SCNAs from targeted re-sequencing of limited gene panels due to uneven coverage of the genome and variation in the efficiency of molecular baits, particularly when no matched normal control is available.
T83366 440772-440780 Sentence denotes Methods:
T41632 440781-441001 Sentence denotes Fifty-four GBM specimens (tumor cellularity ranging from 60 to 97%, average 84%) were subjected to MPS following enrichment of a 516kb target by hybrid capture at Genomics and Pathology Services at Washington University.
T41949 441002-441150 Sentence denotes Target space encompassed the coding regions of 131 genes, selected introns, and a tiled 35kb genomic backbone spaced at 10Mb across all chromosomes.
T90444 441151-441299 Sentence denotes Alignment files generated within the clinically validated pipeline were fed to CNVkit for copy number prediction using both on-and off-target reads.
T2169 441300-441395 Sentence denotes Fifteen cases with no discernable SCNAs were included as an averaged control for normalization.
T80997 441396-441459 Sentence denotes Results were compared with FISH profiles to determine accuracy.
T69482 441460-441468 Sentence denotes Results:
T93573 441469-441631 Sentence denotes Using standard parameters, CNVkit performed robustly on the assay, providing fine resolution of ontarget regions and as well as a broad view of the entire genome.
T93301 441632-441766 Sentence denotes Polysomy of chromosome 7 was detected from MPS data in 45 of 54 GBMs, including 19 of 20 cases with focal EGFR amplification (7p11.2).
T1817 441767-441971 Sentence denotes All cases with EGFR amplification also showed monosomy of chromosome 10 and focal loss of CDKN2A (9p21.3), whereas these aberrations were found in 68% and 56% of cases with unamplified EGFR, respectively.
T49609 441972-442157 Sentence denotes TERTpromoter mutations were common in both EGFR-amplified and unamplified GBM (69% of all cases), whereas TP53 mutations were more common in cases with unamplified EGFR (65% versus 5%).
T21246 442158-442330 Sentence denotes MPS-based detection of focal EGFR amplifications was concordant with FISH in the 39 cases tested with jmd.amjpathol.org ■ The Journal of Molecular Diagnostics both methods.
T70136 442331-442343 Sentence denotes Conclusions:
T57668 442344-442501 Sentence denotes Overall, massive amplifications (e.g., EGFR) were detected with greater accuracy from MPS data compared to lower copy number polysomies and single copy loss.
T96855 442502-442643 Sentence denotes Factors influencing the reliability of SCNA predictions include the quality/quality of input DNA, tumor cellularity, and tumor heterogeneity.
T80414 442644-442648 Sentence denotes I24.
T34980 442649-442787 Sentence denotes An Integrative Multi-Software Approach for FLT3 Internal Tandem Duplication Detection in Hybrid Capture Next-Generation Sequencing Data S.
T4811 442788-442797 Sentence denotes Kadri, I.
T68697 442798-442811 Sentence denotes Mujacic, B.C.
T4205 442812-442820 Sentence denotes Long, C.
T56855 442821-442831 Sentence denotes Zhen, M.N.
T27021 442832-442841 Sentence denotes Wurst, B.
T59740 442842-442851 Sentence denotes Ameti, N.
T89373 442852-442859 Sentence denotes Niu, S.
T84753 442860-442874 Sentence denotes Benhamed, L.V.
T78769 442875-442888 Sentence denotes Furtado, J.P.
T25523 442889-442930 Sentence denotes Segal University of Chicago, Chicago, IL.
T1068 442931-442944 Sentence denotes Introduction:
T27936 442945-443095 Sentence denotes Detection of large insertion mutations with high sensitivity remains a substantial challenge in clinical next-generation sequencing (NGS) diagnostics.
T85099 443096-443214 Sentence denotes At large sizes, it may not be possible for NGS reads to contain the entire insert, leading to frequent failed mapping.
T68898 443215-443419 Sentence denotes Of all the common clinically relevant insertions, FLT3 internal tandem duplications (ITDs) remain potentially the most difficult due to their large size, clinical importance, and sequence characteristics.
T92319 443420-443571 Sentence denotes We have previously presented Amplicon Indel Hunter (AIH), an alignment-independent tool for highly sensitive indel (>5bp) detection in amplicon assays.
T86233 443572-443634 Sentence denotes However, hybrid capture data requires different methodologies.
T3138 443635-443828 Sentence denotes Here, we present an integrated approach combining results from multiple complementary informatics systems, including novel software (ITDHunter) designed to improve sensitivity of ITD detection.
T98599 443829-443978 Sentence denotes Methods: DNA from blood/bone marrow specimens previously tested either by FLT3 fragment length analysis or amplicon-based NGS with AIH were selected.
T89332 443979-444239 Sentence denotes Briefly, DNA was fragmented (Covaris), subjected to library preparation (Kapa Biosystems), pooled and captured (Roche SeqCap EZ) as part of the protocol for UCM-OncoPlus, a 1212 gene hybrid capture sequencing panel which includes 119 clinically reported genes.
T29227 444240-444310 Sentence denotes Captured libraries were sequenced on the HiSeq 2500 system (Illumina).
T74256 444311-444555 Sentence denotes Data processing for FLT3 ITDs included three software systems: (i) indel-realigned software (Abra), (ii) published pattern growth software (Pindel) (iii) ITDHunter, a custom software to discover mis-aligned reads in the FLT3 ITD hotspot region.
T29505 444556-444700 Sentence denotes Results: FLT3 ITDs were detected in all 15 specimens previously observed to contain an ITD via amplicon NGS/AIH or via fragment length analysis.
T72649 444701-444808 Sentence denotes No ITDs were detected in 85 previously tested samples documented to be negative for the presence of an ITD.
T3070 444809-444911 Sentence denotes High concordance between UCM-OncoPlus and previous assay mutant allelic fractions (MAF) were observed.
T82569 444912-445026 Sentence denotes Both perfect and imperfect duplications were identified up to a maximum tested size of 105 bp in clinical samples.
T42829 445027-445170 Sentence denotes In silico simulated ITDs of sizes up to 500 bp were generated at a variety of MAFs as low as 5%, and were all detected via the combined system.
T37530 445171-445240 Sentence denotes At extremely large size, ITDHunter outperformed both Abra and Pindel.
T16378 445241-445335 Sentence denotes Conclusions: FLT3 ITDs are a critical target that requires highly sensitive detection methods.
T90055 445336-445452 Sentence denotes NGS systems may only replace traditional fragment length assays when equivalent or superior performance is attained.
T41275 445453-445554 Sentence denotes Existing published software have weaknesses for detection when ITDs reach very large sizes (>100 bp).
T74773 445555-445720 Sentence denotes ITDHunter fills this performance gap by flagging the existence of large ITDs beyond this threshold, allowing for highly sensitive detection at even extreme ITD size.
T21565 445721-445734 Sentence denotes Introduction:
T45636 445735-445789 Sentence denotes Detection of large indels by NGS is often challenging.
T51503 445790-445853 Sentence denotes Different methods have been adopted to improve indel detection.
T87289 445854-445985 Sentence denotes Here, we demonstrate bioinformatics pipeline enhancements for a TSM assay that lead to reliable detection of a 52 bp CALR deletion.
T61351 445986-445994 Sentence denotes Methods:
T31236 445995-446027 Sentence denotes 1) Update to the alignment tool:
T21702 446028-446107 Sentence denotes The Novoalign aligner (Novocraft Technologies) was upgraded to version 3.04.04.
T86394 446108-446246 Sentence denotes Novoalign version 3.02.07 had required that a deletion of 52 bases be at least ~52 bases from either end of a read to avoid soft-clipping.
T73923 446247-446336 Sentence denotes The CALR deletion is ~ 40 bp from the end of the read based on the Illumina assay design.
T94106 446337-446500 Sentence denotes In the updated Novoalign version, the distance requirement from the end of the read is cut in half, resulting in successful alignment of deletion-containing reads.
T67839 446501-446533 Sentence denotes 2) Update to the variant caller:
T27267 446534-446617 Sentence denotes Freebayes version 0.9.21-19-gc003c1e was used for calling insertions and deletions.
T15925 446618-446810 Sentence denotes Standard Freebayes parameters were modified to lower the minimum base quality requirement, resulting in a more accurate calculation of the variant allele frequency (VAF) of the 52 bp deletion.
T11535 446811-446855 Sentence denotes 3) Updates to the validated filter criteria:
T3634 446856-447042 Sentence denotes The initial TSM Panel used a custom filter that required evidence of an insertion or deletion in both forward and reverse reads if there were overlapping reads in the region of interest.
T10929 447043-447082 Sentence denotes 4) Validation of the improved pipeline:
T39844 447083-447213 Sentence denotes The data from 12 deidentified patients was run through the improved pipeline to evaluate the detection of the CALR 52 bp deletion.
T50853 447214-447222 Sentence denotes Results:
T71873 447223-447319 Sentence denotes In the previously validated TruSight Myeloid pipeline, the CALR 52 bp deletion was not detected.
T62945 447320-447437 Sentence denotes After pipeline updates, the CALR 52 deletion was reliably detected in multiple samples known to exhibit the deletion.
T61585 447438-447590 Sentence denotes Lowering the minimum base quality requirement for indel calling as well as applying less stringent filter criteria may reduce overall assay specificity.
T14242 447591-447852 Sentence denotes To minimize this risk, we evaluated the effect of each of these changes individually and found that most added indel calls lie in non-coding regions and therefore have minimal impact on our clinical reporting, which focuses on coding regions and splicing sites.
T48380 447853-447865 Sentence denotes Conclusions:
T25760 447866-448020 Sentence denotes Large indels may render a drastic change in targeted sequence resulting in soft-clipping by the alignment software or filtering based on quality criteria.
T50767 448021-448143 Sentence denotes Optimization of the match reward feature in Novoalign v.3.04.04 allows for improved alignment with the reference sequence.
T35018 448144-448262 Sentence denotes Optimization of the indel caller Freebayes yields a more accurate determination of the variant allele frequency (VAF).
T64268 448263-448438 Sentence denotes Removing the requirement that the deletion be present in both forward and reverse reads allows the 52 bp CALR deletion to pass preset validated filters for clinical reporting.
T71625 448439-448601 Sentence denotes The combined dataset and all individual institutions had a lower 95% confidence of greater than 99% for both PPA and PPV for all classes of SNV variants examined.
T29907 448602-448814 Sentence denotes Short deletions (< 10 bp) present at a VAF > 20% had a lower 95% confidence interval of 95% for PPA and PPV while short deletions present at a VAF < 20% had a lower 95% confidence interval of 90% for PPA and PPV.
T89401 448815-448933 Sentence denotes There were 11 identified large deletions (> 10 bp) in the combined dataset so analytical performance was not assessed.
T19210 448934-449136 Sentence denotes Short insertions (< 10 bp) present at a VAF > 20% had a point estimate for PPA and PPV of greater than 95%, but the lower 95% confidence interval was determined to be 80% based on the number of samples.
T20709 449137-449273 Sentence denotes There were not enough variants identified for short insertions with a VAF < 20% or larger insertions (> 10 bp) to determine performance.
T71745 449274-449397 Sentence denotes Intermediate precision was determined to be greater than 95% for all variant classes examined containing sample replicates.
T47083 449398-449410 Sentence denotes Conclusions:
T70792 449411-449596 Sentence denotes Analytical performance of the CHP was found to be high for all variant classes examined and met or exceeded requirements as described in the MolDx analytical performance specifications.
T6804 449597-449684 Sentence denotes Performance was found to be concordant across multiple institutions running this panel.
T97048 449685-449854 Sentence denotes This indicates that the CHP can meet stringent analytical performance criteria in a clinical setting such as that set out by the MolDx program for SNVs and short INDELs.
T66424 449855-449868 Sentence denotes Introduction:
T41956 449869-450017 Sentence denotes The identification of disease-associated genes is an important step towards understanding disease mechanisms, diagnosis, and therapy for the future.
T14119 450018-450196 Sentence denotes However, due to the complex and distributed nature of the problem, current scientific knowledge is spread out over several overlapping databases maintained by independent groups.
T35290 450197-450319 Sentence denotes It is unclear how to rank gene-disease research associations due to the distributed and dispersed nature of our knowledge.
T13278 450320-450486 Sentence denotes To fill this gap, we developed Information Genetic Content (IGC), a comprehensive knowledgebase and discovery tool for human genes and genetic disorders research use.
T17645 450487-450516 Sentence denotes IGC is unique in two aspects.
T69228 450517-450583 Sentence denotes First, it integrates data from multiple databases into one system.
T16930 450584-450723 Sentence denotes Second, it provides an unbiased scoring algorithm to rank gene-disease research association at any level of the disease ontology hierarchy.
T25661 450724-450875 Sentence denotes Methods: IGC comprises three components: the Disease-Association Database (DAD), the Gene Scoring Algorithm (GSA), and the Virtual Panel Library (VPL).
T71710 450876-451039 Sentence denotes The DAD module contains over 400,000 associations between over 17,000 genes and 15,000 Mendelian and complex diseases from both expert-curated and text-mined data.
T76606 451040-451217 Sentence denotes The DAD module also features a hierarchical organization of human diseases using a UMLS-controlled vocabulary, permitting queries at any level of the disease ontology hierarchy.
T99485 451218-451293 Sentence denotes The GSA module aims to prioritize genes for a specific disease of interest.
T46376 451294-451469 Sentence denotes This gene scoring algorithm is distinctive in the way it combines the strength of association and the number of associated diseases to provide an unbiased score for each gene.
T25236 451470-451626 Sentence denotes In conjunction with the DAD module, the GSA module is able to produce a list of ranked genes for one or more diseases at any level of the disease hierarchy.
T70179 451627-451752 Sentence denotes The VPL module generates optimal gene grouping by disease classification using hierarchicalclustering-based network analysis.
T74011 451753-451845 Sentence denotes Genes that are involved in the same pathological pathways are grouped into the same cluster.
T52315 451846-451854 Sentence denotes Results:
T61653 451855-451998 Sentence denotes We have successfully applied IGC to select research genes for a variety of diseases, including auto-immune disorders, cleft palate, and autism.
T3009 451999-452175 Sentence denotes Our automated approach achieved more than 70% overlap with the genes prioritized by years of expert selections, providing a crucial verification of our database and algorithms.
T89602 452176-452288 Sentence denotes Moreover, our unbiased selection engine identified disease-related research genes that were missed by subjective
T74020 452289-452361 Sentence denotes The Journal of Molecular Diagnostics ■ jmd.amjpathol.org expert reviews.
T99470 452362-452374 Sentence denotes Conclusions:
T77011 452375-452587 Sentence denotes Taken together, IGC provides both an integrated knowledgebase and a discovery platform that makes it easy for basic and translational researchers to find the most informative genes for their diseases of interest.
T36328 452588-452592 Sentence denotes V.S.
T88389 452593-452609 Sentence denotes Williamson, V.S.
T97451 452610-452623 Sentence denotes Gadepalli, A.
T69436 452624-452639 Sentence denotes Kusmerik 1 , A.
T72605 452640-452648 Sentence denotes Popa, R.
T70879 452649-452656 Sentence denotes Ren, M.
T21003 452657-452677 Sentence denotes SabatoCharreun, C.N.
T61900 452678-452691 Sentence denotes Vlangos, C.I.
T82790 452692-452759 Sentence denotes Dumur Virginia Commonwealth University Health System, Richmond, VA.
T15855 452760-452773 Sentence denotes Introduction:
T93168 452774-452925 Sentence denotes A crucial step in analyzing targeted amplicon sequencing (TAS) data for oncology is the accurate determination of clinically relevant somatic variants.
T55062 452926-453109 Sentence denotes The process is challenging, confounded by tumor heterogeneity and stromal admixture; and many laboratories risk reporting false positives by not sequencing matched tumor-normal pairs.
T61696 453110-453313 Sentence denotes To address this issue, we have developed Onco-Somatik, a program which applies a linear mixed-effects model and expectation maximization (EM)-based clustering of TAS datasets to determine variant origin.
T45709 453314-453535 Sentence denotes Here, we describe the algorithm underlying Onco-Somatik, and present results from a study testing its performance in patient samples and a comparison to variant classifications from the Cancer Genome Atlas (TCGA) dataset.
T89537 453536-453544 Sentence denotes Methods:
T48960 453545-453605 Sentence denotes The algorithm underlying Onco-Somatik is a two-step process.
T36212 453606-453727 Sentence denotes First, purity and ploidy levels are modeled alongside each variant's observed copy number (CN) and allele frequency (AF).
T38736 453728-453818 Sentence denotes CN is calculated using overlapping windows measuring 100 bp which centers on each variant.
T34579 453819-453938 Sentence denotes AF is calculated using the equation where FAO and FRO refer to flow space alternate allele and reference allele counts.
T492 453939-454073 Sentence denotes Model strength and fit is evaluated using Akaike information criterion scores and a "leave-one-variant-out" cross-validation approach.
T72142 454074-454254 Sentence denotes Then, after model selection is complete, all variant AFs are recalculated and groups are determined using EM-based soft clustering with randomized selection of prior probabilities.
T49898 454255-454360 Sentence denotes To test Onco-Somatik, we examined predictions from twenty patients with melanoma, colon, and lung cancer.
T41811 454361-454448 Sentence denotes All samples were sequenced using the OncogenomicDxOnePLUS assay on the Ion Torrent PGM.
T58299 454449-454598 Sentence denotes To test accuracy, we compared Onco-Somatik predictions to those found in the TCGA dataset for matched normal solid-tumor samples of similar diseases.
T96749 454599-454607 Sentence denotes Results:
T27958 454608-454788 Sentence denotes A total of five hundred eighteen variants in 50 genes were evaluated using the software; predictions directly related to known hotspot variants comprised 23.8% of patient variants.
T60508 454789-454913 Sentence denotes The call rate for all variants tested was 92.3% with 94.4% of the variants in patient samples matching TCGA classifications.
T63977 454914-455096 Sentence denotes Allele frequencies recalculated after model selection correlate strongly (Pearson: r = 0.978) with observed values, suggesting that computed purity and ploidy levels are appropriate.
T44857 455097-455241 Sentence denotes A key parameter in the estimation of group membership appears to the variability of CN as its variance is directly related to model performance.
T29357 455242-455254 Sentence denotes Conclusions:
T28362 455255-455380 Sentence denotes Onco-Somatik offers a potential alternative for determining the status of novel variants without a tumor-normal match sample.
T26602 455381-455515 Sentence denotes EM clustering of variant allele frequency and copy number enables users to estimate statistically the probability of group membership.
T18857 455516-455529 Sentence denotes Introduction:
T31035 455530-455633 Sentence denotes Whole-exome sequencing (WES) has been increasingly adopted for the diagnosis of rare genetic disorders.
T49516 455634-455771 Sentence denotes A bioinformatics pipeline that translates raw sequence reads into biologically meaningful information is a key component of the workflow.
T91728 455772-455864 Sentence denotes Bioinformatics tools are constantly being upgraded by the community and commercial entities.
T80203 455865-456018 Sentence denotes Additionally, clinical labs continue to accumulate data that can be used to refine specific quality metrics and related filters in the clinical practice.
T66479 456019-456126 Sentence denotes Therefore, it is essential for clinical labs to review and update their bioinformatics pipelines routinely.
T48279 456127-456310 Sentence denotes Here we present our experience with a major upgrade of our WES pipeline, validation strategy, and defining variant quality criteria that can eliminate unnecessary Sanger confirmation.
T58829 456311-456319 Sentence denotes Methods:
T76020 456320-456424 Sentence denotes Over 200 clinical cases have been signed out since the launch of the Medical Exome Test at CHOP in 2014.
T51904 456425-456517 Sentence denotes To capitalize on the data and to take advantage of latest tools, we have developed CWES 2.0.
T85678 456518-456677 Sentence denotes The new pipeline is set out to improve our indels calling by using GATK HaplotypeCaller and reduce the false de novo variants by joint calling on trio samples.
T81694 456678-456782 Sentence denotes Validation of the pipeline was performed using HapMap sample NA12878 and the Genome in a Bottle dataset.
T17261 456783-456871 Sentence denotes The sample was sequenced in multiple runs to assess inter-and intra-run reproducibility.
T29531 456872-457071 Sentence denotes Additionally, using our Sanger sequenced variants, we have derived quality metrics for highly confident SNVs that do not require Sanger confirmation based on a combination of variant quality metrics.
T38073 457072-457154 Sentence denotes Results: CWES 2.0 achieved sensitivity of 99.7% for SNVs and 98% for indels <20bp.
T46961 457155-457423 Sentence denotes Additionally, a set of quality features including Quality by Depth (QD), strand bias metric of FS (phred-scaled p-value using Fisher's exact test) and allele ratio (AF) were identified to be the most effective features to define variants requiring Sanger confirmation.
T63745 457424-457529 Sentence denotes In contrast, variant quality (QUAL), Read depth (DP) did not separate true variants from false positives.
T63464 457530-457660 Sentence denotes Our experiment also showed GATK VQSR score is not suitable as the score range varies depending on cohort used for variant calling.
T28093 457661-457777 Sentence denotes Our data suggest that a SNV needs to be Sanger confirmed if it meets ANY of these criteria: QD < 13.3; FS > 2.5; AF:
T75327 457778-457798 Sentence denotes Hom <67%, Het < 33%.
T45365 457799-457909 Sentence denotes These criteria captured all confirmed variants and will reduce unnecessary Sanger sequencing by more than 50%.
T49863 457910-457921 Sentence denotes Conclusion:
T43542 457922-458001 Sentence denotes In summary, we presented the evolution of our exome sequence analysis pipeline.
T81812 458002-458126 Sentence denotes In addition, we have also presented the methodology to derive criteria for variants to be excluded from Sanger confirmation.
T62982 458127-458274 Sentence denotes Our strategy and the proposed framework may be beneficial to clinical labs to improve efficiency of their variant detection and analysis workflows.
T92446 458275-458277 Sentence denotes M.
T30504 458278-458286 Sentence denotes Syed, A.
T42737 458287-458296 Sentence denotes Zehir, M.
T85015 458297-458307 Sentence denotes Arcila, J.
T31280 458308-458320 Sentence denotes Casanova, T.
T68333 458321-458330 Sentence denotes Baldi, M.
T55953 458331-458340 Sentence denotes Haque, A.
T24717 458341-458353 Sentence denotes Razumova, I.
T74835 458354-458364 Sentence denotes Kiecka, R.
T52496 458365-458426 Sentence denotes Benayed Memorial Sloan Kettering Cancer Center, New York, NY.
T79802 458427-458440 Sentence denotes Introduction:
T54700 458441-458620 Sentence denotes The Molecular Diagnostics Service in the Department of Pathology at MSKCC processes thousands of samples per year for multiple next-generation sequencing (NGS) and non-NGS assays.
T48749 458621-458865 Sentence denotes Seamless interaction with upstream and downstream systems, interfacing with instruments and storage systems, and capturing important information from sample receiving to sample analysis are critical requirements of high-throughput laboratories.
T18432 458866-459024 Sentence denotes We have achieved significant performance improvement by implementing different LIMS workflows and automating interaction with upstream and downstream systems.
T32806 459025-459033 Sentence denotes Methods:
T73282 459034-459120 Sentence denotes We use Exemplar LIMS to handle sample tracking and processing needs of the laboratory.
T39938 459121-459270 Sentence denotes LIMS receives patient clinical data from another database in a process that is monitored to ensure that all data are received and delays are avoided.
T68250 459271-459338 Sentence denotes Several workflows have been implemented for a seamless integration:
T389 459339-459688 Sentence denotes 1) Sample annotation: users can add annotations such as tumor type and purity for the samples; 2) DNA extraction: allows for sample batching and receives QC data from instruments; 3) various assay specific workflows: these workflows allow batching samples and automatically calculates necessary amounts for DNA and other reagents for the experiment.
T16934 459689-459874 Sentence denotes For NGS assays, sample sheets are also created automatically; 4) Sample storage: interfaces with an automated sample storage system to track samples stored and aids in sample retrieval.
T70275 459875-459976 Sentence denotes Custom plug-ins were written using the JAVA APIs for data transformation and other helpful utilities.
T89136 459977-460071 Sentence denotes Every data point captured in the LIMS is available to downstream systems through web services.
T42334 460072-460233 Sentence denotes Built a dashboard outside Exemplar LIMS, which provides complex data reports, turnaround times, and several user-friendly tools to explore data captured in LIMS.
T43377 460234-460424 Sentence denotes Results: LIMS workflows are used to manage sample registration and annotation, automated DNA extraction and to track several custom NGS assays including MSK-IMPACT as well as non-NGS assays.
T61057 460425-460520 Sentence denotes Collective display of experimental results provides important quality control (QC) information.
T97773 460521-460711 Sentence denotes Providing this information in timely manner using automated APIs, saves time if a sample needs to be reprocessed, often with important consequences for the patient's diagnosis and treatment.
T81969 460712-460826 Sentence denotes For NGS assays, time for gathering information and setting up a batch decreased from 6 hours to less than an hour.
T8530 460827-460919 Sentence denotes Automatic generation of sample sheets eliminated user errors for the sequencing experiments.
T21238 460920-460932 Sentence denotes Conclusions:
T29659 460933-460999 Sentence denotes Our implementation of LIMS has improved performance significantly.
T54764 461000-461088 Sentence denotes Reducing manual typing and adding data checks has decreased errors throughout processes.
T8180 461089-461212 Sentence denotes Seamless communication with upstream and downstream systems has resulted in minimum downtime and faster access to the data.
T19075 461213-461215 Sentence denotes A.
T84344 461216-461226 Sentence denotes O'Hara, A.
T92548 461227-461237 Sentence denotes O'Hara, R.
T18813 461238-461250 Sentence denotes Keshavan, W.
T49951 461251-461262 Sentence denotes Sherman, V.
T14448 461263-461275 Sentence denotes Wasnikar, Z.
T86404 461276-461309 Sentence denotes Che BioDiscovery, El Segundo, CA.
T46689 461310-461323 Sentence denotes Introduction:
T67827 461324-461500 Sentence denotes Targeted panel sequencing has been a popular method to achieve high depth of coverage for certain regions of interest at an affordable cost compared to whole genome sequencing.
T67175 461501-461747 Sentence denotes Shallow whole genome sequencing, where average read-depth can be as low as 0.1x, provides a cost savings-approach for identification of large copy number variant (CNV) events; it has been utilized in various application areas, including oncology.
T57258 461748-461756 Sentence denotes Methods:
T18717 461757-461956 Sentence denotes Here, we introduce a new approach (dCNVSeq) that extends the Pooled Reference algorithm to function with shallow, as well as targeted, sequencing data by introducing a novel dynamic binning approach.
T55036 461957-462353 Sentence denotes The approach uses a Hidden Markov Model to segment the genome into areas forming the "backbone" using the off-target reads and additional areas where targeted reads are present. dCNVSeq uses coarse binning in the backbone area providing base line copy number as well as detection of large CNV events and fine binning in targeted areas to provide high resolution CNV detection in targeted regions.
T77298 462354-462362 Sentence denotes Results:
T44693 462363-462629 Sentence denotes Depending on genomic coverage (whole genome vs. whole exome vs. targeted panel) and sample type (constitutional vs. cancer), different algorithmic approaches may prove to be more or less ideal for estimating copy number from next-generation sequencing (NGS) results.
T58999 462630-462798 Sentence denotes Some approaches require matched normal samples (e.g., ngCGH) while others use the characteristics of the sample itself for normalization (e.g., Wisecondor and QDNASeq).
T50445 462799-462889 Sentence denotes Other methods use a pool of reference samples (e.g., Pooled Reference, xHMM, and CoNFIER).
T72931 462890-463036 Sentence denotes We present a comparison of copy number estimation results from ngCGH, QDNASeq and dCNVSeq using WES, targeted panels, and shallow sequencing data.
T61280 463037-463160 Sentence denotes Whereas each method has strengths for individual samples, only dCNVSeq is functional and reliable across the full data set.
T81595 463161-463531 Sentence denotes Conclusions: dCNVSeq uses a novel dynamic binning approach with a Hidden Markov Model to segment the genome into areas forming the "backbone" using the off-target reads and additional areas where targeted reads are present, providing base line copy number, detection of large CNV events, and high resolution CNV detection in targeted regions within an individual sample.
T12893 463532-463534 Sentence denotes V.
T46181 463535-463551 Sentence denotes Williamson, C.I.
T23883 463552-463561 Sentence denotes Dumur, R.
T84574 463562-463569 Sentence denotes Ren, M.
T98065 463570-463588 Sentence denotes SabatoCharreun, A.
T65827 463589-463603 Sentence denotes Kusmirek, V.S.
T23254 463604-463617 Sentence denotes Gadepalli, A.
T13380 463618-463697 Sentence denotes Ferreira-Gonzalez Virginia Commonwealth University Health System, Richmond, VA.
T7220 463698-463711 Sentence denotes Introduction:
T89761 463712-463903 Sentence denotes The use of targeted amplicon sequencing as a diagnostic tool is increasing and thus creating a need for standardized annotation of variants, scalable, repeatable interpretation and reporting.
T24084 463904-464057 Sentence denotes Qiagen Clinical Insight (QCI) provides a solution for these challenges but must be validated according to guidelines and laboratory's reporting policies.
T90011 464058-464313 Sentence denotes QCI is a web-based clinical decision support platform that provides comprehensive literature references, reported clinical case information, targeted therapy and trial information in support of a lab's annotation, interpretation and reporting of variants.
T99242 464314-464524 Sentence denotes We conducted a QCI platform validation study to determine the concordance rate of variant classifications with cases previously reported by VCU and to assess the relevance and actionability of targeted therapy.
T11305 464525-464533 Sentence denotes Methods:
T74369 464534-464766 Sentence denotes We present the results of a variant classification concordance study comparing the variants from 22 samples with advanced Non-Small Cell Carcinoma, Melanoma, Colorectal Cancer and Thyroid Malignancies analyzed with the QCI platform.
T75114 464767-465046 Sentence denotes Sequencing of samples and primary and secondary analysis of samples were made on the Ion Torrent PGM with the Oncogenomic DX One Assay and patient information and variant calls were manually reviewed for quality prior to upload to the QCI platform where samples were interpreted.
T25803 465047-465085 Sentence denotes The parameters for this study include:
T53404 465086-465355 Sentence denotes 1) the relevance and/or accuracy of pre-computed variant classification before and after QCI rules optimization by an expert user, 2) the consistency with which de-identified patient demographics are uploaded to the QCI annotation pipeline to the final reporting stage.
T21660 465356-465513 Sentence denotes To gauge the impact of the QCI platform training on variant calling, we examined the number of variants for which the results from other subjects were noted.
T64471 465514-465522 Sentence denotes Results:
T90483 465523-465601 Sentence denotes A total of 358 variants encompassing all ACMG categories were analyzed by QCI.
T56242 465602-465734 Sentence denotes A weighted Cohen's Kappa static comparing QCI to our existing knowledgebase dashboard for both preeducation (Kappa = 0.81, Lower CI:
T85601 465735-465753 Sentence denotes 0.75 and Upper CI:
T9016 465754-465800 Sentence denotes 0.87) and post-education (Kappa = 1, lower CI:
T35716 465801-465817 Sentence denotes 1, and upper CI:
T86869 465818-465912 Sentence denotes 1) stages (p <0.05) suggests meaningful improvement of the QCI software after system training.
T32820 465913-466134 Sentence denotes Further, a significant number of variants (Student's t-test p = 0.002, two-tailed) were influenced by the calls of other subjects, suggesting that the system's ability to retain training contributes to increased accuracy.
T4345 466135-466147 Sentence denotes Conclusions:
T138 466148-466405 Sentence denotes Our results suggest that the QCI platform is a robust tool for variant annotation, classification and report generation that allows more scalable and repeatable guidelines interpretation, creation of reports containing insightful and actionable information.
T36968 466406-466564 Sentence denotes In clinical practice and research it is a common challenge to identify disease type or disease subtype based on various types of clinical and biological data.
T33319 466565-466824 Sentence denotes In complex diseases such as cancer where information patterns are complicated due to inherent heterogeneity, involvement of highly complex interaction of biological pathways, continuous multi-level changes with disease progression, this is a challenging task.
T4637 466825-467057 Sentence denotes The challenge with molecular data appears even harder due to very high dimensionality compared to the number of samples, however, unsupervised learning tools may help to quantitatively and more accurately describe a patient's tumor.
T76798 467058-467066 Sentence denotes Methods:
T54146 467067-467269 Sentence denotes We consider quantitative molecular data (such as gene expression, or gene methylation levels) and proceed with feature (e.g., gene or transcript) pre-selection procedure to characterize a given dataset.
T71148 467270-467432 Sentence denotes Next, we apply a variance-based method to identify characteristic distinct subgroups in a given set of samples using an algorithm for optimal partition selection.
T5437 467433-467727 Sentence denotes We applied our method on Level III RNASeq gene expression data from 20531 genes in 889 tumor samples of renal cell carcinoma (RCC) across three subtypes -clear cell renal cell carcinoma, papillary renal cell carcinoma, chromophobe carcinoma, and 129 normal samples from The Cancer Genome Atlas.
T50812 467728-467736 Sentence denotes Results:
T23251 467737-467812 Sentence denotes Our results show close correspondence to known 9 molecular subtypes in RCC.
T8992 467813-467943 Sentence denotes Moreover, we are able to discern novel subsubtypes of interesting patterns, which bear corresponding representative sets of genes.
T82262 467944-468083 Sentence denotes In addition to genes known to play a role in RCC our analysis uncovers important genes not previously associated with renal cell carcinoma.
T96664 468084-468200 Sentence denotes Finally, using pathway enrichment analysis we identified distinctive activation of various pathways across subtypes.
T88371 468201-468213 Sentence denotes Conclusions:
T30139 468214-468354 Sentence denotes In our study, we clearly demonstrated the effectiveness of our computational framework and obtained novel biological characteristics of RCC.
T76971 468355-468462 Sentence denotes Our framework is generic and can be applied in combination with other types of molecular and clinical data.
T67391 468463-468545 Sentence denotes The growth in next-generation sequencing (NGS) and bioinformatics are synchronous.
T8310 468546-468705 Sentence denotes Amplicon based sequencing is one approach to target a small number of genetic regions of interest and can be utilized for a limited number of clinical samples.
T18941 468706-468903 Sentence denotes Primer cross-talk, the finding of overlapping primers generating short reads, can go unnoticed during the initial design, and are found as samples get processed through the bioinformatics pipeline.
T95712 468904-469041 Sentence denotes Thus, the informatics for an ampliconbased bioinformatics pipeline must recognize the insertion artifacts generated by primer cross-talk.
T88555 469042-469256 Sentence denotes In addition, it is necessary to align all the primer concordant reads without removing the primer sequences to detect the end-of-read indels, which may extend into primer sites, which would go undetected otherwise.
T56085 469257-469265 Sentence denotes Methods:
T68427 469266-469489 Sentence denotes An inhouse bioinformatics pipeline was used to map the demultiplexed paired-end primertrimmed reads (fastqs) to human reference genome (hg19), filter the off-target and poor quality reads, detect variants and annotate them.
T86443 469490-469666 Sentence denotes The entire pipeline was coded using Unix-Shell and Python scripts which used the open source tools such as Novoalign, SamTools, Picard, BTrim, GATK, Annovar, SnpEff and Alamut.
T822 469667-469820 Sentence denotes The pipeline also included in house built programs such as "Absolute Var" and "GarbagePicker" to detect SNVs and Indels at as low as 5% allele frequency.
T55779 469821-469829 Sentence denotes Results:
T7613 469830-469958 Sentence denotes False insertions called in our variant call files were investigated thoroughly by reviewing the aligned files (bam) in IGV tool.
T13994 469959-470147 Sentence denotes These insertions were found to be the part of primer sequences that belonged to a different amplicon but bound to the target site of a given amplicon and yeilded shorter amplicon products.
T44452 470148-470251 Sentence denotes These reads also contributed towards a bias in read coverage across positions in a given target region.
T26701 470252-470511 Sentence denotes To address these scenarios Unix shell script was written to identify the reads containing non-concordant primers (i.e. primer cross talk pairs from different amplicons) from the primer-trim-summary files and filter them out from the primertrimmed fastq files.
T61086 470512-470680 Sentence denotes This eliminated the calling of false insertions present in target sites in the bams (i.e. output of alignment of primer-trimmed fastq files with hg19 reference genome).
T30280 470681-470874 Sentence denotes Additionally, a logic was included in the same shell script which filtered all the primer discordant reads from the un-primer-trimmed fastqs and aligned those fastqs with hg19 reference genome.
T68119 470875-470942 Sentence denotes This allowed the detection of indels at the end of target sequence.
T14323 470943-470955 Sentence denotes Conclusions:
T57822 470956-471146 Sentence denotes Removal of primer cross talk insertion artifacts and detection of end-of-the-read indels through the bioinformatics pipeline implemented for Amplicon based Next-generation sequencing assays.
T63920 471147-471273 Sentence denotes The ERG fusion is a key genomic event in prostate cancer development occurring in 40% to 70% of PSA screened prostate cancers.
T1547 471274-471418 Sentence denotes FISH and IHC are the two gold standard ERG-fusion detection methods that are diagnostically available at several centers as stand-alone methods.
T76828 471419-471656 Sentence denotes With the increase use of highresolution microarrays for research or clinical use, predicting ERG fusion using microarray platforms is of valuable clinical importance that hasn't been implemented as part of microarray-based genomic tests.
T1103 471657-471665 Sentence denotes Methods:
T7233 471666-471896 Sentence denotes We previously developed and validated a highly accurate Random-Forest model (m-ERG) to predict ERG FISH status in a multi-institutional radical prostatectomy cohorts using the Decipher assay that is using Human Exon 1.0 ST Arrays.
T94128 471897-472127 Sentence denotes Here, we further validate it in terms on accuracy on genome-wide data generated from Decipher assay on 354 FFPE tissue from RP samples with IHC ERG annotations from John Hopkins Medical Institutions' prostate cancer biorepository.
T82631 472128-472215 Sentence denotes Further, precision and reproducibility of the model was tested in technical replicates.
T36185 472216-472317 Sentence denotes Finally, the model was then evaluated on 3,699 prospective RP tissues from the Decipher GRID program.
T43746 472318-472326 Sentence denotes Results:
T54309 472327-472476 Sentence denotes The m-ERG model was locked and independently validated in 354 RP samples achieving an AUC of 95.5% (93-98%), 95.8% sensitivity and 96.7% specificity.
T34729 472477-472611 Sentence denotes To analytically validate the robustness of the model, the model scores were generated for 5 different samples, each with 5 replicates.
T54743 472612-472707 Sentence denotes The m-ERG model had 100% robustness in terms of agreement on the ERG status within each sample.
T24853 472708-472812 Sentence denotes The model scores were highly reproducible with Intraclass correlation coefficient of 0.97 [0.9 to 0.99].
T57520 472813-472939 Sentence denotes The m-ERG model was also applied on 65 samples from LNCaP cell lines (ERG-) run on the same platform showing 100% sensitivity.
T32139 472940-473047 Sentence denotes These results support implementing it in the Decipher assay; a CLIA test using the Human Exon 1.0 ST Array.
T47792 473048-473139 Sentence denotes Based on the m-ERG model, 41.5% of the prospective cohort samples are predicted to be ERG+.
T97056 473140-473276 Sentence denotes Conclusions: m-ERG model, which is based on the expression of probesets spanning the ERG locus is predictive of the IHC and FISH status.
T92219 473277-473401 Sentence denotes These results support developing and implementing ERG-fusion prediction model in a clinically adopted genomic test pipeline.
T66845 473402-473688 Sentence denotes Despite the lack of impact of ERG fusion on post-RP prognosis, we anticipate that incorporating m-ERG model into a clinically available prognostic assay has several areas of potential near term clinical utility beyond routine pathology such as evaluating multi clonality, clinical trial
T92055 473689-473783 Sentence denotes The Journal of Molecular Diagnostics ■ jmd.amjpathol.org design, evaluating ERG in AS setting.
T31411 473784-473786 Sentence denotes D.
T35173 473787-473800 Sentence denotes Brinza 1 , R.
T60359 473801-473812 Sentence denotes Chen 1 , J.
T81407 473813-473829 Sentence denotes Schageman 2 , E.
T54352 473830-473859 Sentence denotes Ballesteros-Villagrana 2 , R.
T52785 473860-473876 Sentence denotes Chaudhary 1 , J.
T72151 473877-473886 Sentence denotes Gu 2 , V.
T92625 473887-473899 Sentence denotes Bagai 2 , P.
T54809 473900-473916 Sentence denotes Kshatriya 2 , Y.
T69536 473917-473926 Sentence denotes Li 2 , D.
T22058 473927-473941 Sentence denotes Dhingra 1 , J.
T11168 473942-473957 Sentence denotes Au-Young 1 , F.
T96966 473958-473971 Sentence denotes Hyland 1 , K.
T15425 473972-474075 Sentence denotes Bramlett 2 1 Thermo Fisher Scientific, South San Francisco, CA; 2 Thermo Fisher Scientific, Austin, TX.
T46889 474076-474089 Sentence denotes Introduction:
T21828 474090-474278 Sentence denotes Detection and characterization of tumor DNA from circulating cell-free DNA (cfDNA) for the purpose of tracking tumor recurrence and resistance of tumors may improve outcomes in the future.
T45427 474279-474412 Sentence denotes Research studies suggest that somatic DNA mutations in tumor clones can serve as biomarkers that can be tracked in plasma from blood.
T23200 474413-474583 Sentence denotes Tumor DNA comes from different tumor clones, and its abundance in plasma can be very low requiring ability to detect mutation biomarkers at very low frequency from cfDNA.
T76526 474584-474710 Sentence denotes For an efficient low input DNA solution it is necessary to have highly accurate and low-loss interrogation of cfDNA fragments.
T8400 474711-474928 Sentence denotes Here we describe computational and analytical methods used to develop such assay, including a variant calling method that utilizes molecular tagging and enables accurate detection of variants at frequency above 0.05%.
T8354 474929-474937 Sentence denotes Methods:
T70819 474938-475214 Sentence denotes We present computational methods used in development of a multiplex next-generation sequencing assay to optimize cfDNA fragments capturing efficiency, on-target rate, amplification uniformity, primerdimer interactions, molecular tag structure, and number of systematic errors.
T85614 475215-475429 Sentence denotes Followed by an analysis algorithm that models errors accumulated during amplification and sequencing, and accurately reconstructs sequence of tagged DNA molecules based on multiple next-generation sequencing reads.
T93491 475430-475663 Sentence denotes We then developed a variant calling method that uses accurately reconstructed sequences, models offtarget amplification, and PCR polymerase errors to enable sensitive and specific detection of somatic mutations to 0.05% allele ratio.
T57620 475664-475779 Sentence denotes The limit of detection is sample specific and it is dynamically determined by the number of captured DNA molecules.
T15738 475780-476061 Sentence denotes We used our design methods to develop a next-generation sequencing assay that allows interrogation of~150 biomarkers relevant in lung from COSMIC and Oncomine databases, and de-novo variant detection at ~1,700 genomic positions in 11 genes implicated in non-small cell lung cancer.
T55885 476062-476070 Sentence denotes Results:
T88783 476071-476184 Sentence denotes We demonstrate assay performance and accuracy of variant analysis in control and archived cfDNA research samples.
T42784 476185-476404 Sentence denotes The assay delivers >95% on target reads and highly uniform amplification across targeted cfDNA molecules with input DNA of 1ng or higher, and has a fast turnaround time from extracted DNA to variants of less than 24 hr.
T32053 476405-476523 Sentence denotes Observed limits of detection are 0.5%, 0.1%, and 0.05% for amounts of input cfDNA of 1ng, 20ng, and 50ng respectively.
T3281 476524-476603 Sentence denotes This limits are defined for >90% sensitive and >99.5% specific variant calling.
T54026 476604-476684 Sentence denotes For doubled input DNA amount we observe >95% sensitivity and >99.8% specificity.
T68107 476685-476697 Sentence denotes Conclusions:
T78427 476698-476866 Sentence denotes Described computational and analytical methods for assay design and variant calling may facilitate researchers to study relevant biomarkers at 0.05% frequency in cfDNA.
T11302 476867-476923 Sentence denotes University of Pittsburgh Medical Center, Pittsburgh, PA.
T73284 476924-477055 Sentence denotes Introduction: HGVS nomenclature is a de facto method in clinical molecular laboratories and critical component of clinical reports.
T37398 477056-477140 Sentence denotes HGVS representation of variants is a requirement in the CAP accreditation checklist.
T32226 477141-477322 Sentence denotes Conversion of variant representation from genomic to cDNA and protein coordinates using HGVS recommendations for insertion and deletion (indel) variants is particularly challenging.
T31503 477323-477422 Sentence denotes The large number of variants detected in NGS assays requires automation of this conversion process.
T9926 477423-477489 Sentence denotes A python software package, hgvs, was authored by Hart et al (PMID:
T19775 477490-477556 Sentence denotes 25273102) for automated generation of HGVS variant representation.
T80255 477557-477635 Sentence denotes We present our experience with clinical validation of this automated approach.
T48603 477636-477644 Sentence denotes Methods:
T9102 477645-477767 Sentence denotes A total of 50 indel variants across 12 genes from routine clinical somatic mutation testing were evaluated for validation.
T99042 477768-477843 Sentence denotes The indels mapped to both coding and non-coding regions of the above genes.
T83241 477844-477904 Sentence denotes Variants were called by Torrent Variant Caller (TVC) v4.4.3.
T36144 477905-477975 Sentence denotes The hgvs v0.4.5 was used for generating the HGVS nomenclature strings.
T79957 477976-478136 Sentence denotes Custom modules were developed in Python to convert variants in the VCF file to the genomic variant representation (g.) required as an input by the hgvs package.
T53881 478137-478368 Sentence denotes The g. variants were then parsed by hgvs and mapped to coding DNA (c.) and protein (p.) representations with a database of specific RefSeq transcript identifiers and a local instance of Universal Transcript Archive (UTA) v20150827.
T53286 478369-478564 Sentence denotes For each variant the HGVS nomenclature was generated manually by a pathologist by reviewing the sequence pileups in Integrated Genomic Viewer (IGV, Broad Institute) per 2016 HGVS recommendations.
T33017 478565-478642 Sentence denotes Any discrepancies in the automated and manually generated strings were noted.
T82354 478643-478651 Sentence denotes Results:
T55904 478652-478730 Sentence denotes Overall HGVS annotations for 44 of the 50 variants were completely concordant.
T82887 478731-478831 Sentence denotes Six HGVS variant annotations revealed differences between the automated and manually generated c./p.
T52881 478832-478845 Sentence denotes HGVS strings.
T78553 478846-479101 Sentence denotes These differences included miscalculation of the position of the termination codon for frameshift indels, misalignment by 1-2 nucleotides for c. annotation for in-frame indels, and use of abbreviated HGVS format for c., particularly for non-coding indels.
T4348 479102-479258 Sentence denotes In addition, incorrect generation of the input g. annotation is a potential bottleneck and a source of downstream errors for generation of HGVS annotations.
T86912 479259-479271 Sentence denotes Conclusions:
T96467 479272-479392 Sentence denotes Validation of automated HGVS annotation for high volume NGS testing is necessary for optimizing the downstream workflow.
T22044 479393-479528 Sentence denotes Whereas the majority of the annotation using the hgvs package were concordant, specific variants revealed discrepancies in HGVS string.
T32431 479529-479715 Sentence denotes It is desirable to have the ability to accept native VCF variant representation to minimize creating of custom software modules in clinical laboratories with slim bioinformatics support.
T32305 479716-479865 Sentence denotes Introduction:Tumor molecular profiling is rapidly becoming the standard clinical test for selecting targeted therapies in refractory cancer patients.
T88592 479866-479993 Sentence denotes DNA extracted from patient samples is enriched for cancer genes and sequenced to identify actionable somatic mutations therein.
T56972 479994-480131 Sentence denotes A major challenge arises when tumor-derived data is analyzed in the absence of normal tissue data, as it is common in clinical scenarios.
T24865 480132-480262 Sentence denotes The distinction between somatic and germline variants become difficult, leaving clinicians to resort to crude heuristic filtering.
T49615 480263-480271 Sentence denotes Methods:
T94230 480272-480467 Sentence denotes We present here a variant calling software, developed under quality system regulation protocols, capable of accurately identifying somatic mutations from targeted next-generation sequencing data.
T75072 480468-480732 Sentence denotes A novel Bayesian Network approach models the distribution of reads harboring germline and somatic mutations, estimates the contamination from normal tissue in the sample, scores somatic mutations, and imputes germline variants, without matching normal tissue data.
T17260 480733-480891 Sentence denotes This approach also allows joint analysis of multiple specimens from the same patient (e.g., FFPE and ctDNA), when available, improving the limit of detection.
T17599 480892-481087 Sentence denotes To improve specificity, our caller can also utilize prior information from different databases including somatic mutations, germline variation, and healthy controls data, in a principled fashion.
T81309 481088-481096 Sentence denotes Results:
T6939 481097-481210 Sentence denotes We validated our method by analyzing data from the TOMA OS-Seq 131 cancer gene panel using the Illumina platform.
T94435 481211-481362 Sentence denotes Sample inputs ranging from 2-600ng of DNA were sequenced to a depth of Through adaptors with molecular barcodes we measured a median duplicate rate <2.
T61487 481363-481683 Sentence denotes We analyzed somatic mutations simulated at various variant allele fractions on a background of data from reference samples from the Genome-in-a-Bottle consortium, data on a dilution series from two reference samples, and several commercial control and clinical samples, including matched FFPE, PBMC, and ctDNA specimens.
T85330 481684-481810 Sentence denotes In the absence of normal tissue, our method scores each variant with respect to their likelihood of being somatic or germline.
T94806 481811-481971 Sentence denotes We show that, as compared to other commonly used methods, our algorithm can achieve a higher true positive rate whilst controlling a false discovery rate of 1%.
T56368 481972-482084 Sentence denotes We also show that jointly analyzing serial samples (e.g., ctDNA), we can improve sensitivity of shared variants.
T93204 482085-482097 Sentence denotes Conclusions:
T75959 482098-482307 Sentence denotes In conclusion, in contrast to currently used academic software developed for research projects, we observe that our caller outperforms these software and is particularly well suited for the clinical use cases.
T42774 482308-482321 Sentence denotes Introduction:
T39962 482322-482504 Sentence denotes Amicrobial pustulosis (AP) is an aseptic neutrophilic dermatoses characterized by the presence of sterile pustular eruptions affecting cutaneous folds with head and neck involvement.
T60037 482505-482636 Sentence denotes AP may occur as a sporadic disease, or more often, associated with autoimmune diseases (APAD) such as systemic lupus erythematosus.
T10418 482637-482810 Sentence denotes One of the main causes of these cutaneous lesions is an unbalance in the synthesis of inflammatory cytokines, determinant for the development and extension of these lesions.
T33701 482811-482968 Sentence denotes Cytokines are regulating proteins produced by lymphocytes and macrophages, also mediating cell growth, migration, immunity, differentiation and inflammation.
T48746 482969-483076 Sentence denotes For these cellular processes to occur, cytokines need to be transitorily synthesized and tightly regulated.
T36670 483077-483206 Sentence denotes Although this group of diseases have an immunological basis, little is known specifically about their Th1/Th17 cytokine profiles.
T83209 483207-483439 Sentence denotes The aim of this study is to compare the mRNA expression profiles of Th1 (IFN---18, CXCL9), and Th17 (IL-17, IL-23,FOXP3) of AP, APAD as well as skin lesions of systemic erithematous lupus (SEL) and discoid lupus erythematosus (DLE).
T34318 483440-483448 Sentence denotes Methods:
T59343 483449-483606 Sentence denotes We retrospectively reviewed the archives of the Department of Anatomic Pathology of the Instituto Nacional de Ciencias Medicas y Nutrición from 2003 to 2015.
T10425 483607-483724 Sentence denotes We retrieved 5 patients with AP, 7 with APAD, 3 with LED and 5 skin biopsies of healthy individuals (control tissue).
T99228 483725-483865 Sentence denotes Extraction of mRNA was performed with RNA FFPE extraction kit (Promega) and cDNA synthetized with Omniscript Reverse Transcriptase (Qiagen).
T45777 483866-484006 Sentence denotes Primers were design to amplify IL-17, TNF--18, IL-23 e IFN-and FOXP3 using the program Primer Express, based on sequences from ensemble.org.
T88456 484007-484199 Sentence denotes Assessment of mRNA expression using Quanti Tect Master Mix Kit SYBR Green jmd.amjpathol.org ■ The Journal of Molecular Diagnostics (Qiagen) and copy number/ul determined using standard curves.
T50858 484200-484290 Sentence denotes Mean and standard deviation were determined and the results compared using T-Student test.
T83176 484291-484346 Sentence denotes Results: APAD, SLE and DLE show a Th1 cytokine profile.
T46262 484347-484631 Sentence denotes AP show a statistically significant increase in IL-17 (Average -Standard deviation) (8.86 copies/ul -3.63) and IL-23 (38.5copies/ul -6.3) mRNA expression with respect to all other groups, as well as a moderate increase in IL-18 (10 copies/ul -2.82) and loss of FOXP3 (0.23 copies/ul).
T11145 484632-484788 Sentence denotes Conversely, APAD show statistically significant increase in FOXP3 (12 copies/ul) mRNA, and decreased levels of IL-17 (10.76 copies/ul -1.20) compared to AP.
T47066 484789-484801 Sentence denotes Conclusions:
T40263 484802-485050 Sentence denotes These findings indicate different immunological mechanisms subjacent to the pathogenesis of apparently similar morphologic lesions (PA versus APAD), and suggest a possible diagnostic role of Th17 profiling in the discrimination of these conditions.
T12503 485051-485064 Sentence denotes Introduction:
T64193 485065-485217 Sentence denotes Next-generation sequencing (NGS) has quickly become part of standard clinical care and an increasingly important tool in oncology personalized medicine.
T44183 485218-485436 Sentence denotes In 2013, Penn Medicine's Center for Personalized Diagnostics (CPD), a CAP/CLIA laboratory, was an early adopter of NGS technology and needed to navigate transitioning an ever-changing field into a very rigid structure.
T99832 485437-485570 Sentence denotes Starting small and then rapidly becoming a staple to our oncology group, takes good planning, flexibility and learning from mistakes.
T70536 485571-485742 Sentence denotes In an effort to aid and support the molecular diagnostics community, we describe the lessons learned from the establishment and growth of an oncology based NGS laboratory.
T11594 485743-485751 Sentence denotes Methods:
T54839 485752-486011 Sentence denotes Using samples tested via orthogonal methodologies during validation allowed for the establishment of good NGS laboratory practices including defining specimen characteristics, library prep quality control statistics, and true positives for mutation detection.
T19294 486012-486113 Sentence denotes A custom bioinformatics pipeline and integrated database was built to generate and maintain the data.
T43396 486114-486282 Sentence denotes Assays were invented to analyze difficult to sequence or capture genes (e.g., CEBPA) and low quantity or poor quality specimens (e.g., RNA extracted from FFPE tissues).
T4431 486283-486452 Sentence denotes With a growing test menu and increasing sample volume, logistical adjustments were implemented to accommodate the service burden placed on all aspects of the laboratory.
T80015 486453-486461 Sentence denotes Results:
T82952 486462-486614 Sentence denotes Based on thresholds in pre-validation analyses, minimum sample requirements were established at >10% tumor, 100 ng to 250 ng input, and >50% intact DNA.
T48195 486615-486792 Sentence denotes NGS quality controls thresholds were designated at an amplicon minimum depth of 250 reads, average mean coverage of 2000X to 5000X, and minimum reporting allele frequency of 5%.
T57114 486793-487044 Sentence denotes In total, 785 clinical samples were received in year one, 1482 in year two, and 2407 in year three, while staffing went from 8, to 6, to 10 personnel and the total number of assays (library preps and extractions) went from 4, to 7, to 12 respectively.
T96674 487045-487057 Sentence denotes Conclusions:
T59625 487058-487176 Sentence denotes Instituting an oncology NGS center, from inception to a functional clinical laboratory, is complex and multifactorial.
T63017 487177-487376 Sentence denotes The decision to begin the CPD with a limited number of highly specialized personnel and focus on two hotspot oncology panels facilitated the establishment of a robust workflow and clinical reporting.
T85200 487377-487580 Sentence denotes Additional personnel enabled the growth of testing, leading to different iterations of the initial panels, creating RNA based assays, and developing a reflex test to capture low quantity/quality samples.
T67469 487581-487678 Sentence denotes With continued success, the CPD is expanding in personnel, computing power, and clinical utility.
T5868 487679-487856 Sentence denotes Like NGS, the center is changing and evolving, even in a clinical setting, and hopefully lessons learned from our experience may be helpful to others in the molecular community.
T9294 487857-487870 Sentence denotes Introduction:
T30216 487871-487940 Sentence denotes Hepatitis B virus real-time PCR test is a method of quantitative PCR.
T8164 487941-488016 Sentence denotes The quality control limit of HBV real-time PCR was less than 1 log (IU/mL).
T2143 488017-488091 Sentence denotes Considering HBV treatment roadmap, more strict control limit was required.
T20781 488092-488208 Sentence denotes We developed a novel double X bar chart to establish acceptable control limits for a realtime quantitative PCR test.
T28640 488209-488217 Sentence denotes Methods:
T99640 488218-488318 Sentence denotes Two control materials (high and low control) and HBV PCR kits were donated by the Roche Diagnostics.
T86282 488319-488396 Sentence denotes The high and low control material were composed of three kinds of lot number.
T43090 488397-488460 Sentence denotes We tested each control materials two times a week for 6 months.
T60793 488461-488551 Sentence denotes We transformed total 456 results with the Box-Cox, then confirmed the normal distribution.
T15305 488552-488728 Sentence denotes We analyzed the transformed data by X-bar chart, MA (Moving average) chart, EWMA (Exponentially weighted MA) chart, CUSUM (Cumulative sum) chart and a novel double X-bar chart.
T82963 488729-488894 Sentence denotes To create a novel double X-bar chart, we used the jackknife (one-leave out) method and applied the X-bar chart algorithm to each Lower Limit and Upper Limit dataset.
T97517 488895-488902 Sentence denotes Result:
T76441 488903-489053 Sentence denotes The MA, EWMA, CUSUM chart showed good error detection rates, but they could not be utilized as a quality control method due to a narrow control limit.
T42942 489054-489162 Sentence denotes A novel double Xbar chart showed more wide range of control limits than X-bar chart with 3SD control limits.
T79629 489163-489230 Sentence denotes The value of quality rates for X bar chart is 99.7% and were fixed.
T4884 489231-489350 Sentence denotes However those of double X bar chart are 99.96% (n=25) and 99.90% (n=100), so these values depend on the number of data.
T49286 489351-489363 Sentence denotes Conclusions:
T20616 489364-489464 Sentence denotes Double x bar control chart has acceptable control limits compared with original x bar control chart.
T12754 489465-489577 Sentence denotes Therefore, we expect that this method could be utilized for the quality control of a real-time quantitative PCR.
T63025 489578-489591 Sentence denotes Introduction:
T56453 489592-489759 Sentence denotes Minimally invasive diagnostic procedures, such as core biopsies or fine needle aspiration are often the specimen of choice for genomic analysis of actionable variants.
T36895 489760-489882 Sentence denotes There are situations however, where cytologic smears are the only specimen type available for ancillary molecular testing.
T83840 489883-490027 Sentence denotes Next-generation sequencing (NGS) is able to interrogate multiple variants simultaneously from specimens with limited amount of neoplastic cells.
T97503 490028-490138 Sentence denotes The aim of our study was to investigate the minimum cellularity needed on cytologic smears for successful NGS.
T22517 490139-490147 Sentence denotes Methods:
T4219 490148-490326 Sentence denotes Thirty cases from October 2013 to June 2015 of resection-proven primary lung adenocarcinomas previously used for molecular analyses were identified in our institutional archives.
T27299 490327-490424 Sentence denotes Twenty-five cases were known to harbor clinically relevant variants and 5 were negative controls.
T78923 490425-490522 Sentence denotes One Diff-Quik-stained (DQ) and one Papanicolaou-stained (Pap) slide were selected from each case.
T47694 490523-490653 Sentence denotes Based on neoplastic cellularity, cases were categorized as containing < 100 tumor cells, 100-500 tumor cells, or >500 tumor cells.
T97641 490654-490757 Sentence denotes Tumor percentage was estimated as the relative amount of tumor cells vs non-neoplastic nucleated cells.
T31802 490758-490862 Sentence denotes NGS was performed on the Ion Torrent Personal Genome Machine using the Ampliseq cancer hotspot panel v2.
T30559 490863-490871 Sentence denotes Results:
T59503 490872-491017 Sentence denotes Paired DQ and Pap smears from each case showed similar cellularity and cases that differed in cellularity were within one category of each other.
T61008 491018-491110 Sentence denotes Two DQ showed <100 cells, 18 showed 100 to 500 tumor cells, and 10 showed > 500 tumor cells.
T92996 491111-491198 Sentence denotes Four Pap showed <100, 15 showed 100 to 500 tumor cells, and 11 showed >500 tumor cells.
T66544 491199-491277 Sentence denotes Tumor percentages in DQ and Pap were comparable with 46% and 47% respectively.
T41693 491278-491476 Sentence denotes The number of cases with >100 tumor cells had a 93% success rate which was significantly different from, cases with <100 tumor cells that were successfully sequenced only 67% of the time (p= 0.002).
T47208 491477-491606 Sentence denotes All of the DQ cases with <100 tumor cells failed whereas 3 of the 4 Pap cases with <100 tumors cells were successfully sequenced.
T86699 491607-491721 Sentence denotes Overall, NGS was successful in 80% of DQ and 87% of Pap with a total of 31variants identified in DQ and 33 in Pap.
T65695 491722-491774 Sentence denotes There were 3 cases where both DQ and Pap failed NGS.
T94349 491775-491971 Sentence denotes DQ smears failed to identify variants that were successfully identified in 3 cases on Pap whereas there was only 1 case where NGS on Pap failed to identify a variant successfully identified in DQ.
T82616 491972-491984 Sentence denotes Conclusions:
T76929 491985-492091 Sentence denotes There is a significantly higher likelihood of successful NGS using cytologic smears with >100 tumor cells.
T96961 492092-492267 Sentence denotes In cases with <100 tumor cells, Overall, there was a trend for both a higher NGS success rate and a higher likelihood of successful variant identification in Pap smears vs DQ.
T36706 492268-492338 Sentence denotes The advantage of Pap specimens over DQ warrants further investigation.
T17319 492339-492352 Sentence denotes Introduction:
T43414 492353-492490 Sentence denotes Recent technological advancements have reduced the turnaround time and cost of molecular testing and increased access to genetic testing.
T7593 492491-492626 Sentence denotes These changes have enhanced the role genetic tests play in the diagnosis and management of a number of genetic and neoplastic diseases.
T13792 492627-492777 Sentence denotes These advancements have led to an increased demand for genetic testing, which in turn has led to considerable increases in reference laboratory costs.
T43182 492778-492978 Sentence denotes Our institution has not been immune to these cost increases and to address them we previously formed a test utilization review team, consolidated reference laboratory testing and internalized testing.
T14319 492979-493123 Sentence denotes These actions led to a dramatic reduction in annual reference laboratory costs of approximately 2.7 million dollars between FY 2012 and FY 2014.
T20444 493124-493204 Sentence denotes In this report we share the progress we have made to further manage these costs.
T24703 493205-493213 Sentence denotes Methods:
T40241 493214-493417 Sentence denotes The multidisciplinary team consisting of two PhD laboratory medical directors, a PhD clinical chemistry fellow, a rotating pathology resident and a MD medical geneticist review all genetic test requests.
T12315 493418-493540 Sentence denotes The initial review is done by pathology resident or clinical chemistry fellow in consultation with a PhD medical director.
T94945 493541-493730 Sentence denotes The review process incorporates the use of an online genetic test/laboratory selection search engine allowing for price and gene panel composition comparison between reference laboratories.
T89939 493731-493828 Sentence denotes The search engine also enables the retrospective review of orders to look for additional savings.
T52636 493829-493913 Sentence denotes Complex cases or large gene panels elicit a second review by the medical geneticist.
T21947 493914-494073 Sentence denotes Appropriate requests are tiered to cover the most probable genes or directed to the more appropriate panel based upon clinical presentation and family history.
T85455 494074-494149 Sentence denotes The patient's medical record is also reviewed to prevent duplicate testing.
T61412 494150-494392 Sentence denotes All genetic test requests are reviewed and a team member proactively offers consultative services to clinicians to guide test selection based on clinical presentation and a thorough review of the genes associated with the suspected condition.
T68759 494393-494487 Sentence denotes We continue to internalize genetic test panels that are frequently ordered at our institution.
T57390 494488-494496 Sentence denotes Results:
T17245 494497-494606 Sentence denotes Using the review methods highlighted above we have further reduced our costs by a monthly average of $25,000.
T36432 494607-494733 Sentence denotes We have also recently validated a 54 gene myeloid panel that we have used to internalize the sequencing of CALR, JAK2 and MPL.
T28562 494734-494830 Sentence denotes Internalizing these three genes via the myeloid panel has reduced our costs by a monthly average
T30411 494831-494896 Sentence denotes The Journal of Molecular Diagnostics ■ jmd.amjpathol.org $20,000.
T79198 494897-494976 Sentence denotes Combined these approaches account for an average savings of ~$45,000 per month.
T11748 494977-494989 Sentence denotes Conclusions:
T27431 494990-495172 Sentence denotes We have made substantial progress in managing the increased cost of molecular laboratory testing and are on target to reduce our molecular sendout costs by over $1 million this year.
T88294 495173-495361 Sentence denotes Introduction: ACL Laboratories pathologists have been proactively working to improve patient outcomes by helping providers determine the right test at the right time for the right patient.
T88003 495362-495500 Sentence denotes They recognize the importance of staying on top of advances in medicine and improving the ongoing communications with clinical colleagues.
T79069 495501-495708 Sentence denotes Recently, utilization of genetic risk information, particularly Factor V Leiden, to influence patient management in the absence of supporting evidence related to health outcomes has been heavily scrutinized.
T74689 495709-495827 Sentence denotes Current evidence suggests that the Factor V Leiden mutation is not a determinant of the risk of thrombosis recurrence.
T87036 495828-495929 Sentence denotes The risk of recurrence for heterozygous Factor V Leiden was minimal, but significant for homozygotes.
T47675 495930-496051 Sentence denotes Furthermore, compound heterozygotes involving Factor V Leiden and another thrombophilia also meaningfully increases risk.
T50490 496052-496177 Sentence denotes Additionally, since anticoagulation reduces recurrence regardless of mutation status, its presence doesn't change management.
T12191 496178-496393 Sentence denotes To assess our physician usage of Factor V Leiden, the Clinical Effectiveness Utilization Committee at Advocate Health Care System, in conjunction with ACL laboratories, analyzed inpatient orders for Factor V Leiden.
T85653 496394-496402 Sentence denotes Methods:
T59882 496403-496651 Sentence denotes Data from ACL Laboratory "Data Warehouse" was accessed via data collection and analytical software; IBM Cognos Business Intelligence version 10.1.1 from IBM (International Business Machine) and Cube Functions in Microsoft Excel 2010 from Microsoft.
T23545 496652-496900 Sentence denotes Clinical Effectiveness Utilization Committee at Advocate Health Care System implemented system wide testing algorithm, which restricted ordering Factor V as a standing alone order and implemented APC with reflex to Factor V test as the only option.
T67074 496901-496909 Sentence denotes Results:
T66626 496910-497045 Sentence denotes Advocate's baseline positivity rates for wild-type, heterozygous, and homozygous mutant results were 94.7%, 5.2% and 0.1% respectively.
T94692 497046-497215 Sentence denotes Comparison to national positivity rates (89.1% wild-type, 10.5% heterozygous, 0.4% homozygous mutant), suggested an over utilization of the test at Advocate Health Care.
T16183 497216-497407 Sentence denotes With education and communication efforts in conjunction with minor CPOE interventions, physicians were led to order the more appropriate screening test Activated Protein C Resistance (APC-R).
T50903 497408-497496 Sentence denotes Following the changes, Factor V Leiden genetic ordering was successfully reduced by 82%.
T41407 497497-497613 Sentence denotes This resulted in a $50,000 savings after the first 5 months of year 2016 (projected savings for 2106 are ~$120,000).
T32383 497614-497737 Sentence denotes Post implementation Factor V positivity rates were as follow (49.2% wild-type, 50.4% heterozygous, 0.4% homozygous mutant).
T30131 497738-497750 Sentence denotes Conclusions:
T24157 497751-497956 Sentence denotes Our experience illustrates that in a healthcare system designed to encourage active intervention, and more precise patient care, physicians can do a better job of ordering the right test at the right time.
T67261 497957-498162 Sentence denotes This had become a tractable approach for the interrogation of disease progression and immune response due to recent throughput and read length improvements in next-generation sequencing (NGS) technologies.
T95815 498163-498279 Sentence denotes However, structural and sequence complexities of antibody genes have made reliable targeting approaches challenging.
T73845 498280-498288 Sentence denotes Methods:
T36705 498289-498416 Sentence denotes We have developed and optimized a method for accurate sequencing of full-length immune gene repertoires of B-cells and T-cells.
T70423 498417-498883 Sentence denotes The method uses a unique barcoding scheme specifically designed to tag every mRNA molecule with a unique molecule ID (UMI) so that all PCR copies of each mRNA fragment can be collapsed into a single consensus sequence, making the assay extremely accurate by resolving PCR bias and sequencing errors, as well as allowing quantitative assessment of clonal antibody evolution in longitudinal studies in biological samples where disease has triggered an immune response.
T87789 498884-498892 Sentence denotes Results:
T58774 498893-499007 Sentence denotes Immune sequencing libraries were generated from total RNA extracted from PBMCs in duplicate from a single patient.
T84692 499008-499267 Sentence denotes The use of UMIs enabled absolute quantification of starting RNA molecules present in the original sample and therefore accurate ranking of the antibody clone abundance by avoiding the bias incorporated by PCR or sequencing when total reads only were measured.
T23890 499268-499451 Sentence denotes Using the same sequencing method, tumor samples were analyzed for abundance of expanded clones via grouping clones by V gene, J gene and CDR3 similarity and ranking by mRNA abundance.
T6705 499452-499609 Sentence denotes Additionally, the use of isotype-specific primers (IgM, IgD, IgG, IgA and IgE) enabled measurement of the heavy chain isotype proportions within the samples.
T82517 499610-499918 Sentence denotes Further, alignment of full-length heavy chain antibody sequences generated using this method to germline genes from reference databases enabled quantitation of the mutation level of each antibody sequence, thereby providing information on the overall maturity and mutational profile of the sample repertoire.
T99596 499919-499930 Sentence denotes Conclusion:
T45317 499931-500183 Sentence denotes This novel method allows for exhaustive somatic mutation profiling across complete V, D and J segments, full isotype information analysis, and the possibility for synthesis and expression of complete antibody chains for downstream immunological assays.
T29600 500184-500197 Sentence denotes Introduction:
T61907 500198-500338 Sentence denotes With enormous growth in the field of molecular pathology, reporting of results gleaned from this testing is essential to guide patient care.
T70974 500339-500585 Sentence denotes In this study, we examine molecular reports for BRAF mutation testing from labs participating in the College of American Pathologists (CAP) proficiency testing for required elements to convey molecular lab test results to clinicians and patients.
T39422 500586-500634 Sentence denotes This study expands on previously presented data.
T60053 500635-500643 Sentence denotes Methods:
T19155 500644-500845 Sentence denotes Molecular labs participating in the CAP proficiency testing program for BRAF mutation analysis were solicited to submit examples of final reports from two separate proficiency testing reporting cycles.
T13109 500846-500968 Sentence denotes The reports were de-identified at CAP and the data were abstracted by two members of the CAP Molecular Oncology Committee.
T97168 500969-501136 Sentence denotes Reports were reviewed for presence or absence of relevant components based on CAP checklist requirements and published guidelines on molecular reporting (Gulley et al.
T84656 501137-501177 Sentence denotes Arch Pathol Lab Med 2007; 131:852-863) .
T96580 501178-501186 Sentence denotes Results:
T69766 501187-501305 Sentence denotes A total of 116 reports were received (62 demonstrating a positive result for the BRAF V600E mutation and 54 negative).
T23643 501306-501446 Sentence denotes The majority of lab reports (75%) included basic reporting information consisting of lab identity, dates and pertinent specimen information.
T69349 501447-501520 Sentence denotes The test name as the header of the results was present in 98% of reports.
T78465 501521-501673 Sentence denotes Methods for BRAF testing varied, with 95% adequately describing their assay methods and 87% of labs adequately describing the target(s) of their assays.
T63939 501674-501873 Sentence denotes Information on the analytical sensitivity of the assay was present in 74% or reports and 83% reported at least one assay limitation, though only 34% reported on variants not detected by their assays.
T21744 501874-501974 Sentence denotes Analytical and clinical interpretive comments were included in 99% and 90% of reports, respectively.
T51502 501975-502163 Sentence denotes Of participants that perform a laboratory developed test (LDT) only, 88% included language addressing the development of the assay and 62% included all required elements for LDT reporting.
T66340 502164-502176 Sentence denotes Conclusions:
T57187 502177-502336 Sentence denotes Labs participating in BRAF proficiency testing through the CAP are including most of the required reporting elements to unambiguously convey molecular results.
T67171 502337-502430 Sentence denotes Labs should continue to strive to report these results in a concise and comprehensive manner.
T69312 502431-502621 Sentence denotes Close attention should be paid to the required elements for reporting, particularly a clear description of the methods and targets, limitations of the assays, and LDT reporting requirements.
T42538 502622-502742 Sentence denotes Limitations of this study include a relatively small sample size and the fact that it evaluated only one molecular test.
T7664 502743-502966 Sentence denotes Responder bias may have contributed to the overall high rates of reporting compliance, while compliance in some categories may have been underestimated due to report formatting and the nature of proficiency testing samples.
T20851 502967-502980 Sentence denotes Introduction:
T55688 502981-503252 Sentence denotes This tool is born of two frustrations: lost time confirming annotation of insertion and deletion variants from sequencing results and lack of a simple method to demonstrate base and codon changes, transcript variants, and nomenclature to students, residents, and fellows.
T5461 503253-503670 Sentence denotes This tool uses a common spreadsheet program to visualize reference sequence as individual bases in cells numbered according to the given refseq identification number, automatically translates the sequence to numbered codons, allows one to make changes to a parellel copy of the reference sequence, translates the new sequence via macro, and allows one to compare differences between the original and variant sequence.
T47146 503671-503679 Sentence denotes Methods:
T64170 503680-503725 Sentence denotes Microsoft Excel is used as the base platform.
T66970 503726-503828 Sentence denotes An embedded link allows for retrieval of the given FASTA text for the desired NCBI refseq ID as input.
T96345 503829-503950 Sentence denotes Embedded formulae convert the FASTA text into a simple text string and deconcatenates the characters into numbered bases.
T15876 503951-504035 Sentence denotes Translation is performed using a vlookup function and embedded protein coding chart.
T59264 504036-504217 Sentence denotes Re-translation of the manipulated variant sequence is performed using a visual basic macro as is resetting of the sequence if an error is made and the user would like to start over.
T54464 504218-504324 Sentence denotes A formula is used to located sequence of interest which is gleaned from the output of the sequencing data.
T90494 504325-504429 Sentence denotes Search functions can be used to locate base positions, protein codon positions, and stop codons rapidly.
T70962 504430-504769 Sentence denotes A resident with little molecular pathology experience (never previously worked up a sequence variant) was given the "c." coordinates of two variants (an insertion and deletion, both frameshift) and given the task of identifying the "p." information and nomenclature of the changes either manually or with the assistance of the spreadsheet.
T21603 504770-504811 Sentence denotes The methods were staggered to avoid bias.
T65689 504812-504934 Sentence denotes A brief primer on each method, manual and spreadsheet assisted, was given along with printed instructions for each method.
T70001 504935-505048 Sentence denotes A timed trial was also conducted for a molecular faculty member familiar with the manual and spreadsheet methods.
T61339 505049-505057 Sentence denotes Results:
T96369 505058-505192 Sentence denotes The trainee showed manual solution times of 35 and 23 minutes and assisted times of 9 and 9 minutes for problems 1 and 2 respectively.
T55888 505193-505300 Sentence denotes The faculty member demonstrated manual solution times of 13 and 11 and assisted times of 3.3 and 3 minutes.
T16678 505301-505463 Sentence denotes Alternative transcripts from 16 genes were examined to verify that sequences matched reference annotation to validate the translation function of the spreadsheet.
T20231 505464-505476 Sentence denotes Conclusions:
T66725 505477-505571 Sentence denotes This tool can significantly cut down time in verifying annotation of insertions and deletions.
T53709 505572-505684 Sentence denotes It is also useful as a teaching tool when instructing trainees about sequence interpretation and gene structure.
T61560 505685-505751 Sentence denotes It provides visual and interactive feedback to reinforce concepts.
T2810 505752-505765 Sentence denotes Introduction:
T29588 505766-505902 Sentence denotes Circulating cell free (ccf) DNA is a challenging material to work with, the DNA is fragmented and can cause issues in molecular testing.
T197 505903-506075 Sentence denotes There is also an issue with stability where we want to extract the DNA as soon after collection as possible, otherwise increasing the issues with this challenging material.
T52131 506076-506349 Sentence denotes Here we are testing the feasibility of using the PAXgene Blood ccfDNA Tube to increase the stability of ccfDNA when transporting plasma samples across state to our central lab for up to 5 days, in combination with the Therascreen EGFR RGQ PCR Kit, to ensure a valid result.
T21184 506350-506358 Sentence denotes Methods:
T15321 506359-506569 Sentence denotes Collection of multiple blood samples into EDTA and PAXgene Blood ccfDNA Tube, Day 0, and then subsequent isolation of plasma and DNA extraction using the QIAamp DSP Circulating NA Kit at Day 1, Day 3 and Day 5.
T2358 506570-506706 Sentence denotes Followed by testing with the Therascreen EGFR RGQ PCR Kit for DNA assessment using the control assay amplifying exon 2 of the EGFR gene.
T31090 506707-506715 Sentence denotes Results:
T8209 506716-506926 Sentence denotes With all samples collected in the PAXgene Blood ccfDNA, we were able to obtain a valid Ct, as determined by the Therascreen EGFR Kit, at all time points for this study, showing that amplifiable DNA is isolated.
T60377 506927-507160 Sentence denotes Whereas with the samples collected in EDTA, and the plasma isolation and DNA extraction performed at the respective time point showed a decrease in the amount of amplifiable DNA present, with a worsening Ct at each time point tested.
T12203 507161-507173 Sentence denotes Conclusions:
T17254 507174-507384 Sentence denotes Our study found that the PAXgene Blood ccfDNA Tube can be used for collection of blood and subsequent EGFR testing for samples that take an extended period of time, 5 days, to reach our central lab for testing.
T36815 507385-507517 Sentence denotes Compared to blood collecting in EDTA, which would result in an invalid result being reported, and new sample needed to be requested.
T64045 507518-507531 Sentence denotes Introduction:
T81316 507532-507659 Sentence denotes Amplification of the MET gene or increased MET gene copy number has been reported in a subset of non-small cell lung carcinoma.
T46294 507660-507777 Sentence denotes MET amplification has been suggested as a mechanism for resistance to EGFR tyrosine kinase inhibitor-based treatment.
T85486 507778-507921 Sentence denotes Lung cancer patients with MET amplification, defined as MET/CEP7 However, heterogeneous MET amplification in lung tumors has not been examined.
T1807 507922-508022 Sentence denotes Next-generation sequencing (NGS) technology can be used to detect copy number alterations in cancer.
T10727 508023-508306 Sentence denotes The objectives of this study were to assess heterogeneous MET amplification in lung tumors, to compare results of capture-based NGS assay to FISH for detecting MET amplification, and to identify any additional gene alterations in MET amplification-or gene copy number-positive cases.
T28846 508307-508422 Sentence denotes Methods: FISH reports of 696 de-identified lung cancer samples were reviewed retrospectively for MET amplification.
T34789 508423-508500 Sentence denotes At least fifty cells were scored for CEP7 and MET probe signals in each case.
T43373 508501-508577 Sentence denotes FISH data were analyzed as follows: positive for MET amplification (MET/CEP7
T16094 508578-508758 Sentence denotes MET gene copy number (MET/CEP7 ratio of <2.2, average MET MET amplification (presence of 5-<50% of cells with MET/CEP7 conducted with a targeted lung panel (25 genes) in ten cases.
T62059 508759-508882 Sentence denotes Results: MET amplification was detected in 3.5% (n=24) of cases, whereas 9.2% (n=64) showed increased MET gene copy number.
T97184 508883-508968 Sentence denotes Of the 24 MET-amplified cases, 12.5% (n=6) exhibited heterogeneous MET amplification.
T21730 508969-509082 Sentence denotes The MET/CEP7 ratio in these six cases samples that were MET amplification-positive by FISH were subjected to NGS.
T62106 509083-509174 Sentence denotes MET amplification was detected by NGS in four samples in which the MET/CEP7 MET/CEP7 -<3.0.
T71830 509175-509284 Sentence denotes Four samples that were positive for an increased MET gene copy number were MET amplification-negative by NGS.
T76402 509285-509347 Sentence denotes Alterations in the TP53 gene were also detected in 7/10 cases.
T85690 509348-509488 Sentence denotes Pathogenic variants in lung cancer driver genes such as EGFR, KRAS, and PTEN were detected in tumors with an increased MET gene copy number.
T13284 509489-509501 Sentence denotes Conclusions:
T27785 509502-509642 Sentence denotes This study provides evidence for heterogeneous MET amplification in lung tumors and is associated with the tumors exhibiting MET/CEP7 -<3.0.
T71892 509643-509788 Sentence denotes Our NGS assay detected MET amplification in all cases with a MET/CEP7 for detecting MET amplification-positive patients for crizotinib treatment.
T19250 509789-509937 Sentence denotes Driver mutations in EGFR, KRAS, and PTEN genes appear to play a key role in the pathogenesis of lung cancers with an increased MET gene copy number.
T82069 509938-509951 Sentence denotes Introduction:
T74996 509952-510132 Sentence denotes Castration-resistant prostate cancer (CRPC) patients are often treated with drugs that target the androgen receptor (AR) ligand-binding domain (e.g., enzalutamide and abiraterone).
T63580 510133-510313 Sentence denotes Constitutively-active AR splice variant 7 (AR-V7) lacks the ligand-binding domain and, if detected in circulating tumor cells (CTCs), is associated with resistance to these agents.
T47823 510314-510322 Sentence denotes Methods:
T95441 510323-510383 Sentence denotes We validated the AR-V7 assay in a CLIA-certified laboratory.
T72120 510384-510483 Sentence denotes CTCs were isolated from blood by immunomagnetic enrichment, and mRNA reverse transcribed into cDNA.
T35380 510484-510638 Sentence denotes Real-time PCR amplification of reference genes (ACTB and GAPDH), "prostate-specific" genes (PSMA, PSA and AR-full length (AR-FL)) and AR-V7 was performed.
T61798 510639-510823 Sentence denotes Specimens for validation included an AR-V7 expressing prostate cancer cell line (LNCaP95), 38 peripheral blood controls (14 males, 24 females), and 21 blood samples from CRPC patients.
T55131 510824-510832 Sentence denotes Results:
T80493 510833-510951 Sentence denotes The assay detected as few as five LNCaP95 cells spiked into peripheral blood, demonstrating high analytic sensitivity.
T5526 510952-511121 Sentence denotes Multiple inter-and intra-run replicates of LNCaP95 cell line experiments yielded similar Cq values for all genes, showing high analytic precision (AR-V7 Cq CV of 0.67%).
T33912 511122-511227 Sentence denotes All 38 healthy control samples were negative for AR-V7, demonstrating high diagnostic specificity (100%).
T66779 511228-511298 Sentence denotes All healthy control samples were negative for PSA and PSMA expression.
T78565 511299-511547 Sentence denotes The diagnostic accuracy was confirmed by concurrent testing of 21 CRPC samples in the research laboratory and the clinical diagnostic laboratory: concordance in AR-V7 status was achieved in all cases (positive in 4, negative in 17) (100% accuracy).
T20145 511548-511673 Sentence denotes Since, our laboratory is the first CLIA-certified laboratory performing AR-V7 testing, inter-lab proficiency is not possible.
T40665 511674-511795 Sentence denotes In the interim, the clinical samples are subsequently tested by the research laboratory as an additional quality control.
T32477 511796-511867 Sentence denotes Seventeen patient samples were subsequently tested by the research lab.
T73191 511868-511965 Sentence denotes Overall 16 of 17 patients were categorized identically between the two labs (94.11% concurrency).
T71823 511966-512166 Sentence denotes There was one discrepancy between the labs, where the clinical lab detected AR-V7 transcript at a very low level (Cq of 38.10) in one of the two duplicates, which was not detected by the research lab.
T76967 512167-512179 Sentence denotes Conclusions:
T69985 512180-512341 Sentence denotes This first validated clinical assay detects CTC-derived AR-V7 with high analytic sensitivity, analytic precision, diagnostic specificity and diagnostic accuracy.
T75330 512342-512393 Sentence denotes It is currently in use for targeted therapy trials.
T1323 512394-512637 Sentence denotes Within this pathway, BRAF, KRAS, and NRAS as amongst the most frequently mutated genes, with mutation rates as high as 50% and 30% for BRAF and NRAS, respectively, in malignant melanoma, and 40% for KRAS in metastatic colorectal cancer (mCRC).
T65915 512638-512931 Sentence denotes To robustly, rapidly, and reliably detect these mutations across indications, Roche has developed two allele-specific PCR, research use only assays for the detection of mutations in the BRAF, KRAS, and NRAS genes from DNA isolated from formalin-fixed, paraffin-embedded tissue (FFPET) samples.
T89472 512932-513203 Sentence denotes The BRAF/NRAS Mutation Test (life science research only; LSR), covers 36 unique mutations in exons 11 and 15 in the BRAF gene and exons 2, 3 and 4 of NRAS gene; similarly, the KRAS Mutation Test v2 (LSR) detects 28 unique mutations in exons 2, 3, and 4 of the KRAS genes.
T55699 513204-513298 Sentence denotes The assays are designed for use on the userdefined workflow (UDF) of the Cobas z 480 analyzer.
T73048 513299-513567 Sentence denotes To further assess the performance of these assays, 50 FFPET samples of either melanoma, non-small cell lung cancer (NSCLC), or colorectal cancer origin previously characterized with Next-Generation Sequencing were retested with the two assays to determine concordance.
T75630 513568-513703 Sentence denotes Methods: DNA from 50 specimens was isolated using the manual Cobas DNA Sample Preparation Kit and tested by an in-house NGS gene panel.
T81382 513704-513911 Sentence denotes The DNA was tested at Carolinas Medical Center (Charlotte, NC) using the BRAF/NRAS Mutation Test (LSR) and KRAS Mutation Test v2 (LSR) assays and compared to the initial NGS results to determine concordance.
T17099 513912-513920 Sentence denotes Results:
T43748 513921-514024 Sentence denotes The frequency of BRAF, NRAS, and KRAS mutations was 30%, 28%, and 12% respectively using the LSR Tests.
T47589 514025-514096 Sentence denotes Fifteen BRAF mutations were detected -ten in melanoma and five in mCRC.
T96353 514097-514147 Sentence denotes Fourteen NRAS mutations were detected in melanoma.
T14296 514148-514211 Sentence denotes Six KRAS mutations were detected -five in CRC and one in NSCLC.
T41674 514212-514247 Sentence denotes One sample was invalid by KRAS LSR.
T30848 514248-514307 Sentence denotes Another sample was discordant between LSR and NGS for BRAF.
T33422 514308-514372 Sentence denotes The overall agreement between the two methods was 98.0% (48/49).
T57570 514373-514483 Sentence denotes The positive percent agreement (PPA) was 100% (34/34), and negative percent agreement (NPA) was 93.3% (14/15).
T38526 514484-514539 Sentence denotes The overall call rate with all samples was 98% (48/50).
T51869 514540-514552 Sentence denotes Conclusions:
T51673 514553-514672 Sentence denotes We showed excellent concordance between the Roche BRAF/NRAS Mutation Test (LSR) and KRAS Mutation Test v2 (LSR) to NGS.
T35120 514673-514843 Sentence denotes These LSR assays allow for highly sensitive and specific detection of 64 unique BRAF, KRAS, and NRAS mutations in a comparatively simple, streamlined, and rapid workflow.
T63397 514844-514990 Sentence denotes Carolinas Medical Center will be testing an additional 50 mCRC and melanoma samples previously characterized by NGS to further assess concordance.
T5025 514991-515004 Sentence denotes Introduction:
T33927 515005-515199 Sentence denotes Low-grade fibromyxoid sarcoma (LGFMS) and sclerosing epithelioid fibrosarcoma (SEF) were thought to be related before the characteristic FUS-CREB3L2 chimeric gene was identified in both lesions.
T78057 515200-515361 Sentence denotes Once identified, fluorescent in-situ hybridization (FISH) analysis was adopted to detect the t(7;16) and t(11;16) translocations involving the FUS gene in LGFMS.
T73062 515362-515472 Sentence denotes SEF can exist in hybrid or pure forms, with hybrid lesions containing areas of morphologic overlap with LGFMS.
T1711 515473-515609 Sentence denotes Genetically, hybrid tumors have been found to carry the FUS rearrangement whereas pure lesions more frequently show EWSR1rearrangements.
T46869 515610-515690 Sentence denotes We present a case of hybrid SEF that was negative for FUS translocation by FISH.
T95507 515691-515821 Sentence denotes Archer FusionPlex testing revealed an unexpected EWSR1-CREB3L1 fusion that was subsequently confirmed by EWSR1 FISH rearrangement.
T19244 515822-516016 Sentence denotes Methods: FUS and EWSR1 FISH testing was performed via breakapart probe using the LSI FUS 16p11 (Abbott Molecular, Des Plaines, IL) and LSI EWSR1 22q12 probes (Abbott Molecular, Des Plaines, IL).
T33647 516017-516116 Sentence denotes Two hundred cells were counted with a threshold of positivity of 15% (FUS) or 25% (EWSR1) of cells.
T79779 516117-516335 Sentence denotes Fusion detection was performed using the Archer FusionPlex Sarcoma Kit (ArcherDx, Boulder, CO), sequencing was performed on the Miseq (Illumina, San Diego, CA), and final analysis utilized the Archer analysis pipeline.
T36010 516336-516344 Sentence denotes Results:
T85422 516345-516420 Sentence denotes Histology revealed areas consistent with both SEF and LGFMS (hybrid tumor).
T79213 516421-516485 Sentence denotes Immunohistochemical stains were positive for CD99 and MUC4 only.
T40174 516486-516557 Sentence denotes RT-PCR performed at an outside institution for EWSR1-FLI1 was negative.
T9333 516558-516580 Sentence denotes FUS FISH was negative.
T3326 516581-516689 Sentence denotes The Archer analysis identified a strong evidence EWSR1-CREB3L1 (t11;22) fusion supported by 24 unique reads.
T65250 516690-516776 Sentence denotes Follow up testing by FISH for EWSR1 by break-apart probe was positive in 72% of cells.
T77905 516777-516935 Sentence denotes Conclusions: FISH can detect fusion events without previous evidence of the fusion partner; but is hindered by the need to select the appropriate target gene.
T43028 516936-517110 Sentence denotes Nextgeneration sequencing (NGS) does not require previous knowledge of potential fusions, and can be cost effective and more rapid than FISH when multiple studies are needed.
T34220 517111-517316 Sentence denotes The Archer FusionPlex Sarcoma kit uses multiplex anchored-PCR followed by NGS to simultaneously detect 26 different genes and reports any translocation partner, such as in our case detecting EWSR1-CREB3L1.
T56033 517317-517495 Sentence denotes In scenarios with unusual or promiscuous rearrangement, the Archer FusionPlex system offers a way to quickly and affordably identify potential clinically relevant translocations.
T65521 517496-517502 Sentence denotes Inc) .
T8433 517503-517707 Sentence denotes TST 26 provides valuable mutational information to patients, however approximately 40 % of patient samples are referred to pyrosequencing because of poor DNA quality and / or insufficient quantity of DNA.
T53149 517708-517938 Sentence denotes To circumvent this problem, Illumina recently developed the TruSight tumor 15 gene panel (TST 15), which uses next-generation sequencing (NGS) to assess clinically relevant and most prevalent mutations in 15 genes in solid tumors.
T56531 517939-518065 Sentence denotes TST 15 designed to detect low-frequency variants from 20 ng of DNA from formalin-fixed, paraffin embedded (FFPE) tumor tissue.
T58138 518066-518168 Sentence denotes TST 15 will be offered as a single assay for accurate, economical, and rapid analysis of solid tumors.
T61929 518169-518284 Sentence denotes Moffitt Cancer Center was one of the institutions selected by Illumina for a pre-production beta testing of TST 15.
T70065 518285-518293 Sentence denotes Methods:
T46073 518294-518441 Sentence denotes For beta testing of TST15, all the reagents including one control sample with known mutations and the pertinent software were provided by Illumina.
T32880 518442-518514 Sentence denotes The TST15 contains two separate pools of tagged oligonucleotide primers.
T42211 518515-518609 Sentence denotes These pools are used in multiplex PCR to target and amplify regions for a select set of genes.
T58433 518610-518780 Sentence denotes Using adapter primers, templates are indexed and further amplified in a second PCR and then combined into a single tube for paired-end sequencing on the MiSeq Instrument.
T10774 518781-518845 Sentence denotes Data are analyzed by using the on-instrument reporting software.
T7925 518846-518905 Sentence denotes We performed three runs using MiSeq with 8 samples per run.
T17453 518906-519077 Sentence denotes A total of 19 patient samples previously sequenced either by TST 26 gene panel or by pyrosequencing, control sample and horizon diagnostic reference standards were tested.
T48619 519078-519086 Sentence denotes Results:
T18696 519087-519215 Sentence denotes The TST15 demonstrated a remarkably high tolerance to poor DNA quality and / or insufficient quantity when compared with TST 26.
T83362 519216-519358 Sentence denotes All nine patient samples rejected for mutation analysis by TST 26 because of insufficient amount of DNA, were successfully analyzed by TST 15.
T33888 519359-519474 Sentence denotes Mutation profiles obtained for all samples tested were 100% concordant between TST15 and TST 26 and pyrosequencing.
T74871 519475-519614 Sentence denotes TST15 provided uniform coverage of target regions, identifying somatic mutations at frequencies as low as 5% with minimum coverage of 500X.
T76939 519615-519695 Sentence denotes Conclusions: TST15 accurately detected low frequency variants from 20 ng of DNA.
T26586 519696-519783 Sentence denotes The time required for library preparation was approximately 7 h versus 24 h for TST 26.
T91539 519784-519976 Sentence denotes TST 15 offers a rapid, economical and reliable NGS work flow solution that can easily be implemented into laboratories that perform NGS. (SFT) is a mesenchymal tumor rarely seen in the clinic.
T73952 519977-520116 Sentence denotes Conventional treatment for an SFT is a surgical resection and a poor prognosis is frequently observed once postoperative recurrence occurs.
T11696 520117-520229 Sentence denotes However, molecular-targeted therapy provides a new treatment strategy with higher efficiency and lower toxicity.
T96334 520230-520390 Sentence denotes Targeted drugs act on specific gene mutations and their treatment efficiency largely relies on the presence of drug-sensitive mutations within the tumor tissue.
T808 520391-520399 Sentence denotes Methods:
T19727 520400-520591 Sentence denotes This report presents the study of a 50-year-old female with an SFT who was being treated at the Third Affiliated Hospital of Harbin Medical University (Heilongjiang Cancer Hospital) in China.
T66790 520592-520713 Sentence denotes Formalin-fixed paraffin-embedded (FFPE) samples of the SFT tissue were got form the patient prior to pazopanib treatment.
T59537 520714-520835 Sentence denotes Next-generation sequencing (NGS) was performed on the Illumina X platform to analyze a total of 483 cancer-related genes.
T59828 520836-520844 Sentence denotes Results:
T801 520845-520932 Sentence denotes The data of NGS showed that the tumor had mutations in BRCA1 p.Y856H and FGFR2 p.N549K.
T26833 520933-521129 Sentence denotes Based on the results of gene analysis and the clinical evidence of the randomized, placebocontrolled PALETTE trial (Graaf, et al, Lancet 2012) , the patient in this study started taking pazopanib.
T88407 521130-521248 Sentence denotes The size of the lesion continuously decreased from 130.36 × 157.64 mm to 107.5 × 146 mm after two months of treatment.
T92209 521249-521261 Sentence denotes Conclusions:
T96215 521262-521346 Sentence denotes To date, no clear biomarkers have been identified for the targeted treatment of SFT.
T82947 521347-521552 Sentence denotes By combining cancer gene mutations with the clinical research progress of pazopanib treatment, this case report provides new insights into SFT-targeted therapy in terms of clinical research and medication.
T39172 521553-521721 Sentence denotes Since patients with SFT in the liver are rare, a comprehensive genetic analysis offers clinical evidence and directions for further research of the pathogenesis of SFT.
T16373 521722-521735 Sentence denotes Introduction:
T77946 521736-521821 Sentence denotes Identifying actionable mutations is an important aspect of cancer patient management.
T46866 521822-521975 Sentence denotes With the increasing use of fine needle aspiration (FNA) biopsies, it is critical to validate FNA specimens for targeted next-generation sequencing (NGS).
T33956 521976-522111 Sentence denotes In this study, we retrospectively examined NGS performed on FNA samples at a single institution to evaluate their adequacy and utility.
T53213 522112-522120 Sentence denotes Methods:
T16465 522121-522257 Sentence denotes Our database was searched for FNA specimens (between 1/1/2015 to 12/31/2015) with requests for our institutional NGS panel (198 genes) .
T69825 522258-522356 Sentence denotes Clinical data, pathological features, and molecular profiling results were collected and analyzed.
T28741 522357-522487 Sentence denotes Formal histologic review of all cell blocks/core biopsies to evaluate tumor percentage was performed by two independent reviewers.
T1148 522488-522496 Sentence denotes Results:
T21106 522497-522572 Sentence denotes Of the 2752 FNAs performed in 2015, 154 specimens were sent for NGS (5.6%).
T58353 522573-522688 Sentence denotes These included specimens from various anatomical sites, with the majority from lung (51.9%) and lymph node (23.4%).
T34568 522689-522757 Sentence denotes In 49% of the cases, the sample was obtained from the primary tumor.
T21928 522758-523038 Sentence denotes Lung adenocarcinoma was the predominant diagnosis (70.8%, including primary and metastatic lesions), with the remainder representing a spectrum of gastrointestinal, pancreatobiliary, and gynecological carcinomas, large/small cell neuroendocrine carcinomas, and malignant melanoma.
T10117 523039-523102 Sentence denotes There are 21 cases with tumor percentage less than 20% (13.6%).
T99504 523103-523185 Sentence denotes In six cases (3.9%), NGS was cancelled due to insufficient DNA or low DNA quality.
T63978 523186-523276 Sentence denotes Six specimens were also sent out for sequencing and showed concordant mutational profiles.
T67325 523277-523451 Sentence denotes Repeat NGS was performed in subsequent resections in two cases, with both demonstrating identical pathogenic/likely pathogenic variants compared with the primary FNA samples.
T12 523452-523635 Sentence denotes In total, 642 variants were reported with 171 (26.6%) annotated as pathogenic variants, 51 (0.8%) as likely pathogenic variants, and 420 (65.4%) as variants with unknown significance.
T23409 523636-523746 Sentence denotes For pathogenic/likely pathogenic variants, 46 were identified inEGFR, 81 in TP53, 41 in KRAS, and 6 in PIK3CA.
T35974 523747-523842 Sentence denotes Conclusions: FNA samples are reliable and robust for detecting clinically actionable mutations.
T12228 523843-524016 Sentence denotes The NGS data generated from FNA specimens are used to guide targeted therapy, enroll patients into clinical trials, and distinguish a second primary tumor versus metastasis.
T79551 524017-524183 Sentence denotes LGSCs are indolent and evolve via Type I pathway from serous cystadenoma to typical serous borderline tumor (TSBT) and micropapillary serous borderline tumor (MPSBT).
T65289 524184-524240 Sentence denotes KRAS and other oncogenes mutations are frequent in LGSC.
T47058 524241-524351 Sentence denotes In contrast, HGSCs are aggressive, lack KRAS mutations, and arise via Type II pathway in all or none patterns.
T53918 524352-524450 Sentence denotes Exceptionally, rare HGSCs harboring KRAS mutations have beeb reported to arise via Type I pathway.
T29419 524451-524459 Sentence denotes Methods:
T84975 524460-524555 Sentence denotes To contribute to this scant pool of evidence, we present one such case of a 54 years old woman.
T11026 524556-524737 Sentence denotes She was diagnosed with endometrial carcinoma and underwent laparoscopic hysterectomy with bilateral jmd.amjpathol.org ■ The Journal of Molecular Diagnostics salpingo-oophorectomies.
T39170 524738-524783 Sentence denotes Gross and microscopic findings are described.
T52580 524784-524934 Sentence denotes Tissue microdissections were conducted using laser capture microdissection (LCM) for serous cystadenoma, and manually for TSBT, MPSBT, LGSC, and HGSC.
T29843 524935-525112 Sentence denotes DNA was extracted by Life Technology's Arcturus PicoPure DNA Extraction Kit and TrimGen's WaxFree Paraffin DNA Extraction kit from LCM and manual microdissections, respectively.
T60646 525113-525262 Sentence denotes Codon 12/13 of the KRAS gene and flanking DNA sequences were PCR amplified followed by pyrosequencing on Qiagen's PyroMark Q24 pyrosequencing system.
T685 525263-525271 Sentence denotes Results:
T50880 525272-525382 Sentence denotes In addition to the endometrial adenocarcinoma, a left ovarian cyst with papillary excrescences was identified.
T82519 525383-525567 Sentence denotes Microscopically, the ovarian tumor is comprised of an admixture of serous cystadenoma, TSBT and MPSBT with minimal invasion associated to each, LGSC and LGSC in transformation to HGSC.
T40206 525568-525738 Sentence denotes The HGSC portion shows brisk mitoses (16 mitoses/10HPF) and high grade histologic features characterized by nuclear pleomorphism, prominent nucleoli with multinucleation.
T17743 525739-525901 Sentence denotes The same KRAS gene c.38G>A, p.Gly13Asp mutation was detected by pyrosequencing in all of the sampled areas, i.e., serous cystadenoma, TSBT, MPSBT, LGSC, and HGSC.
T64443 525902-525914 Sentence denotes Conclusions:
T69128 525915-526065 Sentence denotes Given the wide variety of KRAS mutations, this same codon mutation serves as a de facto biomarker to illustrate the clonal essence of tumor evolution.
T29513 526066-526196 Sentence denotes The findings that the same KRAS mutation carried from serous cystadenoma, TSBT, MPSBT, LGSC, to HGSC in one single case are novel.
T44923 526197-526367 Sentence denotes The results support the existence of HGSCs evolving via Type I pathway and hence improve our understanding on mechanisms behind tumorigenesis of ovarian serous carcinoma.
T1394 526368-526495 Sentence denotes In clinical settings, preanalytical variables greatly affect downstream technical analysis by next-generation sequencing (NGS).
T75298 526496-526570 Sentence denotes This is pronounced in developing countries lacking standardized protocols.
T46725 526571-526769 Sentence denotes To understand the effect of type and time of fixation on DNA and RNA NGS results, human cell lines and tissues were fixed with commonly available anatomical pathology fixatives for three timepoints.
T92626 526770-526778 Sentence denotes Methods:
T78851 526779-526852 Sentence denotes All experiments were conducted with a developed RNase minimized protocol.
T65322 526853-526993 Sentence denotes Human cell lines A549, MCF-7 and Caco-2 were grown to a confluence of 2x10^6, harvested, pelleted and snap frozen in liquid nitrogen (LiN2).
T83202 526994-527170 Sentence denotes Cell pellets were fixed with neutral buffered formalin (NBF), 100% ethanol (EtOH), 100% methanol (MeOH), zinc buffered formalin (ZBF) or Bouin's Fluid (BF) for 8, 24 or 72 hrs.
T10974 527171-527302 Sentence denotes LiN2 frozen human lung, breast and colon resections were chosen based upon adenocarcinoma subtype, >90% tumor volume, and RIN >8.5.
T88771 527303-527386 Sentence denotes Tissue resections were fixed with NBF, EtOH, MeOH, ZBF and BF for 8, 24, or 48 hrs.
T90545 527387-527523 Sentence denotes Cell pellets and tissue were processed with an 8 hour tissue processing protocol, embedded in paraffin, and cut into 4 and 7μm sections.
T32842 527524-527605 Sentence denotes 4 μm sections were adhered to glass slides and H+E stained for visual comparison.
T55679 527606-527714 Sentence denotes DNA and RNA (NA) were extracted from the 7 μm sections using the RecoverAll Total NA Isolation Kit for FFPE.
T64299 527715-527808 Sentence denotes Quality control (QC) was performed on the extracted NA by fluorometric quantitation and qPCR.
T20500 527809-527918 Sentence denotes NGS was performed on NA using the Ion AmpliSeq Cancer Hotspot Panel v2 and RNA Cancer Panel upon the Ion PGM.
T52842 527919-528031 Sentence denotes NGS success criteria were NA yield, library amplification, low clonality, depth of coverage and number of reads.
T50290 528032-528040 Sentence denotes Results:
T44357 528041-528284 Sentence denotes For fixed and processed (FP) cell lines and tissues, no significant difference was seen between cell type or 8 hour to 24 hour fixation; however NBF, ZBF and BF performed worse with NGS success criteria than EtOH and MeOH samples after 72 hrs.
T70808 528285-528382 Sentence denotes Conversely EtOH and MeOH produced worse morphology compared to NBF, ZBF and BF at all timepoints.
T46887 528383-528395 Sentence denotes Conclusions:
T605 528396-528516 Sentence denotes Alcohol containing compounds are excellent cell and solid tissue NA fixatives but do not preserve cellular architecture.
T83891 528517-528673 Sentence denotes Aqueous formaldehyde containing fixatives, primarily NBF, should be used for 24 hours due to its ability to generate NGS results as well as histomorphology.
T60435 528674-528676 Sentence denotes A.
T7731 528677-528691 Sentence denotes Ordobazari, M.
T47041 528692-528702 Sentence denotes Sharma, Z.
T34604 528703-528781 Sentence denotes Wang Univeristy of Florida College of Medicine Jacksonville, Jacksonville, FL.
T10143 528782-528795 Sentence denotes Introduction:
T59841 528796-528957 Sentence denotes Lung biomarkers testing on patients with non-small cell lung carcinoma (NSCLC) have become routine laboratory practice as companion tests for targeted therapies.
T17305 528958-529040 Sentence denotes EGFR mutations and ALK rearrangement assays are the standard tests as recommended.
T5927 529041-529258 Sentence denotes Although not recommended as a sole determinant of EGFR tyrosine kinase inhibitor (TKI) therapy, KRAS mutations have been determined as mutually exclusive with EGFR mutations and intrinsically resistant to TKI therapy.
T72706 529259-529368 Sentence denotes However, given that negative KRAS mutation results are not helpful, to test or not to test remains a dilemma.
T6880 529369-529581 Sentence denotes This study reports our experience in adopting a modified testing algorithm to ensure testing efficiency by taking advantage of a slew of known parameters that are associated with KRAS mutations or EGFR mutations.
T11437 529582-529692 Sentence denotes Most important parameters include patients' smoking history and mucinous vs. papillary histology of the tumor.
T97711 529693-529701 Sentence denotes Methods:
T47552 529702-529813 Sentence denotes The cohort consists of 111 consecutive cases of surgically resected or biopsied NSCLCs from the past 1.5 years.
T95783 529814-529913 Sentence denotes KRAS codon 12/13 mutation assay was indicated if the patient is a smoker and the tumor is mucinous.
T58679 529914-530004 Sentence denotes Non-smokers, relatively young female patients with papillary type tumor are not indicated.
T22268 530005-530136 Sentence denotes Laboratory work flow, sample volume, turn-around time, patients' clinical conditions, and specimen cellularity are also considered.
T94261 530137-530274 Sentence denotes KRAS codon 12/13 mutation detection was conducted using the pyrosequencing technology with a low limit of detection of 5% mutant alleles.
T7739 530275-530283 Sentence denotes Results:
T59655 530284-530373 Sentence denotes Among the 111 cases of NSCLCs, 64% (71 cases) were tested for KRAS codon 12/13 mutations.
T51649 530374-530444 Sentence denotes Mutations were detected in 26 cases, accounting for 37% of the cohort.
T75899 530445-530530 Sentence denotes Of the 26 positive cases, 24 were codon 12 mutations and two were codon 13 mutations.
T21980 530531-530543 Sentence denotes Conclusions:
T31664 530544-530643 Sentence denotes Our cohort of cases represent a North Florida patient population with most being African Americans.
T11719 530644-530799 Sentence denotes Given that the overall documented frequency of KRAS mutations in such ethnic group ranges from 15%-25%, achieving a 37% detection frequency is significant.
T66665 530800-530897 Sentence denotes The results support and promote KRAS mutation assay for NSCLCs by an efficient testing algorithm.
T63776 530898-531038 Sentence denotes Establishment of one such algorithm eliminates the need to test for EGFR and ALK which are more costly, time consuming, and labor intensive.
T63461 531039-531165 Sentence denotes Introduction: KRAS mutant non-small cell lung cancer (NSCLC) is remarkable for its divergent biological and clinical behavior.
T97869 531166-531299 Sentence denotes Distinct KRAS variants have been identified that engage different downstream effector pathways and vary in drug sensitivity patterns.
T29827 531300-531494 Sentence denotes In particular, G12C and G12V subtypes depend to a greater extent on the RAS/RAF/MEK/ERK signaling cascade and may benefit from the addition of a MEK inhibitor to standard chemotherapeutic agent.
T32889 531495-531614 Sentence denotes The impact of coexisting genetic mutations on treatment responsiveness in KRASmutant NSCLC has not been fully explored.
T2085 531615-531775 Sentence denotes Preclinical studies indicated that concomitant loss of TP53 or LKB1/STK11 tumor suppressor genes impaired the response of KRAS induced lung cancer to docetaxel.
T82217 531776-531955 Sentence denotes The addition of selumetinib provided the greatest benefit for mice bearing tumors with KRAS mutation only, and lesser yet substantial benefit in KRAS tumors with TP53 co-mutation.
T35989 531956-532094 Sentence denotes Conversely, the concurrent LKB1/STK11 loss resulted in primary resistance of KRAS mutant lung cancer to docetaxel and selumetinib therapy.
T54298 532095-532291 Sentence denotes In this study we investigate TP53 and LKB1/STK11 mutation co-occurrence with major subtypes of KRAS as potential biomarkers for efficacy of MEK inhibitors in NSCLC patients within our institution.
T6207 532292-532300 Sentence denotes Methods:
T91570 532301-532519 Sentence denotes A total of 591 NSCLC samples were screened for somatic mutations using the 50 gene AmpliSeq Cancer Hotspot Panel v2 (CHPv2), and samples positive for variants in the KRAS gene (N=198) were included in further analysis.
T38415 532520-532564 Sentence denotes Fisher's exact test was used for statistics.
T6315 532565-532573 Sentence denotes Results:
T66306 532574-532627 Sentence denotes The overall mutation rate in the KRAS gene was 33.5%.
T86253 532628-532741 Sentence denotes The MEK-dependent G12C and G12V KRAS variants were the most common and occurred with a combined frequency of 61%.
T87050 532742-532848 Sentence denotes Across all KRAS subtypes the LKB1/STK11 and TP53 mutations were detected in 26 and 61 cases, respectively.
T69157 532849-533043 Sentence denotes Whereas the TP53 mutations were distributed evenly in-between G12C+V and non-G12C+V cancers (31 vs 30), the LKB1/STK11 mutations clustered with MEK dependent subtypes of KRAS (22 vs 4, p=0.009).
T13546 533044-533262 Sentence denotes As LKB1/STK11and TP53 mutations were found to be mutually exclusive, 3 different subgroups of G12C+V tumors were identified: G12C+V (56%), G12C+V with co-mutated TP53 (26%), and G12C+V with co-mutated LKB1/STK11 (18%).
T79472 533263-533275 Sentence denotes Conclusions:
T48312 533276-533395 Sentence denotes Over a half of KRAS-NSCLCs in our cohort carries G12C or G12V mutation with potential susceptibility to MEK inhibition.
T3603 533396-533632 Sentence denotes As TP53 and LKB1/STK11 co-mutations may impact efficacy of MEK inhibitors, we define 3 subpopulations of patients with predicted: best (G12C+V), moderate (G12C+V and TP53), and minimal (G12C+V and LKB1/STK11) response to such treatment.
T55397 533633-533798 Sentence denotes The concomitant LKB1/STK11 loss in a subset of G12C+V KRASdriven lung cancer may indicate an aggressive phenotype with the need for alternative therapeutic approach.
T57719 533799-533919 Sentence denotes Response to targeted therapies such as Imiatinib, a tyrosine kinase inhibitor, depends on the specific mutation present.
T56824 533920-534050 Sentence denotes A small percentage of GISTs are wild-type for KIT and PDGFRA, but show deficiencies in the succinate dehydrogenase enzyme complex.
T40054 534051-534207 Sentence denotes This study represents an attempt to better characterize the mutations present in GISTs as well as to categorize tumors into prognostic treatment categories.
T37378 534208-534216 Sentence denotes Methods:
T67637 534217-534351 Sentence denotes Histologically diagnosed GISTs were selected from specimens processed at the Dartmouth-Hitchcock Medical Center (DHMC) from 2014-2016.
T82043 534352-534449 Sentence denotes Genomic DNA was extracted from a total of ten formalinfixed and paraffin-embedded (FFPE) tissues.
T98471 534450-534598 Sentence denotes Samples were multiplexed using the AmpliSeq Cancer Hotspot Panel (CHPv2) and sequenced with the Ion Torrent 318v2 chips using the Ion PGM TM System.
T35878 534599-534664 Sentence denotes Variants were identified using the Variant Caller Plugin (v.4.0).
T49788 534665-534673 Sentence denotes Results:
T81648 534674-534749 Sentence denotes Of the ten GIST specimens analyzed, nine had adequate DNA after extraction.
T75093 534750-534895 Sentence denotes The nine sequenced specimens were from the stomach (n=4, 44%), small bowel (n=3, 33%), esophagus (n=1, 11%), and shoulder (metastatic, n=1, 11%).
T47328 534896-534991 Sentence denotes Histologic morphologies were spindled (n=5, 56%), epithelioid (n=1, 11%), and mixed (n=3, 33%).
T17801 534992-535106 Sentence denotes KIT was the most commonly mutated gene (n=6, 67%); five of those mutations were in exon 11 and one was in exon 13.
T33101 535107-535207 Sentence denotes One specimen contained a PDGFRA mutation (11%) and two were wild-type for the sequenced genes (22%).
T30894 535208-535445 Sentence denotes The following KIT mutations were identified: c.1674_1676delGGT, p.K558fs; c.1671_1676delGAAGGT, p.W557_V559delinsC; c.1961T>C, p.V654A; c.1667_1674delAGTGGA, p.Q556_K558delinsQ; c.1676T>A, p.V559D; c.1729_1730insCTTATG, p.Y578_D579insAY.
T90230 535446-535618 Sentence denotes Two patients positive for KIT mutations also had mutations in the MET and IDH1 genes .Sample positive for the PDGFRA mutation (V561D), also had a mutation in the JAK3 gene.
T50184 535619-535631 Sentence denotes Conclusions:
T12229 535632-535777 Sentence denotes As expected, the most common mutation site was in exon 11 of the KIT gene; these tumors generally have an excellent response to Imatinib therapy.
T55306 535778-535890 Sentence denotes One specimen contained a mutation in exon 13 of the KIT gene, which generally show partial response to imatinib.
T14802 535891-536033 Sentence denotes This study demonstrates the importance of somatic mutation detection for multiple genes to better characterize the molecular profile of GISTs.
T55963 536034-536131 Sentence denotes Additional studies are required to elucidate other mutations present in our wild-type GIST cases.
T47933 536132-536145 Sentence denotes Introduction:
T10901 536146-536249 Sentence denotes Clinicians are increasingly relying on tumor profiling via NGS to offer targeted therapies to patients.
T69817 536250-536326 Sentence denotes Quality controls are fundamental for developing and monitoring these assays.
T37428 536327-536589 Sentence denotes However, use of cell lines, either patient-derived or generated through targeted mutation of the genome, has short-comings: the degree of multiplexing and control over allele frequencies are limited, and the background genetic material is not well characterized.
T67820 536590-536782 Sentence denotes We developed a unique technology for generating highly multiplexed, cellular controls for both RNA and DNA sequencing, and demonstrated their compatibility with popular tumor profiling assays.
T43787 536783-536791 Sentence denotes Methods:
T54279 536792-536922 Sentence denotes A biosynthetic construct was generated that contained portions of fifteen (15) genes with clinically actionable somatic mutations.
T67489 536923-537063 Sentence denotes A second construct had twelve (12) clinically actionable RNA fusions, including fusions of ALK, RET and ROS1, as well as rare fusion events.
T59591 537064-537140 Sentence denotes The biosynthetic DNA or RNA was introduced into GM24385 reference cell line.
T8135 537141-537277 Sentence denotes The engineered cells were then characterized, normalized, fixed in formalin, and embedded in paraffin for use as whole-process controls.
T56176 537278-537366 Sentence denotes The DNA reference material was tested by NGS using Ion Ampliseq Cancer Hotspot Panel v2.
T80975 537367-537398 Sentence denotes All 15 mutations were detected.
T21358 537399-537586 Sentence denotes The allele frequencies of mutations could be modulated by mixing engineered cells with wild-type GM24385 cells; a dilution series generated 15%, 7%, and 4% target allele frequencies (AF).
T22065 537587-537711 Sentence denotes Mutation frequencies for the 15% target AF material ranged from 9.6% for ERBB2 p.A775_G776insYVMA to 15.8% for EGFR p.T790M.
T89564 537712-537895 Sentence denotes The RNA reference material was tested by ArcherDx FusionPlex Lung Thyroid Panel, and all twelve fusions were detected as "strong evidence" fusions (per Archer Analysis Software v3.3).
T35524 537896-538034 Sentence denotes There was a ten-fold range observed in the detection levels: SLC34A2-ROS1 had only 34 unique, spanning reads, whereas FGFR3-TACC3 had 328.
T38704 538035-538210 Sentence denotes These differences in breakpoint-spanning reads on NGS were very consistent from one lot of the reference material to the next, and were also observed for digital PCR analysis.
T72701 538211-538292 Sentence denotes One possible explanation is sequence-specific damage caused by formalin fixation.
T79314 538293-538418 Sentence denotes Testing was also successful on the Thermo Fisher OncoMine Focus Assay and Ion Ampliseq RNA Fusion Lung Cancer Research Panel.
T47421 538419-538431 Sentence denotes Conclusions:
T55402 538432-538691 Sentence denotes Highly multiplexed, cellular reference materials with a well characterized background (GM24385) are needed to monitor the whole process of both RNA and DNA NGS tumor profiling assays and to aid in assay optimization and verification of lower detection limits.
T96583 538692-538904 Sentence denotes The ability to create multiple synthetic DNA mutation or RNA fusion targets enables clinical laboratories to ensure their assays perform appropriately even when the variants are rarely observed in the laboratory.
T94693 538905-539043 Sentence denotes The histologic grading of appendiceal mucinous neoplasms into low grade and high grade groups identifies patients with distinct prognosis.
T63950 539044-539132 Sentence denotes The molecular alterations that define these unique subsets, however, are less wellknown.
T62403 539133-539276 Sentence denotes Previous studies were controversial as to the role of GNAS mutations and loss of SMAD4 by immunohistochemistry in correlating with tumor grade.
T58310 539277-539435 Sentence denotes We explored characteristic molecular alterations between two histological groups of appendiceal mucinous neoplasm and their association with overall survival.
T39379 539436-539444 Sentence denotes Methods:
T46607 539445-539629 Sentence denotes Seventy-four surgical resections of primary appendiceal mucinous neoplasms underwent mutation analysis by next-generation sequencing between 2012 and 2015 at MD Anderson Cancer Center.
T72390 539630-539776 Sentence denotes These tumors, classified by degree of differentiation following the WHO grading system, consisted of 21 low grade tumors and 53 high-grade tumors.
T92944 539777-539946 Sentence denotes Substitutions as well small insertions and deletions in 50 cancer-associated genes were reviewed using next-generation sequencing-based AmpliSeq Cancer Hotspot panel v2.
T74707 539947-540072 Sentence denotes We analyzed frequently identified somatic mutations in appendiceal mucinous neoplasms and their association with tumor grade.
T82205 540073-540081 Sentence denotes Results:
T18508 540082-540190 Sentence denotes Tumor grade was significantly associated with GNAS mutations, KRAS and GNAS co-mutation, and TP53 mutations.
T44264 540191-540383 Sentence denotes Low grade appendiceal mucinous neoplasms harbored more frequent GNAS mutations (71%), either alone or in combination with KRAS (67%) than high-grade tumors (30% and 21%, respectively; p<0.01).
T44906 540384-540514 Sentence denotes In contrast, high-grade appendiceal mucinous neoplasms had more frequent TP53 mutations (34%) than low-grade tumors (0%; p=0.002).
T74506 540515-540688 Sentence denotes SMAD4 mutations were found in seven high-grade tumor (13%) and three low-grade tumors (14%); the frequencies of SMAD 4 mutations was not associated with tumor grade (p=1.0).
T11189 540689-540887 Sentence denotes Patients with KRAS and GNAS co-mutation showed a better overall survival than patients without these co-mutations (log-rank test; hazard ratio 0.164; 95% confidence interval, 0.0627 0.8765; p=0.04).
T73179 540888-540900 Sentence denotes Conclusions:
T74925 540901-541105 Sentence denotes Low grade appendiceal mucinous neoplasms have different molecular alterations such as frequent GNAS mutations and co-mutation with KRAS compared to high grade tumors, which harbor frequent TP53 mutations.
T93438 541106-541289 Sentence denotes Identification of these mutations in appendiceal mucinous neoplasms may be helpful as useful diagnostic markers to aid in the distinguishing of these two clinically important subsets.
T91547 541290-541385 Sentence denotes Introduction: NGS is used extensively in research for the molecular characterization of cancer.
T20031 541386-541555 Sentence denotes This has primarily focused on formalin-fixed, paraffin-embedded samples, the use of which is limited by surgical feasibility, tissue availability and patient preference.
T95528 541556-541824 Sentence denotes An emerging field of study has focused on circulating-free DNA (cfDNA), sometimes termed "Liquid Biopsy." These fragments of DNA are released by the tumor mass into the circulatory system, and their mutational information can often shed light on tumor characteristics.
T23634 541825-542092 Sentence denotes Although accepting it has the potential to be a valuable source of clinical information during diagnosis, treatment and monitoring for disease recurrence, the liquid biopsy field is still nascent, and lacks an integrated NGS workflow solution with proven performance.
T17981 542093-542187 Sentence denotes Methods: QIAGEN is an established expert in the field of sample preparation for liquid biopsy.
T56714 542188-542414 Sentence denotes The combination of the QIAamp Circulating Nucleic Acid Kit with the GeneReader NGS System and the GeneRead QIAact Actionable Insights Tumor Panel provides the first true Sample to Insight workflow solution for analyzing cfDNA.
T19355 542415-542595 Sentence denotes A set of 16 liquid biopsy samples from non-small cell lung cancer patients and cfDNA reference standards (Horizon), with allelic frequency at or above 1%, were used for this study.
T4832 542596-542691 Sentence denotes DNA was extracted from the liquid biopsy samples using the QIAamp Circulating Nucleic Acid Kit.
T26192 542692-543084 Sentence denotes The reference standards did not require extraction and were used to confirm detection of low frequency variants (typical of cfDNA samples). cfDNA from the liquid biopsy samples and reference standards were run through the complete QIAGEN GeneReader workflow, including a new QIAGEN Clinical Insight (QCI ) Analyze bioinformatics pipeline developed specifically for liquid biopsy applications.
T54721 543085-543093 Sentence denotes Results:
T66268 543094-543360 Sentence denotes Analysis of the 16 liquid biopsy samples for EGFR-associated mutations, designed to assess system performance on biological samples, identified seven to be EGFR mutation positive (either L858R or deletion mutations) that were verifiable using PCR based technologies.
T25552 543361-543554 Sentence denotes Testing of the fully characterized cfDNA reference standards identified all mutations covered by the Actionable Insights Tumor Panel, with 100% consistence with those reported in ddPCR studies.
T26674 543555-543676 Sentence denotes Both single nucleotide and insertion-deletion variants were correctly identified down to a 1% allele frequency threshold.
T58426 543677-543689 Sentence denotes Conclusions:
T92675 543690-543905 Sentence denotes These data show that high sensitivity and high performance consistency have been achieved with both cfDNA reference standards and liquid biopsy samples with the complete QIAGEN GeneReader workflow for liquid biopsy.
T86391 543906-544072 Sentence denotes This is the first study of its kind to systematically demonstrate the accuracy of a liquid biopsy assay combined with the robustness of a fully integrated NGS system.
T11406 544073-544086 Sentence denotes Introduction:
T40613 544087-544199 Sentence denotes Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease, often presenting with metastasis at diagnosis.
T26622 544200-544375 Sentence denotes Several genetic mutations as well as alterations of miRNA expression have been described in PDAC, however it is unclear how these alterations contribute to metastatic disease.
T36226 544376-544508 Sentence denotes Our goal in this study was to analyze and compare miRNA expression and NGS sequencing results in metastatic and non-metastatic PDAC.
T39561 544509-544571 Sentence denotes Methods: PDAC samples from twenty-four patients were analyzed.
T706 544572-544626 Sentence denotes Five cases were excluded because of inadequate tissue.
T1009 544627-544715 Sentence denotes Eleven cases were from patients with metastatic disease (M:F = 2:9; age 60.4±8.1 years).
T80291 544716-544798 Sentence denotes Eight cases were from patients without metastasis (M:F = 1:1; age 73.1±6.9 years).
T91200 544799-544921 Sentence denotes For each sample, seven unstained FFPE tissue sections were used for miRNA extraction using the miRNeasy FFPE Kit (Qiagen).
T59849 544922-545185 Sentence denotes Expression of miRs was evaluated using the TaqMan MicroRNA Reverse Transcription Kit (Applied jmd.amjpathol.org ■ The Journal of Molecular Diagnostics Biosystems) with primers specific for RNU6B (internal control), miR-10b, miR-21, miR-148a, miR-196a and miR-217.
T54791 545186-545255 Sentence denotes Real-time PCR was performed on the AB 7500 Fast Real-Time PCR System.
T7585 545256-545335 Sentence denotes The expression level of each miRNA was calculated using the formula log2 (2 -).
T54111 545336-545429 Sentence denotes For NGS, DNA was extracted from eight FFPE slides, and quantified using the PicoGreen method.
T70755 545430-545500 Sentence denotes Sequencing was then performed using the IonTorrent PGM on 318v2 chips.
T70913 545501-545562 Sentence denotes Variants were annotated using GoldenHelix SVS software 8.3.4.
T5066 545563-545571 Sentence denotes Results:
T97997 545572-545686 Sentence denotes Metastatic sites included lung (4 cases), liver (3 cases), abdominal wall/peritoneum (3 cases), and bone (1 case).
T93638 545687-545905 Sentence denotes The miRs in both metastatic and non-metastatic groups had similar expression patterns: miR-10b (underexpressed), miR-21 (overexpressed), miR-148a (inconsistent), miR-196a (underexpressed), and miR-217 (underexpressed).
T29192 545906-546005 Sentence denotes Seven cases failed QC for NGS, three due to DNA concentration and/or quality, and four failed qPCR.
T39632 546006-546095 Sentence denotes Like miR expression, NGS results were similar among metastatic and non-metastatic groups.
T94283 546096-546198 Sentence denotes Tumors from both groups contained KRAS (5 cases), TP53 (4 cases), CDKN2A (2 cases), and APC (2 cases).
T66159 546199-546256 Sentence denotes One case of non-metastatic disease showed a RET mutation.
T53094 546257-546339 Sentence denotes Of metastatic disease, one case each showed a SMAD4 mutation, and a JAK3 mutation.
T23638 546340-546367 Sentence denotes Three cases were wild-type.
T11795 546368-546380 Sentence denotes Conclusions:
T60618 546381-546503 Sentence denotes Expression patterns of miR-10b, miR-21, miR-148a, miR-196a, and miR-217 in PDAC were not predictive of distant metastasis.
T98310 546504-546603 Sentence denotes Similarly, NGS results revealed a variety of variants in both patients with and without metastasis.
T76788 546604-546676 Sentence denotes No correlation was seen between variant(s) and miRNA expression pattern.
T43845 546677-546819 Sentence denotes Studies evaluating the genetic basis for metastasis may lead to a better understanding of the pathogenesis and prognosis of pancreatic cancer.
T14550 546821-546998 Sentence denotes Conformation Analysis (CE-SSCA) has been used as a clinical diagnostic assay at the Royal Marsden Hospital (London, UK) for melanoma patients in over 6,000 specimens since 2011.
T21666 546999-547161 Sentence denotes The aim of the study was to assess the performance of the BRAF/NRAS Mutation Test in unselected archived samples and to compare agreement between the two methods.
T84275 547162-547310 Sentence denotes Methods: BRAF exon 15 and NRAS exons 2 and 3 were amplified in a multiplex fluorescent PCR followed by CE-SSCA using an ABI Genetic Analyzer 3130XL.
T10433 547311-547570 Sentence denotes This method has shown a sensitivity of >96% for the common BRAF and NRAS mutations described in malignant melanomas and has a turn-around-time of 4.5 working days for up to 93 samples with a limit of detection between 5-15%, depending on the specific variant.
T89530 547571-547686 Sentence denotes 233 of the 297 samples tested using the BRAF/NRAS Mutation Test were prepared on a Hamilton Starlet liquid handler.
T16871 547687-547877 Sentence denotes The BRAF/NRAS Mutation Test is based on allele-specific, real-time PCR and has been developed for formalin-fixed paraffin-embedded (FFPE) tissue -around-time is about 3 hours for 30 samples.
T73073 547878-547969 Sentence denotes Results: DNA from 304 samples was analyzed by both the BRAF/NRAS Mutation Test and CE-SSCA.
T98803 547970-548038 Sentence denotes Discordant specimens were retested using Illumina's TruSeq protocol.
T74028 548039-548148 Sentence denotes The frequency f BRAF and NRAS mutations was 28.6% and 25.7%, respectively, using the BRAF/NRAS Mutation Test.
T91399 548149-548256 Sentence denotes Seven samples had rare mutations detected by CE-SSCA but were out of scope for the BRAF/NRAS Mutation Test.
T7177 548257-548339 Sentence denotes Seven samples were invalid by the BRAF/NRAS Mutation Test and nineteen by CE-SSCA.
T17690 548340-548406 Sentence denotes The overall agreement between the two methods was 98.6% (273/277).
T93565 548407-548522 Sentence denotes The positive percent agreement (PPA) was 99.4% (165/166), and negative percent agreement (NPA) was 97.3% (108/111).
T70531 548523-548582 Sentence denotes The overall call rate with all samples was 93.3% (277/297).
T89710 548583-548639 Sentence denotes Using Cohen's kappa coefficient the agreement was 0.885.
T48977 548640-548652 Sentence denotes Conclusions:
T86293 548653-548742 Sentence denotes We showed excellent concordance between the Roche BRAF/NRAS Mutation Test (LSR) and SSCA.
T75010 548743-548856 Sentence denotes The BRAF/NRAS Mutation Test offers a sample-to-answer solution, including sample preparation, in 8 hours or less.
T14748 548857-548996 Sentence denotes Furthermore, a cloud-based analysis tool provides a user-friendly data analysis experience and delivers straightforward results in minutes.
T40104 548997-549184 Sentence denotes The mutational status of colorectal carcinoma tumors can provide vital information concerning the prognosis, clinical behavior, and treatment response to specific chemotherapeutic agents.
T72643 549185-549411 Sentence denotes The KRAS and NRAS genes, which are associated with the RAS/RAF/MEK/ERK signaling pathway, are considered consistent predictive biomarkers for monoclonal antibody therapies targeting the epidermal growth factor receptor (EGFR).
T77754 549412-549527 Sentence denotes Traditionally, the efficacy of anti-EFGR therapy was thought to be dependent upon the presence of a wild-type KRAS.
T69996 549528-549691 Sentence denotes However, recent studies have shown that the specific KRAS mutation present may play a more important role in predicting tumor response to EGFR targeted treatments.
T59008 549692-549768 Sentence denotes The value of extended NRAS mutational analysis has not yet been established.
T81224 549769-549951 Sentence denotes The goal of this retrospective study was to identify the specific mutational composition of the RAS gene family (KRAS, NRAS, and HRAS) in primary and metastatic colorectal carcinoma.
T51138 549952-549960 Sentence denotes Methods:
T83868 549961-550089 Sentence denotes Tumor specimens from patients diagnosed with colorectal carcinoma between May 2013 and December 2015 were selected for analysis.
T93639 550090-550229 Sentence denotes Genomic DNA was extracted from formalin-fixed, paraffin-embedded (FFPE) tissues processed at the Dartmouth-Hitchcock Medical Center (DHMC).
T37170 550230-550331 Sentence denotes DNA specimens were multiplexed and sequenced on the Ion Torrent 318v2 chips using the Ion PGM System.
T85933 550332-550507 Sentence denotes Next-generation sequencing was performed using a 50 gene hotspot panel (AmpliSeq Cancer Hotspot Panel v2, Life Technologies) which includes the KRAS, NRAS, and HRAS oncogenes.
T44888 550508-550665 Sentence denotes Variants were identified using the Variant Caller Plugin (v.4.0), and annotation and functional predictions were performed using Golden Helix SVS (v.8.3.4) .
T8731 550666-550674 Sentence denotes Results:
T75528 550675-550757 Sentence denotes We identified a total of 155 colorectal carcinoma tumors harboring a RAS mutation.
T1410 550758-550837 Sentence denotes The most commonly mutated gene was KRAS 93%, followed by NRAS 8% and HRAS 0.6%.
T73894 550838-550924 Sentence denotes KRAS mutations were most prevalent in exons 12 (G12D 24%, G12V 23%) and 13 (G13D 17%).
T11201 550925-551013 Sentence denotes The most common NRAS mutations were identified in exons 12 (G12D 23%) and 61 (Q61H 23%).
T146 551014-551057 Sentence denotes Only one HRAS mutated tumor was identified.
T28227 551058-551070 Sentence denotes Conclusions:
T39871 551071-551194 Sentence denotes Our study indicates that significant variability exists between the RAS mutational profiles of colorectal carcinoma tumors.
T61623 551195-551292 Sentence denotes Further studies to determine the clinical significance of these molecular features are warranted.
T5192 551293-551306 Sentence denotes Introduction:
T37514 551307-551497 Sentence denotes Approximately 8% of non-small cell lung cancer cases feature oncogenic fusions or alternative splicing of cancer-related genes which are amenable to on-market or emerging targeted therapies.
T44787 551498-551647 Sentence denotes Established methods to detect these variants have complex multi-step workflows, restricted target coverage or resolution, and/or limited sensitivity.
T3481 551648-551899 Sentence denotes A new product, the QuantideX NGS RNA Lung Cancer Kit, has potential to address these gaps by covering 110 known oncogenic gene fusions, MET exon 14 skipping, 23 expression markers, and 5 imbalance markers using a streamlined sample-to-answer workflow.
T57476 551900-552036 Sentence denotes In this study, we report consistent results obtained using this technology with well-annotated sample sets in an independent evaluation.
T75418 552037-552045 Sentence denotes Methods:
T82367 552046-552242 Sentence denotes A cohort of 30 study samples were prepared from a collection of total nucleic acid (TNA) isolates derived from formalinfixed, paraffin-embedded (FFPE) residual tumor biopsies and cancer celllines.
T67585 552243-552458 Sentence denotes Target enrichment was performed at Jewish General Hospital and Asuragen on de-identified samples using the QuantideX NGS RNA Lung Kit reagents (Asuragen) and controls, and sequenced on the MiSeq platform (Illumina).
T95862 552459-552676 Sentence denotes Analyses were conducted using QuantideX Reporter (Asuragen), a software suite that includes a FASTQ processing pipeline and incorporates pre-analytical QC information with a fusion-caller algorithm and reporting tool.
T12350 552677-552685 Sentence denotes Results:
T48922 552686-552878 Sentence denotes Targeted RNA-Seq results demonstrated 100% agreement for fusion and splice variant calling across 11 ALK fusions, 1 ROS1 fusion, 1 FGFR3 fusion, 1 MET exon 14 skipping and 16 negative samples.
T59068 552879-553055 Sentence denotes The detection of 3'/5' ALK expression imbalances in known fusion-positive samples indicated potential utility for the detection of fusions not explicitly targeted by the panel.
T4161 553056-553338 Sentence denotes ALK rearrangements were detected down to an input of 400 referencegene amplifiable copies, equivalent to <10 ng of TNA. mRNA expression profiles for the complete set of expression markers covered by the panel were highly correlated (R2>0.98) between intra-and inter-site replicates.
T22805 553339-553449 Sentence denotes Bench procedures from reverse transcription to sequence-ready libraries could be completed in just over 9 hrs.
T32419 553450-553462 Sentence denotes Conclusions:
T87154 553463-553651 Sentence denotes The accuracy of a novel targeted NGS assay for RNA fusions in lung cancer was demonstrated in an independent laboratory evaluation using clinicallyrelevant specimens and low inputs of TNA.
T67643 553652-553993 Sentence denotes The ability of the panel to detect both common and rare gene fusion transcripts and exon splicing events within an integrated wet-bench workflow and companion bioinformatic software provides a foundation for the reliable detection of oncogenic RNA fusions and aberrant splicing events that respond to current and emerging targeted therapies.
T52846 553994-554007 Sentence denotes Introduction:
T194 554008-554255 Sentence denotes Colorectal carcinomas can be separated into two broad categories; 1) those that are microsatellite unstable and associated with hypermutation, and 2) those that are microsatellite stable, chromosomally stable, and associated with nonhypermutation.
T12830 554256-554399 Sentence denotes Amongst the non-hypermutated tumors, studies have reported a striking similarity in the mutational profiles of colon and rectal primary tumors.
T18937 554400-554484 Sentence denotes For this reason, these tumors are typically grouped into a single entity, colorectal
T99838 554485-554576 Sentence denotes The Journal of Molecular Diagnostics ■ jmd.amjpathol.org carcinoma, for molecular purposes.
T43749 554577-554792 Sentence denotes The Cancer Genome Atlas Network identified the most frequently mutated genes in colorectal carcinoma: APC (81%), TP53 (60%), KRAS (43%), TTN (31%), PIK3CA (18%), FBXW7 (11%), SMAD4 (10%), NRAS (9%), and TCF7L2 (9%).
T78120 554793-554980 Sentence denotes Despite their genetic similarities, the clinical management of colon and rectal carcinomas differ due to anatomical location and the higher incidence of local recurrence in rectal tumors.
T7637 554981-555150 Sentence denotes The goal of this retrospective study was to evaluate primary rectal carcinomas and identify mutational characteristics that may differentiate them from colon carcinomas.
T6493 555151-555159 Sentence denotes Methods:
T65063 555160-555284 Sentence denotes Tumor specimens from patients diagnosed with rectal carcinoma between the years of 2013 and 2016 were selected for analysis.
T10694 555285-555365 Sentence denotes Genomic DNA was extracted from formalin-fixed, paraffin-embedded (FFPE) tissues.
T60450 555366-555480 Sentence denotes DNA quantification and quality were assessed using dsDNA Qubit and KAPA hgDNA Quantification and QC, respectively.
T63719 555481-555583 Sentence denotes Barcoded libraries were multiplexed and sequenced on Ion Torrent 318v2 chips using the Ion PGM System.
T92572 555584-555741 Sentence denotes Variants were identified using the Variant Caller Plugin (v.4.0), and annotation and functional predictions were performed using Golden Helix SVS (v.8.3.4) .
T66672 555742-555750 Sentence denotes Results:
T44160 555751-555814 Sentence denotes A total of 66 rectal carcinomas were identified for evaluation.
T28164 555815-555953 Sentence denotes Mutations were most commonly identified in the following genes: TP53 (74%), APC (52%), KRAS (32%), FBXW7 (13%), MET (6%), and PIK3CA (5%).
T11438 555954-556066 Sentence denotes Of the 20 KRAS mutations identified, the most prevalent were in exons 12 (G12D 15%, G12V 15%) and 13 (G13D 20%).
T89827 556067-556079 Sentence denotes Conclusions:
T17737 556080-556253 Sentence denotes Our study indicates that the primary rectal carcinomas encountered at our institution have a mutational profile similar to that previously described in colorectal carcinoma.
T5798 556254-556455 Sentence denotes However, in primary rectal carcinoma, the frequency of mutations in the APC and TP53 genes are inversely proportional to that reported in the combined classification of colorectal carcinomas (p<0.001).
T3801 556456-556547 Sentence denotes Further investigation into the possible clinical significance of this finding is warranted.
T51248 556548-556550 Sentence denotes Y.
T93744 556551-556561 Sentence denotes Lin 1 , R.
T35539 556562-556571 Sentence denotes Yu 2 , W.
T34593 556572-556582 Sentence denotes Hsu 1 , Y.
T25662 556583-556593 Sentence denotes Liu 3 , K.
T99605 556594-556605 Sentence denotes Chiu 2 , Y.
T38821 556606-556756 Sentence denotes Yeh 1 1 General Biologicals Corporation, Hsinchu, Taiwan; 2 Genetics Development Corporation, Lake Bluff, IL; 3 Curiemed Corporation, Hsinchu, Taiwan.
T43296 556757-556770 Sentence denotes Introduction:
T96164 556771-556884 Sentence denotes Determination of HER2 status before administrating therapeutic regimens plays an essential role in breast cancer.
T89001 556885-557004 Sentence denotes Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) are currently working assays for HER2 testing.
T67138 557005-557173 Sentence denotes With the advantages of cost-effectiveness and high accuracy for IHC and FISH respectively, these two assays have been recommended as the routinely-used diagnostic tool.
T52361 557174-557306 Sentence denotes However, several studies have been reported that false signals and lab-to-lab variations are major unsolved issues for HER2 testing.
T96669 557307-557466 Sentence denotes Moreover, both IHC and FISH assays do not have the internal specimen quality control mechanism, which might be the root-causes of the issues that we're facing.
T25617 557467-557475 Sentence denotes Methods:
T44442 557476-557655 Sentence denotes In this study, we developed and evaluated a quantitative HER2 testing assay with built-in internal specimen quality control mechanisms by the principle of multiplex real-time PCR.
T24888 557656-557750 Sentence denotes HER2 messenger RNA copy number could be quantified in real-time using external standard curve.
T6896 557751-557845 Sentence denotes The expression level of reference genes were utilized for monitoring the quality of specimens.
T73 557846-557955 Sentence denotes Eighty FFPE tissues of breast invasive ductal carcinoma were collected and analyzed for the assay validation.
T41927 557956-557964 Sentence denotes Results:
T47810 557965-558092 Sentence denotes The dynamic range of the assay for detecting HER2 mRNA is 5.2E+06 ~ 5.2E+01 copies/mL and showed excellent linearity (R2=0.98).
T57695 558093-558197 Sentence denotes Also, the precision of the assay was demonstrated with inter-operator coefficient of variance (CV) <10%.
T47338 558198-558414 Sentence denotes Most importantly, the validation result showed that the agreement between the developed assay and FDA certified FISH assay is over 90%, suggesting the developed realtime PCR is suitable and reliable for HER2 testing.
T9794 558415-558628 Sentence denotes Furthermore, the quality of FFPE specimens can be distinguished simultaneously with an established cut-off value, which can prevent false signals or lab-to-lab variations that resulted from poor qualified samples.
T17900 558629-558641 Sentence denotes Conclusions:
T57595 558642-558847 Sentence denotes Taken together, the clinical utility of the developed HER2 molecular diagnostic assay shows great FFPE specimen quality control ability and high analytical consistency with current assays for HER2 testing.
T24253 558848-558998 Sentence denotes The ease of use and broad dynamic range makes the developed assay a viable tool for implementation into clinical labs, which may complement IHC tests.
T26365 558999-559114 Sentence denotes Anaplastic Lymphoma Kinase (ALK) represents the only 'druggable' target with a mutation rate of 9-10% in hNB cases.
T76648 559115-559189 Sentence denotes ALK has been implicated as a driver oncogene in a variety of cancer types.
T18037 559190-559330 Sentence denotes As an oncofetal antigen, ALK is an ideal therapeutic target with minimal side effects and it has been simple to target with small molecules.
T47619 559331-559416 Sentence denotes However, resistance to ALK-targeted therapy in other cancers have already been noted.
T61976 559417-559708 Sentence denotes To proactively identify and develop strategies to counter these resistance mechanisms to ALK inhibitors, the present study investigated putative resistance mechanisms identified from our unpublished CRISPR-based genomewide knockout (KO) or overexpression (OV) screens in human NB cell lines.
T24004 559709-559830 Sentence denotes Methods: CRISPR-based whole-genome screening libraries (GeCKO for KO and SAM for OV) were obtained from Zhang Lab at MIT.
T39892 559831-560018 Sentence denotes CRISPR viral libraries were then transduced into SHSY-5Y (ALK+ human NB line) cells after which the cells were cultured in the presence of ALK inhibitors (AP26113 or LDK-378) for 13 days.
T98934 560019-560147 Sentence denotes Enriched CRISPR constructs were identified via HiSeq (RapidRun 100 cycles) of the PCR amplified sgRNAs using custom NGS primers.
T52922 560148-560291 Sentence denotes Resulting FASTQ files were processed and analyzed with MaGECK to identify specific genes/pathways that may induce resistance to ALK inhibitors.
T98357 560292-560413 Sentence denotes Functional validations on each 'hit' identified from the screens were performed in 20 genetically distinct NB cell lines.
T34042 560414-560422 Sentence denotes Results:
T48266 560423-560568 Sentence denotes The KO screen identified 39 candidate genes whereas the OV screen identified 36 candidate genes capable of inducing resistance to ALK inhibitors.
T1039 560569-560678 Sentence denotes The candidate lists included previously reported mechanisms such as RAS pathway genes and mTOR pathway genes.
T22115 560679-560990 Sentence denotes But the lists mostly included novel candidates and Kaplan-Meier analysis (5-year event-free survival) based on publicly available NB patient expression array datasets (n>600) revealed many of those novel candidates (AASDHPPT, CRTC2, TAF12, ETV1, TXLNG, etc.) to be significant (p<0.05) prognostic markers in NB.
T93599 560991-561226 Sentence denotes Some of the putative resistance mechanisms have provided novel therapeutic strategies as PIM1 inhibitors (AZD-1208) produced highly synergistic inhibitory effects against NB cells when applied with ALK inhibitors at low concentrations.
T8308 561227-561239 Sentence denotes Conclusions:
T13225 561240-561341 Sentence denotes The present study produced a list of genes capable of inducing resistance to ALK inhibitors in vitro.
T1074 561342-561553 Sentence denotes As indicated above, this list may be useful as a novel prognostic marker panel for hNB patients and also as a guide to novel therapeutic strategies to ultimately extend the survival periods for the hNB patients.
T18191 561554-561558 Sentence denotes A.M.
T57904 561559-561571 Sentence denotes McDonald, N.
T38598 561572-561583 Sentence denotes Fadra, J.I.
T93495 561584-561596 Sentence denotes Davila, A.A.
T48326 561597-561605 Sentence denotes Nair, J.
T80663 561606-561615 Sentence denotes Jen, R.B.
T24088 561616-561629 Sentence denotes Jenkins, B.R.
T26141 561630-561640 Sentence denotes Kipp, J.L.
T50663 561641-561654 Sentence denotes Winters, B.R.
T33789 561655-561667 Sentence denotes Crusan, W.E.
T21544 561668-561681 Sentence denotes Highsmith, K.
T23088 561682-561695 Sentence denotes Rumilla, J.S.
T40125 561696-561704 Sentence denotes Voss, X.
T18658 561705-561715 Sentence denotes Wang, E.W.
T20513 561716-561726 Sentence denotes Klee, R.N.
T27771 561727-561738 Sentence denotes Wehrs, K.C.
T66605 561739-561774 Sentence denotes Halling Mayo Clinic, Rochester, MN.
T93228 561775-561947 Sentence denotes Introduction: RNASeq is a powerful method for detecting fusions in tumors and can be useful in establishing a diagnosis, determining prognosis and guiding targeted therapy.
T60364 561948-562071 Sentence denotes In this study we describe the verification of an RNASeq assay for the detection of fusions in solid and hematologic tumors.
T89456 562072-562080 Sentence denotes Methods:
T77579 562081-562211 Sentence denotes Total RNA was extracted from solid tumor tissue using a Qiagen miRNeasy Micro kit or whole blood using a Qiagen miRNeasy Mini kit.
T24233 562212-562335 Sentence denotes RIN values were determined using an Agilent 2100 Bioanalyzer and RNA quantity was determined using a Qubit 2.0 fluorometer.
T50236 562336-562605 Sentence denotes Poly-A mRNA selection and cDNA synthesis were performed on a Janus Automated Workstation (Perkin Elmer) and NGS libraries were prepared with a Biomek FXp Liquid Handler (Beckman Coulter) using custom-built protocols and a TruSeq RNASample Preparation v2 Kit (Illumina).
T78081 562606-562700 Sentence denotes Paired-end, 101 bp-read sequencing was performed on a HiSeq 2500 (Illumina) in Rapid Run mode.
T10921 562701-562837 Sentence denotes Data analysis utilized a custom suite of alignment, fusion detection, filtering, and annotation programs to detect fusions in 558 genes.
T99835 562838-563061 Sentence denotes Three known fusion negative tumors and 16 tumors with 15 unique fusions (previously identified by FISH, RT-PCR, or RNA Seq) and 7 normal tissue specimens were analyzed to assess the sensitivity and specificity of the assay.
T2027 563062-563131 Sentence denotes Four samples were analyzed for intra-and inter-assay reproducibility.
T46655 563132-563275 Sentence denotes Seven tumors were used to assess the assay's analytical sensitivity based on the total input mRNA, tumor percentage and total reads per sample.
T45723 563276-563365 Sentence denotes In addition, the impact of RNA degradation on the ability to detect fusions was assessed.
T28038 563366-563374 Sentence denotes Results:
T44294 563375-563541 Sentence denotes Gene fusions were detected in 14/16 tumors with known fusions and 0/10 normal or known fusion negative tumor tissues for a sensitivity of 88% and specificity of 100%.
T60229 563542-563672 Sentence denotes Intra-and inter-assay reproducibility studies revealed 100% concordance for the presence or absence of a fusion in all replicates.
T25402 563673-563815 Sentence denotes Limit of detection thresholds were found to be 62.5ng of input RNA, 25% tumor and 50 million total reads to yield successful fusion detection.
T28097 563816-563944 Sentence denotes Receiver operator curve analysis revealed that 5 or more supporting reads provided the optimal test sensitivity and specificity.
T6765 563945-564136 Sentence denotes We observed that RNA degradation is an important factor found to reduce the ability to detect fusions with a higher impact as the distance of the fusion from the 3' end of the mRNA increases.
T64832 564137-564149 Sentence denotes Conclusions:
T53123 564150-564300 Sentence denotes These studies provide valuable information on the performance characteristics of RNA Seq for the detection of fusions in solid and hematologic tumors.
T35139 564301-564460 Sentence denotes An understanding of the strengths and limitations of this assay will aid in the appropriate interpretation of RNA Seq results obtained during clinical testing.
T30442 564461-564463 Sentence denotes R.
T51154 564464-564476 Sentence denotes Luthra, R.R.
T79552 564477-564488 Sentence denotes Singh, A.J.
T18095 564489-564498 Sentence denotes Lazar, W.
T53355 564499-564509 Sentence denotes Wang, J.M.
T41579 564510-564520 Sentence denotes Meis, K.P.
T54 564521-564532 Sentence denotes Patel, M.J.
T28609 564533-564547 Sentence denotes Routbort, D.D.
T26975 564548-564559 Sentence denotes Panditi, J.
T99091 564560-564569 Sentence denotes Yau, D.Z.
T99498 564570-564582 Sentence denotes Hatfield, J.
T52534 564583-564595 Sentence denotes Medeiros, R.
T49882 564596-564662 Sentence denotes Luthra University of Texas MD Anderson Cancer Center, Houston, TX.
T41118 564663-564676 Sentence denotes Introduction:
T17795 564677-564777 Sentence denotes Sarcomas are a rare heterogeneous group of mesenchymal tumors, which can be challenging to diagnose.
T90272 564778-564911 Sentence denotes Fortunately, a sizeable portion of sarcomas are driven by gene translocations (30% to 40%) and can be useful in classifying sarcomas.
T58211 564912-565027 Sentence denotes A sensitive assay that can identify gene translocations in sarcoma in a timely fashion is of high diagnostic value.
T2901 565028-565245 Sentence denotes In a clinical diagnostic laboratory, we have designed and validated a nanofluidics-based high-throughput RT-PCR platform to screen for fusions in 12 genes using a single input of FFPE RNA from sarcoma tumor specimens.
T77615 565246-565254 Sentence denotes Methods:
T73486 565255-565368 Sentence denotes Taqman-based real-time PCR assays (ThermoFisher Scientific) were designed to cover 38 exonic fusions in 12 genes.
T10261 565369-565413 Sentence denotes GAPDH was used as an RNA-expression control.
T22665 565414-565635 Sentence denotes Synthetic control plasmids with the fusion sequence jmd.amjpathol.org ■ The Journal of Molecular Diagnostics interrogated by the 38 assays were custom ordered (Integrated DNA Technologies) and used to optimize the assays.
T96138 565636-565733 Sentence denotes Sarcoma specimens (n=35), two cancer cell lines (A4573 and RD-ES) and 3 normal samples were used.
T99754 565734-565886 Sentence denotes The tumors were positive for fusions involving genes such as SS18, EWSR1, NAB2, HEY1, PAX3 as tested by RT-PCR, FISH or predicted by the tumor sub-type.
T70465 565887-566053 Sentence denotes RNA was extracted from the FFPE tissue slides using the AllPrep DNA/RNA FFPE kit (Qiagen) and cDNA was prepared using SuperScript VILO kit, (ThermoFisher Scientific).
T50532 566054-566233 Sentence denotes On each Nanofluidics PCR 48X48 Fluidigm chips, 15 samples (14 tumors and A4573 positive control) were simultaneously tested for 38 fusions in triplicates on BioMark HD instrument.
T37895 566234-566380 Sentence denotes Limit-of-detection and reproducibility studies were performed using sequentially diluted FFPE tumor and A4573 cell line RNAs into normal FFPE RNA.
T80642 566381-566389 Sentence denotes Results:
T3724 566390-566467 Sentence denotes The 38 assays were tested and optimized using the synthetic plasmid controls.
T59162 566468-566566 Sentence denotes Out of the 35 samples tested, expected fusions were detected in 32 samples and the two cell lines.
T25261 566567-566655 Sentence denotes In the remaining 3 samples, two were sub-optimal specimens (low RNA and/or decalcified).
T47532 566656-566703 Sentence denotes No fusions were detected in the normal samples.
T74336 566704-566871 Sentence denotes Considering the results from all positive samples, 250ng RNA input for analysis and a Ct value of 20 or lower was found to be optimal cutoff for positive fusion calls.
T2740 566872-566979 Sentence denotes RNA dilution studies demonstrated robust detection sensitivity and intertech and inter-run reproducibility.
T15796 566980-566992 Sentence denotes Conclusions:
T96886 566993-567225 Sentence denotes We have optimized and validated a nanofluidics-based PCR platform which is highly suited for routine high-throughput screening of multiple gene fusions in sarcomas with a single input of FFPE RNA in a clinical diagnostic laboratory.
T61567 567226-567228 Sentence denotes T.
T83355 567229-567246 Sentence denotes Sakamoto 1,2 , S.
T73919 567247-567263 Sentence denotes Matsumoto 2 , T.
T69596 567264-567278 Sentence denotes Saito 2,3 , A.
T19969 567279-567293 Sentence denotes Tsuyada 3 , K.
T30846 567294-567475 Sentence denotes Tsuchihara 2 1 Tottori University, Yonago, Tottori, Japan; 2 National Cancer Center, Kashiwanoha, Kashiwa Chiba, Japan; 3 Riken Genesis Co., Ltd., Tsurumi, Yokohama Kanagawa, Japan.
T89652 567476-567489 Sentence denotes Introduction:
T89205 567490-567642 Sentence denotes Detection of driver gene alterations including gene rearrangements is an essential step for a treatment decision of advanced non-small cell lung cancer.
T97331 567643-567703 Sentence denotes However, genetic testings generally require invasive biopsy.
T48655 567704-567801 Sentence denotes Liquid biopsy is a promising technology which can detect gene alterations with minimal invasions.
T4214 567802-567907 Sentence denotes Circulating cell-free tumor DNA (ctDNA) has been revealed to be useful to detect cancer gene alterations.
T21354 567908-568047 Sentence denotes However, it is difficult to accurately detect gene rearrangements by ctDNA based liquid biopsy even though various methods have been tried.
T53430 568048-568145 Sentence denotes On the other hand, there is cell-free tumor RNA in circulation (ctRNA) as is the case with ctDNA.
T66566 568146-568244 Sentence denotes Therefore, we aimed to develop high sensitivity detection methods for EML4-ALK fusion using ctRNA.
T89622 568245-568253 Sentence denotes Methods:
T47145 568254-568351 Sentence denotes We developed two different high sensitivity detection methods of EML4-ALK fusion based on RT-PCR.
T31557 568352-568471 Sentence denotes The first was multiplexed RT-PCR with fusion specific hydrolysis probes combined with preamplification (OneTube assay).
T87391 568472-568549 Sentence denotes The second was detection of 3' ALK mRNA with nested RT-PCR (NestedPCR assay).
T85548 568550-568690 Sentence denotes Blood samples were collected from patients with EML4-ALK positive lung adenocarcinoma with EDTA blood collection tubes and then centrifuged.
T35819 568691-568743 Sentence denotes Blood plasma samples were stored at -80°C until use.
T74682 568744-568841 Sentence denotes RNA extracted from cell-free culture supernatant of H2228 cell line was used as positive control.
T62220 568842-568936 Sentence denotes RNA was extracted from 1mL stored plasma with Plasma/Serum RNA Purification Midi Kit (Norgen).
T1997 568937-568945 Sentence denotes Results:
T2552 568946-569094 Sentence denotes OneTube assay accurately detected EML4-ALK fusion from diluted samples which contained approximately 2.5 copies of cellfree culture supernatant RNA.
T77741 569095-569223 Sentence denotes OneTube assay was considered to have the ability to detect 1 copy of fusion mRNA considering sample concentration inhomogeneity.
T43529 569224-569286 Sentence denotes NestedPCR assay had equivalent sensitivity with OneTube assay.
T54795 569287-569424 Sentence denotes Both methods could detect EML4-ALK fusion from culture supernatant ctRNA although they could not detect from six clinical plasma samples.
T12943 569425-569437 Sentence denotes Conclusions:
T24309 569438-569535 Sentence denotes We succeeded in development of 2 different high sensitivity detection methods of EML4-ALK fusion.
T67297 569536-569592 Sentence denotes However, we could not detect EML4-ALK from plasma ctRNA.
T84661 569593-569674 Sentence denotes It is expected that there is no or very little amount of ctRNA in plasma samples.
T385 569675-569796 Sentence denotes Development of a novel method enriching ctRNA is needed to detect gene rearrangements by liquid biopsy with plasma ctRNA.
T8317 569797-569994 Sentence denotes Introduction Several guidelines for metastatic colorectal cancer currently recommend genotyping for RAS (KRAS/NRAS) and BRAF mutations to select for patients eligible to anti-EGFR antibody therapy.
T4379 569995-570089 Sentence denotes PIK3CA mutations have been associated with resistance to anti-EGFR therapy in several studies.
T82800 570090-570250 Sentence denotes The frequencies of mutation estimated for KRAS (around 40%), NRAS (2% to 15%), PIK3CA (14.5%) and BRAF (5% to 10%) vary according to methodology and population.
T18316 570251-570765 Sentence denotes In Mexico, colorectal cancer represents the third most common cause of cancer with an estimated incidence of 3.3x100,000 individuals, around 30% presenting as stage IV disease at diagnosis.No data published currently exist to document the status of relevant biomarkers for colorectal cancer therapy in Mexican population.The aim of the present study was to determine the status of these biomarkers in a cohort of patients referred to the Cancer Center of the American-British Cowdray Medical Center in Mexico City.
T79812 570766-570774 Sentence denotes Methods:
T33401 570775-570970 Sentence denotes We performed a retrospective search on the archives of the Pathology Department for cases diagnosed with colorectal adenocarcinoma with KRAS/NRAS/BRAF determination from January 2013 to May 2016.
T67641 570971-571148 Sentence denotes DNA was extracted using QIAamp DNA FFPE kit (Qiagen) and biomarker assessment was performed with Therascreen KRAS, BRAF and PI3KCA kits (Qiagen) using Rotor-Gene Q Thermocycler.
T20530 571149-571324 Sentence denotes NRAS determination was done with Sanger sequencing using consensus primers to amplify regions adjacent to codons 12, 13 and 61 in a 3500 Genetic Analyzer (Applied Biosystems).
T71748 571325-571333 Sentence denotes Results:
T6001 571334-571421 Sentence denotes We retrieved 99 cases, all of them with KRAS determination (59% wild-type/41% mutated).
T91385 571422-571612 Sentence denotes Mutation frequency by type is as follows: c.35G>A(p.G12D)14%, c.35G>T(p.G12V)9%, c.38G>A(p.G13D)7%, c.34G>T(p.G12C)4%, c.34G>A(p.G12S)3%, c.35G>C(p.G12A) and c.34G>A (p.G12R)2% respectively.
T65934 571613-571671 Sentence denotes Of the whole population, 33.3% (n=33) had NRAS assessment.
T69387 571672-571706 Sentence denotes No NRAS mutations were identified.
T40492 571707-571912 Sentence denotes Twenty-four percent (n=24) of the cases had additional BRAF mutation determination, only two cases (2% overall) had a c.1799T>A(V600E) mutation, one of them with a concurrent KRAS mutation c.35G>T(p.G12V).
T32782 571913-572134 Sentence denotes PI3KCA mutation assessment was performed in 14 cases with sufficient DNA to study, 28.6% were mutated, 2 cases with exon 20 mutation c.3140A>G (H1047R)(2% overall) and 2 with exon 9 mutation c.1624G>A (E542K)(2% overall).
T53192 572135-572147 Sentence denotes Conclusions:
T34668 572148-572280 Sentence denotes In our study, KRAS mutation profile of colorectal adenocarcinoma reflects what has been found in other populations around the world.
T56003 572281-572499 Sentence denotes The stepwise-approach of sequentially extend mutation assessment (KRAS-NRAS-BRAF) (based on being mutually exclusive) as well as biased biomarker selection (NRAS versus BRAF), may not fully reflect mutation prevalence.
T17266 572500-572609 Sentence denotes International consensus guidelines to extensively address colorectal predictive biomarker testing are needed.
T42997 572610-572612 Sentence denotes A.
T56982 572613-572630 Sentence denotes Bergamaschi, K.M.
T30077 572631-572650 Sentence denotes Clark-Langone, D.A.
T51097 572651-572663 Sentence denotes Eberhard, J.
T40818 572664-572671 Sentence denotes Han, R.
T32887 572672-572683 Sentence denotes Loverro, P.
T56070 572684-572698 Sentence denotes Harrington, C.
T52014 572699-572705 Sentence denotes Ku, Y.
T15158 572706-572712 Sentence denotes Ma, W.
T84534 572713-572721 Sentence denotes Gibb, A.
T86002 572722-572735 Sentence denotes Dei Rossi, L.
T15715 572736-572746 Sentence denotes Shen, A.D.
T76034 572747-572760 Sentence denotes Goddard, K.M.
T76435 572761-572780 Sentence denotes Clark-Langone, J.M.
T42490 572781-572793 Sentence denotes Anderson, G.
T41318 572794-572843 Sentence denotes Alexander Genomic Health, Inc., Redwood City, CA.
T58280 572844-572857 Sentence denotes Introduction:
T73483 572858-573081 Sentence denotes There is increasing interest in the field of molecular diagnostics to perform liquid biopsies rather than tissue biopsies when tissue is unavailable, or when invasive procedures are associated with high risk to the patient.
T41080 573082-573260 Sentence denotes The 17-Gene Liquid Biopsy Mutation Panel is a Next-generation sequencing (NGS) assay, designed to detect clinically actionable variants in circulating tumor DNA (ctDNA) in blood.
T72655 573261-573452 Sentence denotes Prior to clinical use, analytic performance must be demonstrated to meet the intended use of the assay, and thus requires using clinically relevant variants at levels representative of ctDNA.
T66717 573453-573461 Sentence denotes Methods:
T27081 573462-573657 Sentence denotes Analytical specificity and sensitivity (collectively representing analytical accuracy) were characterized through determination of Limit of Blank (LoB) and Limit of Detection (LoD), respectively.
T68881 573658-573950 Sentence denotes LoB was determined using 73 cell-free DNA (cfDNA) samples from 60 healthy donors, whereas LoD (defined as minimum allelic fraction (AF)/ copy number (CN) required for a 95% detection rate) was determined using a model system of titrated cell line DNA harboring clinically actionable variants.
T63729 573951-574079 Sentence denotes Within and between assay runs, reproducibility was assessed across replicate assays of cfDNA pools derived from cancer patients.
T79739 574080-574284 Sentence denotes The impact of high molecular weight DNA (HWW-DNA) as a potential interfering substance was assessed using 2 approaches; stored blood from healthy controls and HMW-DNA spiked into variant positive samples.
T45013 574285-574293 Sentence denotes Results:
T20650 574294-574383 Sentence denotes Detection thresholds were set above the LoB corresponding to >99% per sample specificity.
T22038 574384-574555 Sentence denotes Mean LoDs on a per sample basis were as follows; single nucleotide variants, 0.56% AF; insertions/deletions, 0.19% AF; fusions, 0.37% AF, and copy number gain, 2.7 copies.
T63746 574556-574661 Sentence denotes The assay was highly robust, demonstrated by detection of >95% of variants near the LoD in 2 cfDNA pools.
T36672 574662-574833 Sentence denotes Increasing amounts of HMW-DNA proportionally impacted intermediate analytical metrics, however, sensitivity of detection of SNVs, indels and SVs was relatively unaffected.
T80770 574834-574958 Sentence denotes For CNV, a decrease in specificity was apparent at longer blood storage times and with increasing amounts of spiked HMW-DNA.
T74855 574959-574971 Sentence denotes Conclusions:
T93264 574972-575078 Sentence denotes Here we report the analytic performance of the 17-Gene Liquid Biopsy Mutation Panel on a per sample basis.
T91174 575079-575221 Sentence denotes In addition to being highly sensitive and specific, we demonstrated that the 17-Gene Liquid Biopsy Mutation Panel yields reproducible results.
T88875 575222-575385 Sentence denotes Finally, we illustrate the importance of characterizing the impact of HMW-DNA resulting from blood storage time on the detection of clinically actionable variants.
T70024 575386-575388 Sentence denotes D.
T39863 575389-575402 Sentence denotes Wieczorek, S.
T51167 575403-575416 Sentence denotes Hermanson, C.
T68344 575417-575425 Sentence denotes Knox, J.
T92358 575426-575434 Sentence denotes Mook, D.
T93339 575435-575446 Sentence denotes Horejsh, E.
T54408 575447-575458 Sentence denotes Vincent, D.
T55602 575459-575469 Sentence denotes Storts, T.
T1492 575470-575499 Sentence denotes Schagat Promega, Madison, WI.
T65093 575500-575513 Sentence denotes Introduction:
T86211 575514-575759 Sentence denotes Quantity and quality of DNA from formalin-fixed, paraffin-embedded (FFPE) tumor tissue samples is highly variable, with degradation and crosslinking due to the fixation process leading to issues with amplification and difficulty in NGS analysis.
T63375 575760-575860 Sentence denotes An alternative to FFPE is circulating cell-free DNA (ccfDNA) from plasma or other biological fluids.
T48635 575861-575971 Sentence denotes Compared to gDNA, ccfDNA yields are typically low, with tumor cell present at significantly lower frequencies.
T62790 575972-576115 Sentence denotes Due to the inherent variability of FFPE and ccfDNA, knowing the quantity of DNA is not in itself reliably predictive of downstream NGS success.
T93683 576116-576234 Sentence denotes In this poster, we describe novel methods for predicting sequencing result quality utilizing a multiplexed qPCR assay.
T12209 576235-576376 Sentence denotes Methods: DNA was purified from four matching tumor and normal FFPE tissue types as well as ccfDNA from plasma samples using multiple methods.
T41366 576377-576534 Sentence denotes DNA quantity was measured via single-target qPCR and used for downstream NGS library construction with a 56 gene oncology panel and subsequent data analysis.
T77230 576535-576565 Sentence denotes Discrepancies between quantity
T25232 576566-576734 Sentence denotes The Journal of Molecular Diagnostics ■ jmd.amjpathol.org of DNA input into library preparation and expected library yield and sequencing coverage uniformity were noted.
T37718 576735-576933 Sentence denotes To investigate if downstream library yield and sequencing quality could be better predicted, a multiplexed qPCR assay was designed that included three different amplicon sizes (75, 150, and 300bp) .
T36542 576934-577206 Sentence denotes The quantitative differences between the increasingly larger amplicon sizes were calculated as a ratio to determine the level of degradation of the DNA from FFPE samples. ccfDNA fragments cluster around 170bp; thus, the 150bp target closely estimates ccfDNA concentration.
T25105 577207-577338 Sentence denotes Since gDNA is expected to be much larger in size, the ratio of 150bp to 300bp targets can help predict the ratio of ccfDNA to gDNA.
T44600 577339-577503 Sentence denotes FFPE samples with high 75/300bp ratios are indicative of highly degraded samples, and ccfDNA samples with low 150/300bp ratios are indicative of gDNA contamination.
T59762 577504-577512 Sentence denotes Results:
T51129 577513-577658 Sentence denotes Retroactive testing with the multiplexed qPCR assay showed a strong correlation of degraded DNA to low library yield and low coverage uniformity.
T51406 577659-577765 Sentence denotes Samples with equal concentration and library input performed much better when degradation ratios were low.
T75156 577766-577778 Sentence denotes Conclusions:
T6861 577779-577886 Sentence denotes Data derived from a multisize target qPCR assay can be very effective in predicting downstream NGS success.
T35901 577887-578022 Sentence denotes Using such a QC method can drive researchers to triage samples and make informed decisions about what downstream library method to use.
T89775 578023-578293 Sentence denotes Concentrating on less complex panels or ddPCR vs. highly multiplexed panels or whole exome sequencing for degraded samples can ensure getting the most useful information out of an individual sample, thus saving time, cost, and loss of information about precious samples.
T39465 578294-578296 Sentence denotes A.
T76873 578297-578305 Sentence denotes Olar, R.
T50030 578306-578318 Sentence denotes Broaddus, M.
T20354 578319-578331 Sentence denotes Routbort, S.
T83928 578332-578351 Sentence denotes Roy-Chowdhuri, K.B.
T77045 578352-578364 Sentence denotes Hodges, R.R.
T25146 578365-578376 Sentence denotes Singh, A.J.
T79862 578377-578386 Sentence denotes Lazar, A.
T18694 578387-578397 Sentence denotes Rashid, H.
T29988 578398-578406 Sentence denotes Chen, A.
T76162 578407-578424 Sentence denotes Yemelyanova, J.L.
T10863 578425-578439 Sentence denotes Medeiros, S.R.
T2860 578440-578452 Sentence denotes Hamilton, R.
T89884 578453-578465 Sentence denotes Luthra, K.P.
T26601 578466-578531 Sentence denotes Patel University of Texas MD Anderson Cancer Center, Houston, TX.
T55023 578532-578545 Sentence denotes Introduction:
T87766 578546-578764 Sentence denotes Broader genomic sequencing by NGS panels pose a unique challenge for oncologic somatic mutation analysis due to identification of variants for which the somatic versus germline origin cannot be definitively determined.
T75872 578765-578874 Sentence denotes The higher number of calls resulting from broader coverage further increases the complexity of data analysis.
T44130 578875-579007 Sentence denotes Bioinformatics tools and public databases can help to a certain extent in identification of germline variants, but, are not perfect.
T63675 579008-579172 Sentence denotes In comparison, a paired normal sample allows definitive identification of variants of germline origin, but, poses challenges in collection, analysis and throughput.
T90816 579173-579261 Sentence denotes We evaluated the value of paired tumor-normal sample analysis in solid tumor genotyping.
T92353 579262-579431 Sentence denotes Methods: NGSbased targeted genotyping of 134 genes (Oncomine Comprehensive Assay, ThermoFisher) was performed on paired tumor-normal (T/N) samples from 103 solid tumors.
T55671 579432-579496 Sentence denotes Sequencing data analysis was performed by TorrentSuite software.
T85457 579497-579674 Sentence denotes Variant calls and coverage information were evaluated using an in-house developed genomic analysis and reporting tool (Oncoseek) and Integrative Genome Viewer (Broad Institute).
T7044 579675-579793 Sentence denotes A combination of variety of database-level and matched T/N filters were applied to identify optimal analysis workflow.
T61160 579794-579924 Sentence denotes We compared the analysis and reporting workflow for the same set of samples under tumor-only (T) vs. paired T/N scenarios.Results:
T83190 579925-580013 Sentence denotes Average number of total variants called per sample was 214.5 (range=141-432;median=208).
T69115 580014-580129 Sentence denotes In a tumor only scenario, database-level filters reduced the average number of calls to 8.6 (range=2-21, median=8).
T61300 580130-580268 Sentence denotes In paired T/N scenario, the germline filter alone reduced the average number of variant calls to 30.6 (range=8-227; median=23) (p<0.0001).
T61306 580269-580373 Sentence denotes The number of germline variants that were filtered out ranged from 130 to 249 (mean=183.85; median=183).
T56383 580374-580599 Sentence denotes A combination of germline and database-level filters in paired T/N setting significantly reduced the average calls for review per sample to 3.6 (range=0-17, median=3) in comparison to T-only or T/N with only germline filters.
T45613 580600-580711 Sentence denotes The final reported somatic mutations averaged 2.8 per sample (range=0-14; median=2) after pathologist's review.
T66536 580712-580889 Sentence denotes As expected, polymorphisms were common in BRCA1 and BRCA2; however, 8 of 118 variants were determined to be of somatic origin, which may be relevant for ongoing clinical trials.
T89917 580890-580902 Sentence denotes Conclusions:
T6142 580903-581071 Sentence denotes Database-level (population and laboratory specific) filters are efficient in identification of majority of artifacts, polymorphisms, non-coding and synonymous variants.
T18476 581072-581325 Sentence denotes The addition of paired normal analysis further improves the somatic mutation analysis significantly by eliminating ambiguity in reporting of calls where the determination of germline versus somatic origin in not feasible through informatics tools alone.
T76081 581326-581339 Sentence denotes Introduction:
T42721 581340-581584 Sentence denotes We previously defined 3 molecular subgroups of High Grade Serous Ovarian Cancer (HGSOC), using gene expression data from 265 FFPE samples obtained from treatment naive patients who received platinum based treatment following surgical resection.
T34164 581585-581811 Sentence denotes The 3 molecular subgroups were Angio: characterised by upregulation of angiogenesis genes; Immune: characterised by upregulation of immune genes and Angio_Immune: characterised by upregulation of angiogenesis and immune genes.
T16787 581812-582008 Sentence denotes Patients within these 3 subgroups respond differently to standard of care treatment with the Immune subgroup having best prognosis and Angio and Angio_Immune groups having similar worse prognosis.
T19256 582009-582093 Sentence denotes A weighted gene signature to identify each of the molecular subgroups was developed.
T38138 582094-582228 Sentence denotes This dataset and associated subgroup designation was used to investigate the effect of chemotherapy on molecular subgroup designation.
T51732 582229-582237 Sentence denotes Methods:
T16449 582238-582390 Sentence denotes To investigate the effect of chemotherapy on predefined molecular subgroups we analysed 35 matched pre-and post-chemotherapy samples by gene expression.
T16828 582391-582488 Sentence denotes The samples were scored with each of the 3 gene signatures and assigned into molecular subgroups.
T20659 582489-582590 Sentence denotes Novel cisplatin resistant HGSOC cell lines were generated to study mechanisms of acquired resistance.
T16211 582591-582599 Sentence denotes Results:
T16612 582600-582737 Sentence denotes Forty-four percent of the treatment naive samples were aligned with the Angio_Immune subgroup and this increased to 71% postchemotherapy.
T22407 582738-582942 Sentence denotes This was especially evident in the Immune group where 67% of cases associated with this good prognosis group pre-chemotherapy, were associated with the bad prognosis Angio_Immune group, post-chemotherapy.
T2651 582943-583147 Sentence denotes Hence platinum chemotherapy selects for the Angio_Immune group, suggesting that this subgroup represents tumours which are innately platinum resistant but also provides a mechanism of acquired resistance.
T34885 583148-583415 Sentence denotes Additionally we demonstrate that the Angio_Immune group is driven by activation of the MAPK pathway and show that cisplatin resistant HGSOC cell lines are specifically sensitive to MEK inhibitors using trametinib suggesting an approach to treating this patient group.
T41817 583416-583428 Sentence denotes Conclusions:
T1454 583429-583513 Sentence denotes The MAPK pathway is a mechanism of innate and acquired platinum resistance in HGSOC.
T19750 583514-583681 Sentence denotes Furthermore the data suggests that original pre-treatment surgical/biopsy samples may fall within a different molecular subgroup to samples taken postplatinum therapy.
T90889 583682-583695 Sentence denotes Introduction:
T42685 583696-583845 Sentence denotes Next-generation sequencing is replacing traditional molecular methods in clinical laboratories to simultaneously detect multiple genetic alterations.
T55316 583846-583977 Sentence denotes At the same time, bioinformatics challenges for handling raw sequence data, through to clinical decision support, must be overcome.
T1923 583978-584131 Sentence denotes We evaluated the Illumina TruSeq Amplicon Cancer Panel (TSACP), along with bioinformatics softwares from Illumina and Philips in our clinical laboratory.
T18141 584132-584410 Sentence denotes Although we found similar sensitivity and specificity to traditional methods, with little difference between the bioinformatics pipelines for detection of most variants, we encountered lower concordance for larger (>15bp) InDels between both analysis pipelines and ground truth.
T63172 584411-584627 Sentence denotes Post variant detection, we have implemented a decision support workflow to streamline variant filtering and annotation, provide assistance with on-and out-of-indication therapy, reporting and clinical trial matching.
T20424 584628-584835 Sentence denotes Although we have not formalized the evaluation of the clinical decision support workflow, our initial experience leads us to believe it will also improve overall laboratory efficiency and patient experience.
T56288 584836-584844 Sentence denotes Methods:
T19539 584845-585064 Sentence denotes In this study, 55 clinical specimens (formalin-fixed, paraffin-embedded; peripheral blood; or bone marrow), 7 human cancer cell lines and HapMap DNA NA12878 samples were used for TSCAP validation on an Illumina MiseqDx.
T6757 585065-585212 Sentence denotes All of these samples were well characterized by traditional methods with mutations found in 7 genes (KRAS, BRAF, EGFR, JAK2, NPM1, FLT3 and IDH1) .
T86461 585213-585298 Sentence denotes All samples successfully processed with the TSACP and sequenced in 4 sequencing runs.
T71377 585299-585365 Sentence denotes The data were analyzed by both VariantStudio and Philips pipeline.
T80058 585366-585454 Sentence denotes The Philips pipeline resulted from indepth comparison of four different variant callers.
T71228 585455-585538 Sentence denotes Results: TSACP was highly repeatable and reproducible with 5% analytic sensitivity.
T70847 585539-585621 Sentence denotes Except for 10 amplicons, all 212 amplicons had >X500 average coverage in all runs.
T28387 585622-585766 Sentence denotes Both VariantStudio and Philips pipeline showed 100% concordance with clinically validated results for point mutation and smaller InDels (<15bp).
T75029 585767-585903 Sentence denotes For larger InDels (>15bp), the detection rates were 56%, 62% and 81% for VariantStudio, Philips pipeline and both methods, respectively.
T5648 585904-586086 Sentence denotes Conclusions: TSACP is a sensitive, reliable pan-cancer mutation panel for sequencing of cancer hot-spot mutations and could be used in clinical laboratory to replace previous method.
T27266 586087-586190 Sentence denotes All bioinformatics softwares demonstrated excellent performance for point mutations and smaller InDels.
T27927 586191-586253 Sentence denotes Howerver, large InDels are much more difficult to be detected.
T47263 586254-586354 Sentence denotes A combination of different data analysis softwares may increase the detection rate for large InDels.
T23748 586355-586570 Sentence denotes In non-small cell lung cancer (NSCLC), the canonical EML4-ALK inversion results in a fusion protein with a constitutively active ALK kinase, and is associated with response to targeted inhibitors such as crizotinib.
T14223 586571-586847 Sentence denotes Clinically, anti-ALK therapy is initiated based on evidence of an ALK rearrangement, detected in tissue jmd.amjpathol.org ■ The Journal of Molecular Diagnostics sections by fluorescent in situ hybridization (FISH) of interphase cells or by anti-ALK immunohistochemistry (IHC).
T16929 586848-587048 Sentence denotes However, FISH cannot differentiate between different ALK partner genes or non-canonical breakpoints, raising the possibility that not all detected ALK fusions may be responsive to targeted inhibitors.
T9712 587049-587286 Sentence denotes Indeed, "ALK positive" NSCLC has only a 60% objective response rate to ALK inhibitors; we sought to determine whether next-generation sequencing (NGS) of targeted DNA regions and/or RNA could refine the diagnosis of ALK rearranged NSCLC.
T28004 587287-587295 Sentence denotes Methods:
T36788 587296-587565 Sentence denotes From a set of 1532 cases at our institution evaluated by interphase FISH for ALK rearrangement, we identified 20 positive cases and sequenced them using a targeted, capture-based clinical NGS panel that included ALK introns 16-22 among other other cancer-related genes.
T10406 587566-587663 Sentence denotes DNA level rearrangements were identified using the Breakdancer and ClusterFast software packages.
T71913 587664-587835 Sentence denotes Strand-specific RNA-seq libraries were created from FFPE-derived RNA on 12 cases with remaining tissue and analyzed for fusion transcripts using the Tophat fusion package.
T48520 587836-587912 Sentence denotes Immunohistochemistry (IHC) was performed on 18 cases with the D5F3 antibody.
T92046 587913-587921 Sentence denotes Results:
T42164 587922-588038 Sentence denotes Of the 20 cases positive for ALK rearrangement by FISH, 17 (85%) showed evidence of rearrangement by DNA sequencing.
T30916 588039-588143 Sentence denotes These included four different canonical rearrangements: three EML4-ALK variants and a KIF5B-ALK variant.
T23359 588144-588230 Sentence denotes Non-canonical breakpoints and rearrangement partners were identified in 6 cases (35%).
T64449 588231-588355 Sentence denotes RNA-seq results were largely consistent with DNA sequencing, failing to detect expected fusion transcripts in 2 of 12 cases.
T97551 588356-588537 Sentence denotes In contrast, ALK IHC was positive in 16 of 18 cases, including 3 cases in which neither DNA nor RNA sequencing provided evidence of a fusion transcript capable of producing protein.
T34203 588538-588550 Sentence denotes Conclusions:
T71697 588551-588696 Sentence denotes Current FISH and IHC testing for "ALK positive" lung adenocarcinoma underestimate the biologic and genomic heterogeneity among ALK driven NSCLCs.
T52463 588697-588790 Sentence denotes In contrast, NGS-based testing can resolve novel partner genes and non-canonical breakpoints.
T59869 588791-589066 Sentence denotes The genomic heterogeneity observed in this study may account for differences in therapeutic response to targeted ALK inhibitors; however, future clinical trials will be needed to ascertain the exact significance of non-canonical and non-expressed ALK rearrangements in NSCLC.
T6193 589067-589080 Sentence denotes Introduction:
T54463 589081-589221 Sentence denotes The use of targeted gene panels in cancer to identify somatic mutations has the potential to inform prognosis and guide treatment decisions.
T10238 589222-589432 Sentence denotes One challenge is that selection of genes for such panels and the designation of actionable findings will continue to evolve as new therapies are developed and new mechanisms of tumor progression are discovered.
T31167 589433-589625 Sentence denotes In this regard, ongoing genomic profiling efforts have recently identified recurrent mutations in the exonuclease POLE) that are associated with high mutational burden in several cancer types.
T3843 589626-589815 Sentence denotes Whereas mismatch repair status has been shown to predict immunotherapy response in clinical trials, tumors with POLE mutations have yet to be broadly included for evaluation in such trials.
T39312 589816-590047 Sentence denotes Here, we describe two cases of colon cancer found to harbor POLE mutations after initial testing revealed an unusually high mutational burden in the setting of microsatellite stability and intact mismatch repair protein expression.
T70453 590048-590056 Sentence denotes Methods:
T47235 590057-590194 Sentence denotes Extended genomic profiling was performed on two cases of microsatellite stable colorectal carcinoma found to have high mutational burden.
T90151 590195-590344 Sentence denotes DNA was extracted from formalin-fixed tumor samples and used to prepare hybrid-capture libraries for genomic profiling by next-generation sequencing.
T28818 590345-590485 Sentence denotes A clinically validated capture panel of 198 cancer-related genes and a research panel of 142 genes undergoing clinical validation were used.
T55883 590486-590634 Sentence denotes Data analysis was performed using an internally developed and clinically validated bioinformatics pipeline followed by expert review and annotation.
T7958 590635-590708 Sentence denotes All testing was performed in a CLIA-certified, CAP-accredited laboratory.
T34765 590709-590717 Sentence denotes Results:
T13032 590718-590936 Sentence denotes Extended evaluation of these two cases of hypermutated, microsatellite-stable colon cancer revealed nonsynonymous changes in the POLE exonuclease domain at known mutational hotspots (p.P286R and p.V411L, respectively).
T62938 590937-591094 Sentence denotes This testing demonstrated high reproducibility in regards to variant identification and allele frequency between two distinct but overlapping capture panels.
T49938 591095-591328 Sentence denotes Notably, these findings led to discussion at a molecular tumor board and initiation of the approval process for immune checkpoint therapy on a compassionate use basis for these patients with POLE mutations and high mutational burden.
T28298 591329-591341 Sentence denotes Conclusions:
T90851 591342-591492 Sentence denotes These cases exemplify the impact of molecular testing in clinical decision-making and further support the inclusion of POLE on targeted cancer panels.
T69438 591493-591637 Sentence denotes Evaluation of patients with POLE mutated tumors in immunotherapy trials will further guide the best use of such testing for treatment decisions.
T76851 591638-591834 Sentence denotes These results highlight the evolving nature of actionable findings in cancer profiling and underscore the importance of thoughtful interpretation and design when working with targeted gene panels.
T82064 591835-591837 Sentence denotes B.
T99226 591838-591845 Sentence denotes Das, S.
T87726 591846-591890 Sentence denotes Bhaumik SRL Ltd, Mumbai, Maharashtra, India.
T89471 591891-591904 Sentence denotes Introduction:
T35328 591905-591973 Sentence denotes Lung cancer is the leading causes of cancer related death worldwide.
T92468 591974-592086 Sentence denotes In India, the incidence of lung cancer is rising at alarming rates which constitutes around 14.4% of all cancer.
T56084 592087-592284 Sentence denotes Molecular markers such as EGFR, KRAS, BRAF, HER2, and PTEN play a considerable role in lung carcinoma through their involvement in cell proliferation, apoptosis, and assist in therapeutic decision.
T8919 592285-592388 Sentence denotes Unlike EGFR, KRAS mutations are more common males, smokers and frequently found in Western populations.
T54604 592389-592514 Sentence denotes BRAF, HER2, PTEN gene mutation is found in a small proportion of lung cancers with activating mutations in 1% to 4% of NSCLC.
T51817 592515-592703 Sentence denotes In the current study, we evaluate the frequency, distributional pattern of these gene mutations and their association with the clinicopathological characteristics in Indian NSCLC patients.
T96527 592704-592712 Sentence denotes Methods:
T88891 592713-592807 Sentence denotes The present study was performed on 400 formalin-fixed, paraffin-embedded (FFPE) tumor samples.
T22701 592808-592888 Sentence denotes Extracted genomic DNA was amplified by nested PCR followed by direct sequencing.
T66736 592889-592897 Sentence denotes Results:
T93347 592898-592948 Sentence denotes The frequency of EGFR, KRAS, BRAF, HER2 mutations:
T55183 592949-593021 Sentence denotes 29% (116/400), 6.4 % (14/204), 1.5% (3/204), 1.5% (3/204), respectively.
T40558 593022-593096 Sentence denotes There were 3 different BRAF mutations were detected (L584P, V600E, K601E).
T27814 593097-593230 Sentence denotes The presence of HER2 insertion and deletion were observed in 3 (1.5%) cases, whereas remaining 201 cases confirmed wild-type alleles.
T36207 593231-593275 Sentence denotes HER2 mutations were preponderant in exon 20.
T23583 593276-593416 Sentence denotes Sequencing analysis revealed two c.2324-2325ins12 (ATACGTGATGGCduplication, p.Ala775_Gly776insTyrValMetAla) and one c.2305delC in HER2 gene.
T91356 593417-593511 Sentence denotes Interestingly, the study cohort demonstrated wildtype PTEN allele for all EGFR negative cases.
T59115 593512-593524 Sentence denotes Conclusions:
T71329 593525-593600 Sentence denotes To date, present study consist the largest number of Indian NSCLC patients.
T69690 593601-593694 Sentence denotes Several novel variations added new insights into the genetic heterogeneity of NSCLC patients.
T8783 593695-593834 Sentence denotes Further exploration with clinical follow up will strengthen the association of these mutations with disease outcome in Indian ethnic group.
T72629 593835-593839 Sentence denotes N.E.
T78872 593840-593856 Sentence denotes Stachowicz, N.E.
T14976 593857-593873 Sentence denotes Stachowicz, M.B.
T52180 593874-593887 Sentence denotes Campion, K.C.
T10377 593888-593901 Sentence denotes Halling, E.W.
T13634 593902-593917 Sentence denotes Highsmith, B.R.
T20051 593918-593926 Sentence denotes Kipp, B.
T22166 593927-593939 Sentence denotes Crusan, L.A.
T86983 593940-593956 Sentence denotes Kottschade, S.N.
T52296 593957-593971 Sentence denotes Markovic, C.J.
T99409 593972-593984 Sentence denotes Nelson, J.M.
T44961 593985-594001 Sentence denotes Cunningham, J.S.
T27498 594002-594012 Sentence denotes Voss, K.E.
T24199 594013-594027 Sentence denotes Hasselkorn, A.
T24333 594028-594041 Sentence denotes Rajeswari, D.
T78283 594042-594054 Sentence denotes Walker, M.C.
T10681 594055-594086 Sentence denotes Liu Mayo Clinic, Rochester, MN.
T15515 594087-594100 Sentence denotes Introduction:
T15130 594101-594270 Sentence denotes The proposed assay was validated for the detection of BRAF V600E and K/R mutations in cfDNA from double spun, platelet poor plasma using the RainDrop Digital PCR system.
T54812 594271-594366 Sentence denotes This blood-based assay is intended to be utilized as a substitute for invasive tissue biopsies.
T52224 594367-594536 Sentence denotes Reliable mutation detection in melanoma is critical, as patients with BRAF V600 mutant positive tumors benefit significantly from small molecule BRAF and MEK inhibitors.
T70127 594537-594545 Sentence denotes Methods:
T88536 594546-594956 Sentence denotes Clinical peripheral blood samples were collected in 10 mL Streck Cell-Free DNA BCT tubes from 20 healthy volunteers and 47 patients with metastatic melanoma of known BRAF mutation status. cfDNA was extracted from plasma using the Qiagen QIAamp Circulating Nucleic Acid Kit. cfDNA was processed with the RainDrop Digital PCR System using Taqman probes designed to detect wild-type BRAF and BRAF V600E mutations.
T80771 594957-594965 Sentence denotes Results:
T26274 594966-595217 Sentence denotes Cross hybridization of the V600E probe to V600K and V600R mutated DNA allows for the detection of all three mutations; however, the close proximity of V600K to V600R renders them indistinguishable. considered positive for a BRAF V600E or K/R mutation.
T67906 595218-595284 Sentence denotes The LOD for a BRAF V600E and K/R mutation is 5% at 1 ng input DNA.
T16865 595285-595407 Sentence denotes Results were 100% concordant for two variant calls among 3 samples (within and across runs) for the reproducibility study.
T45651 595408-595511 Sentence denotes BRAF RainDrop and tumor biopsy results were compared to determine the agreement between the two assays.
T46956 595512-595624 Sentence denotes Among the 47 melanoma patients, 20 were found to have a BRAF mutation in their tissue specimen, and 27 were not.
T72694 595625-595825 Sentence denotes The sensitivity, specificity, positive and negative predictive value of the RainDrop BRAF assay when compared to biopsy results was 9/20 (45%), 27/27 (100%), 9/9 (100%), and 27/38 (71%), respectively.
T19179 595826-596034 Sentence denotes Notably, the 11 patients with a negative cfDNA BRAF result but a positive tissue result had either well-controlled, low burden metastatic disease or an isolated local recurrence at the time of the blood draw.
T25647 596035-596046 Sentence denotes Conclusion:
T75248 596047-596162 Sentence denotes The cfDNA BRAF Mutation Detection assay was developed to assess for clinically relevant V600 mutations in melanoma.
T51498 596163-596365 Sentence denotes The test was found to have high specificity and positive predictive value such that patients with a positive cfDNA result may avoid invasive tumor biopsies for the determination of BRAF mutation status.
T89241 596366-596516 Sentence denotes However, patients with a negative BRAF result may still require a tissue biopsy for the determination of BRAF mutation status if clinically indicated.
T99439 596517-596762 Sentence denotes This blood-based assay may serve as a reliable substitute for determining BRAF status when tissue biopsies are not feasible or practical, and it may provide an effective means of serial monitoring for patients receiving relevant targeted agents.
T58656 596763-596765 Sentence denotes S.
T49458 596766-596782 Sentence denotes Kongkarnka, P.E.
T28183 596783-596796 Sentence denotes Oberstein, S.
T25834 596797-596808 Sentence denotes Hsiao, A.T.
T8749 596809-596819 Sentence denotes Turk, A.N.
T42864 596820-596832 Sentence denotes Sireci, B.M.
T44371 596833-596843 Sentence denotes Kurtis, H.
T13660 596844-596855 Sentence denotes Varma, A.I.
T19068 596856-596866 Sentence denotes Neugut, M.
T20049 596867-596883 Sentence denotes Mansukhani, A.R.
T65562 596884-596928 Sentence denotes Sepulveda Columbia University, New York, NY.
T80301 596929-596942 Sentence denotes Introduction:
T12605 596943-597189 Sentence denotes Molecular testing of colorectal cancer (CRC) for mutations in exons 2, 3, and 4 of KRAS and NRAS, and BRAF p.600 is recommended as predictive markers of response to anti-EGFR therapy and as a marker of prognosis in advanced disease, respectively.
T72106 597190-597312 Sentence denotes DNA mismatch repair (MMR)/MSI testing is emerging as a predictive marker of response to immune checkpoint therapy for CRC.
T9348 597313-597421 Sentence denotes Next-generation sequencing (NGS) multiplex cancer panels allow simultaneous detection of multiple mutations.
T67555 597422-597716 Sentence denotes We evaluated the clinical utility of molecular testing of all CRC cases received in our Pathology Department as a standing order, whether received as initial biopsy or resection specimens of primary or metastatic disease, tested with a CRC molecular panel and by IHC for MMR and/or MSI testing.
T99097 597717-597725 Sentence denotes Methods:
T87611 597726-597848 Sentence denotes Ninety CRC samples and patients who had CRC molecular panel testing at Columbia University Medical Center were identified.
T6785 597849-598024 Sentence denotes FFPE sections were macro or microdissected, NGS was performed with the TruSeq cancer panel (Illumina) and extended RAS and BRAF p.600 and PIK3CA mutation status were reported.
T63356 598025-598092 Sentence denotes Sequencing data was analyzed with NextGene software (SoftGenetics).
T46540 598093-598153 Sentence denotes IHC for MMR proteins and/or MSI testing were also performed.
T41805 598154-598162 Sentence denotes Results:
T16861 598163-598241 Sentence denotes Patients' mean age was 63 years (24 to 92) years; 45 patients were male (50%).
T81712 598242-598368 Sentence denotes Seven of 90 (7.8%) cases were tested from a metastatic lesion and the remaining 83, from biopsy or resection of primary tumor.
T51102 598369-598417 Sentence denotes RAS mutation was identified in 48 cases (53.3%).
T5315 598418-598493 Sentence denotes 3 cases had NRAS mutation, including one case with KRAS and NRAS mutations.
T89632 598494-598564 Sentence denotes RAS mutations other than KRAS exon 2 were observed in 6 cases (12.5%).
T35815 598565-598616 Sentence denotes PIK3CA mutations were detected in 12 cases (13.3%).
T7863 598617-598696 Sentence denotes BRAF mutation was present in 11 cases, with concurrent RAS mutation in 2 cases.
T91181 598697-598773 Sentence denotes Deficient DNA MMR or MSI-High were present in 12 of 88 cases tested (13.6%).
T3729 598774-598850 Sentence denotes Thirty-two of 90 cases (35.5%) had advanced metastatic or recurrent disease.
T24002 598851-598984 Sentence denotes RAS testing provided useful information for therapy selection in 29 of 90 (32.2%) cases and MMR in 3 cases, for biological therapies.
T47311 598985-599172 Sentence denotes The remaining 58 patients were tested on primary site biopsy or resection and received adjuvant therapy, were early stage disease, were treated elsewhere or were not eligible for therapy.
T48028 599173-599185 Sentence denotes Conclusions:
T72862 599186-599250 Sentence denotes Extended RAS testing increased mutation detection rate by 12.5%.
T28992 599251-599598 Sentence denotes Molecular testing of CRC cases performed as a standing order in all initial biopsy or resection specimens of primary or metastatic CRC, tested for RAS/BRAF mutations with a TruSeq cancer panel and for dMMR/ MSI, showed frequent clinical utility (greater than one third of cases tested) in the selection of patients for biological targeted therapy.
T95955 599599-599720 Sentence denotes For early stage disease at presentation, mutational data may still be of utility at the time of recurrence or metastasis.
T29796 599721-599725 Sentence denotes E.M.
T22995 599726-599736 Sentence denotes Azzato, M.
T83492 599737-599746 Sentence denotes Rusch, S.
T14154 599747-599760 Sentence denotes Shurtleff, J.
T21974 599761-599775 Sentence denotes Nakitandwe, Z.
T71185 599776-599785 Sentence denotes Zhang, D.
T46730 599786-599796 Sentence denotes Hedges, S.
T89867 599797-599807 Sentence denotes Newman, M.
T44480 599808-599820 Sentence denotes Edmonson, A.
T88235 599821-599833 Sentence denotes Thrasher, J.
T44697 599834-599847 Sentence denotes Becksfort, C.
T92427 599848-599861 Sentence denotes Kesserwan, S.
T42372 599862-599876 Sentence denotes Raimondi, K.E.
T44111 599877-599890 Sentence denotes Nichols, D.W.
T41049 599891-599902 Sentence denotes Ellison, J.
T93717 599903-599912 Sentence denotes Zhang, J.
T3953 599913-599972 Sentence denotes Downing St. Jude Children's Research Hospital, Memphis, TN.
T89870 599973-599986 Sentence denotes Introduction:
T23098 599987-600343 Sentence denotes As part of a prospective clinical research study, Genomes for Kids, we are performing a clinical feasibility study of three-platform next-generation sequencing (NGS) for molecular diagnostics, utilizing an integrative whole-genome (WGS), whole-exome (WES) and transcriptome (RNA-Seq) analysis on fresh/frozen tumor samples from pediatric oncology patients.
T94996 600344-600352 Sentence denotes Methods:
T77160 600353-600460 Sentence denotes Eligible patients receive parallel standard-of-care cytogenetic and molecular evaluations prior to consent.
T24075 600461-600899 Sentence denotes Matched tumor/normal samples undergo WGS and WES at 45x coverage and RNA-Seq at 20-30x coverage; the assay pipeline integrates genetic lesions detected by all three NGS platforms to cross-validate and characterize single nucleotide variations (SNVs), short insertions and deletions (indels), structural variations (SVs) including fusions, karyotypes, focal copy number alterations (CNVs), and copy neutral loss of heterozygosity (CN-LOH).
T59474 600900-601018 Sentence denotes After calls are manually confirmed, a multidisciplinary committee determines variant categorization and reportability.
T27046 601019-601115 Sentence denotes An integrative report is generated through Molecular Pathology and placed in the medical record.
T70008 601116-601124 Sentence denotes Results:
T43851 601125-601268 Sentence denotes Over the first 10 months, we analyzed 73 tumor samples from 68 patients, which included 35 leukemia, 19 brain tumor and 19 solid tumor samples.
T88911 601269-601418 Sentence denotes Seventy-one specimens (97.3%) had reportable pathologic or likely pathologic lesions, whereas two tumor samples (2.7%) had no reportable alterations.
T29074 601419-601543 Sentence denotes We identified 36 SVs, 53 focal CNVs, 81 SNVs/Indels, 53 ploidy alterations and 17 instances of CN-LOH of clinical relevance.
T80780 601544-601832 Sentence denotes Fifty-five of these tumors (75%) had cytogenetic or molecular testing performed as part of their routine diagnostic workup; 107 clinically significant alterations, including 22 (61.1%) SVs, 36 (44.4%) SNV/Indels, and 49 (92.5%) focal CNVs, had not been detected by traditional evaluation.
T66944 601833-601928 Sentence denotes This included 10 novel SVs that resulted in in-frame fusion transcripts or aberrant expression.
T18471 601929-602038 Sentence denotes We observed perfect correlations between NGS and fusion detection by RT-PCR, single gene sequencing and FISH.
T29714 602039-602166 Sentence denotes Karyotyping was performed on 33 leukemia samples; hyperdiploidy and hypodiploidy were properly categorized in all cases (n=13).
T99772 602167-602354 Sentence denotes Minor karyotype discrepancies were observed in 12 samples, the majority of which were explainable by marker chromosomes, subclonality or confirmed by additional FISH analyses.Conclusions:
T32524 602355-602563 Sentence denotes Use of an integrative, multi-platform NGS approach is feasible in the clinical setting, improving detection of clinically relevant alterations, while matching performance with gold-standard diagnostic assays.
T27851 602564-602765 Sentence denotes Optimizing laboratory, computational and review processes will improve turn-around-time, allow real-time reporting and ideally streamline multiple laboratory approaches into one comprehensive platform.
T60348 602766-602779 Sentence denotes Introduction:
T10443 602780-602945 Sentence denotes The concept of exclusive mutation of certain driving genes during oncogenesis is facing challenge due to the availability of more and more next gene sequencing data.
T49448 602946-603117 Sentence denotes Although there is usually one critical mutation within one oncogene on majority of the tumors sequenced in our institute, the exception of such general observation exists.
T57788 603118-603248 Sentence denotes Here, we reported that even within one oncogenic gene, KRAS, in the colonic adenocarcinoma, multiple point mutations were present.
T7185 603249-603257 Sentence denotes Methods:
T70934 603258-603367 Sentence denotes The colonic adenocarcinoma was diagnosed by staff at University of Miami and confirmed by second pathologist.
T3214 603368-603450 Sentence denotes The adenocarcinoma was macro dissected and DNA was extracted using QIAamp DNA kit.
T13837 603451-603527 Sentence denotes Illumina truseq cancer panel was employed to perform the next gene sequence.
T7322 603528-603617 Sentence denotes Unusual results such as multiple mutation in same gene were confirmed by pyro-sequencing.
T8384 603618-603626 Sentence denotes Results:
T10285 603627-603745 Sentence denotes After nextgeneration sequencing of 432 colonic adenocarcinoma, 214 tumors were found to carry KRAS mutation (214/432).
T69422 603746-603815 Sentence denotes We found 5 adenocarcinoma cases had multiple point mutations (5/214).
T89770 603816-603854 Sentence denotes The mutation sites varied among cases.
T26337 603855-603978 Sentence denotes Pyro-sequencing data confirmed that multiple mutations were present in the oncogenic gene KRAS within all five cases (5/5).
T75315 603979-603991 Sentence denotes Conclusions:
T5282 603992-604115 Sentence denotes Although it is rare event, multiple mutations could be present in the single oncogenic gene KRAS in colonic adenocarcinoma.
T15133 604116-604299 Sentence denotes Whether it represents multiple mutations with different sites in one allele or in different alleles in the tumor cell or in different tumor cells needs to be elucidated in the future.
T73874 604300-604302 Sentence denotes L.
T88466 604303-604320 Sentence denotes Ritterhouse, B.E.
T24844 604321-604331 Sentence denotes Howitt, V.
T46603 604332-604351 Sentence denotes Rojas-Rujilla, F.C.
T36501 604352-604361 Sentence denotes Kuo, L.M.
T38513 604362-604409 Sentence denotes Sholl Brigham and Women's Hospital, Boston, MA.
T13567 604410-604423 Sentence denotes Introduction:
T42545 604424-604499 Sentence denotes A subset of endometrial carcinomas (EMC) is characterized by hypermutation.
T13914 604500-604716 Sentence denotes Mismatch repair (MMR) defects are a well-recognized mechanism of hypermutation in EMC and may be due to germline or somatic alterations in MMR POLE) have been associated with an ultramutated phenotype in ~10% of EMC.
T54052 604717-604864 Sentence denotes Both MMR and POLE-related EMC have characteristic mutational signatures, and other EMC signatures have been described but are poorly characterized.
T3678 604865-604924 Sentence denotes This study sought to further characterize hypermutated EMC.
T11224 604925-604933 Sentence denotes Methods:
T48641 604934-605075 Sentence denotes Targeted massively parallel sequencing of 275 or 300 known cancer genes was performed on tumor tissue using an Illumina HiSeq 2500 sequencer.
T58129 605076-605192 Sentence denotes Hypermutated tumors were defined as those having > mean number of mutations (21.9 per Mb) present in the EMC cohort.
T34480 605193-605276 Sentence denotes Hotspot genotyping of POLE (exons 9/13) was performed by PCR and Sanger sequencing.
T10711 605277-605385 Sentence denotes Mismatch repair immunohistochemistry (IHC) and MLH1 promoter methylation was performed in a subset of cases.
T77024 605386-605488 Sentence denotes Mutational signatures were defined by the substitution class and sequence context of the mutated base.
T77187 605489-605497 Sentence denotes Results:
T96678 605498-605732 Sentence denotes Of 170 total EMCs sequenced on the targeted panel, 35 (21%) met the criteria for hypermutation, and included 12 grade 1, 11 grade 2, and 7 grade 3 endometrioid, as well as 1 mixed endometrioid/serous and 4 undifferentiated carcinomas.
T52100 605733-605873 Sentence denotes DNA was available for POLE sequencing in 34/35, of which POLE mutations were identified in 7 (20%), including 3 P286R and 4 V411L mutations.
T54135 605874-605959 Sentence denotes Mutational signature analysis identified three distinct patterns in hypermutated EMC:
T20110 605960-606169 Sentence denotes 1) POLE EDM (n=8) (Tp(C>T)>50% and (C>A)pT>20%), 2) Signature 12 "S12" (n=3) (Np(T>C)>40%), and 3) MMR (n=18) (% of frameshift>20% or Np(A>G)>20% or Gp(C>T)+(C>T)pG>50% and frameshift>9% and neither of above).
T99541 606170-606230 Sentence denotes 6 cases were not classifiable based on mutational signature.
T65800 606231-606322 Sentence denotes All 7 confirmed POLE-mutated cases had the POLE signature and intact MMR expression by IHC.
T82439 606323-606536 Sentence denotes 9 cases with an MMR signature had IHC available with abnormal MMR protein expression in 100% (7 MLH1/PMS2 loss (confirmed MLH1 promoter methylation), 1 MSH6 loss (MSH6 germline), 1 MSH2/MSH6 loss (MSH2 germline)).
T20020 606537-606616 Sentence denotes An additional 2 MMR signature cases were known to have MSH6 germline mutations.
T64324 606617-606703 Sentence denotes The S12 signature cases exhibited unusual subclonal changes in MMR protein expression.
T7842 606704-606716 Sentence denotes Conclusions:
T79974 606717-606854 Sentence denotes A subset of EMC exhibit a hypermutated molecular profile, of which ~20% exhibit POLE mutations and a characteristic mutational signature.
T74520 606855-606940 Sentence denotes In addition, ~10% of EMC have a unique signature (S12) with unusual MMR IHC patterns.
T41103 606941-607077 Sentence denotes The remaining hypermutated EMC include those with germline or somatic defects in MMR, as well as those with an unclassifiable signature.
T43991 607078-607080 Sentence denotes J.
T85847 607081-607089 Sentence denotes Saab, S.
T20695 607090-607100 Sentence denotes Mathew, P.
T13630 607101-607110 Sentence denotes Zhang, H.
T74686 607111-607120 Sentence denotes Zia, M.J.
T97257 607121-607129 Sentence denotes Kluk, H.
T27087 607130-607208 Sentence denotes Fernandes NewYork-Presbyterian Hospital, Weill Cornell Medicine, New York, NY.
T52280 607209-607222 Sentence denotes Introduction:
T80451 607223-607380 Sentence denotes Oncogenic driver mutations in KRAS have been identified in a third of lung adenocarcinomas and predict a lack of response to EGFR tyrosine kinase inhibitors.
T52726 607381-607518 Sentence denotes Approximately 5% to 15% of lung adenocarcinomas show amplification of the KRAS gene and 37% of these tumors harbor somatic KRAS variants.
T49537 607519-607703 Sentence denotes The impact of KRAS amplification or polysomy on patient survival is not clear with some studies suggesting a potential tumorigenic effect, particularly with concomitant KRAS mutations.
T18310 607704-607904 Sentence denotes The objective of this study was to evaluate for KRAS amplification in lung adenocarcinomas demonstrating increased relative coverage of the KRAS gene as determined by next-generation sequencing (NGS).
T673 607905-608065 Sentence denotes Methods: NGS results for 140 lung adenocarcinomas with clinically relevant variants were examined for evidence suggestive of KRAS copy number alterations (CNA).
T59093 608066-608237 Sentence denotes NGS was performed on the Ion Torrent platform using the AmpliSeq Cancer Hotspot Panel v2 and sequencing data was analyzed using the Variant Caller v4.4 (Thermofisher, CA).
T85604 608238-608410 Sentence denotes Tumors were suspected of having CNA when the relative coverage of KRAS amplicons showed a fold increase >1.3 after normalization to total coverage of pooled normal samples.
T76210 608412-608531 Sentence denotes The presence of KRAS variants with concomitant amplification of the KRAS gene in lung adenocarcinoma is not infrequent.
T28796 608532-608692 Sentence denotes KRAS copy number alterations including amplifications and polysomy could potentially be flagged using an algorithm that utilizes the relative amplicon coverage.
T46811 608693-608757 Sentence denotes FISH can further differentiate KRAS amplification from polysomy.
T90007 608758-608911 Sentence denotes The clinical relevance in terms of disease progression and outcome in patients with KRAS variants with concomitant KRAS CNA needs to be further explored.
T23096 608912-608970 Sentence denotes Extended RAS Profiling Due to Insufficient DNA Material H.
T73067 608971-608982 Sentence denotes Shojaei, B.
T10611 608983-608994 Sentence denotes Sundman, L.
T88482 608995-609089 Sentence denotes Zhou University Hospitals Case Medical Center, Case Western Reserve University, Cleveland, OH.
T11514 609090-609254 Sentence denotes Introduction: RAS molecular profiling has become important companion test to guide the use of anti-EGFR antibody treatment in metastatic colorectal cancer patients.
T32237 609255-609545 Sentence denotes Although sequencing offers better coverage of the coding area and identifies specific locations of genetic alterations in the gene, the mutant/wild-type allele ratio present in the tumor affects its diagnostic sensitivity and makes the detection of point mutations particularly challenging.
T33222 609546-609714 Sentence denotes Real-time polymerase chain reaction (PCR) based methods are more sensitive but do not test for less frequently occurring mutations with uncertain clinical implications.
T14641 609715-609926 Sentence denotes Sequencing is not successful in all cases, the aim of this study is to evaluate the overall failure rate of testing caused by "insufficiency" by sequencing method and reevaluating PCR results in these specimens.
T69779 609927-609935 Sentence denotes Methods:
T7461 609936-610064 Sentence denotes At our center, a series of 121 patients who had been enrolled in extended RAS gene profiling between 2014 to 2015 was evaluated.
T53598 610065-610182 Sentence denotes All samples were sent out to a molecular reference laboratory for extended RAS analysis (Next-generation sequencing).
T82640 610183-610287 Sentence denotes Sixteen of these samples were returned to our institution due to insufficient DNA material for analysis.
T25895 610288-610587 Sentence denotes These samples were reevaluated in our lab by a real-time qualitative PCR assay (Therascreen KRAS RGQ PCR Kit, used on the Rotor-Gene Q MDx instrument) for the detection of seven somatic mutations in the human KRAS oncogene (Gly12Ala, Gly12Asp, Gly12Arg, Gly12Cys, Gly12Ser, Gly12Val, and Gly13Asp) .
T68310 610588-610641 Sentence denotes This assay has an analytical sensitivity of 1% to 5%.
T80637 610642-610650 Sentence denotes Results:
T23129 610651-610754 Sentence denotes The overall success rate for PCR was 75% (12/16), in returned samples due to insufficient DNA material.
T46478 610755-610990 Sentence denotes PCR evaluation for KRAS mutations in sixteen cases, revealed eight patients with KRAS mutations (one Gly12Ser, four Gly12Val, two Gly12Asp, and one Gly12Cys), three patients with no KRAS mutations and one case had indeterminate result.
T2539 610991-611070 Sentence denotes Four remaining samples did not have sufficient DNA material for PCR evaluation.
T12842 611071-611148 Sentence denotes Extended RAS testing was successful in 105/121 (86.7%) of send out specimens.
T67776 611149-611413 Sentence denotes There were 45 cases with positive results for KRAS mutations (six Gly12Val, two Gly12Ser, four Gly12Cys, three Gly12Ala, sixteen Gly12Asp, eleven Gly13Asp, two Gln61His and one Gln61Lys) and 4 cases had NRAS mutations (two Gly12Asp, one Gly61Leu and one Gln61Lys).
T38601 611414-611426 Sentence denotes Conclusions:
T45290 611427-611554 Sentence denotes The majority of patient samples submitted for extended RAS molecular cancer genomic profiling have successful results reported.
T17708 611555-611740 Sentence denotes Reevaluating specimens for KRAS hotspot point mutations by realtime PCR, who had insufficient DNA material for extended RAS analysis may improve the success rate of molecular profiling.
T44476 611741-611754 Sentence denotes Introduction:
T55944 611755-611915 Sentence denotes Serous, endometrioid, and clear cell subtypes of ovarian and endometrial carcinomas have similar embryological origins and share common molecular abnormalities.
T28380 611916-612084 Sentence denotes Distinction between these subtypes has prognostic and potentially therapeutic implications, but is often challenging based on traditional histopathological examination.
T54447 612085-612153 Sentence denotes Different DNA methylation profiles are associated with each subtype.
T26421 612154-612294 Sentence denotes We sought to identify a panel of DNA methylation biomarkers that could be used to complement histopathology and improve diagnostic accuracy.
T30617 612295-612303 Sentence denotes Methods:
T64500 612304-612577 Sentence denotes Genome-scale DNA methylation data for 196 serous, 49 endometrioid, and 17 clear cell subtypes of ovarian carcinomas were retrieved from a previous study, and from 210 serous, 40 endometrioid, and 14 mixed subtypes of endometrial cancers from The Cancer Genome Atlas (TCGA).
T85047 612578-612698 Sentence denotes These data sets were screened to identify markers that could distinguish each subtype based on their methylation status.
T6188 612699-612917 Sentence denotes The selected biomarkers were further tested on formalin-fixed, archival tissue sections from 18 serous, 16 endometrioid, and 10 clear cell uncontentious ovarian carcinomas using real-time PCR-based MethyLight analysis.
T54767 612918-613006 Sentence denotes The tissue sections were macrodissected to ensure a cancer cell content of at least 50%.
T16039 613007-613103 Sentence denotes We built a three-group classification model using Lasso regression and 10-fold cross validation.
T10381 613104-613250 Sentence denotes Predicted probabilities of each subtype were calculated on a set of 12 tumors, and the percentage of true calls/correct classification summarized.
T55274 613251-613259 Sentence denotes Results:
T53631 613260-613403 Sentence denotes We identified 21 potential subtype specific DNA methylation markers, of which 9 were further verified on independent ovarian carcinoma samples.
T25329 613404-613696 Sentence denotes Four of the 9 markers tested (OvaCA1, OvaCA3, OvaCA4 and OvaCA6) correctly predicted the serous tumor subtype, 3 (OvaCA6, OvaCA8 and OvaCa10) predicted the endometrioid subtype, and 2 (OvaCA10 and OvaCA11) predicted the clear cell subtype with probabilities of 100%, 94% and 90% respectively.
T77241 613697-613780 Sentence denotes Two markers with poor predictive probabilities were excluded from further analysis.
T35596 613781-613793 Sentence denotes Conclusions:
T3255 613794-613932 Sentence denotes Our panel of 7 biomarkers can distinguish between ovarian serous, endometrioid and clear cell carcinomas based on DNA methylation profile.
T59266 613933-614056 Sentence denotes The performance of this panel to help distinguishing between subtypes of endometrial carcinomas has not yet been evaluated.
T66263 614057-614312 Sentence denotes This panel may be useful in establishing a molecular classification that may complement histopathological diagnosis of ovarian and endometrial carcinomas and increase our ability to predict clinical outcome and therapeutic responsiveness in these cancers.
T77804 614313-614315 Sentence denotes D.
T28456 614316-614327 Sentence denotes Dunaway, C.
T39950 614328-614339 Sentence denotes Merritt, J.
T79317 614340-614348 Sentence denotes Jung, P.
T4846 614349-614360 Sentence denotes Webster, C.
T63990 614361-614373 Sentence denotes Ngouenet, G.
T18973 614374-614381 Sentence denotes Ong, I.
T98404 614382-614393 Sentence denotes Sprague, C.
T21055 614394-614404 Sentence denotes Warren, G.
T65957 614405-614414 Sentence denotes Geiss, S.
T85276 614415-614425 Sentence denotes Warren, A.
T47556 614426-614436 Sentence denotes Cesano, J.
T35355 614437-614482 Sentence denotes Beechem NanoString Technologies, Seattle, WA.
T57152 614483-614496 Sentence denotes Introduction:
T3167 614497-614731 Sentence denotes As intra-tumoral heterogeneity has emerged as a challenge in development of targeted cancer therapeutics, the tissue context of biomarker levels has become an increasingly important aspect of patient classification and stratification.
T68295 614732-614907 Sentence denotes Historically, immunohistochemistry and in situ hybridization have been used to assess spatial heterogeneity of proteins and RNA transcripts, respectively, in clinical samples.
T10742 614908-614988 Sentence denotes These approaches, however, have limited multiplexing capacity and dynamic range.
T68452 614989-615198 Sentence denotes Here, we report the development of a spatially resolved approach for quantifying up to 800 protein or RNA targets with over 5 logs of dynamic range in a single formalin-fixed, paraffin-embedded (FFPE) section.
T89513 615199-615207 Sentence denotes Methods:
T18206 615208-615321 Sentence denotes An automated prototype capable of imaging and sample collection was developed by modifying a standard microscope.
T46784 615322-615533 Sentence denotes For protein detection, a multiplexed cocktail of 30+ primary antibodies, each with a unique, photocleavable oligo tag, and 1-2 fluorescently labeled antibodies was applied to a slide-mounted FFPE tissue section.
T27797 615534-615697 Sentence denotes Regions of interest, selected based on a fluorescence imaging scan of the entire tissue, were illuminated sequentially with focused UV light to release the oligos.
T13218 615698-615900 Sentence denotes Following each illumination cycle, eluent was collected from the local region, moved to a microtiter plate, hybridized to NanoString barcodes, and subsequently analyzed with an nCounter Analysis System.
T69351 615901-616012 Sentence denotes The resulting digital counts corresponded to the abundance of each targeted protein in the regions of interest.
T85243 616013-616127 Sentence denotes For RNA detection, a cocktail of multiple UV-cleavable in situ hybridization probes were used in a similar manner.
T65614 616128-616136 Sentence denotes Results:
T19127 616137-616233 Sentence denotes In control samples, we found expected levels of protein and RNA targets, including HER2 and CD3.
T98389 616234-616412 Sentence denotes We demonstrate multiplexed detection from discrete regions within a tumor and adjacent normal tissue, enabling systematic interrogation of a heterogeneous tumor microenvironment.
T62670 616413-616586 Sentence denotes We also demonstrate a linear correlation (R 2 > 0.99) between observed counts and area of UV illumination with a resolution limit of 500 μm^2, or approximately four T-cells.
T80589 616587-616716 Sentence denotes We are testing methods for analyzing target abundance from individual cells and from noncontiguous cells with the same phenotype.
T94967 616717-616729 Sentence denotes Conclusions:
T34322 616730-616900 Sentence denotes With further development, our novel approach to capture the spatial context of protein and RNA levels will have many applications in biomarker and translational research.
T74790 616901-617085 Sentence denotes The ability to digitally measure RNA and protein at up to 800-plex from FFPE tissues could facilitate drug mechanism-of-action and resistance studies within the tumor microenvironment.
T44689 617086-617259 Sentence denotes Quantitative, high-plex data should also greatly accelerate the discovery of immune biomarkers in tumors and the development of companion diagnostics for targeted therapies.
T45018 617260-617379 Sentence denotes Introduction: RAS gene mutations play a significant prognostic and predictive role in colorectal cancer (CRC) patients.
T21175 617380-617480 Sentence denotes RAS-mutated CRC patients exhibit a poor response to anti-EGFR therapy resulting in adverse outcomes.
T68757 617481-617637 Sentence denotes In the current study, we sought to investigate the incidence and patterns of RASmutations in a cohort of histopathologically proven CRC patients from India.
T52223 617638-617646 Sentence denotes Methods:
T16088 617647-617767 Sentence denotes The study included formalin fixed paraffin embedded tissue samples of CRC patients for KRAS and NRAS mutational testing.
T2750 617768-618018 Sentence denotes The extracted DNA from the samples were further analyzed to investigate the mutational status of KRAS, covering exons 2, 3, and 4 (codons 12, 13, 61,117, and 146) andNRAS exon 2, 3, and 4 (codon 12, 13, 61, and 146) using a real-time PCR-based assay.
T4700 618019-618027 Sentence denotes Results:
T64288 618028-618129 Sentence denotes A total of 1109 and 1010 CRC patient samples were screened for KRAS and NRAS mutations, respectively.
T89915 618130-618159 Sentence denotes 388 (35%) of the cases (Male:
T97309 618160-618264 Sentence denotes Female = 1.4:1; 20 years to 85 years) presented with a mutation of the KRAS gene whereas 26 (2.6%; Male:
T95807 618265-618345 Sentence denotes Female = 0.8:1; 25 years to 80 years) patient samples harbored an NRAS mutation.
T85249 618346-618442 Sentence denotes For the KRAS gene, 88% of the mutations were found in exon 2, 27% in exon 3, and 4.4% in exon 4.
T14943 618443-618532 Sentence denotes For NRAS, 16% of the mutations were observed in exon 2, 55% in exon 3, and 28% in exon 4.
T23338 618533-618616 Sentence denotes Majority of the KRAS mutations were detected in CRC patients above 40 years of age.
T82255 618617-618742 Sentence denotes G12D was the most common mutation observed in the KRAS gene whereas Q61H mutation was predominant in NRAS positive CRC cases.
T86051 618743-618755 Sentence denotes Conclusions:
T36343 618756-618862 Sentence denotes This is the largest study to report the frequency of the KRAS and NRAS mutations in the Indian population.
T62123 618863-618989 Sentence denotes In this study, the KRAS mutation pattern in Indian CRC patients was comparable to that reported from other parts of the world.
T7424 618990-619081 Sentence denotes This is in contrast to the previously reported Indian studies where the incidence is lower.
T60533 619082-619178 Sentence denotes We attribute this to the use of a more sensitive assay with a broader coverage of the two genes.
T16561 619179-619273 Sentence denotes A high incidence of G12D and Q61H mutations were found in the KRAS and NRAS gene respectively.
T56169 619274-619413 Sentence denotes Further analysis with clinical correlation would help better understand the role of RAS mutations in the CRC patient treatment and outcome.
T25290 619414-619416 Sentence denotes F.
T186 619417-619428 Sentence denotes Ahmad, B.R.
T49435 619429-619469 Sentence denotes Das SRL Ltd, Mumbai, Maharashtra, India.
T49843 619470-619483 Sentence denotes Introduction:
T35864 619484-619595 Sentence denotes Mutations in exon 9 and 20 of the PIK3CA gene represent to most common genetic alterations in breast carcinoma.
T14096 619596-619676 Sentence denotes These mutations are reported in around 20% to 40% of the breast cancer patients.
T36201 619677-619783 Sentence denotes Recent studies suggest that PIK3CA mutations are associated with predicting prognosis in breast carcinoma.
T4997 619784-619879 Sentence denotes In the current study we explored frequency of PIK3CA mutation in Indian breast cancer patients.
T62188 619880-619888 Sentence denotes Methods:
T5578 619889-619967 Sentence denotes The study was performed on 190 breast cancer patients using direct sequencing.
T74000 619968-619976 Sentence denotes Results:
T47154 619977-620065 Sentence denotes Sequencing analysis revealed PIK3CA mutations in 22.6% (43/190) of breast tumor samples.
T30321 620066-620135 Sentence denotes Exon 20 mutations were more common in comparison to exon 9 mutations.
T12552 620136-620239 Sentence denotes Mutations were predominantly spotted between nucleotide positions 1624 to 1636 or between 3129 to 3140.
T9084 620240-620449 Sentence denotes Overall four different missense mutations (E542K, E545K, E545A and E545G) were detected in the helical domain, and two different amino acid substitutions at codon 1047 (H1047R and H1047L) in the kinase domain.
T72333 620450-620545 Sentence denotes PIK3CA mutations were predominantly seen in older age patients with grade II ductal carcinomas.
T71229 620546-620665 Sentence denotes Furthermore, PIK3CA mutations were common in hormone positive cases and a comparativel lesser in triplenegative tumors.
T29622 620666-620678 Sentence denotes Conclusions:
T73868 620679-620767 Sentence denotes This is one of the largest study evaluating PIK3CA mutation in breast cancer from India.
T91553 620768-620853 Sentence denotes This study confirms that PIK3CA mutations are quite prevalent in Indian subcontinent.
T65671 620854-620974 Sentence denotes Furthermore, identification of prognostic utility in Indian scenario is the way forward and warrants further evaluation.
T83135 620975-621221 Sentence denotes To evaluate whether a single targeted next-generation sequencing-based (NGS) assay yields sufficient molecular information necessary to classify LGG, we retrospectively surveyed 50 cases of LGG and reclassified them based on the updated criteria.
T52598 621222-621230 Sentence denotes Methods:
T69136 621231-621577 Sentence denotes A targeted, hybridization capturebased, clinical NGS assay was used to assess at least 25 genes and somatic copy number alterations (SCNA) in 50 LGG cases, consisting of 36 mixed oligoastrocytomas (MOA), 11 oligodendrogliomas (OD), 2 astrocytomas (AC), and 1 low-grade glioma NOS (LGG NOS), all originally classified by the 2007 CNS WHO criteria.
T39647 621578-621799 Sentence denotes SCNA detection was performed following alignment using CopywriteR, an off-the-shelf, open source tool that utilizes off-target reads in hybridization capture-based sequencing data, including data from limited gene panels.
T35346 621800-622003 Sentence denotes The NGS results were compared with immunohistochemistry (IHC) and fluorescence in-situ hybridization (FISH) and also used to reclassify the tumors based on the updated 2016 CNS WHO classification scheme.
T47869 622004-622012 Sentence denotes Results:
T8197 622013-622202 Sentence denotes Our NGS-based approach was able to re-classify the 36 MOAs into 30 AC (20 IDH-mutant and 10 IDH-wildtype) and 6 OD (IDH-mutant and 1p/19q codeleted), and 1 LGG NOS into an AC (IDHwildtype).
T10232 622203-622376 Sentence denotes 9 of the 11 OD remained in their original category; one of the remaining 2 ODs was reclassified as an IDH-mutant AC, the other had findings consistent with a "pediatric" OD.
T51660 622377-622483 Sentence denotes The 2 ACs remained in their original categories, both yielding an integrated diagnosis of IDH-wildtype AC.
T61883 622484-622583 Sentence denotes IDH1-R132H IHC was performed on all 50 cases and was concordant with NGS when the IHC was positive.
T61162 622584-622716 Sentence denotes Additionally, IDH1/2 NGS molecular results yielded an additional 5 cases of non-R132H IDH1 mutations and one case with IDH2 p.R172K.
T9946 622717-622843 Sentence denotes FISH and NGS based testing for 1p/19q deletions were concordant for 45 of the 48 cases with informative FISH results (93.75%).
T22003 622844-623053 Sentence denotes NGS-based SCNA detection revealed segmental, partial losses of 1p and or 19q in the regions of the FISH probes in three discordant cases, which would not meet the 2016 CNS WHO criteria for whole arm deletions.
T55666 623054-623066 Sentence denotes Conclusions:
T19238 623067-623235 Sentence denotes Here we show a single targeted NGS assay can serve as the sole molecular testing modality necessary to categorize LGG by the updated 2016 CNS WHO classification scheme.
T1437 623236-623418 Sentence denotes This increased diagnostic efficiency enables the potential for greater accuracy and cost-efficiency while reducing the specimen tissue requirements compared to multimodal approaches.
T65350 623419-623421 Sentence denotes C.
T46299 623422-623431 Sentence denotes Huang, R.
T58629 623432-623442 Sentence denotes Vemula, P.
T76906 623443-623455 Sentence denotes Kamineni, J.
T10104 623456-623462 Sentence denotes Wu, B.
T80530 623463-623513 Sentence denotes Anekella SeraCare Life Sciences, Gaithersburg, MD.
T81242 623514-623527 Sentence denotes Introduction:
T67351 623528-623667 Sentence denotes Developing, optimizing, and running somatic mutation detection assays is challenging due to variability in tumor samples and NGS workflows.
T27788 623668-623773 Sentence denotes Monitoring these assays using appropriate reference materials is critical for laboratory quality control.
T50063 623774-624173 Sentence denotes A mix of purified DNA containing well characterized, clinically actionable tumor-specific somatic mutations at three different allele frequencies was developed to assess detection of a wide range of single nucleotide variants (SNVs), insertion/deletion mutations (indels), mutations within homopolymers and structural variants (SVs) to fulfill unmet needs for accurate, multiplexed quality controls.
T75310 624174-624182 Sentence denotes Methods:
T73002 624183-624347 Sentence denotes Human genomic DNA was extracted from the well-characterized GM24385 cell line and blended with biosynthetic constructs bearing forty (40) cancer-relevant mutations.
T12169 624348-624587 Sentence denotes Blending was performed to achieve three levels of allele frequencies within the mix: approximately one third of the forty targets were at 10% allele frequency (AF), approximately one third at 7% AF, and the remaining targets were at 4% AF.
T17088 624588-624781 Sentence denotes These levels make it possible to detect mutations well above many assays' stated limit of detection (LOD), right above the LOD, and just below the LOD, within the same quality control material.
T79493 624782-624859 Sentence denotes Allelespecific digital PCR was used to verify the frequency of each mutation.
T96391 624860-624976 Sentence denotes Testing was also performed using Ion Ampliseq Cancer Hotspot Panel v2 and the Illumina TruSeq Amplicon Cancer Panel.
T39551 624977-624985 Sentence denotes Results:
T42752 624986-625181 Sentence denotes Average allele frequency by dPCR was 10.43% (range 9.33% to 11.53%) for the AF10 tier of mutations, 6.97% (range 6.07% to 7.88%) for the AF7 tier, and 3.93% (range 3.52 to 4.34) for the AF4 tier.
T34490 625182-625353 Sentence denotes Variant allele frequencies detected by NGS were generally consistent with dPCR results, and all discordances could be explained by limitations of commonly used NGS panels.
T56254 625354-625713 Sentence denotes For example, TruSeq Amplicon Cancer Panel did not detect PIK3CA c.3204_3205insA, ATM c.1058_1059delGT, and ERBB2 c.2324_2325ins12 mutations at the expected frequency, as these mutations are either under a primer used for amplification of target regions, or are at the very end of a sequencing read such that the strand bias filter prevents positive detection.
T15140 625714-625726 Sentence denotes Conclusions:
T48093 625727-625898 Sentence denotes We developed a highly multiplexed and accurate reference material that allows simultaneous monitoring of a broad range of somatic mutation types at a large number of loci.
T51377 625899-626129 Sentence denotes This material may aid in optimization and verification of detection limits for NGS-based oncology tests and provide laboratories greater assurance in their ability to correctly call various types of mutations on a daily run basis.
T79453 626130-626134 Sentence denotes H.J.
T60863 626135-626148 Sentence denotes Dubbink, P.N.
T127 626149-626181 Sentence denotes Atmodimedjo, R. van Marion, P.H.
T99187 626182-626195 Sentence denotes Riegman, J.M.
T64279 626196-626225 Sentence denotes Kros, M.J. van den Bent, W.N.
T18634 626226-626286 Sentence denotes Dinjens Erasmus MC Cancer Institute, Rotterdam, Netherlands.
T22462 626287-626300 Sentence denotes Introduction:
T22176 626301-626471 Sentence denotes Cancer cells are genomic unstable and accumulate tumor typespecific molecular aberrations, which may represent hallmarks for predicting prognosis and targets for therapy.
T57332 626472-626621 Sentence denotes Co-deletion of chromosomes 1p and 19q marks gliomas with an oligodendroglioma component and predicts a better prognosis and response to chemotherapy.
T72936 626622-626630 Sentence denotes Methods:
T34536 626631-626895 Sentence denotes In the current study, we present a novel method to detect chromosome 1p/19q co-deletion or loss of heterozygosity (LOH) in a diagnostic setting, based on single nucleotide polymorphism (SNP) analysis and next-generation sequencing (NGS) on an Ion Torrent platform.
T73980 626896-626977 Sentence denotes We selected highly polymorphic SNPs evenly distributed over both chromosome arms.
T6024 626978-627259 Sentence denotes To experimentally determine the sensitivity and specificity of targeted SNP analysis, we used DNAs extracted from 49 routine formalin-fixed, paraffin-embedded (FFPE) glioma tissues and compared the outcome with diagnostic microsatellite-based LOH analysis and calculated estimates.
T49896 627260-627268 Sentence denotes Results:
T29280 627269-627517 Sentence denotes We show that targeted SNP analysis by NGS allows reliable detection of 1p and/or 19q deletion in a background of 70% normal cells according to calculated outcomes, is more sensitive than microsatellitebased LOH analysis, and requires much less DNA.
T97712 627518-627529 Sentence denotes Conclusion:
T16288 627530-627718 Sentence denotes This specific and sensitive SNP assay is broadly applicable for simultaneous allelic imbalance analysis of multiple genomic regions and can easily be incorporated in NGS mutation analyses.
T76565 627719-627903 Sentence denotes The combined mutation and chromosomal imbalance analysis in a single NGS assay is perfectly suited for routine glioma diagnostics and other diagnostic molecular pathology applications.
T49654 627904-627933 Sentence denotes J Mol Diagn 2016, 18:775-786.
T45217 627934-628105 Sentence denotes One of the most commonly mutated genes in GBMs is the epidermal growth factor receptor (EGFR), and one of the most common mutations in EGFR is EGFR variant III (EGFRvIII).
T8849 628106-628375 Sentence denotes EGFR is a transmembrane receptor tyrosine kinase, and the EGFRvIII mutant is characterized by a deletion of 267 amino acids in the extracellular domain, resulting in a novel residue at the newly formed junction and leading to ligand-independent constitutive activation.
T36925 628376-628559 Sentence denotes The University of Pennsylvania initiated a pilot study of autologous T cells re-directed to the EGFRvIII mutation using a lentiviral vector encoding a chimeric antigen receptor (CAR).
T18331 628560-628568 Sentence denotes Methods:
T80108 628569-628786 Sentence denotes One hundred and ninety-nine patient samples with newly diagnosed or recurrent GBMs were screened for genetic abnormalities, including EGFRvIII, MGMT methylation, and/or next-generation sequencing analysis of 47 genes.
T56774 628787-628970 Sentence denotes EGFRvIII was detected from RNA extracted from FFPE tissue by a custom library jmd.amjpathol.org ■ The Journal of Molecular Diagnostics methodology and sequenced on the Illumina MiSeq.
T71556 628971-629108 Sentence denotes Correlation analysis was performed using a custom program in R to detect positive and negative association of co-occurrence of mutations.
T51054 629109-629117 Sentence denotes Results:
T70117 629118-629367 Sentence denotes One hundred and ninety tumor specimens from patients with histologically confirmed GBMs were tested for EGFRvIII at our institution as standard-of-care; of these, 37 tested as high positive (>30% reads from EGFRvIII/total EGFR) for EGFRvIII (19.5%).
T66257 629368-629514 Sentence denotes Eleven patients were infused and 5 of these 11 had post infusion resections, which were analyzed for EGFRvIII, gene mutations, and amplifications.
T86272 629515-629691 Sentence denotes Loss of some or all of EGFRvIII was detected in 3 of 5 specimens, with the remaining 2 not having shown expansion of the CAR T-EGFR cells in the peripheral blood post-infusion.
T13685 629692-629913 Sentence denotes Comparison of pre-and post-infusion studies in patients with peripheral expansion showed complete or partial loss of EGFRvIII; however other mutations including EGFR amplification remained, suggesting tumor heterogeneity.
T11088 629914-630031 Sentence denotes Patients with EGFR amplification and/or EGFRvIII show significant overlap, and often harbor additional EGFRmutations.
T96100 630032-630230 Sentence denotes The most significant positive co-mutation correlations were between EGFR amplification and EGFR mutations, and the strongest negative co-occurrence was between IDH1 mutations and EGFR amplification.
T6208 630231-630243 Sentence denotes Conclusions:
T26607 630244-630422 Sentence denotes The complexity observed in our clinical specimens suggests that late stage GBMs are heterogeneous tumors with EGFRvIII representing a late mutation in the development of disease.
T67738 630423-630635 Sentence denotes We also observe that amplification and mutation of EGFR tends to occur in tumors without prognostically favorable mutations in IDH1, supporting that EGFR-driven tumors constitute a more aggressive subset of GBMs.
T84708 630636-630640 Sentence denotes M.N.
T41679 630641-630655 Sentence denotes Nikiforova, M.
T15948 630656-630667 Sentence denotes Durso, K.M.
T43595 630668-630682 Sentence denotes Callenberg, A.
T9560 630683-630693 Sentence denotes Wald, Y.E.
T4601 630694-630760 Sentence denotes Nikiforov University of Pittsburgh Medical Center, Pittsburgh, PA.
T40854 630761-630774 Sentence denotes Introduction:
T27759 630775-630940 Sentence denotes Whole transcriptome (RNA-Seq) analysis is mostly used as a discovery tool for detection of gene fusions and gene expression, typically in freshfrozen tissue samples.
T61888 630941-631048 Sentence denotes Its use for clinical samples with limited quantity and quality of RNA has not been previously demonstrated.
T33957 631049-631242 Sentence denotes We have developed and validated an RNA-Seq approach that can be effectively used for detection of novel driver gene fusions and gene expression in clinical thyroid FNA and fixed tissue samples.
T24371 631243-631492 Sentence denotes Methods: RNA-Seq was performed on 41 thyroid samples with sequencing parameters optimized for use in clinical samples with limited quantity and quality of RNA (thyroid fine needle aspirates (FNA) and formalin-fixed paraffin-embedded (FFPE) tissues).
T65551 631493-631642 Sentence denotes As low as 30 ng of RNA was used for library generation with the TruSeq Stranded Total RNA kit (Illumina) and sequencing on the HiSeq 2500 (Illumina).
T62191 631643-631904 Sentence denotes A custom bioinformatics pipeline was developed that uses both existing bioinformatics tools (TopHat, Chimerascan, FusionCatcher, SOAPfuse) and also inhouse developed scripts and filters for accurate detection of driver gene fusions and gene expression analysis.
T20323 631905-631996 Sentence denotes All samples were previously analyzed by ThyroSeq v2 amplification-based targeted NGS panel.
T44782 631997-632005 Sentence denotes Results:
T2843 632006-632160 Sentence denotes Initially, clinical RNA-Seq analysis was performed in a set of 20 thyroid tumors with known fusion types and showed 100% sensitivity for fusion detection.
T93061 632161-632364 Sentence denotes Next, we performed RNA-Seq on 21 clinical samples (9 FNA, 12 FFPE tissue) that showed suspicious differential expression profile (n=10) and/or absence of all known driver mutations (n=11) by ThyroSeq v2.
T69210 632365-632474 Sentence denotes Novel driver fusions or new breakpoints of the known fusions were detected in 15 (71%) cases (7 FNA, 8 FFPE).
T91882 632475-632691 Sentence denotes They included 2 novel RET fusions, 2 NTRK3, 1 PPARG, 2 NTRK1 novel fusion breakpoints, 3 novel gene partners for BRAF, two for THADA, and three novel fusions involving genes not previously reported in thyroid cancer.
T54729 632692-632888 Sentence denotes This included identification of fusions in 8 (80%) of samples that showed suspicious expression profile on ThyroSeq v2 consistent with upregulation of a known oncogene, but no known fusions types.
T18981 632889-632952 Sentence denotes Each fusion demonstrated 3 to 300 reads spanning a break point.
T45559 632953-633030 Sentence denotes Functional driver status of fusions was confirmed by gene expression profile.
T92931 633031-633146 Sentence denotes All samples positive for gene fusions were papillary carcinomas and a follicular carcinoma on a surgical follow up.
T81692 633147-633159 Sentence denotes Conclusions:
T35552 633160-633331 Sentence denotes Clinically modified whole transcriptome (RNA-Seq) analysis can be successfully used to detect oncogenic fusions in small thyroid FNA samples and in fixed tissue specimens.
T28657 633332-633478 Sentence denotes Our results demonstrate that novel gene fusions account for a large proportion of thyroid carcinomas lacking all currently known driver mutations.
T8224 633479-633492 Sentence denotes Introduction:
T3964 633493-633603 Sentence denotes Advances in Next-Generation Sequencing (NGS) have facilitated lower input sample volumes, and sample material.
T13864 633604-633725 Sentence denotes These advances are not without the challenges of library bias and library complexity increasingly affecting data quality.
T88366 633726-633932 Sentence denotes Accel-NGS 2S DNA Library kits (Swift Biosciences) have been designed to overcome these challenges providing uniform and more complete coverage, resulting in improved sequencing efficiency and reduced costs.
T55287 633933-634143 Sentence denotes Additional cost and time savings can be found in automating library preparation, leading to increased throughput while maintaining high-quality data and reproducibility with little to no variation across wells.
T62984 634144-634152 Sentence denotes Methods:
T13692 634153-634360 Sentence denotes In this study, we demonstrate high-throughput automated NGS library preparation for targeted capture and WGS from 10ng FFPE and 10ng cfDNA on the PerkinElmer Sciclone NGSx with Accel-NGS 2S DNA Library kits.
T96248 634361-634522 Sentence denotes An Alu 247/115 repeat assay was used to assess contamination of high molecular weight DNA, quantity and integrity of cfDNA and FFPE prior to library preparation.
T57103 634523-634648 Sentence denotes Reproducibility and quality were determined by qPCR, LabChip GX analysis and subsequent sequencing on the Illumina HiSeq X10.
T95924 634649-634657 Sentence denotes Results:
T61312 634658-634795 Sentence denotes As DNA extracted from FFPE exhibits varying degrees of damage, the Alu repeat assay is an accurate way to assess and quantify usable DNA.
T12200 634796-634876 Sentence denotes Our results show a DNA integrity cfDNA, sufficient for 10ng input into the prep.
T85295 634877-635018 Sentence denotes Library yields were sufficient for sequencing, and LabChip analysis validated the targeted insert size of 350bp for FFPE and 165bp for cfDNA.
T70826 635019-635261 Sentence denotes Sequencing of manual versus automated library preparations yielded similar results: ultra-low duplication, no adapter dimer formation, median insert size in-line with the LabChip results and library complexity as expected for 10ng human gDNA.
T25249 635262-635274 Sentence denotes Conclusions:
T52186 635275-635440 Sentence denotes Accel-NGS 2S DNA Library kits on the Sciclone NGSx provide sequence-ready libraries, with equal or better reproducibility and quality to manually prepared libraries.
T62601 635441-635668 Sentence denotes Highly efficient library preparation driven by end repair of both 3' and 5' DNA termini delivers a more complex library requiring less sequencing, enabling comprehensive analysis of low input DNA samples such as FFPE and cfDNA.
T40337 635669-635873 Sentence denotes Significant cost savings and improvement in lab efficiencies were achieved combining the use of PerkinElmer automation for higher throughput library prep with Swift's highly efficient library preparation.
T65544 635874-635887 Sentence denotes Introduction:
T16946 635888-636098 Sentence denotes Accumulating evidence suggests the relevance of PARP inhibitors and BRCA-like phenotype and PARP inhibitors have become a part of clinical practice in the ovarian cancer patients with BRCA1/2 germline mutation.
T33362 636099-636316 Sentence denotes Germline and somatic sequence variants of DNA repair genes are thought to be predictors of BRCA-like phenotype and recent advances in sequencing technology enable us to effectively detect variants in DNA repair genes.
T27565 636317-636428 Sentence denotes We are trying to identify germline and somatic sequence variants of DNA repair genes in female cancer patients.
T19917 636429-636437 Sentence denotes Methods:
T93122 636438-636536 Sentence denotes Genomic sequencing of investigative biomarkers was prospectively offered to patients with cancers.
T82608 636537-636676 Sentence denotes Normal and tumor DNA libraries were prepared separately from peripheral blood and a retrieved archival FFPE tumor sample from each patient.
T96453 636677-636792 Sentence denotes Relevant targets were enriched by custom designed Agilent SureSelect hybrid capture baits using standard protocols.
T39223 636793-636838 Sentence denotes Samples were sequenced on Illumina platforms.
T54040 636839-636993 Sentence denotes We analyzed germline and somatic pathogenic variants of DNA repair genes using Freebayes (Cornell), Oncotator (Broad Institute) and UNC-created pipelines.
T54189 636994-637002 Sentence denotes Results:
T59223 637003-637143 Sentence denotes Four hundred and ninety-six patients' samples were analyzed with 201 breast, 201 uterine endometrial, 68 ovarian, and 26 colorectal cancers.
T17930 637144-637341 Sentence denotes Pathogenic germline mutations were detected in 91 (18.3%) patients and somatic mutations were present in 151 (30.4%) patients with both germline and somatic mutations present in 24 (4.8%) patients.
T95743 637342-637456 Sentence denotes In total, 218 (44.0%) out of 496 female cancer patients had germline and/or somatic mutations in DNA repair genes.
T60671 637457-637618 Sentence denotes Genes with pathogenic germline variants included ATM, ATR, BARD1, BLM, BRCA1, BRCA2, BRIP1, CHEK1, CHEK2, ERCC2, FANCD2, FANCG, MLH1, MSH6, NBN, PMS2 , and POLE.
T54505 637619-637849 Sentence denotes Somatic variants were observed in genes such as ATM, ATR, BLM, BRCA1, BRCA2, BRIP1, CHEK1, CHEK2, ERCC2, FANCA, FANCB, FANCD2, FANCE, FANCG, FANCI, FLCN, MLH1, MRE11A, MSH2, MSH6, NBN, PALB2, PMS2, PTEN, RAD50, SLX4, and SMARCA4 .
T63912 637850-637862 Sentence denotes Conclusions:
T48194 637863-638100 Sentence denotes We demonstrate that almost half of female cancers are characterized by germline and/or somatic mutations in DNA repair genes, which could make these patients potential candidates for PARP inhibitor trials in a clinic or research setting.
T45377 638101-638275 Sentence denotes Targeted sequencing using normal and tumor samples with focus on DNA repair genes could be a useful strategy to identify patients with BRCA-like phenotype in female patients.
T72522 638276-638278 Sentence denotes S.
T35145 638279-638294 Sentence denotes Gunn 1 , 2 , C.
T5286 638295-638310 Sentence denotes Sims 1 , 2 , S.
T22001 638311-638326 Sentence denotes Govender 1 , A.
T95499 638327-638341 Sentence denotes Khurana 1 , M.
T14357 638342-638354 Sentence denotes Moore 1 , P.
T1466 638355-638437 Sentence denotes Cotter 1 1 ResearchDx/PacificDx, Irvine, CA; 2 Targeted Genomics, San Antonio, TX.
T19697 638438-638451 Sentence denotes Introduction:
T21921 638452-638759 Sentence denotes In breast cancer with amplification of the CEP17 probe region and equivocal HER2 results by FISH, alternative HER2 probe sets (representing the 17p13, 17p12, and/or 17q21 regions) have been proposed as a method for establishing a stable denominator to reveal the tumor's "true" HER2 gene copy number status.
T27914 638760-639159 Sentence denotes However, the chromosome 17 regions represented by these alternative probes can themselves contain genomic loci that are commonly rearranged in cancer (TP53 loss at 17p13), (RARA amplification at 17q21), and inherited polyneuropathy (CMT duplications, deletions, or translocations at 17p12) resulting in false skewing of the HER2 gene/alternative probe ratio towards negative, positive, or equivocal.
T55990 639160-639168 Sentence denotes Methods:
T15107 639169-639346 Sentence denotes In the current study, DNA was extracted from IHCtargeted HER2 receptor "hot-spots" representing 25 FFPE breast tumors previously characterized by FISH for HER2 gene copy number.
T5032 639347-639448 Sentence denotes Tumor DNA (test) and human genomic DNA (reference with known diploid HER2 gene copy number) (Promega,
T66128 639449-639767 Sentence denotes The Journal of Molecular Diagnostics ■ jmd.amjpathol.org Madison WI) were differentially labeled with Alexa Fluor dyes, and competitively hybridized to a custom-designed oligonucleotide genomic DNA microarray with highdensity probe coverage of the HER2 amplicon on chromosome 17 (Agilent Technologies, Santa Clara CA).
T83368 639768-639940 Sentence denotes The array design includes over 4,600 chromosome 17 probes representing the p arm, q arm, telomeric and centromeric regions with 66 tiling probes over the HER2 (ERBB2) gene.
T16966 639941-640216 Sentence denotes Following hybridization, average HER2 gene copy number was calculated for each tumor sample by converting mean log2 signal intensity ratio value into genomic region copy number adjusted for % clonal fraction and experimentally established log2 ratio compression of the assay.
T48496 640217-640225 Sentence denotes Results:
T64256 640226-640398 Sentence denotes All samples yielded adequate DNA for array CGH analysis and HER2 gene copy number was scored as: HER2-Negative, < 4 copies, HER2-Low, 4-6 copies, HER2-Positive, > 6 copies.
T12974 640399-640489 Sentence denotes In 25/25 (100%) of cases, HER2 gene copy number results were consistent with FISH results:
T71443 640490-640502 Sentence denotes Conclusions:
T17841 640503-640841 Sentence denotes Calculation of average HER2 gene copy number using array CGH derived mean log2 ratios with reference DNA of known diploid HER2 gene copy number circumvents ratio skewing associated with unstable chromosome 17 reference probes, and provides an alternative high-resolution method for determining HER2 status in FISH equivocal breast cancer.
T94402 640842-640844 Sentence denotes R.
T70830 640845-640861 Sentence denotes Ruiz-Cordero, A.
T68085 640862-640872 Sentence denotes Khanna, G.
T89556 640873-640882 Sentence denotes Lyons, R.
T55230 640883-640894 Sentence denotes Bassett, M.
T28767 640895-640902 Sentence denotes Guo, J.
T32310 640903-640914 Sentence denotes Heymach, R.
T22368 640915-640925 Sentence denotes Luthra, S.
T73719 640926-640979 Sentence denotes Roy Chowdhuri MD Anderson Cancer Center, Houston, TX.
T38110 640980-640993 Sentence denotes Introduction:
T64409 640994-641254 Sentence denotes Comprehensive molecular profiling of lung adenocarcinomas (LADC) has identified three different molecular subtypes: terminal respiratory unit (TRU) based on EGFR, proximal-inflammatory (PI) based on TP53, and proximalproliferative (PP) based on KRAS mutations.
T18194 641255-641393 Sentence denotes Herein we correlate results of FISH and Next-Generation Sequencing (NGS) in LADC cytology samples to stratify by these molecular subtypes.
T84817 641394-641591 Sentence denotes Methods: LADC patients with fine needle aspirations between May 2010 and October 2015 for diagnosis and staging were identified to collect demographic, clinical, FISH (ALK, ROS1, MET) and NGS data.
T18268 641592-641675 Sentence denotes Frequency tables with number of patients and proportions for categorical variables.
T33642 641676-641862 Sentence denotes To assess relationship between the molecular subtypes and age, sex, ethnicity, smoking, vital and aneuploidy statuses, cross-tabulations and Chi-square or Fisher's exact tests were used.
T9452 641863-641871 Sentence denotes Results:
T72040 641872-642006 Sentence denotes 283 LADC patients were included (mean age=65y, female=53%, Caucasian=76%, Asian=9%, other=14%, current/former smokers=77%, Alive=65%).
T3497 642007-642118 Sentence denotes 82% of cases were run on NGS, 14.5% on Sanger or Pyrosequencing platforms and 3.5% did not have molecular data.
T30490 642119-642250 Sentence denotes NGS yielded positive mutations in 93% of cases with TP53 (43%), KRAS (34%), and EGFR (25%) being the most frequently mutated genes.
T85584 642251-642396 Sentence denotes Molecular results showed TRU=14%, PI=16% and PP=16%, EGFR/TP53 and TP53/KRAS co-mutation=19%, other genes mutated=12%, no mutations detected=18%.
T86287 642397-642640 Sentence denotes In keeping with the Cancer Genome Atlas Research Network (TCGA) results, we also found that TRU subtype is more frequent in Asian women (p=0.01) and never-smokers (p<0.0001), while least associated with ALK/ROS1/MET positive tumors (p=0.0089).
T97443 642641-642921 Sentence denotes The PI subtype did not show any significant differences by ethnicity (p=0.90), gender (p=0.18), smoking (p=0.17), vital status (p=0.74), or FISH results (p=0.12); however this group had a significantly higher number of cases with aneuploidy and ALK/ROS1/MET positivity (p=0.0012).
T32300 642922-643107 Sentence denotes The PP subtype was significantly associated with Caucasian women (p=0.003) and smokers (p=0.0002), but was not associated with ALK/ROS1/MET positivity (p=0.0161) or aneuploidy (p=0.61).
T50312 643108-643265 Sentence denotes Among patients with mutations detected by NGS, about 25% were deceased, compared with 76% of patients with no mutations detected by our NGS panel (p=0.0135).
T92955 643266-643277 Sentence denotes Conclusion:
T49704 643278-643365 Sentence denotes Our study confirms that cytology specimens are suitable for FISH and molecular testing.
T82699 643366-643542 Sentence denotes Whereas the clinical significance of aneuploid cells found in FISH in cases of LADC is not completely understood, aneuploidy seems to be associated to the PI molecular subtype.
T73563 643543-643642 Sentence denotes Our results confirm previous associations identified by TCGA as well as new additional information.
T58010 643643-643776 Sentence denotes Further studies evaluating the possible worse scenario of NGS-mutation-negative patients are in progress to corroborate this finding.
T74566 643777-643855 Sentence denotes Skipping Using nanoString Digital nCounter Technology in a Clinical Setting L.
T93136 643856-643865 Sentence denotes Borsu, K.
T37288 643866-643878 Sentence denotes Mullaney, R.
T25384 643879-643890 Sentence denotes Benayed, P.
T14626 643891-643898 Sentence denotes Chi, K.
T41542 643899-643908 Sentence denotes Busam, P.
T70655 643909-643917 Sentence denotes Paik, A.
T16024 643918-643928 Sentence denotes Drilon, M.
T47981 643929-643990 Sentence denotes Ladanyi Memorial Sloan Kettering Cancer Center, New York, NY.
T55169 643991-644004 Sentence denotes Introduction:
T39990 644005-644250 Sentence denotes There is an increasing need to screen patient tumors for certain oncogenic alterations that are manifested exclusively or most clearly at the RNA level, such as ALK Alternative Transcription Initiation and mutations causing MET exon 14 skipping.
T31126 644251-644435 Sentence denotes A novel isoform of ALK gene initiating from a de novoalternative transcription initiation (ATI), ALK ATI , has recently been shown to be oncogenic in melanomas and other cancers (PMID:
T18846 644436-644446 Sentence denotes 26444240).
T15501 644447-644582 Sentence denotes Lung cancers harboring a wide variety of mutations causing MET exon 14 skipping have shown excellent responses to MET inhibitors (PMID:
T81444 644583-644593 Sentence denotes 25971939).
T75976 644594-644792 Sentence denotes Here, we report our clinical validation of the nanoString nCounter RNA detection technology for RNA-based detection of these alterations and its benchmarking against targeted RNA and DNA NGS assays.
T68708 644793-644801 Sentence denotes Methods:
T89909 644802-645034 Sentence denotes To evaluate the digital nCounter RNA detection technology, 8 cell lines, 12 melanoma and 5 lung adenocarcinoma FFPE specimens also tested by Archer targeted RNA-Seq sequencing were selected to evaluate ALK ATI transcript expression.
T36599 645035-645119 Sentence denotes Twelve FFPE lung carcinoma specimens previously testedby MSK-IMPACT NGS assay (PMID:
T33982 645120-645186 Sentence denotes 25801821) were selected to assess MET transcripts lacking exon 14.
T90124 645187-645236 Sentence denotes RNAs were extracted from unstained FFPE sections.
T30578 645237-645374 Sentence denotes The digital nCounter RNA detection 250ng RNA with barcoded Elements Reporter, biotin-labeled Capture Tags and two target-specific probes.
T50294 645375-645559 Sentence denotes After hybridization, excess tags and probes were removed while hybridized complexes bound to streptavidin were counted for RNA acid target to calculate fold-changes in gene expression.
T87332 645560-645568 Sentence denotes Results:
T65508 645569-645782 Sentence denotes Archer targeted RNA-Seq data for 25 ALK ATI samples (TAT 3.5 days) and targeted DNA NGS data for 12 MET Exon 14 samples (TAT 21 days) were compared to digital RNA detection results for the same cases (TAT 2 days).
T66739 645783-645931 Sentence denotes The expected ALK ATI and MET Exon 14 results were found in 23 (92%) and 11 (91.5%) samples using the digital RNA detection technology, respectively.
T31233 645932-646159 Sentence denotes In addition, 8 (17%) ALK ATI FFPE RNA samples that failed quality control metrics for the Archer RNA-Seq assay, nonetheless passed quality thresholds for nanoString and generated satisfactory data by digital nCounter detection.
T72067 646160-646172 Sentence denotes Conclusions:
T28946 646173-646317 Sentence denotes The digital RNA detection technology identified the correct variant transcripts in 34 (92%) samples tested by other clinical laboratory methods.
T97212 646318-646553 Sentence denotes This platform does not require cDNA synthesis, amplification, or library preparation thereby decreasing technical variation and enabling greater tolerance of RNA degradation and inhibitors associated with the formalin fixation process.
T81060 646554-646732 Sentence denotes Our results show that the digital nCounter expression technology can rapidly and accurately detect and quantify aberrant, oncogenic transcripts in FFPE RNA in a clinical setting.
T96376 646733-646735 Sentence denotes M.
T99255 646736-646743 Sentence denotes Zhu, B.
T55632 646744-646755 Sentence denotes Sundman, M.
T54457 646756-646767 Sentence denotes Atchley, D.
T30803 646768-646778 Sentence denotes Winner, N.
T12394 646779-646874 Sentence denotes Sadri University Hospitals Case Medical Center, Case Western Reserve University, Cleveland, OH.
T80400 646875-646888 Sentence denotes Introduction:
T18904 646889-647074 Sentence denotes About 40% to 50% of metastatic melanomas harbor a mutation in BRAF, with V600E and V600K being the most common mutations which are targeted by several first line FDA-approved therapies.
T83865 647075-647275 Sentence denotes In addition, non-V600E/K mutations in BRAF and mutations in KIT and NRAS have been shown to respond to targeted therapy in case studies and are currently being better characterized in clinical trials.
T43977 647276-647459 Sentence denotes Although allele-specific PCR can be used for detection of BRAF V600E mutation, some clinicians have more recently advocated for use of targeted-NGS in patients with advanced melanoma.
T73566 647460-647468 Sentence denotes Methods:
T10343 647469-647661 Sentence denotes To help guide the testing on metastatic melanoma cases in our institution, we analyzed all samples sent to UHCMC molecular lab for BRAF V600E PCR and targeted-NGS testing during 2015 and 2016.
T49770 647662-647740 Sentence denotes Test results, turnaround times, and costs were compared between the two tests.
T1303 647741-647922 Sentence denotes Samples which were negative for BRAF V600E by allele-specific PCR were tested by NGS using the Ion Torrent PGM and Ampliseq Cancer Hotspot Panel v2 to look for additional mutations.
T15344 647923-647931 Sentence denotes Results:
T82008 647932-648154 Sentence denotes Lab-developed allele-specific BRAF V600E PCR is less expensive with shorter turnaround time, 3.2 +/-0.3 days as compared to targeted-NGS assay that is roughly 4x more expensive with longer turnaround time, 6.6 +/-1.6 days.
T64612 648155-648238 Sentence denotes In the samples sent for testing by allele-specific PCR, 25% are positive for V600E.
T91703 648239-648344 Sentence denotes In samples sent for targeted-NGS, 24% are V600E positive, 16% harbor other actionable BRAF mutations (ie.
T51754 648345-648471 Sentence denotes V600K, complex mutations involving V600, and L597R), and 16% harbor other potentially actionable mutations in other genes (ie.
T83804 648472-648484 Sentence denotes NRAS, GNAQ).
T17487 648485-648618 Sentence denotes Of the mutations detected by NGS testing, 57% of potentially actionable mutations would not be detected by allele-specific PCR alone.
T91326 648619-648927 Sentence denotes Similarly, of the samples negative for BRAF V600E by allele-specific PCR, 72% harbor potentially relevant mutations when analyzed by targeted-NGS (12% harbor another V600 BRAF mutations and 60% harbor mutations in KIT or NRAS), all of which have either an FDA-approved therapy or an available clinical trial.
T1633 648928-648940 Sentence denotes Conclusions:
T25233 648941-649124 Sentence denotes Although allele-specific BRAF V600E PCR is less expensive with a shorter turnaround time, it does not detect over two-third of potentially actionable mutations in metastatic melanoma.
T30526 649125-649310 Sentence denotes These findings need be viewed in the context of the institution's participation in clinical trials and oncologist preference for targeted therapy, immunotherapy, and off-label drug use.
T43337 649311-649497 Sentence denotes Within our institution, we recommend screening patients with allele-specific PCR and reflexing negative cases for targeted-NGS, or using targeted-NGS upfront to maximize patient benefit.
T65526 649498-649649 Sentence denotes This study provides guidance on choosing appropriate molecular test for metastatic melanoma in a tertiary medical center harboring on-site NGS testing.
T83574 649650-649753 Sentence denotes Introduction: KIAA1549-BRAF rearrangements occur in approximately two thirds of pilocytic astrocytomas.
T52872 649754-649946 Sentence denotes A duplication within 7q34 encompassing a portion of both genes results in fused copies of the 5' end of KIAA1549 and the 3' end of BRAF and the constitutive activation of BRAF kinase activity.
T68413 649947-650105 Sentence denotes The detection of this rearrangement can be useful in the diagnostic distinction of pilocytic astrocytoma from diffuse astrocytoma and other low-grade gliomas.
T91007 650106-650297 Sentence denotes We sought to compare two methods -fluorescence in situ hybridization (FISH) and reverse transcription PCR (RT-PCR) -for the clinical detection of BRAF rearrangements in pilocytic astrocytoma.
T35764 650298-650306 Sentence denotes Methods:
T37659 650307-650509 Sentence denotes We designed an RT-PCR assay to detect the three most common KIAA1549-BRAF fusion transcripts -exon 16/exon 9, exon 15/exon 9, and exon 16/exon 11 -as well as the more unusual exon 15/exon 11 transcript.
T77610 650510-650578 Sentence denotes Together these encompass approximately 98% of KIAA1549-BRAF fusions.
T19995 650579-650745 Sentence denotes In addition, we developed a FISH assay with probes flanking all breakpoints described jmd.amjpathol.org ■ The Journal of Molecular Diagnostics in BRAF rearrangements.
T82543 650746-650908 Sentence denotes A total of 31 cases of pilocytic astrocytoma were evaluated by both methods, 9 of which were previously found to have a BRAF rearrangement at another institution.
T31678 650909-651140 Sentence denotes Results: BRAF rearrangements were identified by RT-PCR and/or FISH in 18/22 (81.8%) of pilocytic astrocytomas without a previously identified rearrangement and in all 9 cases found to have a rearrangement at an outside institution.
T85578 651141-651185 Sentence denotes One of 31 cases failed both RT-PCR and FISH.
T1774 651186-651395 Sentence denotes Among the remaining cases, 3 cases were not scorable by FISH but were positive by RT-PCR (with a weak control product) and one case was positive by FISH but was inadequate by RT-PCR due to a very weak control.
T38211 651396-651556 Sentence denotes Among cases positive by RT-PCR, 20/25 (80%) demonstrated an exon 16/exon 9 fusion, 4/25 (16%) an exon 15/exon 9 fusion, and 1/25 (4%) an exon 15/exon 11 fusion.
T39471 651557-651702 Sentence denotes No exon 16/exon 11 fusions were identified; however, evaluation of a plasmid control confirmed the ability of RT-PCR to identify this transcript.
T10003 651703-651797 Sentence denotes Concordance was observed between FISH and RT-PCR for 25/26 cases evaluable by both techniques.
T67596 651798-651986 Sentence denotes One case was negative by RT-PCR but positive by FISH with the typical doublet pattern, suggestive of an uncommon KIAA1549-BRAF fusion transcript not detectable by the RT-PCR primer design.
T1156 651987-652162 Sentence denotes Conclusions: FISH and RT-PCR both represent accurate and complimentary methods for detection of BRAF rearrangements which are useful in the diagnosis of pilocytic astrocytoma.
T15563 652163-652416 Sentence denotes Whereas RT-PCR is more specific for KIAA1549-BRAF fusions and had slightly fewer failures, FISH is more comprehensive and may be useful for the detection of rare breakpoints, less common BRAF fusion partners, and BRAFrearrangements in other tumor types.
T33294 652417-652419 Sentence denotes H.
T21880 652420-652431 Sentence denotes Mellert, L.
T83924 652432-652443 Sentence denotes Jackson, W.
T24959 652444-652452 Sentence denotes Hahn, N.
T96050 652453-652463 Sentence denotes Dupuis, A.
T75281 652464-652474 Sentence denotes Weaver, J.
T17629 652475-652484 Sentence denotes Greer, G.
T8207 652485-652520 Sentence denotes Pestano Biodesix Inc., Boulder, CO.
T15317 652521-652534 Sentence denotes Introduction:
T24187 652535-652676 Sentence denotes Blood-derived proteomic signatures have clinical utility, with one example, VeriStrat (VS), measuring acute phase reactant proteins in serum.
T88474 652677-652804 Sentence denotes Patients with a POOR (VS-P) prognostic classification have worse clinical outcomes relative to those with a GOOD (VS-G) status.
T18779 652805-652906 Sentence denotes A VS-P result limits standard of care therapeutic options, thus clinical trials may be a good option.
T80084 652907-653078 Sentence denotes A global Phase II study, FOCAL is being conducted that examines the addition of ficlatuzumab to erlotinib for first line EGFR sensitizing mutation positive, VS-P patients.
T94677 653079-653183 Sentence denotes In contrast, treatment options for patients with NSCLC and EGFR wild-type (wt), VS-P status are limited.
T89572 653184-653293 Sentence denotes For this study, we first profiled serum using MALDI-ToF for VeriStrat-POOR classification in donor specimens.
T13338 653294-653473 Sentence denotes Circulating-free DNA (cfDNA) was then profiled in matched plasma specimens with a targeted next-generation sequencing (NGS) panel to identify actionable somatic variant mutations.
T20176 653474-653482 Sentence denotes Methods:
T32092 653483-653622 Sentence denotes Plasma samples determined to be EGFR wt by droplet digital PCR and VS-POOR by MALDI-ToF were analyzed by NGS with a targeted 15-gene panel.
T82652 653623-653731 Sentence denotes Screening of cfDNA isolated from the plasma of 11 VS-P patients with advanced stages of NSCLC are presented.
T99206 653732-653740 Sentence denotes Results:
T90748 653741-653881 Sentence denotes As expected, EGFR sensitizing variants were detected only in the positive control but not among any of the EGFR wt pre-screened VS-P cohort.
T13734 653882-653991 Sentence denotes Six of the 11 plasma samples contained TP53 mutations, which included missense, splice-site and frame-shifts.
T99961 653992-654252 Sentence denotes Although targeted therapies for p53 are not currently approved by the FDA, several pre-clinical and clinical trials are underway with either compounds such as the p53 re-activating agent APR-246 (a PRIMA-1 analogue), or with wt p53 gene-therapy such as SGT-53.
T32195 654253-654265 Sentence denotes Conclusions:
T64875 654266-654424 Sentence denotes Targeted profiling using NGS on cfDNA from plasma can identify actionable mutations in patient samples with a poor prognosis as determined the VeriStrat test.
T2729 654425-654532 Sentence denotes It is likely that other targets associated with current therapy may be uncovered with additional profiling.
T97588 654533-654683 Sentence denotes Expanded profiling of EGFR wt/VS-P patients with a 35-gene NGS panel that measures additional somatic variants, indels and amplifications is underway.
T22298 654684-654802 Sentence denotes We expect that these studies will yield targets that could provide options for patients with advanced stages of NSCLC.
T77013 654803-654805 Sentence denotes A.
T85117 654806-654815 Sentence denotes Cheng, N.
T92165 654816-654829 Sentence denotes Hernandez, R.
T27471 654830-654845 Sentence denotes Sunnadeniya, D.
T71906 654846-654854 Sentence denotes Meza, J.
T60055 654855-654866 Sentence denotes Whiting, W.
T56941 654867-654879 Sentence denotes Roberson, M.
T82674 654880-654890 Sentence denotes Carter, X.
T17799 654891-654933 Sentence denotes Fang Thermo Fisher Scientific, Austin, TX.
T15859 654934-654947 Sentence denotes Introduction:
T10615 654948-655119 Sentence denotes As personalized cancer care evolves, the patient's nucleic acid becomes ever so important to provide valuable information regarding their genetic makeup and disease state.
T13455 655120-655291 Sentence denotes Common sample types for these analyses include biopsies, which can be very limited in material making the downstream measurement of more than one analyte rather difficult.
T45864 655292-655418 Sentence denotes Obtaining another biopsy, using a different section or splitting the sample can be problematic because of tumor heterogeneity.
T46506 655419-655695 Sentence denotes Even adjacent areas of the same tumor tissue can result in different RNA/DNA profiles so the ability to isolate multiple analytes from the same sample offer a number of benefits, which include preserving samples and data consistency eliminating any sample to sample variation.
T93778 655696-655918 Sentence denotes As more tests are developed to simultaneously monitor genetic alterations, there is a strong need to efficiently isolate both DNA and RNA from the same starting sample in a format compatible with highthroughput processing.
T69714 655919-655927 Sentence denotes Methods:
T92162 655928-656113 Sentence denotes Here we describe a novel chemistry using magnetic beads and robust workflow that will eliminate extraction variabilities and process a large number of samples with consistency and ease.
T42450 656114-656224 Sentence denotes We've developed both manual and automated protocols on the KingFisher Flex and Duo Prime purification systems.
T47225 656225-656527 Sentence denotes We have extracted from a number of different FFPE cancer tissues including breast, lung, colorectal, and melanoma from tumor resections, core needle biopsies (CNBs) and fine needle aspirates (FNAs) for various downstream applications such as real-time PCR and targeted sequencing with different panels.
T75916 656528-656536 Sentence denotes Results:
T17961 656537-656639 Sentence denotes High sequencing metrics were met from tumor resections aged 5 to 25 years old, and from CNBs and FNAs.
T36041 656640-656734 Sentence denotes DNA sequencing metrics include >95% uniformity, >90% end to end reads and >95% no strand bias.
T6343 656735-656839 Sentence denotes RNA sequencing metrics include expression of all 5 endogenous controls and high mean read lengths >80bp.
T81353 656840-656989 Sentence denotes FFPE samples with known variants or fusions were extracted and analysis for variant calling or fusion detection confirmed the corresponding variants.
T11978 656990-657110 Sentence denotes Extractions processed manually or automated on the KingFisher yielded a high concordance of hotspot variants identified.
T27836 657111-657297 Sentence denotes We have also titrated tissue inputs from 200mm 2 down to 5mm 2 showing sensitivity of variant detection is not affected when working with small pieces of tissue or low amounts of tissue.
T29258 657298-657310 Sentence denotes Conclusions:
T54217 657311-657420 Sentence denotes In summary, we have a robust workflow that allow for the extraction of DNA and RNA from the same FFPE sample.
T80320 657421-657547 Sentence denotes Samples can be processed manually or with automation on the KingFisher purification systems with concordant variant detection.
T7194 657548-657730 Sentence denotes From tiny amounts of material such as CNB's or FNA's to bigger tissues such as tumor resections, samples generated quality sequencing data meeting all of our core sequencing metrics.
T69046 657731-657831 Sentence denotes The KRAS oncogene is mutated in approximately 35% to 45% of colorectal cancers and NRAS in 1% to 6%.
T68815 657832-657960 Sentence denotes Studies have shown that EGFR monoclonal antibody therapy is unlikely to be beneficial in tumors with any KRAS or NRAS mutations.
T12324 657961-658130 Sentence denotes NCCN guidelines have recommended that cetuximab and panitumumab for the first-line treatment be given only to patients who are negative for both KRAS and NRAS mutations.
T87953 658131-658322 Sentence denotes Our KRAS, NRAS extended gene mutation analysis assays will detect mutations in exons 2 (codons 12, 13), 3 (codons 59, 61) and 4 (codons 117, 146), allowing the determination of drug response.
T40517 658323-658419 Sentence denotes In this study, we have evaluated the clinical and analytical performance features of this assay.
T62583 658420-658428 Sentence denotes Methods:
T102 658429-658480 Sentence denotes Genomic DNA was isolated from FFPE tumor specimens.
T29773 658481-658610 Sentence denotes Exons 2, 3 and 4 of the KRAS and NRAS genes were subjected to SNaPshot multiplex PCR and primer extension for mutation detection.
T70703 658611-658788 Sentence denotes DNA from colorectal cancer FFPE specimens and synthetic oligonucleotides were used to evaluate accuracy, repeatability, reproducibility and analytical sensitivity of the assays.
T76710 658789-658797 Sentence denotes Results:
T6671 658798-658954 Sentence denotes Of the specimens tested during validation, 21 specimens with known mutations in KRAS and 14 in NRAS had SNaPshot analysis results that were 100% concordant.
T85688 658955-659064 Sentence denotes The 21 synthetic oligonucleotides positive for mutations in KRAS and 40 in NRAS also showed 100% concordance.
T94927 659065-659120 Sentence denotes Repeatability and reproducibility were 100% concordant.
T51925 659121-659235 Sentence denotes This assay can detect 5% of mutant DNA in a background of wildtype genomic DNA when the input DNA is 25ng or more.
T2333 659236-659342 Sentence denotes The KRAS and NRAS assays have been offered as clinical tests based on the successful performance features.
T41149 659343-659467 Sentence denotes From the 1272 KRAS specimens tested for the past year, 55.58% had no mutation detected, 39.31% had a KRAS mutation detected.
T59295 659468-659574 Sentence denotes Among the 500 specimens with a KRAS mutation, 87% were found in Exon 2, 6.8% in Exon 3 and 5.6% in Exon 4.
T45876 659575-659668 Sentence denotes Three specimens (0.6%) had double mutations that were most likely due to tumor heterogeneity.
T63773 659669-659787 Sentence denotes From the 1496 NRAS specimens tested for the past year, 91.24% had no mutation detected, 4.55% had a mutation detected.
T11644 659788-659898 Sentence denotes Among the 68 specimens with an NRASmutation, 41.18% were found in Exon 2, 58.82% in Exon 3 and none in Exon 4.
T57302 659899-660046 Sentence denotes In this data set, 236 specimens were tested for both KRAS and NRAS, 50% had no mutation, 39.41% had a KRAS mutation and 5.93% had an NRAS mutation.
T77922 660047-660134 Sentence denotes The mutation distribution of our data set is in agreement with other published studies.
T72080 660135-660264 Sentence denotes There were 1.63% specimens that had partial results and 3% that had no results due to FFPE specimen degradation or low DNA yield.
T19115 660265-660277 Sentence denotes Conclusions:
T14794 660278-660500 Sentence denotes The KRAS and NRAS extended gene mutation detection assay using multiplex SNaPshot method is a robust, reproducible, sensitive, and fast assay for molecular diagnostic utilization on targeted therapies in colorectal cancer.
T40412 660501-660514 Sentence denotes Introduction:
T66925 660515-660694 Sentence denotes The PathVysion, FDA-approved dual probe HER2 fluorescence insitu hybridization (FISH) assay provides the ratio of HER2 to CEP17, a centromeric enumeration probe for chromosome 17.
T65786 660695-660834 Sentence denotes Several reports have suggested that CEP17 copies are increased in many breast cancers which were traditionally labeled as "polysomy" cases.
T31128 660835-661075 Sentence denotes However, recent data questioned the existence of true polysomy in breast cancer and increased CEP17 signals were thought to be due to pericentromeric amplication which might then skew the ratio of HER2/CEP17 underestimating the HER2 status.
T88125 661076-661276 Sentence denotes The aim of the current study was to analyze the utility of an alternative chromosome 17 reference locus (D17S122) to assess HER2 gene status accurately in cases with unusual FISH signal patterns, viz.
T86615 661277-661337 Sentence denotes CEP17 aneusomy, average HER2 th borderline HER2/CEP17 ratio.
T68960 661338-661346 Sentence denotes Methods:
T18641 661347-661633 Sentence denotes We retrospectively reevaluated the HER2 FISH status of 25 invasive breast cancers accessioned in the year 2016 in the Division of Molecular Pathology, The Journal of Molecular Diagnostics ■ jmd.amjpathol.org Tata Memorial Centre, which displayed a mean CEP17 copy number greater than 3.
T76646 661634-661764 Sentence denotes -<6 copy numbers or with HER2/CEP17 ratio >1.8-2.2 by PathVysion (Abbott Molecular Inc., Des Plaines, IL, USA) HER2 DNA Probe Kit.
T82464 661765-661898 Sentence denotes The cases were reflex tested using alternate, non centromeric FISH probe by ZytoLightSPEC/D17S122 (Zytovysion, Bremerhaven, Germany).
T27948 661899-661975 Sentence denotes HER2 status was interpreted in accordance with the ASCO/CAP 2013 guidelines.
T265 661976-661984 Sentence denotes Results:
T53776 661985-662088 Sentence denotes Twenty five cases comprised of mean CEP17 -<6 copy numbers (n=8), and HER2/CEP17 ratio >1.8 -2.2 (n=8).
T77207 662089-662145 Sentence denotes Of 9cases of polysomy with mean CEP17 centromeric probe.
T65902 662146-662247 Sentence denotes Two of nine polysomy cases displayed an unusual HER2/CEP17 colocalization with coamplication pattern.
T71263 662248-662474 Sentence denotes Though the amplified HER2 status was retained in the latter cases, the colocalization pattern was not seen with the alternate probe.Out of 8 cases with mean HER2 cases HER2 gene status was upgraded from equivocal to amplified.
T59384 662475-662532 Sentence denotes In rest of the cases (n=11), the results were concordant.
T46935 662533-662545 Sentence denotes Conclusions:
T95720 662546-662718 Sentence denotes Our results support the finding that polysomy of chromosome 17 is indeed a rare event in breast cancers and increased CEP17 signals indicates pericentromeric amplification.
T76883 662719-663031 Sentence denotes Our data also highlight the limitations of currently used centromeric probe (CEP17) for HER2 FISH testing and demonstrates that the usage of non-centromeric chromosome 17 reference probe alters the HER2 status thereby increasing the eligibility for antiHER2 based therapy in a significant proportion of patients.
T41905 663032-663162 Sentence denotes We thus recommend the use of non centromeric chromosome 17 reference probe in cases with CEP17 aneusomy and equivocal HER2 status.
T55892 663163-663176 Sentence denotes Introduction:
T56075 663177-663312 Sentence denotes Human papillomavirus (HPV) is known to be associated with squamous cell carcinomas of the head and neck (HNSCC), especially oropharynx.
T4740 663313-663539 Sentence denotes Whereas patients whose tumors are HPV positive tend to have a better response to treatment, most of the clinical tests for HPV detection are DNA-based and use chromogenic in situ hybridization (CISH) as the preferred modality.
T20793 663540-663675 Sentence denotes Whereas HPV DNA test (CISH) has good sensitivity, its specificity is poor and it does not correlate with biologically active infection.
T52659 663676-663887 Sentence denotes The APTIMA HPV Assay uses transcription mediated amplification (TMA), thereby targeting the E6/E7 mRNA of 14 high-risk HPV types and has shown good sensitivity and specificity in liquid-based cytology specimens.
T30636 663888-664049 Sentence denotes Therefore, the objective of this study was to evaluate the performance of this assay on formalin-fixed paraffin embedded (FFPE) samples from patients with HNSCC.
T2410 664050-664058 Sentence denotes Methods:
T79533 664059-664104 Sentence denotes Twenty-two FFPE samples of HNSCC were tested.
T96888 664105-664197 Sentence denotes Two non-SCC cases and 10 papillomas that were secondary to low-risk HPV were also evaluated.
T52773 664198-664311 Sentence denotes Macrodissection was performed on all the specimens followed by RNA extraction using the Promega LEV FFPE RNA kit.
T43743 664312-664394 Sentence denotes Purity and concentrations were measured using Nanodrop and QuantiFluor on Quantus.
T87479 664395-664524 Sentence denotes All samples were run in duplicate with an input of at least 40ng/tube using the APTIMA HPV Assay on Panther instrument (Hologic).
T27124 664525-664673 Sentence denotes In addition to sensitivity and specificity, samples were also tested for accuracy, precision, tech-to-tech and instrument-to instrument variability.
T95217 664674-664761 Sentence denotes Results were compared to a national reference laboratory's HPV high-risk DNA ISH assay.
T72243 664762-664770 Sentence denotes Results:
T59925 664771-664820 Sentence denotes Most common site of involvement was tonsil (n=8).
T94716 664821-664904 Sentence denotes Majority of the tumors were moderate to poorly differentiated and non-keratinizing.
T58403 664905-664972 Sentence denotes 21 of the 22 cases were positive for high-risk HPV by APTIMA assay.
T46429 664973-665047 Sentence denotes Both non-SCC and all papillomas were negative for high-risk HPV by APTIMA.
T27809 665048-665165 Sentence denotes In contrast, the HPV DNA ISH assay was positive in 12 of the 22 samples, with 4 negative and 6 indeterminate results.
T61684 665166-665217 Sentence denotes One case was negative by both ISH and APTIMA assay.
T72119 665218-665366 Sentence denotes Serial dilutions revealed a minimum RNA input of 2.5ng/tube and a viable tumor cut-off of at least 10% to achieve a positive result by APTIMA assay.
T6116 665367-665443 Sentence denotes Thus, sensitivity and specificity of APTIMA was 95.5% and 100% respectively.
T19385 665444-665617 Sentence denotes Tech-to-tech and instrument-to-instrument variability was 100% at 40ng/tube input, however at lower inputs there were frequent occurrences of variability between replicates.
T49466 665618-665738 Sentence denotes Conclusions: APTIMA assay is highly sensitive and specific for detection of highrisk HPV subtypes associated with HNSCC.
T95999 665739-665834 Sentence denotes It offers improved specificity compared to ISH and can be used reliably even with FPPE tissues.
T904 665835-665945 Sentence denotes It also correlates with biologically active infection, consequently improving clinical management of patients.
T18153 665946-666048 Sentence denotes and PIK3CA in Pulmonary Adenocarcinoma Using iPLEX HS, a New Highly Sensitive Assay for MassARRAY R.T.
T1112 666049-666061 Sentence denotes Birse 1 , D.
T83076 666062-666156 Sentence denotes Irwin 2 1 Agena Bioscience, San Diego, CA; 2 Agena Bioscience, Herston, Queensland, Australia.
T75490 666157-666170 Sentence denotes Introduction:
T87970 666171-666370 Sentence denotes Increased early detection and personalized therapy for lung cancer have coincided with greater use of minimally invasive sampling techniques such as endobronchial ultrasound and needle core biopsies.
T29557 666371-666439 Sentence denotes These procedures offer lower risk alternatives for tissue diagnosis.
T12677 666440-666638 Sentence denotes However, they also generate analytical challenges such as a requirement for robust detection of low level somatic mutations, particularly when the starting sample is small or has sparse tumor cells.
T26663 666639-666972 Sentence denotes In this study we assessed 43 clinical cases of pulmonary adenocarcinoma (PA) previously tested for EGFR, KRAS, NRAS, and BRAF mutations using a novel multiplexed analytic approach that reduces wild-type signal and allows for detection of low mutation load approaching 1%, the iPLEX HS for MassARRAY (Agena Bioscience, San Diego, CA).
T44253 666973-666981 Sentence denotes Methods:
T29098 666982-667183 Sentence denotes Archived frozen deoxyribonucleic acid (DNA) samples were searched for PA cases previously tested for EGFR, KRAS, NRAS and BRAF mutations using the OncoFOCUS Panel v1.0 or v2.0 and the MassARRAY system.
T67101 667184-667242 Sentence denotes Specimens were deidentified prior to entry into the study.
T35309 667243-667311 Sentence denotes DNA from formalin-fixed, paraffin-embedded (FFPE) PA tissue samples.
T54720 667312-667420 Sentence denotes All histologic diagnoses were confirmed by a pathologist and minimum tumor cellularity for analysis was 20%.
T14687 667421-667497 Sentence denotes DNA was extracted using the QIAamp DNA FFPE Tissue Kit (Qiagen, Boston, MA).
T16517 667498-667851 Sentence denotes Prior to repeat testing, all specimens were assessed for DNA integrity using the iPLEX Pro Sample ID, and all specimens with adequate amplifiable DNA were then interrogated with a new high sensitivity, single PCR reaction iPLEX HS panel that includes more than 34 common mutations in BRAF, EGFR, KRAS, NRAS and PIK3CA, both using the MassARRAY platform.
T84608 667852-667860 Sentence denotes Results:
T42203 667861-668015 Sentence denotes In 43 samples, mutations in KRAS (n=11; 11/43= 26%), BRAF (n=1; 1/43 = 2%), EGFR (n=6; 6/43 = 14%) and NRAS (n=1; 1/43 = 2%) were detected using iPLEX HS.
T89557 668016-668260 Sentence denotes When compared to previous results from the OncoFOCUS assay (sensitivity of 5% to 10% mutated allele burden), 2 additional EGFRmutations (L858R, G719A), 2 additional KRAS mutations (G12A, G12D) and one more BRAF mutation (V600E) were identified.
T8890 668261-668427 Sentence denotes The majority of these cases had smaller, more limited tissue specimens (three needle core biopsies, one pleural fluid cytology cell block, and one excision specimen).
T40428 668428-668509 Sentence denotes Previously undetected mutations in PIK3CA were identified in 4 cases (4/43 = 9%).
T13956 668510-668522 Sentence denotes Conclusions:
T62819 668523-668671 Sentence denotes 1) Use of a highly sensitive assay such as iPLEX HS and MassARRAY increases detection of clinically significant mutations in challenging PA samples:
T84943 668672-668759 Sentence denotes 12% more KRAS, EGFR and BRAF mutations were identified in this pilot study of 43 cases.
T48707 668760-668839 Sentence denotes This study is currently being expanded to include 185 archived patient samples.
T31211 668840-668941 Sentence denotes 2) PIK3CA mutations were detected in 9% of PA cases, one of which also had a mutation in NRAS (A59D).
T55120 668942-668946 Sentence denotes E.M.
T93220 668947-668961 Sentence denotes Hissong 1 , P.
T50608 668962-668974 Sentence denotes Zhang 1 , J.
T95495 668975-668988 Sentence denotes Shia 2 , R.K.
T7874 668989-669003 Sentence denotes Yantiss 1 , H.
T92223 669004-669083 Sentence denotes Fernandes New York, NY; 2 Memorial Sloan Kettering Cancer Center, New York, NY.
T53592 669084-669097 Sentence denotes Introduction:
T2233 669098-669275 Sentence denotes The TCGA data identified four molecular signatures among gastric cancers: EBV positivity (EBV+), microsatellite instability (MSI), genomic stability, and chromosome instability.
T11585 669276-669364 Sentence denotes However, these data are heavily skewed toward signatures of common morphologic variants.
T59310 669365-669519 Sentence denotes Gastric carcinoma with lymphoid stroma (GCLS) is an uncommon subtype that shows a solid, or medullary, growth pattern with a dense lymphocytic infiltrate.
T29161 669520-669581 Sentence denotes Our aim is to characterize the molecular alterations in GCLS.
T11853 669582-669694 Sentence denotes The study group consisted of formalin fixed, paraffin embedded tissue sections from 30 GCLS resection specimens.
T33018 669695-669794 Sentence denotes All cases were subjected to in situ hybridization for EBER and immunohistochemistry (IHC) for HER2.
T80468 669795-669895 Sentence denotes DNA mismatch repair status was assessed using IHC (MLH1, MSH2, PMS2, MSH6) and PCR analysis for MSI.
T37882 669896-670072 Sentence denotes DNA was extracted from tumor and matched-normal specimens and sequenced on the Ion Proton using Comprehensive Cancer Panel (ThermoFisher) that targets 409 cancer-related genes.
T4854 670073-670162 Sentence denotes Data were analyzed using the Variant caller 4.4 software and Ion Reporter (ThermoFisher).
T48112 670163-670171 Sentence denotes Results:
T91406 670172-670314 Sentence denotes Five (17%) cases were EBV+ and microsatellite stable (EBV+), 12 were EBV-negative with MSI (MSI), and 13 were EBV-negative and MSS (EBV-/MSS).
T87045 670315-670447 Sentence denotes PIK3CA variants within the kinase domain were common in EBV+ tumors (43%), whereas 36% of MSI tumors showed helical domain variants.
T4589 670448-670518 Sentence denotes ERBB2 variants were more prevalent in EBV+ (43%) and MSS (27%) groups.
T16068 670519-670578 Sentence denotes Somatic alterations in KRAS were detected in 45% MSI cases.
T72298 670579-670696 Sentence denotes EBV-/MSS tumors often harbored TP53 variants (>80%). and 3 (23%) were genetically unstable with numerous alterations.
T15499 670697-670919 Sentence denotes Copy Number Alterations (CNA) were more common among MSS and EBV+ tumors than EBV-/MSS cancers, with frequent amplification of PIK3CA, JAK2, and MYC in EBV+ cases and amplifications in EGFR, JAK2 and BAI3 in the MSS group.
T90018 670920-670989 Sentence denotes Several tumors in all groups had amplifications and deletions in APC.
T81978 670990-671086 Sentence denotes Amplification of ERBB2 was lacking in GCLS, and only one case (3%) showed HER-2 staining by IHC.
T55136 671087-671099 Sentence denotes Conclusions:
T37946 671100-671221 Sentence denotes Molecular changes in GCLS are heterogeneous, but seem to cluster in three major groups: EBV+/MSS, EBV-/MSI, and EBV-/MSS.
T12091 671222-671437 Sentence denotes EBV+ tumors commonly show PIK3CA variants and high-frequency CNA, raising the possibility that affected patients may benefit from PI3-kinase inhibitors, or have a better prognosis than patients with EBV-/MSS tumors.
T19070 671438-671442 Sentence denotes R.N.
T25136 671443-671455 Sentence denotes Ptashkin, C.
T16237 671456-671465 Sentence denotes Pagan, S.
T87563 671466-671476 Sentence denotes Middha, R.
T98757 671477-671487 Sentence denotes Yaeger, M.
T72918 671488-671499 Sentence denotes Ladanyi, M.
T16098 671500-671510 Sentence denotes Berger, A.
T25363 671511-671522 Sentence denotes Zehir, J.F.
T32378 671523-671585 Sentence denotes Hechtman Memorial Sloan Kettering Cancer Center, New York, NY.
T16241 671586-671599 Sentence denotes Introduction:
T19635 671600-671678 Sentence denotes Recurrent copy number gains of 20q have been observed in several malignancies.
T52624 671679-671929 Sentence denotes Despite several studies investigating 20q gain on a functional level, 20q gain in colorectal carcinoma (CRC) has not been methodically investigated in relation to clinicopathologic and molecular features including prognosis and response to treatment.
T36565 671930-672029 Sentence denotes In this study, we aimed to elucidate the molecular and clinical features of CRC harboring 20q gain.
T82475 672030-672038 Sentence denotes Methods:
T36948 672039-672202 Sentence denotes Tumor and matched normal DNA from patients with advanced CRC were sequenced using the MSK-IMPACT assay which detects somatic alterations in 410 cancer genes (PMID:
T88042 672203-672213 Sentence denotes 25801821).
T71616 672214-672450 Sentence denotes jmd.amjpathol.org ■ The Journal of Molecular Diagnostics Microsatellite instability (MSI) status was calculated using MSIsensor, a clinically validated assay that assesses microsatellite regions in next-generation sequencing (NGS) data.
T41279 672451-672548 Sentence denotes We calculated the Tumor/Normal log2 ratio (lr) for all 20q genes in the (0.45 -0.45 < lr < 0.45).
T29090 672549-672670 Sentence denotes Associations with clinical and molecular data were assessed by Fisher's test with multiple hypothesis testing correction.
T18005 672671-672731 Sentence denotes A Cox proportional hazard model was used to assess survival.
T75070 672732-672740 Sentence denotes Results:
T2379 672741-672853 Sentence denotes Of the 413 CRC samples from 401 patients, 150 (36.3%) had 20q gain, and 30 samples (7.3%) had 20q amplification.
T57312 672854-673154 Sentence denotes CRCs with 20q gain or amplification were more often KRAS WT (67% vs. 42%, p=<0.0001), BRAF WT (94% vs. 80%, p=0.001), TP53 mutant (86% vs. 61%, p<0.0001), arising as left sided primaries, (78% vs. 52%, p<0.0001) and microsatellite stable (>99% vs. 84%, p<0.001) in comparison to CRC with diploid 20q.
T69921 673155-673369 Sentence denotes For 390 patients for which survival data were available, 237 (60.6%) presented with stage IV metastatic disease and 117 (30%) progressed to stage IV during the course of study (median time to progression = 15.9mo).
T8831 673370-673593 Sentence denotes 20q amplification or gain was associated with longer survival from date of diagnosis with metastatic disease (Hazard Ratio = 0.52, p=0.004) on multivariate analysis controlling for age at diagnosis, RAS/ RAF and MSI status.
T89162 673594-673606 Sentence denotes Conclusions:
T8385 673607-673703 Sentence denotes Our data show that 20q gain or amplification is frequent, occurring in approximately 40% of CRC.
T86714 673704-673945 Sentence denotes This molecular event defines a specific subgroup with distinctive clinicopathologic features including left sided primary tumors, microsatellite stability, RAS/ RAF WT status, and better overall survival in the setting of metastatic disease.
T65815 673946-673959 Sentence denotes Introduction:
T62058 673960-674088 Sentence denotes Molecular profiling of DNA alterations and mutations has offered unprecedented insight into the causes and treatments of cancer.
T76138 674089-674218 Sentence denotes However, a comprehensive understanding of how these DNA alterations affect the expression of tumor-related genes remains unknown.
T52880 674219-674227 Sentence denotes Methods:
T82237 674228-674387 Sentence denotes We analyzed the expression of 315 cancer related genes across 7 different tumor types using RNA-seq data obtained from The Cancer Genome Atlas (TCGA) database.
T10992 674388-674582 Sentence denotes We compared the expression data to single nucleotide variant (SNV) and structural variants including copy number alterations (CNA) and translocations detected in the genomes of the same samples.
T10166 674583-674591 Sentence denotes Results:
T56845 674592-674758 Sentence denotes We found that, among the cancer-related genes analyzed, 5% of the time when a SNV is detected in the DNA, there is no detectable expression of that allele in the RNA.
T84308 674759-674873 Sentence denotes We further analyzed the correlation between somatic gene amplifications to the expression levels of the same gene.
T34142 674874-675104 Sentence denotes We found that, overall, CNA status was a very weak predictor of gene expression (r-squared = .00012) with CNA amplification only associated with expression in 8 genes correlated with CNA amplification at a level of r-squared >0.3.
T22283 675105-675347 Sentence denotes Finally, we examined the expression of ALK, a known oncogene involved in an intrachromosomal fusion (EML4-ALK) in 4-6% of lung adenocarcinomas that results in overexpression of ALK, and is associated with increased response to ALK-inhibitors.
T41621 675348-675474 Sentence denotes We confirmed that the EML4-ALK fusion resulted in ALK overexpression in lung cancer compared to cases with non-rearranged ALK.
T42607 675475-675648 Sentence denotes However, we found that in a subset of patients, particularly in breast cancer, ALK was overexpressed to the same degree (Z-score>10) as seen in lung cancer EML4-ALK fusions.
T50110 675649-675661 Sentence denotes Conclusions:
T61602 675662-675856 Sentence denotes Our analysis indicates that not all somatic DNA mutations are expressed in tumors, suggesting that concurrent RNA expression profiling would provide additional clinically-actionable information.
T43084 675857-676040 Sentence denotes We further conclude that increased ALK expression may be seen in cancer cases that do not harbor EML4-ALK fusions, raising the possibility of treating such tumors with ALK-inhibitors.
T29218 676041-676043 Sentence denotes R.
T80035 676044-676056 Sentence denotes Birse 1 , D.
T6219 676057-676151 Sentence denotes Irwin 2 1 Agena Bioscience, San Diego, CA; 2 Agena Bioscience, Herston, Queensland, Australia.
T61132 676152-676165 Sentence denotes Introduction:
T44240 676166-676436 Sentence denotes The 2016 National Comprehensive Cancer Network treatment guidelines (NCCN Guidelines) recommend that patients with metastatic colorectal adenocarcinomas (mCRC) have tumor tissue genotyped for RAS (KRAS and NRAS) mutations, and suggest BRAF V600E testing be done as well.
T30658 676437-676579 Sentence denotes In the United States, clinical laboratories offer mutation testing using many different platforms with a wide range of analytical sensitivity.
T23946 676580-676800 Sentence denotes Despite considerable progress, analytical challenges remain to be resolved such as the need for reliable detection of low abundance somatic mutations, particularly in small specimens with a low percentage of tumor cells.
T12073 676801-677105 Sentence denotes In this study we assessed 46 clinical CRC cases previously tested for EGFR, KRAS, NRAS, and BRAF mutations using a novel multiplexed analytic approach that reduces wild-type signal and allows for detection of low mutation load approaching 1%, the iPLEX HS for MassARRAY (Agena Bioscience, San Diego, CA).
T58899 677106-677114 Sentence denotes Methods:
T5720 677115-677293 Sentence denotes Archived frozen DNA samples were searched for CRC cases previously tested for EGFR, KRAS, NRAS and BRAF mutations using the OncoFOCUS Panel v1.0 or v2.0 and the MassARRAY system.
T42896 677294-677352 Sentence denotes Specimens were deidentified prior to entry into the study.
T21759 677353-677493 Sentence denotes DNA originated from formalin fixed paraffin embedded (FFPE) CRC tissue samples and all histologic diagnoses were confirmed by a pathologist.
T61850 677494-677570 Sentence denotes DNA was extracted using the QIAamp DNA FFPE Tissue Kit (Qiagen, Boston, MA).
T63041 677571-677923 Sentence denotes Prior to repeat testing, all specimens were assessed for DNA integrity using the iPLEX Pro Sample ID and all specimens with adequate amplifiable DNA were then interrogated with a new high sensitivity, single PCR reaction iPLEX HS panel that includes more than 34 common mutations in BRAF, EGFR, KRAS, NRAS and PIK3CA, both using the MassARRAY platform.
T78572 677924-677932 Sentence denotes Results:
T90339 677933-678078 Sentence denotes The OncoFOCUS assay has a mutation detection limit of 5% to 10% mutant allele burden, whereas the iPLEX HS is more sensitive at approximately 1%.
T25191 678079-678266 Sentence denotes In this pilot study of 46 patient samples, the iPLEX HS panel confirmed all previously identified CRC mutations in KRAS (n=10; 10/46=22%), NRAS (n=1; 1/46=2%), and BRAF (n=8; 8/46 = 17%).
T60859 678267-678484 Sentence denotes Previously undetected mutations in PIK3CA were also identified (n=10; 10/46=22%), and 5 of the PIK3CA mutations coexisted with other driver mutations, including KRAS G12D and G12C; NRAS A59T; and BRAF V600E (2 cases).
T51874 678485-678497 Sentence denotes Conclusions:
T20654 678498-678720 Sentence denotes 1) In this small sample set, lowering the assay sensitivity from 5% to about 1% mutant alleles detected all previously known mutations in KRAS, NRAS and BRAF and 10 previously undetected mutations in Pi3KCA in CRC samples.
T56004 678721-678800 Sentence denotes This study is currently being expanded to include 286 archived patient samples.
T48980 678801-678902 Sentence denotes 2) PIKC3A mutations may coexist with other driver mutations in RAS or BRAF in about 11% of CRC cases.
T11306 678903-679127 Sentence denotes Histologically, these entities may present a diagnostic challenge and the usage of molecular methods such as FISH, RT-PCR/next-generation sequencing to detect specific translocations is currently a routine part of diagnosis.
T99502 679128-679136 Sentence denotes Methods:
T35811 679137-679294 Sentence denotes In this study, we evaluated the presence of EWSR1 translocation with FISH and RTnext-generation sequencing using the anchored-multiplex PCR technology (AMP).
T12086 679295-679303 Sentence denotes Results:
T82940 679304-679502 Sentence denotes Among the 18 SBRCTs where there was strong histologic/immunehistochemical evidence suggesting the presence of a EWSR1 translocation, concordant positivity was seen by FISH and AMP in 13 cases (72%).
T97433 679503-679592 Sentence denotes In the 5 negative EWSR1 FISH cases, EWSR1 translocations were detected by AMP in 4 cases.
T21897 679593-679710 Sentence denotes Additional orthogonal methods such as partner FISH and/or RT-PCR also confirmed the presence of EWSR1 rearrangements.
T71475 679711-679723 Sentence denotes Conclusions:
T96564 679724-679933 Sentence denotes Consistent with the reported sensitivity of 82% to 97% of EWSR1 FISH in the literature, our results demonstrate the potential for false negativity when using EWSR1 FISH as the only molecular diagnostic method.
T95709 679934-680062 Sentence denotes In cases with classic morphology and immunehistochemical profile, additional molecular testing such as AMP should be considered.
T10236 680063-680076 Sentence denotes Introduction:
T71181 680077-680241 Sentence denotes Premalignant epithelial lesions are commonly diagnosed yet current medical practice is limited in its ability to stratify them for risk of progression to carcinoma.
T94454 680242-680459 Sentence denotes We cataloged the genomic mutation spectrum in lesions adjacent to invasive gastric adenocarcinoma to explore how frequently premalignant lesions shared mutation signatures in common with those found in the malignancy.
T86144 680460-680468 Sentence denotes Methods:
T56129 680469-680685 Sentence denotes Macrodissected cancers and 30 premalignant lesions from 25 gastric carcinoma patients' paraffin embedded tissue weresequenced across hotspots in 26 human cancer genes (Illumina TruSight Tumor 26 reagents on a MiSeq).
T35586 680686-680816 Sentence denotes Also sequenced were an additional 4 premalignant gastric lesions from 4 patients without subsequent cancer on long-term follow-up.
T29736 680817-680929 Sentence denotes Non-synonymous mutations and small indels at allele frequency >5% with population frequency < 1% were cataloged.
T65610 680930-680938 Sentence denotes Results:
T96152 680939-681098 Sentence denotes 24/25 invasive carcinomas had a detectable mutation (range 1 to 4 mutations per tumor) in TP53, KRAS, APC, PIK3CA, FBXW7, CDH1, SMAD4, PTEN, MSH6, MET, or ALK.
T14585 681099-681213 Sentence denotes All 30 dissected premalignant lesions yielded adequate read depth (>500x) for interpretation of hotspot mutations.
T80322 681214-681439 Sentence denotes Whereas 23 premalignant lesions had no detectable mutation, 7 (23%) had at least one mutation (range 1 to 2 mutations of TP53, APC, MET, or CDH1) that nearly always matched mutation(s) present in the adjacent invasive cancer.
T14804 681440-681527 Sentence denotes Average mutant allele fraction in these 7 premalignant lesions was 31 (range 8 to 53%).
T89523 681528-681676 Sentence denotes In two patients there was evidence of clonal evolution whereby the premalignant lesion harbored only one of two mutations detected in the carcinoma.
T9317 681677-681894 Sentence denotes Only 1/30 premalignant lesions had a mutation that was absent in the matched cancer, suggesting that variants in these 26 cancer-related genes may be uncommon unless the patient has cancer or cancer precursor lesions.
T83770 681895-682067 Sentence denotes A non-canonical NRASmutation was also identified in an intestinal metaplasia (IM) lesion from one of four IM patients who did not progress to cancer on long term follow-up.
T83119 682068-682079 Sentence denotes Conclusion:
T62042 682080-682156 Sentence denotes Successful sequencing of dissected premalignant mucosal lesions is feasible.
T85634 682157-682531 Sentence denotes Genomic findings reflect intrinsic biology of precursor lesions and suggest they can be clonally related to cancer tissue of the study provides evidence that real-time PCR assays enhanced by ARMS, Scorpions or PANClamp technologies despite occasional lack of reproducibility (in more challenging samples) could serve well as a more sensitive testing option in EGFR analysis.
T17325 682532-682536 Sentence denotes O.A.
T54900 682537-682549 Sentence denotes Shetty, M.R.
T86614 682550-682564 Sentence denotes Ramadwar, M.Y.
T95856 682565-682576 Sentence denotes Gurav, T.D.
T37620 682577-682584 Sentence denotes Pai, V.
T18359 682585-682595 Sentence denotes Kamble, G.
T14142 682596-682611 Sentence denotes Chinnaswamy, T.
T68334 682612-682620 Sentence denotes Vora, S.
T71216 682621-682632 Sentence denotes Qureshi, S.
T61516 682633-682646 Sentence denotes Dhanavade, S.
T83794 682647-682658 Sentence denotes Tambe, S.B.
T36535 682659-682715 Sentence denotes Desai Tata Memorial Hospital, Mumbai, Maharshtra, India.
T56408 682716-682729 Sentence denotes Introduction:
T52897 682730-682922 Sentence denotes Neuroblastoma (NB) is a primitive neuroectodermal tumor and the most common neoplasm of childhood presenting with genetic instability in which prognosis and response to therapy varies greatly.
T46641 682923-683089 Sentence denotes Genomic amplification of MYCN oncogene has been used to predict outcome in NB for over 30 years, however clinical behaviour of MYCN non-amplified cases are ambiguous.
T93895 683090-683353 Sentence denotes Recent advances enables detailed analysis of NB genome, leading to the identification of new prognostic markers such as loss of heterozygosity at chromosome arms 1p, 3p, and 11q, unbalanced gain of 1q, 17q and numerous mutations in key genes such as ALK and DDX1.
T2103 683354-683568 Sentence denotes Analysis of these will in turn help in better patient stratification.The present study is aimed at characterizing the genetic abnormalities in MYCN non amplified NBs along with clinical and histological parameters.
T26044 683569-683577 Sentence denotes Methods:
T72577 683578-683683 Sentence denotes The present study was conducted on 26 cases of NB reported in Tata Memorial Hospital during 2014 to 2016.
T79202 683684-683815 Sentence denotes MYCN amplification was assessed by fluorescence in situ hybridization (FISH) using Vysis LSI N-myc dual colour probe (Abbott, USA).
T11968 683816-683979 Sentence denotes Multiplex Ligation Probe Amplification assay (MLPA) was used to assess the genetic profile of cases using probe mixes P251, P252 and P253 (MRC Holland, Amsterdam).
T65628 683980-684144 Sentence denotes MLPA is used to study segmental chromosomal aberrations at chromosomes 1p, 3p, 14q and 11q, unbalanced gain of 1q, 11p and 17q and genomic aberrations in ALK, DDX1.
T32372 684145-684241 Sentence denotes Coffalyser software was used for data analysis and SIOPEN guidelines were used for nomenclature.
T67290 684242-684250 Sentence denotes Results:
T62685 684251-684386 Sentence denotes The study included patient's in the age group between 1 month to 2 years with a median age of 1 year and male to female ratio of 2.5:1.
T83860 684387-684509 Sentence denotes Interpretable MLPA results were obtained in 22 cases of which 18 (81%) were MYCN non amplified and 4 (19%) were amplified.
T30227 684510-684715 Sentence denotes Among the MYCN non amplified (n=18), 11q loss in 5 (28%) cases, 17q amplification in 7 cases (39%), 17q gains were observed in 4 (22%) cases, followed by 3p loss in 5 (28%) cases, 1p loss in 3 (17%) cases.
T1849 684716-684864 Sentence denotes ALK gene gains were seen in 7 (39%) cases.17q amplification was found to be mutually exclusive with MYCN non amplification in this series (p=0.014).
T90744 684865-685031 Sentence denotes 17q amplification was found to be correlated with unfavourable histology, high MKI index and poor clinical response to treatment though statistically not significant.
T75945 685032-685043 Sentence denotes Conclusion:
T63583 685044-685155 Sentence denotes 17q amplification was associated with aggressive behaviour with poor clinical outcome in MYCN non amplified NB.
T59367 685156-685294 Sentence denotes This indicates the need to study the genetic profile of MYCN non amplified cases to predict prognosis and clinical management of patients.
T39167 685295-685503 Sentence denotes MLPA analysis hence becomes an important tool which can be used effectively and simultaneously to detect multiple genomic imbalances and these changes are being utilized to classify NB by prognostic subtypes.
T92221 685504-685506 Sentence denotes E.
T73471 685507-685522 Sentence denotes Golomb 1 , I.B.
T31818 685523-685537 Sentence denotes Achache 1 , R.
T66133 685538-685550 Sentence denotes Beeri 1 , T.
T47362 685551-685566 Sentence denotes Grenader 1 , I.
T46809 685567-685662 Sentence denotes Haviv 2 1 Shaare Zedek Medical Center, Jerusalem, Israel; 2 Bar Ilan University, Safed, Israel.
T51407 685663-685676 Sentence denotes Introduction:
T48921 685677-685911 Sentence denotes Differences in the prevalence of RAS/BRAF somatic mutations in colon cancers among ethnic groups have been suggested, raising the possibility that epigenetic environmental and genetic factors govern the selection of somatic mutations.
T20952 685912-685998 Sentence denotes The population examined in Jerusalem consists of two main populations, Jews and Arabs.
T54549 685999-686104 Sentence denotes We compared the spectrum of RAS/BRAF mutation in metastatic colorectal cancers between these populations.
T97801 686105-686113 Sentence denotes Methods:
T35438 686114-686259 Sentence denotes During the period between 2014 and May 2016, 244 extended RAS / BRAF analyses were performed at Shaare Zedek Medical Center in Jerusalem, Israel.
T3542 686260-686317 Sentence denotes Seventy of these patients (25.6%) were Palestinian Arabs.
T68760 686318-686386 Sentence denotes The rest of the patients were Jews and tourists from Eastern Europe.
T96014 686387-686474 Sentence denotes KRAS codons 12/13and BRAF V600E mutations were analyzed by allele discrimination assay.
T52988 686475-686530 Sentence denotes The other mutations were detected by Sanger sequencing.
T98218 686531-686539 Sentence denotes Results:
T98807 686540-686623 Sentence denotes The overall incidence of WT versus mutated RAS/BRAF was similar between the groups.
T26402 686624-686827 Sentence denotes However, the Arab population of Jerusalem showed a specifically high prevalence of KRAS mutations in exons 3 or 4 (15.7%) compared to the Jewish / non Arab population (2.9%, p<0.01 by Fisher exact test).
T73672 686828-686913 Sentence denotes This was balanced by a lower rate of NRAS and KRAS exon 2 mutations in Arab patients.
T45722 686914-686925 Sentence denotes Conclusion:
T2719 686926-687084 Sentence denotes Prevalence of somatic driver mutations may differ between ethnic groups, so that a mutation that is considered rare in one population may be common in others.
T64472 687085-687188 Sentence denotes The genetic / epigenetic basis for these differences may shed light on the mechanism of carcinogenesis.
T34565 687189-687334 Sentence denotes This may stem from a different genetic background among populations, or from different environmental factors, such as dietary and smoking habits.
T23418 687335-687348 Sentence denotes Introduction:
T69902 687349-687448 Sentence denotes Next-generation sequencing has enabled large-scale studies of cancer genomes to identify mutations.
T85209 687449-687617 Sentence denotes These tumor-specific somatic changes offer great potential as biomarkers to inform clinical practice, including detection, personalized medicine and disease monitoring.
T61981 687618-687815 Sentence denotes The ability to detect the mutational landscape of tumor cells using non-invasive approaches (liquid biopsies) could enable rapid, personalized variant screening and efficient monitoring of therapy.
T38948 687816-688049 Sentence denotes Unfortunately, limitations of current standard technologies include lack of sensitivity and the scalability for a rapid analysis, especially in the case of small amounts of circulating cell-free DNA (cfDNA) from blood plasma samples.
T22999 688050-688058 Sentence denotes Methods:
T90595 688059-688310 Sentence denotes Here we present a streamlined, DNA library preparation workflow that enables sample multiplexing for Illumina next-generation sequencing with high sensitivity and reproducibility for variant detection using minuscule amounts of circulating plasma DNA.
T22778 688311-688479 Sentence denotes Mixed plasma from healthy female and male donors were used to generate different ratios of the X and Y chromosomes for determining sensitivity of copy number detection.
T47221 688480-688606 Sentence denotes Exome enrichment of the libraries was done using Nimblegen's VCRome kit for in-depth analysis of single nucleotide variations.
T37115 688607-688615 Sentence denotes Results:
T68108 688616-688763 Sentence denotes With as little as 500 pg of cfDNA we were able to generate sequencing libraries for whole genome (WGS) and whole exome (WES) with good GC coverage.
T97925 688764-688882 Sentence denotes All samples with 10ng input DNA have >99.5% reads mapping back to the reference genome and >99% have unique sequences.
T24690 688883-688965 Sentence denotes Even at the lowest input of 500pg >98% of reads map and 99% have unique sequences.
T66287 688966-689071 Sentence denotes We accurately determined the ratio of sex chromosomes in a mixed sample down to 5% of Y chromosome input.
T73821 689072-689291 Sentence denotes Exome target enrichment allowed recovery of known variants (>80%), showed good correlation with expected allele frequencies, and detection was consistent and reproducible across different starting concentrations of DNA.
T68105 689292-689304 Sentence denotes Conclusions:
T58969 689305-689402 Sentence denotes The simple and rapid workflow allows a quick and sensitive approach to detect genomic variations.
T79465 689403-689523 Sentence denotes This method could facilitate biomarker identification to supplement diagnostic, prognostic and therapeutic applications.
T87418 689524-689633 Sentence denotes We also achieved sufficient library complexity for highconfidence copy number detection and variant recovery.
T83856 689634-689636 Sentence denotes J.
T95376 689637-689648 Sentence denotes Tull 1 , J.
T28154 689649-689660 Sentence denotes Yang 2 , W.
T63693 689661-689672 Sentence denotes Song 2 , S.
T3686 689673-689778 Sentence denotes Zhang 2 1 Upstate Medical University Hospital, Syracuse, NY; 2 Upstate University Hospital, Syracuse, NY.
T8579 689779-689792 Sentence denotes Introduction:
T69284 689793-689977 Sentence denotes Activating mutations in the epidermal growth factor receptor (EGFR) gene in non-small-cell lung cancers (NSCLC) are predictive of response to treatment with tyrosine kinase inhibitors.
T38838 689978-690067 Sentence denotes Therefore a sensitive assay to detect EGFR mutations is recommended in clinical practice.
T58022 690068-690190 Sentence denotes The real-time PCR based Therascreen EGFR RGQ kit is FDA approved for the detection of the most frequent EGFR alternations.
T30671 690191-690331 Sentence denotes During validation in our laboratory, up to 25% of clinical samples could not pass the kit's DNA sample assessment assay for amplifiable DNA.
T37128 690332-690409 Sentence denotes Obtaining amplifiable DNA presents challenging issues for mutation detection.
T68604 690410-690566 Sentence denotes These include the poor quality of DNA in formalin fixed paraffin embedded (FFPE) tissue and the low quantity of DNA as most tissue are from needle biopsies.
T72215 690567-690698 Sentence denotes In this study, we modified the DNA isolation method to obtain more high-quality DNA optimizing the Therascreen EGFR mutation assay.
T64849 690699-690787 Sentence denotes Methods: DNA was extracted in a total of 152 cases using QIAamp DSP DNA FFPE Tissue Kit.
T8130 690788-690933 Sentence denotes The isolation method was modified with pre-digestion heat treatment, overnight proteinase-K digestion, and double elution of the provided column.
T32925 690934-691067 Sentence denotes The Therascreen EGFR PCR kit was used to test the presence of amplifiable DNA using the kit's assessment assay or a control reaction.
T8115 691068-691186 Sentence denotes The control reaction mix uses a Scorpion primer and an unlabeled primer to amplify a short sequence of exon 2 of EGFR.
T27921 691187-691335 Sentence denotes The cycle threshold (Ct) values were used to assess the amount of amplifiable DNA obtained (the lower the Ct value, the more amplifiable DNA input).
T9489 691336-691567 Sentence denotes Three groups were included in this study: non-modification control group (n=48), pre-heat treatment with overnight digestion group (n=67), and combination of pre-heat treatment, overnight digestion and double elution group (n= 37).
T9763 691568-691727 Sentence denotes In addition the success rate, defined as % cases passing the DNA assessment, was compared between non-modification (n=48) and post-modification groups (n=104).
T81473 691728-691816 Sentence denotes The data was analyzed by repeated-measures ANOVA followed by Neuman-Keuls post hoc test.
T40284 691817-691825 Sentence denotes Results:
T85438 691826-691940 Sentence denotes A higher PCR success rate was obtained after method modifications (91.13%) compared to the control group (66.25%).
T38539 691941-692025 Sentence denotes There was a significant difference in Ct values between the three groups (p=0.0036).
T47671 692026-692224 Sentence denotes The control group had significantly higher Ct values than the other two groups (Newman-Keuls post hoc test, control versus overnight, p <0.05; control versus overnight with double elution, p <0.05).
T74516 692225-692387 Sentence denotes The lowest Ct values were obtained from the group with the combination of pre-heat treatment, overnight proteinase K digestion and double elution (mean Ct=27.12).
T31948 692388-692400 Sentence denotes Conclusions:
T76098 692401-692533 Sentence denotes Our modifications in DNA isolation from FFPE samples have significantly optimized Therascreen EGFR mutation assay in NSCLC patients.
T60101 692534-692536 Sentence denotes S.
T87334 692537-692547 Sentence denotes Jain, M.A.
T38076 692548-692559 Sentence denotes Melan, A.I.
T95151 692560-692568 Sentence denotes Wald, R.
T77422 692569-692583 Sentence denotes Hamilton, M.N.
T54884 692584-692651 Sentence denotes Nikiforova University of Pittsburgh Medical Center, Pittsburgh, PA.
T99435 692652-692665 Sentence denotes Introduction:
T74357 692666-692857 Sentence denotes Methylation status of MGMT (O 6 -methylguanine-DNA methyltransferase) is used as a prognostic/predictive marker for glioma patients treated with alkylating agents, such as temozolomide (TMZ).
T70926 692858-693125 Sentence denotes Conventional methylation specific PCR (MSP) methods provide evaluation of CpGs in the MGMTpromoter region; however, some patients with methylated MGMT status show poor survival and response to TMZ, likely due to aberrant MGMT expression not evaluated by these assays.
T38575 693126-693350 Sentence denotes Using targeted NGS panel (GlioSeq), we analyzed MGMT mRNA expression and correlated it with the MGMT promoter methylation status to determine if MGMT expression plays role in mediating tumor sensitivity to alkylating agents.
T31125 693351-693359 Sentence denotes Methods:
T31282 693360-693591 Sentence denotes GlioSeq, a targeted in-house developed NGS panel was used for assessing MGMT mRNA expression in addition to SNVs, copy number changes and gene fusions in 53 glioblastoma (GBM) patients and in 10 normal temporal lobe brain biopsies.
T81354 693592-693722 Sentence denotes MGMT promoter methylation was determined by methylation-specific PCR and gel electrophoresis (MSP-G) and MethyLight qPCR (MLqPCR).
T92465 693723-693853 Sentence denotes MGMT expression was compared to expression of GUSB, HPRT1 and PGK1 housekeeping genes and correlated with MGMT methylation status.
T25755 693854-693862 Sentence denotes Results:
T39756 693863-693935 Sentence denotes Normal brain tissue showed a high level of MGMT expression (mean=51.73).
T41020 693936-694056 Sentence denotes Thirty GBMs negative for methylation showed MGMT mRNA expression by GlioSeq that ranged from 5.02 to 35.27 (mean=16.32).
T73913 694057-694182 Sentence denotes Twenty-three MGMT methylation positive GBMs showed mRNA expression levels that ranged from 0.26 to 18.77 with a mean of 6.81.
T66662 694183-694499 Sentence denotes Using an mRNA MGMT expression cut off >10 for high level of expression and <10 for low level of expression, the MGMT promoter methylation status showed correlations with MGMT mRNA expression in 42/53 (79%) of cases, including 78% concordance in the methylated GBM group and 83% in the unmethylated GBMs. Conclusions:
T82444 694500-694677 Sentence denotes GlioSeq targeted NGS panel allows to detect MGMT mRNA expression in fixed brain tissue specimens during routine NGS analysis for most common genetic alterations in brain tumors.
T85208 694678-694769 Sentence denotes MGMT mRNA expression was concordant with the MGMT methylation status in 79% of GBMs tested.
T73226 694770-694922 Sentence denotes Discordant findings may indicate methylation-independent pathways for MGMT expression regulation and explain aberrant responses to temozolomide therapy.
T80151 694923-694927 Sentence denotes C.L.
T11697 694928-694939 Sentence denotes Sims 1 , S.
T95832 694940-694955 Sentence denotes Govender 1 , A.
T21704 694956-694970 Sentence denotes Khurana 1 , M.
T85875 694971-694983 Sentence denotes Moore 2 , P.
T78862 694984-694997 Sentence denotes Cotter 2 , S.
T11779 694998-695055 Sentence denotes Gunn 2 1 PacificDx, Irvine, CA; 2 ResearchDx, Irvine, CA.
T4902 695056-695188 Sentence denotes Introduction: DNA-based microarray technology is useful for providing information about gene copy-number and chromosomal aneuploidy.
T150 695189-695311 Sentence denotes Formalin-fixed, paraffinembedded (FFPE) tissue is an important yet difficult sample type to work with in molecular assays.
T30330 695312-695502 Sentence denotes The chemical fixation process intended to preserve specimens for long term storage causes genetic information to be lost through DNA/protein cross linking and the creation of apurinic sites.
T74482 695503-695629 Sentence denotes Further compounding the problem, longterm storage of blocks before molecular analysis contributes to nucleic acid degradation.
T64505 695630-695851 Sentence denotes Here we describe a method for reference size matching, amplification and fluorescent labeling of FFPE specimens that yields high quality material for downstream DNA-based microarray analysis of this difficult sample type.
T47743 695852-695860 Sentence denotes Methods:
T24148 695861-696160 Sentence denotes In the current study, nucleic acid extraction was performed for 50 FFPE samples using the QIAmp DNA FFPE Tissue Kit either manually or on the QIAcube, followed by fragmentation using an Episonic 2000 sonicator to a mean library size of 500bp (with a +/-100bp cutoff interval) tapering off to 2000bp.
T2896 696161-696312 Sentence denotes Mean library size was determined electrophoretically using a Bioanalyzer 2100 (Agilent); samples were matched to an appropriately fragmented reference.
T38245 696313-696465 Sentence denotes Whole genome amplification and incorporation of 5-(3 aminoallyl)-dUTP was performed using the GenomePlex Whole Genome Amplification kit (Sigma-Aldrich).
T7984 696466-696655 Sentence denotes To allow for size selection of DNA libraries, while maintaining the mean library size, amplification products were cleaned up using Agencourt Ampure XP DNA magnetic beads (Beckman Coulter).
T89065 696656-696726 Sentence denotes Fluorophore (NHS Ester) coupling was performed under basic conditions.
T87495 696727-696828 Sentence denotes Final labeled products were cleaned up using the NucleoSpin Gel and PCR clean up kit (Machery Nagel).
T87976 696829-696992 Sentence denotes Degree of labeling (DOL), dye molecules per 100 bases, and total recovery was measured spectrophotometrically using a Nanodrop 2000 instrument (Thermo Scientific).
T55215 696993-697001 Sentence denotes Results:
T5170 697002-697263 Sentence denotes All samples yielded labeled DNA of sufficient quality and quantity for downstream analysis on the Agilent SureScan G4900DA using the following QC metrics: optimal DOL of 2.5, and at least 1000ng to 2000ng of labeled DNA required for an Agilent 8x60k microarray.
T92860 697264-697341 Sentence denotes Across the 50 samples tested, mean DOL was 2.2 (acceptable range 1.5 to 3.5).
T41984 697342-697412 Sentence denotes Mean labeled DNA yield was 3000ng (acceptable range 2000ng to 4000ng).
T24149 697413-697425 Sentence denotes Conclusions:
T34452 697426-697631 Sentence denotes As technology and our ability to ask and answer genetic questions about solid tumors improve, FFPE solid tumor specimens will become central to the molecular diagnosis, treatment and monitoring of disease.
T86406 697632-697796 Sentence denotes Given the use of commercially available kits and easily sourced reagents, the method described could be optimized for use on any microarray based analysis platform.
T2275 697797-697810 Sentence denotes Introduction:
T99334 697811-697973 Sentence denotes Salivary duct carcinoma (SDC) is a rare type of salivary gland cancer (SGC), typically refractory to treatment and with a particularly aggressive clinical course.
T94937 697974-698182 Sentence denotes Whereas specific gene fusions have been conclusively associated with other types of salivary gland malignancies, it remains to be determined whether genetic rearrangements are a characteristic feature of SDC.
T19729 698183-698243 Sentence denotes In the current study, we sought to explore this possibility.
T98245 698244-698252 Sentence denotes Methods:
T62992 698253-698446 Sentence denotes Nucleic acid from 42 SDC archival specimens was evaluated for structural rearrangements by a targeted gene-fusion assay based on anchored multiplex PCR and next-generation sequencing (AMP-NGS).
T48167 698447-698576 Sentence denotes In addition, tumor genotyping, focused on a panel of clinically-relevant genes, was performed using a DNA-based AMP-NGS platform.
T77100 698577-698662 Sentence denotes HER2 gene amplification was assessed using fluorescence in situ hybridization (FISH).
T80992 698663-698671 Sentence denotes Results:
T35774 698672-698722 Sentence denotes Oncogenic fusions were detected in 8 (19%) tumors.
T7607 698723-698834 Sentence denotes Four SDC harbored RET gene rearrangements, including 3 NCOA4-RET fusions and one case with a TRIM33-RET fusion.
T53087 698835-698994 Sentence denotes Additional translocations included one case each of: CHCHD7-PLAG1 and FGFR2-KIAA1598 fusions, and previously unreported CCDC6-EGFR and ETV6-MET rearrangements.
T82127 698995-699291 Sentence denotes Out of these translocations, only two had been previously associated with salivary gland neoplasms: CHCHD7-PLAG1 fusions have been reported in pleomorphic adenomas of the salivary gland, and ETV6 rearrangements (with NTRK3, not MET) have been associated with mammary analogue secretory carcinoma.
T19633 699292-699566 Sentence denotes By contrast, similar rearrangements involving the RET oncogene have been described in papillary thyroid tumors and in lung adenocarcinoma, and FGFR2-KIAA1598 fusions have been reported in intrahepatic cholangiocarcinoma, but were never associated with salivary gland tumors.
T84393 699567-699698 Sentence denotes Targeted genotyping identified additional mutations in TP53 (63%), PIK3CA (17%), HRAS (11%), CDH1 (7%), PIK3R1 (7%), and BRAF (5%).
T34286 699699-699756 Sentence denotes HER2 gene amplification was observed in 31% of the cases.
T99123 699757-699879 Sentence denotes With the exception of mutations in TP53, oncogenic rearrangements in SDC did not overlap with other genetic abnormalities.
T33008 699880-699892 Sentence denotes Conclusions:
T13279 699893-699986 Sentence denotes Our study demonstrates, for the first time, that genetic rearrangements are prevalent in SDC.
T35287 699987-700132 Sentence denotes Molecular analysis detected clinically-relevant alterations in 86% of the cohort and implicated novel genetic drivers in the pathogenesis of SDC.
T27768 700133-700252 Sentence denotes Importantly, due to the availability of targeted inhibitors, EGFR, MET and FGFR fusions may have therapeutic potential.
T24426 700253-700383 Sentence denotes Our findings highlight the utility of using multiple testing modalities in the discovery of actionable genetic alterations in SDC.
T51774 700384-700511 Sentence denotes The high prevalence of potential targets identified in our study may also expand the therapeutic strategies for these patients.
T19933 700512-700525 Sentence denotes Introduction:
T38467 700526-700642 Sentence denotes Next-generation sequencing (NGS) has become a critical technology in guiding patient treatment in clinical oncology.
T45332 700643-700883 Sentence denotes As laboratories are increasingly challenged to reduce testing time while managing increased sample volumes, there is a high demand for targeted panels that offer rapid library preparation and the ability to highly multiplex patient samples.
T2127 700884-701026 Sentence denotes Here we evaluate the Pillar SLIMamp Lung and Colon Hot Spots Panel and compare the results to the Ion Torrent Cancer Hotspot Panel v2 (CHPv2).
T41813 701027-701035 Sentence denotes Methods:
T18494 701036-701164 Sentence denotes A total of 15 samples were included in this evaluation: six non-small cell lung carcinoma (NSCLC) and nine colon adenocarcinoma.
T22924 701165-701255 Sentence denotes All samples had DNA concentration higher than 50 ng/μL and high DNA quality (Q129bp/Q41bp:
T19947 701256-701323 Sentence denotes 0.8 to 0.92) according to the KAPA hgDNA Quantification and QC Kit.
T88943 701324-701402 Sentence denotes Library preparation was performed using 50 ng and 5 ng of gDNA of each sample.
T90622 701403-701526 Sentence denotes A total of 30 samples were normalized using Qubit, pooled and sequenced on the v3 cartridge on the Illumina's MiSeq system.
T21931 701527-701667 Sentence denotes For data analysis, FASTq files were uploaded to the Pillar, where sequence alignment, annotation, and variant classification were performed.
T53789 701668-701742 Sentence denotes Variant calls within genomic regions covered by both panels were compared.
T53391 701743-701751 Sentence denotes Results:
T92927 701752-701915 Sentence denotes For the 15 FFPE samples, there was a high degree of concordance between the SLIMamp Lung and Colon Hot Spots Panel and CHPv2 variant calls (90.0%, 27/30 variants).
T18983 701916-702056 Sentence denotes Three variants that were called by the Pillar panel were not called using the CHPv2 (two single base-pair deletions and one-point mutation).
T10478 702057-702211 Sentence denotes In addition, variant calls for the Pillar panel were highly reproducible using both 50 ng and 5 ng of input material (100.0% concordance, 30/30 variants).
T15707 702212-702368 Sentence denotes Allelic frequencies for the variants detected in the 50 ng and 5 ng replicates were also highly reproducible (average deviation of 1.5% between replicates).
T2259 702369-702381 Sentence denotes Conclusions:
T14532 702382-702579 Sentence denotes As NGS tumor profiling becomes an increasingly integral component in determining patient treatment, clinical laboratories will need to accommodate high sample volumes and variable specimen quality.
T18500 702580-702742 Sentence denotes The Pillar SLIMamp Lung and Colon Hot Spots sequencing panel allows laboratories to perform accurate, highly-multiplexed, targeted NGS using benchtop instruments.
T4778 702743-702883 Sentence denotes In addition, this panel demonstrates a high degree of reproducibility in variant calls using both average and extremely low FFPE DNA inputs.
T660 702884-702954 Sentence denotes jmd.amjpathol.org ■ The Journal of Molecular Diagnostics Introduction:
T86440 702955-703023 Sentence denotes The PI3K/AKT/mTOR pathway is frequently activated in ovarian cancer.
T86581 703024-703184 Sentence denotes Mutations and copy number alterations (CNAs) involving genes within this pathway may have clinical significance in predicting chemoresistance in ovarian cancer.
T24351 703185-703284 Sentence denotes Further, a number of inhibitors are currently in development that target PI3K pathway deregulation.
T26039 703285-703541 Sentence denotes To evaluate whether a single targeted next-generation sequencing-based (NGS) assay can fully characterize the spectrum of mutations and CNAs that may deregulate the PI3K pathway in ovarian cancer, we retrospectively analyzed a series of ovarian carcinomas.
T60689 703542-703550 Sentence denotes Methods:
T28446 703551-703709 Sentence denotes Clinical next-generation sequencing (NGS) was used to assess the coding regions of 131 genes and select introns in a series of 47 ovarian carcinoma specimens.
T80442 703710-703864 Sentence denotes For analysis, Varscan 2 and Genome Analysis Toolkit (GATK 1.2) were used to call single nucleotide variants (SNVs) and small indel variants, respectively.
T80359 703865-703913 Sentence denotes Pindel (0.2.4.d) was used to call larger indels.
T34130 703914-704004 Sentence denotes Genome-wide CNA prediction was extracted using the open source CNV detection tool, CNVkit.
T5812 704005-704104 Sentence denotes Fifteen non-tumor cases lacking CNAs were used as a reference, averaged, control for normalization.
T57660 704105-704207 Sentence denotes PI3K pathway gene CNAs of interest to this study included gains in PIK3CA and AKT1/2/3, and PTEN loss.
T39347 704208-704216 Sentence denotes Results:
T37407 704217-704531 Sentence denotes Of the 47 ovarian carcinomas, 41 were high grade serous carcinomas (HGSC), 3 were clear cell carcinomas (CC), 1 was a high grade carcinoma, mixed epithelial type with both HGSC and CC histologies, 1 case was a malignant mixed mullerian tumor (MMMT) of the ovary, and 1 case was a low grade serous carcinoma (LGSC).
T9613 704532-704611 Sentence denotes The genome-wide CNA landscape in all of the cases was suggestive of aneuploidy.
T29516 704612-704751 Sentence denotes Twenty-two of the 41 HGSC (53%) harbored PIK3CA copy number (CN) gains, 11 of which also harbored other co-existing PI3K deregulating CNAs:
T35957 704752-704853 Sentence denotes 7 harbored AKT1/2/3 gains, 1 harbored aPTEN loss, and 3 harbored both AKT1/2/3 gains and a PTEN loss.
T56913 704854-704954 Sentence denotes An activating PIK3CA mutation was identified in 1 of the HGSC cases that was negative for PI3K CNAs.
T643 704955-704996 Sentence denotes None of the 3 pure CC harbored PI3K CNAs.
T11317 704997-705093 Sentence denotes The mixed HGSC/CC case harbored an isolated AKT1 gain and the MMMT showed an isolated PTEN loss.
T83936 705094-705153 Sentence denotes Forty-two of 47 total cases (89%) harbored a TP53 mutation.
T87521 705154-705218 Sentence denotes Cases without a TP53 mutation included 2 CC, 1 LGSC, and 2 HGSC.
T83490 705219-705330 Sentence denotes None of these 5 cases harbored any PI3K pathway CNAs; however, one (CC) harbored an activating PIK3CA mutation.
T66782 705331-705343 Sentence denotes Conclusions:
T7161 705344-705490 Sentence denotes Ovarian cancers with genomic alterations affecting the PI3K pathway, including CNAs, can be identified from a single clinical, targeted NGS assay.
T19565 705491-705637 Sentence denotes Such assays increase diagnostic efficiency and are particularly useful in cases where limited material is available for multiple diagnostic tests.
T91535 705638-705651 Sentence denotes Introduction:
T15799 705652-705781 Sentence denotes Cancer is a complex disease caused by the accumulation of genomic alterations including mutations, CNVs, and gene rearrangements.
T61108 705782-705903 Sentence denotes Integrated approaches are required to generate comprehensive genomic profiles to meet the needs of precision cancer care.
T3459 705904-706013 Sentence denotes This is extremely important in childhood cancers due to the higher long-term survival rate of these patients.
T22797 706014-706201 Sentence denotes Targeted-panel NGS enables testing of many cancer-related genes and different genomic alterations simultaneously and has become the method of choice for clinical cancer genomic profiling.
T80814 706202-706210 Sentence denotes Methods:
T45950 706211-706462 Sentence denotes Four large custom designed DNA-or RNA-based NGS panels (HeredPanel, HemePanel, SolidPanel and FusionPanel) were developed to detect different types of mutations (SNV, indel, CNV, LOH, and gene fusion) associated with hereditary and/or somatic cancers.
T24064 706463-706606 Sentence denotes These panels interrogate 402 genes, 5506 exons, 232 intronic regions, and 586 known fusions and can potentially detect many more novel fusions.
T894 706607-706615 Sentence denotes Results:
T37834 706616-706748 Sentence denotes The performance of the three DNA panels were evaluated using six cancer cell lines, one HapMap cell line, and three normal controls.
T58344 706749-706842 Sentence denotes The results showed 100% sensitivity, specificity, and reproducibility for mutation detection.
T85212 706843-706922 Sentence denotes The 1% detection limit was established using serially diluted tumor cell lines.
T1676 706923-707041 Sentence denotes The average sequencing depth for the targeted regions was >650X for HerePanel and >1600X for HemePanel and SolidPanel.
T12533 707042-707132 Sentence denotes Thirty-three RNA samples were tested blindly to assess the performance of the FusionPanel.
T12862 707133-707212 Sentence denotes The panel correctly identified all 32 (100%) known fusions and two new fusions.
T54383 707213-707288 Sentence denotes The detection limit of FusionPanel is 1% (1 tumor cell in 99 normal cells).
T37580 707289-707508 Sentence denotes These panels were further validated using 28 discarded clinical samples including blood; bone marrow; and fresh, frozen, or FFPE tissues and showed concordant results with those of Sanger, FISH, microarray, and RT-qPCR.
T65010 707509-707705 Sentence denotes These panels have been offered as clinical services at the Children's Hospital of Philadelphia and have provided genomic data important for clinical decision-making in over 80% of patients tested.
T25130 707706-707718 Sentence denotes Conclusions:
T44999 707719-707862 Sentence denotes We reported the development, validation, and assessment of the performance and clinical utility of four large NGS panels for childhood cancers.
T39831 707863-708115 Sentence denotes Our experience demonstrated that these NGS-based panels provide comprehensive genomic information of pediatric cancers, which plays a significant role in clinical cancer management, and can be incorporated as part of routine clinical oncology practice.
T65790 708116-708120 Sentence denotes J.L.
T96846 708121-708133 Sentence denotes Dillon, J.D.
T86767 708134-708145 Sentence denotes Marotti, H.
T56250 708146-708156 Sentence denotes Jung, J.A.
T78373 708157-708171 Sentence denotes Lefferts, L.J.
T44393 708172-708225 Sentence denotes Tafe Dartmouth-Hitchcock Medical Center, Lebanon, NH.
T32386 708226-708239 Sentence denotes Introduction:
T41839 708240-708446 Sentence denotes Using the updated 2013 ASCO/CAP HER2 testing and scoring guidelines, we and other institutions identified an increased detection in the number of breast cancer (BC) cases equivocal for HER2 (ERBB2) by FISH.
T73212 708447-708590 Sentence denotes By definition, these cases have a HER2 copy number of 4 to less than 6 copies per cell (equivocal range) with a HER2:CEP17 ratio less than 2.0.
T46235 708591-708694 Sentence denotes Some laboratories utilize alternative chromosome 17 gene probes in instances of HER2 equivocal by FISH.
T87247 708695-708845 Sentence denotes Here, we evaluate the utility of the OncoScan SNP array to evaluate chromosome copy number (CN) as an alternative assessment inHER2 FISH equivocal BC.
T80697 708846-708854 Sentence denotes Methods:
T18034 708855-708994 Sentence denotes Our laboratory performs primary HER2 FISH testing on all breast cancer cases with reflex to immunohistochemistry (IHC) for equivocal cases.
T22464 708995-709108 Sentence denotes Ten cases of BC that were HER2 equivocal by FISH (PathVysion) were included; all cases also had HER2 IHC results.
T11199 709109-709211 Sentence denotes DNA was extracted from 8 unstained, 4 micron FFPE slides with the QIAamp DNA FFPE Tissue Kit (Qiagen).
T32473 709212-709387 Sentence denotes The samples were processed using the Affymetrix OncoScan FFPE Array according to the manufacturer's protocol and analyzed using Nexus Express and Affymetrix software packages.
T52705 709388-709396 Sentence denotes Results:
T23095 709397-709529 Sentence denotes For all 10 cases, the HER2:CEP17 FISH ratio was <2.0 and the average number of HER2 signals per cell by FISH ranged from 4.1 to 5.4.
T4973 709530-709625 Sentence denotes The SNP array identified aneuploidy of the HER2 locus in all cases ranging from a CN of 3 to 6.
T59307 709626-709813 Sentence denotes In addition, multiple additional chromosomal CN abnormalities were seen in a majority of tumors including amplifications of CCND1(2 cases), FGFR1(4 cases), GNAS (4 cases) or MYC (1 case).
T24875 709814-709826 Sentence denotes Conclusions:
T57067 709827-709923 Sentence denotes Low CN gains at the HER2 locus were able to explain our equivocal FISH results in 9 of 10 cases.
T90345 709924-709970 Sentence denotes In one case (EQ8) a HER2 CN of 3 was detected.
T66065 709971-710117 Sentence denotes However, the SNP array provides a summary result of all cells tested so this sample may consist of a heterogenous population with an average CN 3.
T16889 710118-710319 Sentence denotes Our findings support the utility of SNP array analysis to further evaluate the CN state of BC that is HER2 equivocal by FISH and in the process, detect additional potentially meaningful CN alterations.
T47269 710320-710464 Sentence denotes Case The proto-oncogene MET is known to harbor donor/acceptor splice site mutations that can give rise to transcripts lacking exon 14 (METex14).
T22260 710465-710715 Sentence denotes Although this "skipping" event occurs at frequencies comparable to ALK rearrangements in lung adenocarcinomas, current METex14 detection technologies are underdeveloped and burdened by a combination of high costs, low resolution or complex workflows.
T16637 710716-710911 Sentence denotes Herein we report the development of a novel, accurate, and accessible 2-channel droplet digital PCR (ddPCR) assay that can quantify the fraction of skipped METex14 transcripts in patient samples.
T76744 710912-711053 Sentence denotes Successful identification ofthis event correlates with MET inhibitor responsiveness, potentially benefiting treatment decisions and outcomes.
T50820 711054-711062 Sentence denotes Methods:
T67410 711063-711265 Sentence denotes A METex14 RT-ddPCR assay was developed using optimized reverse transcription and PCR reagents, with an optional pre-amplification step incorporated for samples with low nucleic acid quantity or quality.
T56471 711266-711447 Sentence denotes Gene-specific primers and junction-spanning hydrolysis probes were designed for MET exons 13/14 or 14/15 (wildtype (WT), HEX labeled) and 13/15 (METex14, FAM labeled), respectively.
T35156 711448-711538 Sentence denotes Droplets were generated and run on the QX200 ddPCR platform (Bio-Rad) with a 4-bin output.
T33463 711539-711745 Sentence denotes Samples consisted of cell lines and a set of 125 adenocarcinomas (MD Anderson), which were additionally sequenced for RNA fusions and METex14 skipping using the QuantideX NGS RNA Lung Cancer Kit (Asuragen).
T71696 711746-712047 Sentence denotes Results: MET exon 14 skipping status was determined with 100% accuracy in representative cell lines from as little as 2 ng RNA inputs.Known copy number titrations demonstrated analytical sensitivity down to 1% METex14 skipped/WT transcripts, exceeding levels typically achieved by NGSbased approaches.
T33488 712048-712247 Sentence denotes METex14 positive and negative samples were correctly identified from previously characterized FFPE specimens, including two FFPE samples that showed 27% and 95% METex14 skipping levels, respectively.
T45271 712248-712349 Sentence denotes Compatibility of the RT-ddPCR assay for both RNA and total nucleic acid inputs was also demonstrated.
T75832 712350-712362 Sentence denotes Conclusions:
T23868 712363-712587 Sentence denotes The prevalence of METex14 skipping events in NSCLC, combined with reports of patient responses to MET inhibitors such as crizotinib and cabozantinib, makes the lesion a high-priority diagnostic target for precision medicine.
T84042 712588-712718 Sentence denotes The described assay provides absolute copy number jmd.amjpathol.org ■ The Journal of Molecular Diagnostics and other solid tumors.
T40950 712719-712875 Sentence denotes Quantification of RNA variants can complement DNA analyses to capture the dynamic molecular state of tumor cell populations and inform treatment strategies.
T16469 712876-713085 Sentence denotes We describe a novel targeted next-generation sequencing (NGS) technology that offers both modularity and systems-level integration to achieve accurate RNA and DNA profiling from challenging clinical specimens.
T12823 713086-713094 Sentence denotes Methods:
T32527 713095-713279 Sentence denotes Formalin-fixed, paraffin-embedded (FFPE) or fine-needle aspirate (FNA) specimens from three cohorts, including the BATTLE-2 clinical trial, were collected at MD Anderson Cancer Center.
T71533 713280-713422 Sentence denotes Pre-analytical QC was performed using novel qPCR assays that quantify distinct populations of amplifiable RNA and DNA from total nucleic acid.
T79288 713423-713595 Sentence denotes RNA or DNA was enriched by PCR using the QuantideX NGS RNA Lung Cancer Kit, or a prototype DNA panel with common non-primer-based reagents, and sequenced on a MiSeq System.
T99889 713596-713819 Sentence denotes Data analysis was achieved using QuantideX NGS Reporter, an analysis suite that directly incorporates pre-analytical QC data into the variant calling algorithm to improve the identification of DNA mutations and RNA fusions.
T66194 713820-713828 Sentence denotes Results:
T4818 713829-714028 Sentence denotes We evaluated the QuantideX NGS RNA Lung Cancer Kit, which targets 110 gene fusions, MET exon 14 skipping, 23 mRNA expression markers, and 3'/5' imbalance markers for ALK, RET, ROS1, NTRK1 and PGDFRA.
T45633 714029-714199 Sentence denotes A total of 224 NSCLC FFPE specimens were characterized to reveal multiple fusions involving ALK, RET, ROS1 along with evidence of 3' expression imbalances in ALK and RET.
T16603 714200-714317 Sentence denotes Cases of partially and fully skipped MET exon 14 were also identified and quantitatively confirmed by digital RT-PCR.
T43397 714318-714427 Sentence denotes A majority of the specimens were also evaluated with a DNA panel that targets 20 genes associated with NSCLC.
T43618 714428-714634 Sentence denotes Mutations were observed in 77% of samples with profiles that were qualitatively consistent with previous reports but with quantitative molecular features that reflected cohort-specific clinical annotations.
T99442 714635-714780 Sentence denotes In addition, the DNA panel identified mutations in blinded BATTLE-2 clinical trial FNA specimens with 95% agreement to an independent NGS method.
T6485 714781-714793 Sentence denotes Conclusions:
T75413 714794-715002 Sentence denotes We describe an efficient and accurate workflow that reports prognostic and theranostic markers in lung cancer and leverages the integration of common wet and dry bench components for targeted RNA-and DNA-Seq.
T71383 715003-715103 Sentence denotes The flexibility of these components means that new NGS panels can be rapidly developed and verified.
T50066 715104-715326 Sentence denotes We also show that a "sampleaware " approach can unify the analysis of SNVs, indels, RNA fusions, and aberrant transcripts within a single NGS run to support comprehensive and streamlined characterization of tumor biopsies.
T4593 715327-715452 Sentence denotes Traditionally, gene fusions are detected by fluorescence in situ hybridization (FISH) and reverse transcription PCR (RT-PCR).
T29379 715453-715657 Sentence denotes Both methods require prior knowledge of the fusion partners and specific probes or primer sets for different fusions, have no or limited multiplex capacity, and lack the ability to identify novel fusions.
T93132 715658-715881 Sentence denotes We evaluated the performance of a next-generation sequencing (NGS)-based assay utilizing Anchored Multiplex PCR technology for detection of gene fusions from formalin-fixed, paraffin-embedded (FFPE) lung and thyroid tumors.
T3752 715882-715890 Sentence denotes Methods:
T71103 715891-716034 Sentence denotes Total RNA was extracted from 13 cases of microdissected FFPE tissues including adenocarcinoma of the lung (9), PTC (2) and FC (2) Introduction:
T57997 716035-716289 Sentence denotes There is increased demand for clinical genome-wide copy number assessment of pediatric solid tumors, given the established clinical significance of certain chromosomal aberrations and its use for prognostic classification and treatment of these patients.
T62029 716290-716518 Sentence denotes Neuroblastomas characterized by whole chromosome changes tend to act favorably, whereas tumors with MYCN gene amplification and/or segmental chromosomal aberrations, including loss of heterozygosity (LOH), exhibit poor outcomes.
T56708 716519-716730 Sentence denotes Given the clinical relevance, our aim was to verify the accuracy of OncoScan FFPE SNP Array (Affymetrix) in assessing genome-wide copy number changes and LOH in tumor samples, for use in the clinical laboratory.
T50027 716731-716739 Sentence denotes Methods:
T68567 716740-716855 Sentence denotes Thirty-six neuroblastic tumors diagnosed in our pediatric hospital were analyzed using the OncoScan FFPE SNP Array.
T74539 716856-716946 Sentence denotes Tumor genomic DNA was isolated and arrays hybridized according to manufacturer's protocol.
T763 716947-717068 Sentence denotes Data was analyzed using the OncoScan Console and viewed using Chromosome Analysis Suite (ChAS) and Nexus Copy Number 7.5.
T3380 717069-717198 Sentence denotes Copy number results determined by SNP array were compared to ploidy results obtained by karyotype analysis and/or flow cytometry.
T42488 717199-717370 Sentence denotes Additionally, chromosome 2, 12, 22, X and Y aneuploidy data by FISH were used as a surrogate ploidy marker in cases where cytogenetics and flow cytometry were unavailable.
T39581 717371-717480 Sentence denotes MYCN and ALK FISH were performed for gene copy number assessment and 1p36 and 11q23 LOH were assessed by PCR.
T36598 717481-717489 Sentence denotes Results:
T86473 717490-717602 Sentence denotes The results for MYCN and ALK gene amplification both showed 100% concordance (33/33 and 8/8 cases respectively).
T65869 717603-717694 Sentence denotes The 1p36 and 11q23 LOH also showed 100% (3/3) and 67% (2/3) concordance, in selected cases.
T36297 717695-717798 Sentence denotes Karyotype and FISH for chromosome aneuploidy showed the highest concordance for assessing tumor ploidy.
T91121 717799-717916 Sentence denotes After initial analysis, 2 cases showed discordance with karyotype, flow cytometry or FISH results (2/36 cases or 6%).
T80985 717917-718060 Sentence denotes Both specimens were confirmed as near tetraploid by FISH and/or flow cytometry and required the OncoScan baseline to be adjusted appropriately.
T88842 718061-718073 Sentence denotes Conclusions:
T26269 718074-718242 Sentence denotes Our results demonstrate that the OncoScan SNP array is useful in the clinical laboratory for accurate assessment of gene amplification, LOH and genome-wide copy number.
T82650 718243-718413 Sentence denotes In 2 cases, a second method was necessary to correctly assign tumor ploidy baseline, despite complex algorithms used by the OncoScan software to assign copy number calls.
T20405 718414-718442 Sentence denotes The complex chromosomal copy
T72607 718443-718608 Sentence denotes The Journal of Molecular Diagnostics ■ jmd.amjpathol.org number changes present in tumors can challenge the ability of the software to correctly assign ploidy state.
T55821 718609-718823 Sentence denotes Therefore, we conclude that a second independent method may be necessary in complex tumor cases to correctly assign ploidy that truly reflects tumor biology and that may be necessary for correct patient management.
T48320 718824-718837 Sentence denotes Introduction:
T59354 718838-718882 Sentence denotes Genomic medicine has been advancing rapidly.
T54153 718883-719039 Sentence denotes The complexity of delivering precision medicine to oncology patients across a university health system suggested the need for a Molecular Tumor Board (MTB).
T87889 719040-719169 Sentence denotes Unlike MTBs reported elsewhere, we decided to discuss the cases before molecular tumor profiling for appropriateness of the test.
T81443 719170-719255 Sentence denotes When the results were available, further treatment options were discussed in the MTB.
T75978 719256-719337 Sentence denotes The objective of this study was to analyze our progress over the first two years.
T55578 719338-719346 Sentence denotes Methods:
T44876 719347-719496 Sentence denotes Patients were reviewed in the MTB for appropriateness prior to the submission for comprehensive next-generation sequencing (NGS) cancer gene testing.
T75341 719497-719950 Sentence denotes A set of criteria were in place to determine appropriateness, in brief, 1) the patient has treatment resistant or recurrent measurable tumor, which has failed at least one standard therapy or no standard therapy options are available, 2) the patient has a good performance status and 3) the cases with a driver gene mutation that can be found by routine single gene testing such as those in lung adenocarcinoma, melanoma and colon cancer, were excluded.
T44924 719951-720051 Sentence denotes In this study, we analyzed the cases discussed and sent for testing with their results and outcomes.
T52777 720052-720060 Sentence denotes Results:
T4483 720061-720160 Sentence denotes 138 cases were discussed at the MTB, 92 of them were approved for testing and 46 were not approved.
T47007 720161-720254 Sentence denotes The most common reason for rejection was the availability of a standard therapy option (n=8).
T80996 720255-720305 Sentence denotes 22 cases did not have adequate tissue for testing.
T3300 720306-720407 Sentence denotes Most common tumor types tested were gynecologic (43%), central nervous system (15%) and breast (11%).
T25589 720408-720620 Sentence denotes Among the available results (n=88) (4 cases did not have enough DNA), 29 cases (31.5%) had driver gene mutations associated with an active targeted therapeutic agent, includingBRAF, PIK3CA, IDH1, KRAS, and BRCA1.
T443 720621-720681 Sentence denotes An additional 40 cases (43.5%) had cancer-related mutations.
T43474 720682-720751 Sentence denotes 19 cases (20.7%) did not have any clinically significant alterations.
T4541 720752-720863 Sentence denotes Alterations in over 40 different genes were identified, TP53 alteration being the highest in incidence (54.5%).
T62396 720864-720994 Sentence denotes One third of the patients with an actionable mutation benefited from the test with a good or partial response to targeted therapy.
T79772 720995-721006 Sentence denotes Conclusion:
T14333 721007-721096 Sentence denotes The MTB has provided the opportunity to offer precision medicine for our cancer patients.
T5424 721097-721226 Sentence denotes The application of precision medicine and molecular genetic testing for cancer patients remains a continuous educational process.
T85715 721227-721462 Sentence denotes Since our MTB patient population excluded lung adenocarcinoma, melanoma and colon cancer, the fact that the rate of finding actionable mutations is comparable to other studies suggests the case selection through the MTB is appropriate.
T61705 721463-721616 Sentence denotes A well-designed MTB will evolve along with the technology to ensure that patients receive the best possible treatment without unnecessary costs or risks.
T61213 721617-721630 Sentence denotes Introduction:
T8828 721631-721809 Sentence denotes Mutation detection in the EGFR, KRAS, NRAS and BRAF genes is standard of care for targeted therapy of metastatic non-small cell lung cancers (NSCLC) and colorectal cancers (CRC).
T40251 721810-721996 Sentence denotes Mutations within these genes are usually mutually exclusive since they are in the same signal transduction pathway, though a low incidence of such coexisting mutations has been reported.
T57717 721997-722117 Sentence denotes In the clinical diagnostic setting, encounter of unexpected coexisting mutations raises a concern for quality assurance.
T43945 722118-722126 Sentence denotes Methods:
T26104 722127-722298 Sentence denotes We studied 510 NSCLC and 304 CRC consecutive cases submitted to a clinical molecular diagnostic laboratory for mutation profiling by a next-generation sequencing platform.
T44300 722299-722307 Sentence denotes Results:
T39791 722308-722525 Sentence denotes We identified coexisting EGFR p.G917A mutation and KRAS p.G12C mutations in one NSCLC, co-existing activating KRAS and NRAS mutations in one NSCLC and 3 CRC, and two activating KRAS mutations in one NSCLC and one CRC.
T96662 722526-722745 Sentence denotes Although kinase-deficient BRAF mutation can be seen in specimens with an activating RAS mutation (Zheng, et al., BMC Cancer 2015) , no coexisting kinase-activated BRAF p.V600E and activating RAS mutations were observed.
T74051 722746-722967 Sentence denotes Retrospective tissue identification testing and repeated NGS assay of multiple subareas confirmed that these coexisting mutations are valid and may occur in a single tumor population or separately in tumor subpopulations.
T6842 722968-723038 Sentence denotes One mistaken case of dual KRAS and NRAS mutations was also identified.
T12162 723039-723051 Sentence denotes Conclusions:
T47230 723052-723216 Sentence denotes This retrospective study for quality assessment confirmed true coexisting mutations in the EGFR, KRAS and NRAS genes, which may also be caused by laboratory errors.
T54816 723217-723380 Sentence denotes A standard operating protocol is proposed to confirm unexpected coexisting mutation within the same signal transduction pathway in the clinical diagnostic setting.
T87936 723381-723394 Sentence denotes Analysis R.T.
T11332 723395-723405 Sentence denotes Taylor, J.
T54482 723406-723414 Sentence denotes Hill, A.
T21032 723415-723426 Sentence denotes Fuentes, C.
T20167 723427-723437 Sentence denotes Heiser, Z.
T81083 723438-723454 Sentence denotes Abdulateef, J.R.
T37716 723455-723495 Sentence denotes Hill Spot On Sciences, Inc., Austin, TX.
T89297 723496-723509 Sentence denotes Introduction:
T91366 723510-723624 Sentence denotes Robust preservation of tissue biospecimens for molecular analysis is essential for personalized disease diagnosis.
T94233 723625-723834 Sentence denotes Current methods for the preservation of tissue, such as formalin-fixed, paraffin-embedded (FFPE) or flash freezing in liquid nitrogen, are labor intensive and present challenges for collection and maintenance.
T81438 723835-723995 Sentence denotes Additionally, analyte integrity can become compromised due to cross-linking and freeze/thaw degradation which poses challenges to downstream molecular analysis.
T992 723996-724069 Sentence denotes We demonstrate an alternative method that is more streamlined and robust.
T37813 724070-724190 Sentence denotes Leveraging proven dried blood spot technology to preserve tissue homogenate by drying on HemaSpot-HF collection devices.
T25324 724191-724367 Sentence denotes We seek to identify the optimal homogenization and extraction buffers as well as demonstrate suitable recovery for common molecular analytes including proteins and nucleotides.
T37774 724368-724480 Sentence denotes The simple procedure provides long-term analyte stability and recovery with minimal infrastructure requirements.
T38255 724481-724489 Sentence denotes Methods:
T30645 724490-724625 Sentence denotes Small samples (50 mg) of Wistar-Hannover Rat tissue were added to a Dounce homogenizer in the presence of 0.7 mL of stabilizing buffer.
T49450 724626-724739 Sentence denotes 88 uL of homogenate was applied to a HemaSpot-HF sample storage collection device and stored at room temperature.
T96096 724740-724795 Sentence denotes Aliquots of homogenate were frozen and stored at -20°C.
T58998 724796-725015 Sentence denotes Dried and wet homogenates were extracted using common methods and representative protein (BCA protein assay kit), Beta-galactosidase activity (LC-MS/MS), DNA (PicoGreen assay), and RNA (Taqman) markers were quantitated.
T67259 725016-725024 Sentence denotes Results:
T25100 725025-725168 Sentence denotes High recovery (>85%) of total proteins from dried rat liver, lung, spleen, and kidney homogenates were observed compared to frozen homogenates.
T76032 725169-725308 Sentence denotes Beta-galactosidase activities from dried liver homogenates were comparable (>90% activity) to corresponding wet, frozen homogenate samples.
T54888 725309-725470 Sentence denotes The presence of Triton X-100 in the homogenization buffer is shown to inhibit DNA recovery while protease inhibitors are shown to improve total protein recovery.
T41589 725471-725616 Sentence denotes PicoGreen assay results demonstrate high recovery (75%) of double stranded DNA extracted from dried liver homogenates compared to frozen samples.
T58497 725617-725749 Sentence denotes Levels of mRNA (AUF1, LCN2, b-actin, p53 and TNF-a) were equivalent or slightly higher for dried samples as compared to wet samples.
T2282 725750-725762 Sentence denotes Conclusions:
T27578 725763-725893 Sentence denotes These results demonstrate feasibility for solid tissue preservation as a dried homogenate on filter paper at ambient temperatures.
T87673 725894-726019 Sentence denotes Subsequent quantitation of protein and nucleotide biomarkers without significant degradation or loss of activity is observed.
T4826 726020-726142 Sentence denotes This method offers an efficient and cost-effective alternative for tissue biospecimen preservation and molecular analysis.
T52464 726143-726314 Sentence denotes They are almost exclusively seen in pediatric high grade midline tumors with the most frequent mutation being the K27M mutation whereas the G34R mutation is less frequent.
T86067 726315-726369 Sentence denotes They have been suggested as sensitive markers of HGGs.
T69524 726370-726594 Sentence denotes We sought to i) determine the frequency of these mutations in our cohort of pediatric gliomas and to ii) explore the utility of a single pyrosequencing dispensation protocol for the detection of both K27M and G34R mutations.
T45171 726595-726603 Sentence denotes Methods:
T29900 726604-726945 Sentence denotes We analyzed fifty two (52) pediatric brain tumors including 29 pilocytic astrocytomas (PA), grade I, 3 anaplastic PAs, grade III, 3 diffuse astrocytomas (DA) grade II, anaplastic DA, grade III, 10 glioblastoma, grade IV, 2 dysembryoplastic neuroepithelial tumor (DNET), grade I, 1 ganglioglioma (GG), grade II and 1 anaplastic GG, grade III.
T67462 726946-727056 Sentence denotes 27 adult tumors including 23 glioblastomas, 3 anaplastic DAs, grade III and 1 DA, grade II were also analyzed.
T12902 727057-727065 Sentence denotes Results:
T52207 727066-727252 Sentence denotes Using appropriate and known controls as reference, it was possible to use a modified pyrosequencing protocol that allowed for the detection of the respective mutations in a single assay.
T35523 727253-727323 Sentence denotes All of the adult tumors were negative for the K27M and G34R mutations.
T63452 727324-727439 Sentence denotes A total of 5 pediatric tumors were positive for mutation including 2/10 glioblastoma, 1/3 anaplastic PA and 2/3 DA.
T65911 727440-727480 Sentence denotes The 2 DA and 1 GBM were thalamic masses.
T32126 727481-727534 Sentence denotes The other positive GBM was a right frontal lobe mass.
T25173 727535-727720 Sentence denotes The 2 positive thalamic masses that were initially diagnosed as DA, grade II were worrisome for higher grade due to elevated proliferation index and may have been inadequately biopsied.
T68991 727721-727789 Sentence denotes We demonstrated H3F3A mutations in pediatric high grade tumors only.
T21595 727790-727930 Sentence denotes This finding is consistent with a correlation between the presence of an H3F3A mutation and high grade histology in the pediatric age group.
T20714 727931-728111 Sentence denotes We recommend analysis of histologically low grade appearing midline diffuse astrocytomas for H3F3A mutations as biopsies may inadequately represent the true biology of such tumors.
T43066 728112-728308 Sentence denotes We also confirm the utility of a previously described single dispensation pyrosequencing protocol that allows for the simultaneous identification of both K27M and G34R mutations in a single assay.
T91124 728309-728353 Sentence denotes RNA-seq Using Unique in vitro Transcripts K.
T4715 728354-728366 Sentence denotes Lawrence, R.
T81012 728367-728380 Sentence denotes Leonard, D.R.
T49268 728381-728389 Sentence denotes Hout, S.
T82012 728390-728401 Sentence denotes Qadri, B.L.
T98564 728402-728449 Sentence denotes Schweitzer Insight Genetics Inc, Nashville, TN.
T61661 728450-728463 Sentence denotes Introduction:
T9463 728464-728650 Sentence denotes The potential for technical errors such as cross-contamination and/or misidentification of individual samples can occur while performing high-throughput next-generation RNA-seq analysis.
T35971 728651-728796 Sentence denotes To mitigate this risk, a method was developed to add individually bar-coded in vitro transcripts (IVTs) to samples prior to library construction.
T90923 728797-728907 Sentence denotes Therefore, any such errors can be identified after processing, thereby improving the quality of reported data.
T99837 728908-729064 Sentence denotes This IVT set may be incorporated into highthroughput RNA-seq workflows to serve as a quality control check on sample identification and cross-contamination.
T2183 729065-729293 Sentence denotes The IVTs are compatible with multiple library preparation methods including both positive and negative enrichment strategies as well as methods used for preparation of formalin-fixed paraffin embedded (FFPE)derived nucleic acid.
T99597 729294-729302 Sentence denotes Methods:
T52578 729303-729411 Sentence denotes Bar-coded IVT RNA was spiked into 1 microgram of FFPE RNA extracted from archival surgical resection tissue.
T18880 729412-729551 Sentence denotes To establish a proper IVT input, various quantities (2x10 11 to 2x10 7 copies) were spiked prior to mRNA enrichment and library generation.
T7987 729552-729663 Sentence denotes The RNA was processed using ScriptSeq Complete Gold H/M/R Low input kit and ScriptSeq v2 library prep reagents.
T58339 729664-729821 Sentence denotes The standard protocol for degraded samples was utilized, which involves no RNA fragmentation prior to cDNA synthesis, and 15 cycles of library amplification.
T85967 729822-729987 Sentence denotes To quantify the IVT present in the NGS data, the sample fastq files were aligned to the IVT sequence to calculate the number of reads that align to the IVT sequence.
T76887 729988-730145 Sentence denotes To quantify the relative IVT transcript abundance in the IVT libraries, three Taqmanbased qPCR assays were designed to target the unique sequence in the IVT.
T55618 730146-730380 Sentence denotes Results: FFPE RNA-seq libraries were successfully constructed without noticeable interference from the addition of IVT as evidenced by the fragment distribution of IVT-spiked samples being indistinguishable from non-spiked replicates.
T68101 730381-730639 Sentence denotes We observed a consistent relationship between IVT spike amounts and relative transcript quantity detected by qPCR, and between IVT spike amounts and the number of IVT-aligned reads per total reads in both FFPE RNA-seq libraries and poly-A enriched libraries.
T10705 730640-730652 Sentence denotes Conclusions:
T95621 730653-730966 Sentence denotes The feasibility of an IVT spike as a quality control measure in FFPE RNA-seq protocols was demonstrated by the lack of interference in standard RNA-seq protocols, the successful detection and qualification with post-sequencing bioinformatics tools, and the correlation of IVTaligned read counts with qPCR results.
T74101 730967-731077 Sentence denotes The resulting IVT design could be utilized as a novel QC to monitor technical errors in RNA-seq based studies.
T4569 731078-731080 Sentence denotes A.
T83287 731081-731089 Sentence denotes Iyer, R.
T71698 731090-731100 Sentence denotes Haigis, M.
T78685 731101-731111 Sentence denotes Porter, D.
T26550 731112-731119 Sentence denotes Lee, S.
T27248 731120-731129 Sentence denotes Smith, S.
T86101 731130-731141 Sentence denotes Dhillon, T.
T48011 731142-731150 Sentence denotes Dunn, S.
T57694 731151-731162 Sentence denotes Murillo, N.
T43432 731163-731198 Sentence denotes Udar Illumina, Inc., San Diego, CA.
T5406 731199-731212 Sentence denotes Introduction:
T22594 731213-731356 Sentence denotes Next-generation sequencing (NGS) assays allow for the simultaneous detection of numerous somatic mutations in multiple samples in a single run.
T48226 731357-731602 Sentence denotes Ensuring that an assay is performing correctly and optimally is of critical importance in any setting. accurate identification is therefore of utmost importance with regard to therapeutic decision making in non-small cell lung carcinoma (NSCLC).
T75937 731603-731755 Sentence denotes Here we assess the differential abilities of a variety of clinical assays, to identify and appropriately annotate these complex variants in lung cancer.
T45477 731756-731764 Sentence denotes Methods:
T59341 731765-731948 Sentence denotes A systematic review of clinical lung cancer cases, which were tested by next-generation sequencing using AmpliSeq HotSpot Cancer Panel v2, was undertaken at two major medical centers.
T64307 731949-732023 Sentence denotes Samples that demonstrated complex indels in exon 19 were further examined.
T30471 732024-732150 Sentence denotes Sequence alignments were visualized in Integrated Genomic Viewer (IGV) to evaluate the presence of possible complex variation.
T32681 732151-732366 Sentence denotes Cases with identifiable complex indels were then subjected to a multi-platform review including alternative pipelines, Sanger sequencing, PCR-CE, and an FDA approved assay for the detection of EGFR exon 19 variants.
T52741 732367-732375 Sentence denotes Results:
T6787 732376-732543 Sentence denotes Of 531 NSCLC tested, 11% (n=57) demonstrated EGFR exon 19 deletions and 22% of these (n=13) were found on alignment visualization to actually represent complex indels.
T84741 732544-732676 Sentence denotes PCR followed by fragment analysis using capillary electrophoresis, confirmed variable product sizes in EGFR exon 19 in all 13 cases.
T54329 732677-732786 Sentence denotes Current pipeline with validated settings, failed to identify any feature whatsoever in 15% of complex indels.
T80748 732787-732881 Sentence denotes The FDA approved Therascreen assay had a clinical sensitivity of 78% for these complex indels.
T12967 732882-733184 Sentence denotes Pipelines optimized for complex indel detection did identify the presence of some form of variant in all samples, however, the left alignment normalization strategy they employed for annotation typically produced several separate variant calls as a result of microhomology within the inserted sequence.
T29204 733185-733300 Sentence denotes The best performing pipeline produced an HGVS acceptable indel annotation for only 38.5% of these complex variants.
T42219 733301-733313 Sentence denotes Conclusions:
T13394 733314-733398 Sentence denotes Complex indels within exon 19 of EGFR may be more common than previously documented.
T63435 733399-733573 Sentence denotes Several factors affect proper identification and annotation of these variants and even a FDA approved assay demonstrates limitations in identification of all possible indels.
T16385 733574-733730 Sentence denotes Localized microhomology of the inserted sequence may lead many NGS pipelines to treat complex mutations as a series of separate events (deletions and SNPs).
T12527 733731-733935 Sentence denotes Recognition and visualization of entirely phased variants should prompt careful review to assess the need for a more accurate annotation of these clinically actionable complex indels within the EGFR gene.
T89347 733936-733948 Sentence denotes Conclusions:
T78277 733949-734059 Sentence denotes Evaluation of MLH1 promoter methylation by MethyLight is highly sensitive, specific, reproducible, and robust.
T80307 734060-734227 Sentence denotes Our data support that MLH1 promoter methylation testing may reduce the number of unnecessary cases sent for germline sequencing given the appropriate clinical context.
T224 734228-734230 Sentence denotes Z.
T47689 734231-734247 Sentence denotes Shajani-Yi, L.N.
T32196 734248-734260 Sentence denotes Nguyen, W.F.
T60428 734261-734273 Sentence denotes Hickey, S.J.
T19473 734274-734288 Sentence denotes Deharvengt, F.
T64394 734289-734313 Sentence denotes Blumental de Abreu, J.D.
T71795 734314-734328 Sentence denotes Peterson, T.L.
T39526 734329-734344 Sentence denotes Gallagher, A.J.
T86728 734345-734358 Sentence denotes Erskine, J.A.
T14553 734359-734371 Sentence denotes Lefferts, G.
T37273 734372-734430 Sentence denotes Tsongalis Dartmouth-Hitchcock Medical Center, Lebanon, NH.
T7870 734431-734444 Sentence denotes Introduction:
T48425 734445-734596 Sentence denotes Gliomas are composed of a heterogeneous group of neoplasms with respect to malignancy grade, histological subtype, invasiveness and treatment response.
T62251 734597-734889 Sentence denotes Histology is the gold standard for grading and typing of gliomas; however, molecular biomarkers such as MGMT hypermethylation, co-deletion of the chromosome arms 1p and 19q (1p/19q co-deletion) and IDH1/IDH2 mutations are generally recognized to be associated with a more favorable prognosis.
T10207 734890-735110 Sentence denotes In high grade gliomas, these markers have also been indicated in tumors that have an improved response to alkylating chemotherapy and information on the molecular status of the tumor could be used to direct patient care.
T24289 735111-735256 Sentence denotes Here we retrospectively examined patient tumor samples to determine the frequency of co-occurrence of these biomarkers in our patient population.
T75617 735257-735270 Sentence denotes Introduction:
T94860 735271-735383 Sentence denotes Microarray analysis can accurately assess genome-wide copy number variations in DNA extracted from FFPE tissues.
T23360 735384-735510 Sentence denotes This type of testing may be beneficial in cases with both neoplastic and non-neoplastic disease in the differential diagnosis.
T70207 735511-735595 Sentence denotes For example, a 7-year-old male presented with several months of worsening headaches.
T2853 735596-735727 Sentence denotes He had had a history of severe head trauma, and shunts had been placed for bilateral subdural fluid collections at 4 months of age.
T21707 735728-735845 Sentence denotes MRI identified a large complex collection; however, the artifact from the shunt device made interpretation difficult.
T6389 735846-735980 Sentence denotes The histologic appearance of surgical biopsy specimens was atypical with spindled and pleomorphic cells in a myxoid matrix background.
T4996 735981-736062 Sentence denotes Epithelioid cells with eccentric nuclei and eosinophilic cytoplasm were observed.
T9862 736063-736148 Sentence denotes Scattered multinucleated neoplastic cells and abundant necrosis were also identified.
T769 736149-736334 Sentence denotes Reactive subdural membranes, however, are also known to have an atypical histologic phenotype; therefore, it was difficult to differentiate a neoplastic process from a reactive process.
T65534 736335-736473 Sentence denotes The differential included an unclassifiable myxoid sarcoma and reactive subdural membranes with organized clot adjacent to the shunt site.
T20284 736474-736554 Sentence denotes An accurate diagnosis is critical because of the different treatment approaches.
T41176 736555-736661 Sentence denotes We performed genome-wide detection of alterations in copy number using the Affymetrix OncoScan FFPE assay.
T62531 736662-736670 Sentence denotes Methods:
T85998 736671-736738 Sentence denotes Tissue samples from the lesion were collected through a craniotomy.
T93729 736739-736801 Sentence denotes Cytogenetic analyses were performed at a reference laboratory.
T95789 736802-736888 Sentence denotes DNA was extracted from unstained tissue sections using the QIAamp DNA FFPE Tissue kit.
T38744 736889-736980 Sentence denotes The sample was analyzed using the OncoScan assay kit following the manufacturer's protocol.
T72374 736981-737068 Sentence denotes The TuScan algorithm employed in the assay was used for copy number variation analysis.
T16902 737069-737077 Sentence denotes Results:
T24225 737078-737273 Sentence denotes The OncoScan assay demonstrated multiple chromosomal copy number gains in chromosomes 8 and 14q, and copy number losses in chromosomes 1p, 3, 4p, 5, 6, 10q, 12, 13, 15, 17, 18, 21, 22 , X, and Y.
T4598 737274-737346 Sentence denotes Additionally, chromosomes 11 and 20 contained multiple gains and losses.
T18732 737347-737461 Sentence denotes These alterations appeared to be relative to a state of tetraploidy and were consistent with a neoplastic process.
T69138 737462-737725 Sentence denotes FISH analysis with NR4A3 (9q31.1) gene probes ruled out a diagnosis of extraskeletal myxoid chondrosarcomas (no rearrangements/fusion detected) and also supported the suspected tetraploidy with 3 to approximately 5 copies of the gene in 62% (31/50) of the nuclei.
T34800 737726-737807 Sentence denotes The OncoScan assay generated the discernible diagnosis between the differentials.
T26898 737808-737820 Sentence denotes Conclusions:
T84829 737821-737887 Sentence denotes This case represents a rare diagnostic challenge for pathologists.
T94048 737888-737976 Sentence denotes The OncoScan assay confirmed a neoplastic lesion and helped rule out a reactive process.
T80202 737977-738060 Sentence denotes The OncoScan microarray may be used for confirmatory diagnosis of rare tumor types.
T74150 738061-738354 Sentence denotes The molecular analysis of cell-free DNA (cfDNA) has gained increasing attention during recent years. cfDNA is an easily accessible source for disease monitoring at the molecular level, particularly in patients with disseminated metastatic cancer who are receiving molecular targeted therapies.
T46958 738355-738485 Sentence denotes The underling mechanisms by which cfDNA are released in blood under normal and pathologic conditions are not yet fully understood.
T79845 738486-738684 Sentence denotes However, multiple studies hypothesize that pathologic conditions like cancer will yield a higher amount of cfDNA in the circulation compared to the amount of cfDNA obtained from healthy individuals.
T4560 738685-738768 Sentence denotes There is significant variability in the amount of cfDNA obtained from human plasma.
T83265 738769-738906 Sentence denotes It is uncertain if this difference is related to the variation among patients, preanalytic conditions and/or different isolation methods.
T49216 738907-739034 Sentence denotes In this study, we isolated cfDNA from plasma samples from patients with lung adenocarcinomas using two different methodologies.
T78111 739035-739113 Sentence denotes Next, we PCR amplified a region of the EGFR gene exon 20 harboring T790 codon.
T23486 739114-739204 Sentence denotes Sequencing analysis was then performed on Ion Torrent next-generation sequencing platform.
T63854 739205-739213 Sentence denotes Methods:
T79762 739214-739290 Sentence denotes Plasma from EDTA-drawn samples was collected after two centrifugation steps.
T84643 739291-739530 Sentence denotes The column-based system from Qiagen (QIAmp Circulating Nucleic Acid Kit) was used in 16 samples and the Maxwell 16 instrument (Promega Circulating DNA Purification kit) was used in 7 samples, using protocols supplied by both manufacturers.
T2088 739531-739597 Sentence denotes Cell-free DNA concentrations were measure using Qubit Fluorometer.
T37957 739598-739707 Sentence denotes The sizing and quantification of cfDNA were analyzed by High Sensitivity DNA chip (2100 Agilent Bioanalyser).
T74768 739708-739838 Sentence denotes Ten samples using Qiagen and 3 samples using Promega with >1ng/μL DNA were selected, for further PCR amplification and sequencing.
T20429 739839-739847 Sentence denotes Results:
T38289 739848-740002 Sentence denotes Three out of 13 samples with >1 ng/μL DNA revealed T790M mutation at low allele frequency <1% detectable only visually on integrated genomic viewer (IGV).
T58919 740003-740072 Sentence denotes The depth of reads in all 13 samples was >23,000 with no strand bias.
T97631 740073-740142 Sentence denotes The allele frequency in three T790M harboring samples were as follow:
T93542 740143-740241 Sentence denotes 0.14% (33 out of 23411 reads), 0.17% (42 out of 24983), and 0.085% (21 out of 24836) respectively.
T86777 740242-740254 Sentence denotes Conclusions:
T79915 740255-740381 Sentence denotes The results of our study shows that successful PCR amplification can be achieved even with very low cfDNA concentration yield.
T6470 740382-740528 Sentence denotes Several preanalytic factors impact the cfDNA yield including the type of collection tube, age of sample and plasma volumes used for DNA isolation.
T87138 740529-740754 Sentence denotes Given the extremely low level of positive reads that are typically bellow the detection sensitivity; target enrichment such as digital PCR appears to be an essential step in successful mutation detection in clinical settings.
T3902 740756-740758 Sentence denotes A.
T54869 740759-740766 Sentence denotes Ras, S.
T46934 740767-740775 Sentence denotes Helm, K.
T53717 740776-740785 Sentence denotes Kelly, V.
T87576 740786-740799 Sentence denotes Spotlow, H.V.
T11655 740800-740845 Sentence denotes Reddi The Jackson Laboratory, Farmington, CT.
T41444 740846-740948 Sentence denotes Introduction: FFPE tissues have long been considered a valuable resource in the study of solid tumors.
T27687 740949-741111 Sentence denotes The extraction of nucleic acids from FFPE specimens is challenging due to cross-linking and the damaging effects of the paraffin embedding process to genomic DNA.
T62591 741112-741350 Sentence denotes The quality of purified DNA related to degree of fragmentation and yield depends highly on the sample type, age, archiving process and storage conditions of the FFPE tissue, but the method of extraction can also have a significant impact.
T63148 741351-741464 Sentence denotes Selection of the proper FFPE DNA extraction technique is critical to generate DNA adequate for genetic profiling.
T8605 741465-741473 Sentence denotes Methods:
T32938 741474-741617 Sentence denotes In this study, DNA was purified from five 5μm tissue scrolls, each from 5 FFPE blocks, using 8 commercially available FFPE DNA extraction kits.
T9087 741618-741700 Sentence denotes All purifications were executed manually according to the manufacturers' protocol.
T52138 741701-741777 Sentence denotes DNA was also subjected to subsequent removal of any residual PCR inhibitors.
T18470 741778-741987 Sentence denotes Aspects of purification including tissue deparaffinization with various solvents, enzymatic digestion time and extraction technique were assessed based on DNA quality and quantity using the Nanodrop and Qubit.
T86829 741988-741996 Sentence denotes Results:
T33013 741997-742115 Sentence denotes Final DNA extraction results differ significantly between kits in terms of DNA quantity and purity (OD 260/280 ratio).
T75344 742116-742300 Sentence denotes Whereas all 8 kits produced measurable amounts of DNA, only 5 recovered DNA of sufficient yield and purity to proceed effectively to library preparation for next-generation sequencing.
T32189 742301-742440 Sentence denotes The 3 FFPE kits that performed the best were QIAamp, Bio-Chain, and E.Z.N.A, which yielded a minimum of 700ng and a purity of at least 1.4.
T36410 742441-742540 Sentence denotes The ReliaPrep and Nucleospin extractions generated satisfactory DNA quality, but with lower yields.
T57550 742541-742756 Sentence denotes The purity obtained with MasterPure DNA, UltraRapid and GenElute kits were considerably lower, suggesting highly impure DNA, perhaps caused by insufficient digestion time allowing for residual protein contamination.
T7298 742757-742882 Sentence denotes Lysis time has a significant impact on DNA purity with enzymatic digestions less than 2 hours resulting in inadequate purity.
T15904 742883-743006 Sentence denotes Also, utilization of xylene as a paraffin dissolver has harmful effects on the tissue, resulting in lower yield and purity.
T57351 743007-743019 Sentence denotes Conclusions:
T54027 743020-743093 Sentence denotes In summary, all kits evaluated allowed for isolation of quantifiable DNA.
T20506 743094-743170 Sentence denotes However, only 5 recovered sufficient DNA yields for downstream applications.
T44192 743171-743310 Sentence denotes The performance of the QIAamp kit was superior, producing the highest average DNA yield within acceptable purity range and turnaround time.
T31950 743311-743579 Sentence denotes In addition, purification kits using nonxylene paraffin solvents and those having lysis time more than 2 hours yielded significantly better results than those that did not, suggesting that these steps have significant impact on the outcome of the purification process.
T62761 743581-743595 Sentence denotes Prostatectomy:
T70302 743596-743637 Sentence denotes Initial Results from the Decipher GRID D.
T53997 743638-743650 Sentence denotes Dolginow, J.
T63101 743651-743666 Sentence denotes Chelliserry, M.
T11012 743667-743681 Sentence denotes Alshalalfa, N.
T8459 743682-743690 Sentence denotes Erho, H.
T23587 743691-743709 Sentence denotes Al-Deen Ashbab, M.
T15123 743710-743720 Sentence denotes Takhar, P.
T31459 743721-743729 Sentence denotes Wood, L.
T51208 743730-743737 Sentence denotes Lam, E.
T2184 743738-743794 Sentence denotes Davicioni GenomeDx, Vancouver, British Columbia, Canada.
T19878 743795-743808 Sentence denotes Introduction:
T33047 743809-743959 Sentence denotes Prostate cancer patient management has been enhanced with several commercial genomic prognostic tests such as the Decipher prostate cancer classifier.
T21667 743960-744044 Sentence denotes Unlike other tests, Decipher generates genome-wide expression data for each patient.
T20632 744045-744128 Sentence denotes This data has been anonymized and made available for research in the Decipher GRID.
T85796 744129-744303 Sentence denotes Here we report an initial analysis further characterizing the genomic landscape of localized prostate cancer patients most at risk for recurrence after radical prostatectomy.
T83446 744304-744312 Sentence denotes Methods:
T28425 744313-744430 Sentence denotes We conducted a literature search to identify genes and signatures of potential clinical relevance to prostate cancer.
T33698 744431-744724 Sentence denotes The expression and distribution of 697 genes and 31 prostate cancer disease signatures for metastasis risk, proliferation, luminal/basal, small cell and AR signalling were examined across 2,978 radical prostatectomy tumors with adverse pathology tested with Decipher and available in the GRID.
T28895 744725-744904 Sentence denotes For the 697 genes, expression distribution was characterized and high and low expression were defined using thresholds based on median +/-1.5*1.48*MAD (median absolute deviation).
T18292 744905-745021 Sentence denotes Genes from 31 published prostate cancer signatures were adapted to the Decipher platform and scores were calculated.
T23501 745022-745030 Sentence denotes Results:
T56759 745031-745221 Sentence denotes For various druggable targets including immune checkpoint inhibitors (PD1, PDL1) and growth receptors (c-MET, EGFR, HER), between 2-11% of patients had high expression above right threshold.
T75687 745222-745306 Sentence denotes Clustering of scores for 31 signatures revealed several clear groupings of patients.
T2818 745307-745434 Sentence denotes About 20% of patients consistently had high scores for all the metastasis risk signatures and low-average AR signalling scores.
T68860 745435-745537 Sentence denotes About 10% of patients had high scores for the proliferation signatures but low metastasis risk scores.
T11364 745538-745690 Sentence denotes Tumors with low AR signalling scores were enriched with high basal and small cell signature scores whereas most luminal tumors had higher AR signalling.
T50803 745691-745703 Sentence denotes Conclusions:
T23443 745704-745949 Sentence denotes Since every patient who has received the Decipher test also has a genome-wide expression profile, the Decipher GRID allows researchers to evaluate on a systematic, population-level the expression of genes and signatures that may guide therapies.
T64037 745950-746083 Sentence denotes Such information may be useful for selection of optimal systemic therapy and inclusion into clinical trials of novel targeted agents.
T75601 746084-746288 Sentence denotes This rich genomic resource is being made available on a research use only basis to prostate cancer researchers and to clinicians seeking to better understand prostate cancer to advance precision medicine.
T15368 746289-746291 Sentence denotes V.
T90405 746292-746303 Sentence denotes Thodima, A.
T8206 746304-746318 Sentence denotes Guttapalli, R.
T8544 746319-746334 Sentence denotes Padmanabhan, S.
T51898 746335-746350 Sentence denotes Kamalakaran, J.
T48660 746351-746366 Sentence denotes Houldsworth, B.
T24454 746367-746425 Sentence denotes Gowrishankar Cancer Genetics Incorporated, Rutherford, NJ.
T36917 746426-746439 Sentence denotes Introduction:
T68920 746440-746689 Sentence denotes There is a growing body of evidence in the literature showing that mutations, copy number changes and a few single nucleotide polymorphisms (SNPs) consistently correlate with prognosis and therapy response in clear cell renal cell carcinoma (ccRCC).
T5961 746690-746842 Sentence denotes Here, we describe analytical validation of a custom targeted next-generation sequencing (NGS) assay for assessing genomic alterations relevant in ccRCC.
T390 746843-746972 Sentence denotes The assay has been validated in fresh-frozen/formalin-fixed paraffin embedded (FFPE) resected specimens and core needle biopsies.
T12788 746973-746981 Sentence denotes Methods:
T29793 746982-747242 Sentence denotes The targeted hybrid capture-based NGS panel (FOCUS::Renal) comprised of 76 genes mutated in ccRCC (including 7 targets for FDA-approved drugs), 15 SNPs of prognostic relevance, and a 3Mb-spaced SNP backbone for copy number assessment (Nimblegen, 2400 targets).
T97668 747243-747374 Sentence denotes Sequencing was performed (MiSeq/NextSeq, Illumina) and variants identified using CLCbio Biomedical Genomics Workbench 2.1 (Qiagen).
T32972 747375-747500 Sentence denotes Genomic gain/loss estimated using Nexus Copy Number Algorithm 8.0 (BioDiscovery) and CNVkit (https://github.com/etal/cnvkit).
T75996 747501-747584 Sentence denotes Performance metrics were set on 152 ccRCC (128 fresh-frozen and 24 FFPE) specimens.
T46314 747585-747664 Sentence denotes An additional set of 50 ccRCC were used for analytical validation of the panel.
T61217 747665-747719 Sentence denotes Diluted Jurkat cell line was used as positive control.
T10025 747720-747728 Sentence denotes Results:
T86520 747729-747852 Sentence denotes For quality control, each sample was expected to achieve >95% of targets with at least 60X coverage for 95% of each target.
T36019 747853-747932 Sentence denotes Among targets, ten were identified as lowperforming and excluded from analysis.
T82131 747933-748097 Sentence denotes About 100 ng (fresh-frozen) and 250ng (FFPE) DNA were used as input routinely, whereas as low as 25 ng (fresh-frozen) and 50 ng (FFPE) yielded reproducible results.
T84634 748098-748198 Sentence denotes Average read depth of 750X and 1143X was achieved across fresh-frozen and FFPE samples respectively.
T28120 748199-748422 Sentence denotes Accuracy of variant detection (identified by Sanger sequencing or CLIA-approved NGS assay) and gain/loss (detected by array-CGH) were evaluated in 15 and 25 specimens respectively and >95% concordance was observed for both.
T12141 748423-748647 Sentence denotes For variant detection, analytical sensitivity was established using Jurkat cell line and assay limit of detection (LOD) was found to be 5% allele variant frequency (AVF) at >200X read depth and 10% AVF at 60-200X read depth.
T46205 748648-748704 Sentence denotes LOD was also confirmed using diluted clinical specimens.
T62195 748705-748819 Sentence denotes For gain/loss detection, analytical sensitivity was 40% for single copy gains/losses using diluted A498 cell line.
T75458 748820-748904 Sentence denotes Reproducibility/precision analysis also showed high intra-and inter-run concordance.
T69675 748905-748917 Sentence denotes Conclusions:
T86704 748918-749124 Sentence denotes Overall, the Focus::Renal NGS assay developed by us has undergone rigorous validation to detect genomic alterations relevant in ccRCC and thereby assist in the overall clinical management of ccRCC patients.
T40905 749125-749127 Sentence denotes H.
T34978 749128-749141 Sentence denotes Fernandes, H.
T98107 749142-749149 Sentence denotes Zia, H.
T48584 749150-749161 Sentence denotes Rennert, M.
T38086 749162-749204 Sentence denotes Kluk Weill Cornell Medicine, New York, NY.
T81103 749205-749336 Sentence denotes Introduction: NGS assays for detection of tumor variants have replaced conventional methodologies at several major medical centers.
T94714 749337-749527 Sentence denotes However, due to the lack of consensus guidelines in variant classification and reporting of assays, the data presented in NGS-based diagnostic reports varies considerably among laboratories.
T88612 749528-749771 Sentence denotes We compared the clinical NGS reports from an academic laboratory at Weill Cornell Medicine (WCM) with a commercially derived report from QIAGEN's Clinical Insight Interpret (QCI) platform generated from the same variant call format (VCF) file.
T75686 749772-749780 Sentence denotes Methods:
T45341 749781-749931 Sentence denotes 50 FFPE lung and colorectal cancer specimens that were sequenced using the Ion Torrent platform (Life Technologies), were selected for the comparison.
T136 749932-750149 Sentence denotes The VCF files from the specimens were found to harbor single nucleotide variants (SNVs) and indels with COSMIC ID's that were annotated at WCM, using the Torrent suite variant caller software v4.4 (Life Technologies).
T7708 750150-750268 Sentence denotes The same VCF files were subsequently parsed through QCI software for decision support of clinically relevant variants.
T77206 750269-750390 Sentence denotes Variants from 5 additional lung adenocarcinoma specimens with complex EGFR variants including indels, were also compared.
T65013 750391-750523 Sentence denotes Data present in the molecular pathology reports generated for clinical purposes at WCM, were compared to the reports created by QCI.
T42108 750524-750532 Sentence denotes Results:
T21108 750533-750751 Sentence denotes At WCM, the validity of positive variant calls is based on assigned QC metrics and the classification of variants into 3 tiers is based on relevance to therapeutic actionability, diagnostic and prognostic significance.
T17545 750752-750860 Sentence denotes Using QCI, the rationale for reporting a variant and classification based on pathogenicity is user assigned.
T10045 750861-750981 Sentence denotes Using comparable metrics 83 variants in 12 genes, all having COSMIC ID's were evaluated in 50 specimens, by WCM and QCI.
T92164 750982-751170 Sentence denotes Among these were several clinically relevant cancer-related hotspot variants pertinent in lung adenocarcinoma and colorectal cancer including EGFR exon 19 deletions and exon 20 insertions.
T78698 751171-751381 Sentence denotes WCM reported the variant allelic frequency, coverage and COSMIC ID for each variant while QCI provided detailed information on approved therapies and current clinical trials related to the variant in the tumor.
T18820 751382-751519 Sentence denotes Only QCI was able to annotate targetable complex indels present in exons 19 and 20 of the EGFR gene from 5 lung adenocarcinoma specimens.
T35563 751520-751667 Sentence denotes The NGS-based reports generated using laboratory developed tools may have limitations in providing supplemental information on the clinical report.
T15446 751668-751755 Sentence denotes QCI reports provided an evidence-based approach to targeted therapy and trial matching.
T18442 751756-752021 Sentence denotes The dedicated resources in a commercial setting offer clinical evidence to enhance Target with known fusions from colon, brain, bladder and lung tumors, 13 previously uncharacterized tumor samples of thyroid carcinoma, prostate carcinoma, and sarcoma were screened.
T9458 752022-752030 Sentence denotes Methods:
T39005 752031-752281 Sentence denotes The libraries were prepared using the Archer Universal RNA Reagent Kits v2 and the FusionPlex Solid Panel probe mix, quantified using KAPA Biosystems Library Quantification Kit and then sequenced on the Illumina MiSeq using v2 Reagent Kit/300 cycles.
T73408 752282-752407 Sentence denotes Data were analyzed using the Archer Analysis 4.0 software followed by Sanger sequencing for confirmation of detected fusions.
T77536 752408-752535 Sentence denotes The fusion status of 13 clinical cases was determined using RNA extracted from formalin-fixed paraffin-embedded tissues (FFPE).
T15910 752536-752676 Sentence denotes Samples tested included 5 papillary thyroid carcinomas (PTC), 4 anaplastic thyroid carcinomas (ATC), 3 prostate carcinomas, and one sarcoma.
T90114 752677-752685 Sentence denotes Results:
T24805 752686-752898 Sentence denotes All expected fusions including CCDC6-RET (thyroid), ELM4-ALK (lung), NPM1-ALK (lymph node), TPM3-NTRK1 (colon), FGFR3-TACC3 (bladder), GOPC-ROS1 (brain) were successfully detected during the technical validation.
T35969 752899-753143 Sentence denotes Of the 13 previously uncharacterized tumor samples, two PTC cases were positive for the common CCD6-RET fusion, one was positive for the NCOA4-RET fusion with a novel breakpoint in NCOA4 gene, and one was positive for a novel fusion PEX14-BRAF.
T79257 753144-753204 Sentence denotes One ATC case was positive for a novel fusion, WHSC1L1-NUTM1.
T72444 753205-753264 Sentence denotes The sarcoma case was positive for the EWSR1-CREB3L2 fusion.
T87402 753265-753350 Sentence denotes Two of the cases ofprostate carcinoma cases were positive for the TMPRSS2-ERG fusion.
T46638 753351-753558 Sentence denotes Conclusions: NGS fusion assays enable simultaneous screening and detection of known gene fusions using FFPE tissue, and allows detection of novel translocations which would be missed by targeted FISH assays.
T81511 753559-753764 Sentence denotes This multigene NGS assay can be implemented in routine clinical practice at a lower cost than comparable FISH panels, utilizes tissue more efficiently and allows detection of rare and/or novel breakpoints.
T73524 753765-753876 Sentence denotes Barcoded libraries were prepared using 10ng of DNA; sequencing was performed on the Ion Torrent PGM (318 chip).
T98598 753877-753972 Sentence denotes Variants were identified using the Ion Torrent Variant Caller Plugin and reference genome hg19.
T99743 753973-754076 Sentence denotes Golden Helix's SVS software was used for annotation and prediction of the significance of the variants.
T5158 754077-754085 Sentence denotes Results:
T57423 754086-754251 Sentence denotes Seven samples from six patients (1.2%) were positive for IDH1 (p.R132C and p.132L in two patients each) or, IDH2 mutations (p.R140W and p.R172S in one patient each).
T62410 754252-754302 Sentence denotes All patients' tumors had adenocarcinoma histology.
T69893 754303-754398 Sentence denotes The patients' age ranged from 72 years to 83 years and two of the patients were female (33.3%).
T80245 754399-754451 Sentence denotes All patients were current (2) or former (4) smokers.
T93506 754452-754527 Sentence denotes Four patients presented with stage IV disease and two with stage I disease.
T36897 754528-754604 Sentence denotes Three are deceased, two are alive with disease and one is lost to follow up.
T35016 754605-754703 Sentence denotes In five instances (83%), the tumors also had a KRAS mutation (p.G12D (2), p.G12V, p.G12C, p.Q61H).
T51551 754704-754784 Sentence denotes In the sixth patient, KIT p.M541L and APC p.T175fs mutations were also detected.
T10580 754785-754797 Sentence denotes Conclusions:
T69956 754798-754886 Sentence denotes Known cancer associated IDH1/IDH2 mutations are rarely identified in a subset of NSCLCs.
T63615 754887-755008 Sentence denotes In our population, these were associated with patient age greater than 70 years, a history of smoking and KRAS mutations.
T56454 755009-755263 Sentence denotes Additional studies are needed to understand the role of IDH1/IDH2 mutations in the development of NSCLC but such patients appear to be of more advanced age and may be eligible for IDH1/IDH2 targeted therapies such as the multi-kinase inhibitor dasatinib.
T93487 755264-755277 Sentence denotes Introduction:
T64376 755278-755413 Sentence denotes Human papillomavirus (HPV) is known to be associated with squamous cell carcinomas of the head and neck (HNSCC), especially oropharynx.
T63815 755414-755539 Sentence denotes Patients whose tumors are HPV positive tend to have a significantly better response to treatment and better overall survival.
T49740 755540-755673 Sentence denotes Also, transcriptionally-active HPV-related HNSCC is clinically, biologically, morphologically and molecularly distinct form of HNSCC.
T86616 755674-755787 Sentence denotes Therefore it is critical to choose the best testing modality for an accurate diagnosis of high-risk HPV in HNSCC.
T41360 755788-755890 Sentence denotes Both, p16 IHC and HPV DNA ISH, lack specificity and do not correlate with biologically active disease.
T11937 755891-756034 Sentence denotes We have validated the APTIMA HPV assay which targets E6/E7 mRNA of 14 high-risk HPV on FFPE samples with excellent sensitivity and specificity.
T63888 756035-756175 Sentence denotes Objective of this study was to correlate these 3 assays on formalin-fixed paraffin embedded (FFPE) to identify single best testing modality.
T2513 756176-756184 Sentence denotes Methods:
T7389 756185-756235 Sentence denotes Twenty two (22) FFPE samples of HNSCC were tested.
T43171 756236-756288 Sentence denotes One mucoepidermoid carcinoma (MC) was also included.
T81594 756289-756355 Sentence denotes P16 IHC was performed on Leica Bond III per manufacturer protocol.
T83916 756356-756520 Sentence denotes P16 slides were independently reviewed by 2 authors who were blinded to the HPV ISH and APTIMA jmd.amjpathol.org ■ The Journal of Molecular Diagnostics HPV results.
T90777 756521-756593 Sentence denotes HPV DNA ISH results were available from a national reference laboratory.
T28284 756594-756758 Sentence denotes For in-house molecular testing (APTIMA HPV Assay), macrodissection was performed on all the specimens followed by RNA extraction using the Promega LEV FFPE RNA kit.
T97299 756759-756841 Sentence denotes Purity and concentrations were measured using Nanodrop and QuantiFluor on Quantus.
T91344 756842-756971 Sentence denotes All samples were run in duplicate with an input of at least 40ng/tube using the APTIMA HPV Assay on Panther instrument (Hologic).
T16381 756972-756980 Sentence denotes Results:
T13841 756981-757030 Sentence denotes Most common site of involvement was tonsil (n=8).
T9706 757031-757114 Sentence denotes Majority of the tumors were moderate to poorly differentiated and non-keratinizing.
T90886 757115-757236 Sentence denotes P16 IHC was positive in all the cases, including MC, with percentage positivity ranging from 20% up to 100% (median 60%).
T14216 757237-757331 Sentence denotes HPV DNA ISH was positive in 12 of the 22 samples, with 4 negative and 6 indeterminate results.
T14937 757332-757411 Sentence denotes Twenty one (21) of the 22 cases were positive for highrisk HPV by APTIMA assay.
T63068 757412-757490 Sentence denotes One HNSCC was negative by both ISH and APTIMA assay but positive by p16 (40%).
T33942 757491-757540 Sentence denotes ME was negative by both DNA ISH and APTIMA assay.
T13023 757541-757714 Sentence denotes There was no significant correlation between histologic subtype, keratinization and/or percent p16 positivity with the presence of high-risk HPV as seen by the APTIMA assay.
T59723 757715-757845 Sentence denotes Conclusions: APTIMA HPV mRNA assay is highly sensitive and specific for detection of high-risk HPV subtypes associated with HNSCC.
T15635 757846-758131 Sentence denotes Since the presence of transcriptionally active HPV in HNSCC is highly and independently predictive of better patient survival, we propose that the mRNA based assays, preferably using PCR, should be used as the sole testing modality for an accurate determination of HPV status in HNSCC.
T33292 758132-758310 Sentence denotes The ability to construct a library is critically dependent on the preanalytical tissue selection as well as the quality and quantity of nucleic acid extracted from FFPE material.
T84685 758311-758477 Sentence denotes Whereas there are general guidelines for NGS, most of the preanalytical and sample preparation steps are set by individual institutions based on a validation process.
T97096 758478-758654 Sentence denotes Here we reviewed clinically tested FFPE samples submitted for solid tumor NGS testing to determine factors that contribute to a successfully constructed library and sequencing.
T49905 758655-758663 Sentence denotes Methods:
T55216 758664-758780 Sentence denotes We performed a retrospective review of 592 FFPE samples that were submitted for solid tumor by NGS clinical testing.
T91023 758781-758982 Sentence denotes Our primary objective was to identify pre-analytic factors that contribute to the overall success of extracting DNA and preparing a sequenceable library from FFPE tissue for next-generation sequencing.
T21688 758983-759172 Sentence denotes Pre-analytic factors reviewed were sample type (resections, biopsies, and cytology cell blocks), tumor percentage, tissue fixation, percent necrosis, percent mucin, and tumor heterogeneity.
T29455 759173-759295 Sentence denotes Additionally, extracted samples were quantified for DNA concentration with the Qubit fluorometer using the dsDNA BR Assay.
T71323 759296-759426 Sentence denotes In a subset of the samples, quality of the extracted DNA was assessed with the QuantideX DNA Assay to amplifiable DNA copy number.
T78312 759427-759435 Sentence denotes Results:
T61932 759436-759632 Sentence denotes There was significant correlation (p < 0.001) for successful library construction in four of the observed variables: extracted DNA concentration, quality of DNA, tumor percentage, and sample type.
T3180 759633-759734 Sentence denotes Samples with an extraction concentration > 10ng/μL had a 98% success rate whereas 61.9% respectively.
T15578 759735-759931 Sentence denotes In assessing the quality of the extracted DNA, libraries above a cut-off of 300 amplifiable copies per μL were five times more likely to produce a successful library than those below this cut-off.
T34833 759932-760002 Sentence denotes Samples with a tumor percentage > produced a successful library 84.4%.
T7709 760003-760156 Sentence denotes Lastly, the rate of successful libraries produced for resection samples was 93.1%, for biopsies was 83.6%, and for cytology cell block samples was 90.9%.
T82567 760157-760338 Sentence denotes Understanding the underlying factors as they relate to sample preparation of the NGS workflow enabled us to construct tree models for troubleshooting clinical samples in the future.
T76439 760339-760350 Sentence denotes Conclusion:
T95303 760351-760556 Sentence denotes The results highlight the value of understanding pre-analytical and early sample preparation factors that contribute to the ability to extract DNA, construct successful libraries and subsequent sequencing.
T3775 760557-760785 Sentence denotes Furthermore, this study has allowed us to implement a review process clinically to predict whether sample failure may be due to technical issues or sample integrity thus guiding repeat testing and improving overall patient care.
T29511 760786-760799 Sentence denotes Introduction:
T11959 760800-760953 Sentence denotes Next-generation sequencing panels are becoming increasingly important in cancer diagnosis for determining treatment as well as prognosis for the patient.
T47406 760954-761091 Sentence denotes Assays to detect point mutations in oncogenes are common, but detection of translocations and copy number variation may also be critical.
T15239 761092-761313 Sentence denotes In collaboration with oncologists and pathology medical directors, a comprehensive solid tumor NGS panel was developed, consisting of actionable and prognostic genes covering SNVs, translocations, and copy number changes.
T62370 761314-761384 Sentence denotes Methods: DNA was isolated from blood, FFPE tissue, or FFPE cell lines.
T33085 761385-761523 Sentence denotes Libraries were prepared using 10 ng to 50 ng of DNA and the KAPA Hyper Prep Kit, and then sequenced on the Illumina NextSeq500 instrument.
T64003 761524-761689 Sentence denotes Analysis for single nucleotide variants (SNVs) used LoFreq, translocations used Delly, copy number used a modified Contra/DNAcopy, and LOH used B-allele frequencies.
T188 761690-761768 Sentence denotes This panel contained full coding exon coverage of 126 genes for SNV detection.
T5193 761769-761838 Sentence denotes For translocation detection, 18 genes had selected intronic coverage.
T11582 761839-762029 Sentence denotes The 79 CNV genes had exon as well as intronic coverage over select known population SNVs; to gain B-allele frequency data for LOH detection, as well as providing additional copy number data.
T60108 762030-762355 Sentence denotes Samples consisting of three sets of tumor/normal pairs for CNV analysis, four cell lines with ALK, RET, ROS1, and NTRK1 translocations, and the Horizon FFPE Quantitative Multiplex reference standard were tested for proof of principle to determine if multiple types of mutations could be detecting using this NGS assay design.
T71601 762356-762364 Sentence denotes Results:
T54254 762365-762470 Sentence denotes All three types of variations (SNVs, translocations, and copy number changes) were successfully detected.
T69512 762471-762560 Sentence denotes The Horizon standard had all SNVs called as expected above a 3% variant allele frequency.
T7634 762561-762673 Sentence denotes The NGS copy number and B-allele frequency data correlated well with results using the Oncoscan microarray data.
T25584 762674-762727 Sentence denotes The translocations were identified in all cell lines.
T27420 762728-762740 Sentence denotes Conclusions:
T45139 762741-762924 Sentence denotes This comprehensive cancer panel is successfully able to capture regions of interest for somatic cancer and can identify translocation, copy number, LOH and SNVs in the same NGS assay.
T12003 762925-763048 Sentence denotes 1 1 Thermo Fisher Scientific, South San Francisco, CA; 2 Thermo Fisher Scientific, Carlsbad, CA; 3 CosmosID, Rockville, MD.
T68241 763049-763062 Sentence denotes Introduction:
T31184 763063-763166 Sentence denotes At this time next-generation sequencing (NGS) is hindered by slow and often manual workflow procedures.
T64868 763167-763371 Sentence denotes Decreasing overall workflow times is critical for the widespread adoption of targeted and whole genome sequencing (WGS) for many time-sensitive applications, in particular for infectious disease analysis.
T44456 763372-763543 Sentence denotes To this end, we describe improvements to the four main steps of the NGS workflow: i) library preparation; ii) template preparation, iii) sequencing; and iv) data analysis.
T91234 763544-763620 Sentence denotes Together, these advances dramatically decrease the overall turnaround times.
T98483 763621-763629 Sentence denotes Methods:
T60989 763630-764035 Sentence denotes The new rapid workflow innovations were applied to two different library preparation protocols: i) targeted libraries created using a highly-multiplexed PCR approach consisting of 1200 amplicons targeting the 16S rRNA gene as well as speciesspecific identification targets and antimicrobial resistance determinants; and ii) an unbiased WGS approach using a MuA transposon-based library preparation method.
T15220 764036-764282 Sentence denotes To assess the accuracy of detection with the improved workflows, nucleic acid from six bacterial cultures (A. baumannii, E. cloacae, E. faecium, K. pneumoniae, P. aeruginosa, and S. aureus) were extracted as input for targeted and WGS sequencing.
T40130 764283-764451 Sentence denotes Targeted libraries were optimized for speed by reducing amplification and incubation times as well as substitution of the polymerase used in standard library protocols.
T6805 764452-764543 Sentence denotes The MuSeek-based WGS library preparation times were improved by eliminating a cleanup step.
T1925 764544-764711 Sentence denotes Both targeted and WGS libraries were clonally amplified (template preparation) using an isothermal amplification approach that saves 3 hours over the standard methods.
T69505 764712-764807 Sentence denotes Sequencing times were improved by reducing nucleotide flow times and the total number of flows.
T74783 764808-764930 Sentence denotes The implementation of On-Instrument Analysis (OIA) enabled near real-time base calling reducing the primary analysis time.
T37688 764931-764939 Sentence denotes Results:
T73557 764940-765151 Sentence denotes Targeted and WGS libraries were generated, sequenced, and analyzed in 6.5 hours with sequencing and analysis taking 50 minutes compared to 2.5 hours and 1 hour for standard sequencing and analysis, respectively.
T13439 765152-765267 Sentence denotes Analysis of sequencing accuracy revealed a raw read accuracy >99.5%, comparable to data from the standard workflow.
T56440 765268-765414 Sentence denotes The read length distribution for the targeted libraries was unaffected by speed improvements with a distribution similar to the standard workflow.
T51501 765415-765622 Sentence denotes Further, 100% specificity for species identification and antimicrobial resistance determinants was observed for targeted and WGS libraries indicating rapid sequencing without compromising detection accuracy.
T30935 765623-765635 Sentence denotes Conclusions:
T14790 765636-765792 Sentence denotes For the targeted and WGS methods described, the total turnaround time from isolated nucleic acid to sequencing data could be completed in a typical workday.
T8781 765793-765806 Sentence denotes Introduction:
T45549 765807-766012 Sentence denotes ChimerMarker (SoftGenetics) is a commercially available software package designed to integrate analysis, genotyping, and chimerism calculations by short-tandem repeat (STR) polymerase chain reaction (PCR).
T30583 766013-766180 Sentence denotes We present the evaluation, validation, and implementation of ChimerMarker into the workflow of a clinical laboratory for post-transplant chimerism analysis by STR PCR.
T3342 766181-766189 Sentence denotes Methods:
T9496 766190-766338 Sentence denotes Thirteen cases ranging from 0% to 100% recipient were selected for software validation with comparison to a manual, single locus calculation method.
T98523 766339-766470 Sentence denotes PCR was performed using Powerplex 16 HS System (Promega), followed by capillary gel electrophoresis on a 3500 (Applied Biosystems).
T75351 766471-766630 Sentence denotes Pre-transplant projects were created using a novel algorithm developed to include or exclude informative loci based on stutter positions and signal background.
T29396 766631-766699 Sentence denotes Special features, such as deconvolution were incorporated as needed.
T93081 766700-766794 Sentence denotes Post-transplant samples were analyzed using the selected loci from the pre-transplant project.
T33452 766795-767024 Sentence denotes Data from 12 recipient/donor pairs were gathered to calculate the percent stutter at each locus and allele in the cohort (n-1, n-2, and n+1 positions), which were then used to set marker-specific stutter filtering and adjustment.
T62399 767025-767109 Sentence denotes Varied DNA input was also tested in 9 patients to identify optimal assay conditions.
T4397 767110-767309 Sentence denotes An additional 120 patients were examined to compare the program's calculated % chimerism result with the manual, single locus method as well as to determine cut-offs for the lower limit of detection.
T89394 767310-767381 Sentence denotes Criteria for interpreting 100% donor and 100% recipient were developed.
T70612 767382-767390 Sentence denotes Results:
T48493 767391-767479 Sentence denotes On average, 4 loci were used (range 2 to 12) per sample for calculation in ChimerMarker.
T56313 767480-767596 Sentence denotes Correlation with the manual single locus calculation method was strong (R 2 =0.99) across all levels of engraftment.
T72847 767597-767726 Sentence denotes DNA inputs of 2 ng were more reliable, with lower % CV, less bias, and less allelic imbalance or allele dropout compared to 1 ng.
T7758 767727-767830 Sentence denotes The average stutter percentage varied at each locus from 1% to 10%, with highest % in the n-1 position.
T78440 767831-768058 Sentence denotes Since the stutter adjustment chimerism calculation performed by the software only corrects for n-1 stutter, manually excluding loci with n-1, n-2, and n+1 stutter from the analysis markedly improved the accuracy of the results.
T23074 768059-768161 Sentence denotes The analytic sensitivity of the software reliably detected donor or recipient DNA down to at least 1%.
T46488 768162-768174 Sentence denotes Conclusions:
T60432 768175-768407 Sentence denotes We developed a novel algorithm for the selection of loci in the ChimerMarker software that minimizes stutter positions and background, providing a reliable, sensitive, and reproducible approach to post-transplant chimerism analysis.
T85432 768408-768534 Sentence denotes These alterations empower the software to detect complete engraftment equal to or better than the manual, single locus method.
T60135 768535-768537 Sentence denotes H.
T91480 768538-768546 Sentence denotes Achi, F.
T57217 768547-768556 Sentence denotes Abbas, R.
T56719 768557-768573 Sentence denotes Abdel Khalek, R.
T66930 768574-768644 Sentence denotes Mahfouz American University of Beirut Medical Center, Beirut, Lebanon.
T5172 768645-768658 Sentence denotes Introduction:
T9008 768659-768905 Sentence denotes Quantitative measurements of HIV viremia in the peripheral blood have shown that higher virus levels may be correlated with increased risk of clinical progression of HIV disease, and reduction in viremia level is associated with a decreased risk.
T53924 768906-769298 Sentence denotes Virus levels can be quantified by measurement of the HIV p24 antigen, by quantitative culture of HIV, or by direct measurement of viral RNA using nucleic acid amplification technologies, such as the polymerase chain reaction (PCR) that achieve high sensitivity and dynamic range for the quantitative detection of HIV RNA, to assess prognosis and monitor the effects of antiretroviral therapy.
T14214 769299-769538 Sentence denotes This pilot study compares the performance and the results of two HIV qPCR platforms, COBAS Ampliprep/COBAS TaqMan (Roche Molecular Diagnostics) and GeneXpert (CEPHEID), for a total of 34 patients referred for HIV testing at a major center.
T94730 769539-769547 Sentence denotes Methods:
T73802 769548-769744 Sentence denotes The HIV-1 Quant Assay kit, constitutes a readyto-use system for the rapid quantification of HIV-1 group M, N and O, in infected individuals using polymerase chain reaction on GeneXpert Instrument.
T29453 769745-769894 Sentence denotes The system automates sample purification, nucleic acid amplification, and detection of the target sequence using real-time reverse transcriptase PCR.
T98742 769895-770009 Sentence denotes The process require the use of single-use disposable GeneXpert cartridges that hold the reagents and host the PCR.
T81169 770010-770384 Sentence denotes It includes reagents, 2 internal controls for quantitation of HIV-1 RNA and for identifying possible PCR inhibitors, and a Probe Check Control that verifies the probe integrity.The COBAS AmpliPrep/COBAS TaqMan HIV-1 Test permits automated specimen preparation followed by PCR amplification, detection of HIV-1 target RNA and HIV-1 Quantitation Standard (QS) RNA for group M.
T12771 770385-770481 Sentence denotes The Master Mix reagent contains primers and probes specific for both HIV-1 RNA and HIV-1 QS RNA.
T1819 770482-770678 Sentence denotes The detection of amplified DNA is performed using target-specific and QSspecific dual-labeled oligonucleotide probes that permit independent identification of HIV-1 amplicon and HIV-1 QS amplicon.
T85994 770679-770747 Sentence denotes The quantitation of HIV-1 viral RNA is performed using the HIV-1 QS.
T3362 770748-770913 Sentence denotes It compensates for effects of inhibition and controls the preparation and amplification processes, allowing a more accurate quantitation of HIV RNA in each specimen.
T23606 770914-771007 Sentence denotes The results of 32 patients run using both kits were compared using the EP evaluator software.
T83029 771008-771016 Sentence denotes Results:
T92634 771017-771159 Sentence denotes The difference between the 2 methods was within the allowable error for 28 out of 32 specimens (87.5%), with a correlation coefficient R=0.85.
T16617 771160-771171 Sentence denotes Conclusion:
T64241 771172-771523 Sentence denotes The HIV-1 GeneXpert PCR Kit and the COBAS AmpliPrep/COBAS TaqMan HIV-1 kit are 2 acceptable assays that can be used for the sensitive detection of HIV in a wide variety of clinical specimens. (QIAGEN) and the platform used by the College Of American Pathologists on respiratory samples for a total of 9 patients at AUBMC, using two extraction methods.
T36873 771524-771532 Sentence denotes Methods:
T9782 771533-771767 Sentence denotes The principle of the FTD respiratory pathogens 33 RG PCR Kit is based on the transcription of viral RNA into cDNA using a specific primer mediated reverse transcription step followed by amplification of the DNA of different pathogens.
T24542 771768-771996 Sentence denotes The presence of specific pathogen sequences in the reaction is detected by an increase in fluorescence observed from the relevant dual-labeled probe, and is reported as a cycle threshold value (Ct) by the Real-Time thermocycler.
T8087 771997-772147 Sentence denotes The assay uses Equine arteritis virus as an internal control, which is introduced into each sample and the negative control at the extraction process.
T27598 772148-772309 Sentence denotes Nucleic acid material was manually extracted from the sample using QIAamp UltraSens Virus kit for 4 of the patients and QIAamp Viral RNA Mini kit for the others.
T31629 772310-772456 Sentence denotes We studied overall 18 samples and the results of 14 were compared to the College of American Pathologists results using the EP evaluator software.
T26528 772457-772550 Sentence denotes The remaining 4 were compared to the meridian platform results, and awaiting the CAP results.
T87006 772551-772559 Sentence denotes Results:
T45760 772560-772643 Sentence denotes The samples extracted using QIAamp UltraSens gave "no signal" result on Rotor Gene.
T1488 772644-772757 Sentence denotes When retested using QIAamp Viral RNA mini kit, the results of the 14 patients were identical to those of the CAP.
T65039 772758-772853 Sentence denotes As for the 4 pending patients we had similar results between Rotor Gene and Meridian platforms.
T5520 772854-772986 Sentence denotes Difference between the methods was within the allowable error for 18 out of 18 specimens (100%), with a correlation coefficient R=1.
T64557 772987-772998 Sentence denotes Conclusion:
T40822 772999-773234 Sentence denotes The FTD respiratory pathogens 33 RG PCR Kit is an acceptable assay that can be used for the sensitive detection of a wide variety of respiratory pathogens using, as recommended, the QIAamp Viral RNA mini kit for nuclei acid extraction.
T64115 773235-773248 Sentence denotes Introduction:
T35418 773249-773387 Sentence denotes Next-generation sequencing is a powerful technique capable of detecting a variety of genetic alterations in multiple genes simultaneously.
T35395 773388-773546 Sentence denotes Focused cancer hotspot panels are widely used and can detect single nucleotide variants and small insertions/deletions (in/dels) that are recurrent in cancer.
T60111 773547-773796 Sentence denotes Although focused ampliseq panels are not typically used to detect copy number alterations, we sought to determine if the data from such an assay could be used to infer copy number alteration status of specific genes in glioblastoma multiforme (GBM).
T29671 773797-773805 Sentence denotes Methods:
T67992 773806-773850 Sentence denotes A total of 41 GBM cases were tested to date.
T69333 773851-774088 Sentence denotes DNA was extracted from the tumorenriched area of FFPE sections and was sequenced on the Ion Torrent Personal Genome Machine (PGM) after library preparation and enrichment using AmpliSeq Cancer Hotspot Panel v2 (Thermo Fisher Scientific).
T75615 774089-774301 Sentence denotes Copy number alteration status was determined by an algorithm to calculate the Log2 values of the fractional coverage of EGFR, CDKN2A, PDGFRA and PTEN for each tumor after normalization with pooled normal samples.
T30968 774302-774546 Sentence denotes The copy number status from the ampliseq panel was then compared to results from other platforms, i.e., whole exome sequencing (WES), targeted hybrid-capture panels, EGFR chromogenic in situ hybridization (CISH) and digital droplet PCR (ddPCR).
T37261 774547-774555 Sentence denotes Results:
T77667 774556-774717 Sentence denotes Thirty-seven percent of cases (15/41) were positive for EGFR amplification (log2 ratio >3), consistent with the reported prevalence of EGFR amplification in GBM.
T95084 774718-774819 Sentence denotes Of these, 100% (15/15) were confirmed to show EGFR amplification by other methods (CISH, WES, ddPCR).
T38305 774820-775021 Sentence denotes 63% of cases (26/41) were negative for EGFR amplification by ampliseq; of these, 11/26 cases had data available from other methods and 100% (11/11) were confirmed to be negative for EGFR amplification.
T27633 775022-775181 Sentence denotes 39% of cases (16/41) showed CDKN2A deletion (log 2 ratio <-2) and 100% (12/12) cases with data available from other methods were confirmed for CDKN2A deletion.
T6498 775182-775294 Sentence denotes PDGFRA amplifications (Log2 ratio >2) were found in 5% of cases (2/41) and were confirmed by orthogonal testing.
T5093 775295-775370 Sentence denotes Lastly, PTEN deletion (Log2 ratio <-2) was observed in 20% of cases (8/41).
T97690 775371-775383 Sentence denotes Conclusions:
T41765 775384-775684 Sentence denotes These findings demonstrate that selective copy number alteration status can be successfully determined in glioblastoma multiforme using the data acquired during the routine use of a targeted cancer hotspot panel, thereby broadening the scope of genetic alterations that can be detected by this panel.
T69630 775685-775698 Sentence denotes Introduction:
T24529 775699-775987 Sentence denotes Next-generation sequencing (NGS) has been rapidly adopted in clinical laboratories as highly complex tests that can substantially vary in both their design and application, bringing challenges that can be associated with sample handling, library preparation, data analysis, and reporting.
T98680 775988-776201 Sentence denotes To be able to monitor the multi-step NGS workflow and consistently provide accurate results, quality control materials should be included both during the validation phase, as well as during routine clinical tests.
T67872 776202-776450 Sentence denotes The goal of this multi-laboratory study was to evaluate the Seraseq AF10 and AF20 reference materials by verifying their performance as a quality control material, and by evaluating their ability to measure quality metrics vital to a clinical test.
T15624 776451-776535 Sentence denotes These characteristics were assessed within and between the cooperating laboratories.
T46252 776536-776544 Sentence denotes Methods:
T15066 776545-776641 Sentence denotes Six CLIA certified clinical laboratories and one research laboratory participated in this study.
T6410 776642-776831 Sentence denotes The Seraseq AF10 and AF20 reference materials consist of 26 biosynthetic DNA plasmids, each containing a specific variant or Mutation of Interest (MOI) and an Internal Quality Marker (IQM).
T96723 776832-776944 Sentence denotes Both reference materials were included in their respective routine clinical NGS pipelines over an 8-week period.
T33116 776945-777137 Sentence denotes Six of the seven laboratories ran a target amplicon based assay, and four of these laboratories used the Ion Torrent PGM instrument, whereas the other two laboratories used the Illumina MiSeq.
T12728 777138-777220 Sentence denotes One laboratory used a target hybrid capture based assay on the Illumina NexSeq500.
T34638 777221-777364 Sentence denotes Five out of six NGS panels used by laboratories during this study covered all 26 variants present in Seraseq AF10 and AF20 reference materials.
T78086 777365-777373 Sentence denotes Results:
T14676 777374-777517 Sentence denotes Both reference materials generated reproducible results when performed by different operators, using different sequencing panels and platforms.
T35775 777518-777758 Sentence denotes Although the laboratories identified most of MOI and their corresponding IQM present in the reference materials, some discrepancies involving either the MOIs (MiSeq platform only) or IQMs (MiSeq and Ion Torrent PGM platforms) were observed.
T19106 777759-777895 Sentence denotes No significant difference was observed between expected and observed allelic frequency in the Seraseq AF10 and AF20 reference materials.
T20772 777896-778073 Sentence denotes When comparing allelic frequency in each variant type (deletion, insertion, SNVs in homopolymer regions, and SNVs) for each laboratory, no significant differences were observed.
T41796 778074-778158 Sentence denotes Allelic frequency was also used to indicate reagent stability over the study period.
T693 778159-778170 Sentence denotes Conclusion:
T50427 778171-778454 Sentence denotes The Seraseq AF10 and AF20 reference materials showed characteristics of an ideal quality control material (high DNA quality, high stability, high flexibility, and genomic complexity) that could be used during validation and routine clinical sequencing processes of NGS cancer panels.
T39104 778455-778468 Sentence denotes Introduction:
T420 778469-778575 Sentence denotes Fusion proteins created by gene rearrangements are common in cancer and are often attractive drug targets.
T39013 778576-778714 Sentence denotes However, the clinical detection of gene rearrangements can be challenging, particularly if performed via next-generation sequencing (NGS).
T76960 778715-778857 Sentence denotes This becomes even more difficult in cases of complex or noncanonical rearrangement events and requires sophisticated bioinformatic approaches.
T29006 778858-779070 Sentence denotes Here we demonstrate a clinical case of a difficult to detect EML4-ALK fusion in which the genomic breakpoint occurred within exon 20 of ALK and the resulting fusion transcript included intronic sequence fromEML4.
T69146 779071-779079 Sentence denotes Methods:
T52331 779080-779178 Sentence denotes The rearrangement was initially detected by break-apart fluorescence in-situ hybridization (FISH).
T95318 779179-779286 Sentence denotes Anchored multiplex PCR (AMP) was performed on total nucleic acid via the Archer FusionPlex Solid Tumor kit.
T93197 779287-779440 Sentence denotes Two separate bioinformatic approaches were used for analysis of AMP NGS data: an alignment based fusion detection method and a de novo assembly strategy.
T21533 779441-779570 Sentence denotes The de novo assembly method took advantage of the anchored nature of the AMP assay to allow for anchored clustering and assembly.
T40216 779571-779644 Sentence denotes Findings from the AMP assay were confirmed via reverse-transcriptase PCR.
T7455 779645-779716 Sentence denotes The genomic breakpoint was confirmed via a DNA-based capture NGS assay.
T88199 779717-779725 Sentence denotes Results:
T66913 779726-779924 Sentence denotes The ALK FISH-positive lung cancer patient responded to crizotinib and then developed the well characterized ALK F1174C resistance mutation, confirming the presence of a productive ALK rearrangement.
T17000 779925-780011 Sentence denotes Tumor material from the patient was subsequently used for validation of the AMP assay.
T39069 780012-780101 Sentence denotes Initial attempts to detect the fusion using the alignment-based method were unsuccessful.
T68887 780102-780376 Sentence denotes However, upon adoption of the updated analysis algorithm that employed de novo assembly prior to annotation, the fusion transcript was detected, and it was revealed that the ALK breakpoint occurred within exon 20 and that the transcript included intronic sequence from EML4.
T4565 780377-780499 Sentence denotes The de novo assembly was required because the two breakpoints could not be detected solely via the alignment-based method.
T69265 780500-780713 Sentence denotes Additionally, the intron retention event created a large insertion between the two exonic regions, which was difficult to capture in a single read but could easily be assembled from a collection of anchored reads.
T58169 780714-780787 Sentence denotes The complex transcript and exonic breakpoint were orthogonally confirmed.
T12423 780788-780896 Sentence denotes Conclusions: NGS is becoming a platform of choice for detection of gene rearrangements in many laboratories.
T29044 780897-781011 Sentence denotes However, complex and non-canonical rearrangement events present additional challenges for bioinformatic detection.
T47101 781012-781161 Sentence denotes Here we report that de novo assembly of sequence reads is critical for the detection of fusion transcripts that do not conform to canonical patterns.
T47805 781162-781166 Sentence denotes L.Z.
T37168 781167-781178 Sentence denotes Hong 1 , L.
T98108 781179-781190 Sentence denotes Zhou 2 , R.
T38553 781191-781201 Sentence denotes Zou 2 , A.
T1522 781202-781213 Sentence denotes Chen 1 , S.
T38383 781214-781225 Sentence denotes Shih 1 , C.
T20624 781226-781295 Sentence denotes Chin 1 1 Merck Sharp & Dohme, Singapore; 2 MiRXES Pte Ltd, Singapore.
T31000 781296-781472 Sentence denotes Introduction: microRNA (miRNA) profiling in biofluids has the potential for identifying biomarkers that are informative for early diagnosis or predictive of treatment response.
T41219 781473-781672 Sentence denotes However, quantifying miRNA levels in biofluids is technically challenging due to their low abundance, the small size of mature miRNAs, and the high degree of sequence homology between family members.
T94074 781673-781843 Sentence denotes Using a set of reference samples, we describe a systematic evaluation on which miRNA profiling was performed using three different platforms: qPCR, NanoString, miRNA-Seq.
T62331 781844-781906 Sentence denotes Methods: RNA extraction from 200 μl of a Reference Serum (Ref.
T23426 781907-781980 Sentence denotes Serum) sample was performed using the miRNeasy Serum/Plasma Kit (Qiagen).
T35788 781981-782262 Sentence denotes FirstChoice Human Brain Reference RNA (Thermo Fisher Scientific) was used as a QC sample. miRNA profiling was performed on the MiRXES qPCR platform by splitting the RNA sample for reverse transcription using pools of gene-specific primers, followed by multiplex cDNA amplification.
T64244 782263-782353 Sentence denotes Amplified cDNA was used as template for gene-specific quantitation in a single-plex assay.
T14662 782354-782464 Sentence denotes The expression level of each miRNA was interpolated from a standard curve generated from a synthetic sequence.
T42760 782465-782567 Sentence denotes NanoString analysis was performed using the nCounter Human v3 miRNA Expression Assay Kit (NanoString).
T29543 782568-782859 Sentence denotes Raw counts were background subtracted using the negative controls, normalized to the positive controls, and normalized to the top 100 genes. miRNA-Seq library preparation was performed using the TruSeq Small RNA Library Prep Kit (Illumina), followed by 1 x 40 bp sequencing on a NextSeq 500.
T53406 782860-782969 Sentence denotes Within each sample, expression levels for each miRNA were normalized to reads per million mapped reads (RPM).
T64697 782970-782978 Sentence denotes Results:
T96209 782979-782986 Sentence denotes In Ref.
T41229 782987-783187 Sentence denotes Serum, the qPCR and miRNA-Seq platforms had almost perfect concordance between runs (concordance correlation coefficient, ccc = 0.99) but the NanoString platform had moderate concordance (ccc = 0.82).
T82275 783188-783402 Sentence denotes There were significant differences in the number of miRNAs detected above the lower limit of quantification (LLOQ)-NanoString only detected 84 miRNAs but qPCR and miRNA-Seq detected 440 and 372 miRNAs respectively.
T26983 783403-783459 Sentence denotes Further, 591 miRNAs were detected above the LLOQ in Ref.
T53392 783460-783553 Sentence denotes Serum by at least one platform, but only 48 miRNAs (8%) were detected by all three platforms.
T68398 783554-783728 Sentence denotes The highest inter-platform correlation was observed between miRNA-Seq and qPCR (r = 0.69), followed by NanoString and qPCR (r = 0.46) and miRNA-Seq and NanoString (r = 0.17).
T58186 783729-783869 Sentence denotes Fourteen novel miRNAs detected by miRNA-Seq were validated using qPCR and their expression levels were measured in six different cell lines.
T88278 783870-783882 Sentence denotes Conclusions:
T2248 783883-784088 Sentence denotes Our results suggest that using miRNA-Seq for discovery and targeted qPCR for validation is a rational strategy for miRNA biomarker development in clinical samples that involve limited amounts of biofluids.
T43914 784089-784102 Sentence denotes Introduction:
T66705 784103-784272 Sentence denotes It is known that due to adverse effects of formalin fixation and paraffin embedding on DNA quality, FFPE specimens are often not compatible with molecular genetic tests.
T78207 784273-784452 Sentence denotes Nevertheless, FFPE-derived DNA has been reliably used for analysing gene copy number status in a plethora of Multiplex Ligation-dependent Probe Amplification (MLPA)-based studies.
T62409 784453-784601 Sentence denotes MLPA probes target relatively short sequences of up to 100 base-pairs and thereby formalin treatment-induced DNA fragmentation does not hinder MLPA.
T61250 784602-784735 Sentence denotes However, formalin-induced DNA crosslinking and base modifications can affect MLPA if not eliminated or reduced during DNA extraction.
T28112 784736-784952 Sentence denotes To date, there has been no detailed investigation on the effects of tissue fixation conditions and DNA extraction methods on MLPA, so this study focused on selecting the most optimal pre-analytic conditions for MLPA.
T65072 784953-784961 Sentence denotes Methods:
T22770 784962-785154 Sentence denotes To compare tissue fixation conditions, healthy colon tissue was fixed in 1) buffered or nonbuffered formalin for 2) one hour, 12h to 24h or 48h to 60h; 3) at 4 o C or at room temperature (RT).
T30615 785155-785258 Sentence denotes DNA extracted from differently fixed and paraffin-embedded tissues was used for MLPA by two probemixes.
T26643 785259-785550 Sentence denotes To compare DNA extraction methods, four commercial kits (RecoverAll Total Nucleic Acid Isolation, QIAamp DNA FFPE tissue, Zymo Research FFPE DNA miniprep, WaxFree DNA extraction kit) and one in-house method were used to extract genomic DNA from eight different FFPE extraction in triplicate.
T55321 785551-785607 Sentence denotes The data were normalised against commercial genomic DNA.
T17988 785608-785728 Sentence denotes Results: MLPA results were greatly influenced by FFPE tissue fixation conditions, DNA extraction method and tissue type.
T17131 785729-785856 Sentence denotes MLPA results with lowest variability were obtained on DNA derived from tissues fixed for 12h to 24h in buffered formalin at RT.
T48771 785857-786170 Sentence denotes For most tissues, in-house DNA extraction method produced DNA with the lowest number of MLPA probes (1% to 27%) deviating from the normal copy number ratio range in all tissues, followed by Zymo Research FFPE DNA miniprep (1% to 41%), WaxFree DNA extraction (1% to 71%) and QIAamp DNA FFPE Tissue kit (9% to 71%).
T63962 786171-786366 Sentence denotes Furthermore, in-house FFPE DNA extraction method was shown to perform as efficient or superior to other methods in suitability for MLPA, DNA yield, time-, cost-efficiency and ease of performance.
T156 786367-786379 Sentence denotes Conclusions:
T90879 786380-786456 Sentence denotes The recovery of DNA from clinical FFPE tissue samples is a challenging task.
T87823 786457-786641 Sentence denotes As the fixation conditions and extraction method impact copy number quantification, it is critical to use the same pre-analytic conditions for all samples, including reference samples.
T76351 786642-786770 Sentence denotes Our study shows that FFPE-derived DNA using optimal tissue fixation and DNA extraction methods is well-suited for MLPA analysis.
T10698 786771-786916 Sentence denotes The Journal of Molecular Diagnostics ■ jmd.amjpathol.org HPV Assay can be used with a Pap specimen to assess the presence of high risk HPV types.
T94954 786917-786968 Sentence denotes This study compares results of 3 HPV PCR platforms:
T65200 786969-787084 Sentence denotes Genomica microarray Hybrid Capture 2 GeneXpert, and a nucleic-acid hybridization assay, for a total of 25 patients.
T23006 787085-787157 Sentence denotes Methods: CLART HPV2 Genomica detects single infections or co-infections.
T99560 787158-787349 Sentence denotes It genotypes 35 HPV types, including the High and Low Risk based on the amplification of specific fragments of the viral genome and their hybridization with specific probes for each HPV type.
T57836 787350-787471 Sentence denotes It is based on the extraction of HPV DNA from swabs, cell suspensions and FFPE tissues, Followed by amplification of DNA.
T53654 787472-787529 Sentence denotes Then, the amplified product is visualized on CLART-strip.
T32074 787530-787687 Sentence denotes The detection is performed by means of a low-density microarray platform: CLART which consists of a microarray printed at the bottom of the microtiter plate.
T17413 787688-787795 Sentence denotes The Xpert HPV Assay for detection and differentiation of HPV, is performed on Cepheid GeneXpert Instrument.
T22633 787796-787938 Sentence denotes The System automate sample processing, nucleic acid amplification, and detection of the target sequences in cervical samples by real-time PCR.
T85258 787939-788021 Sentence denotes A disposable GeneXpert cartridges holds the reagents and carry out the processing.
T37419 788022-788177 Sentence denotes It contains primers and probe for the detection of HPV16 and HPV 18/45 in two distinct detection channels, and 11 other high risk types in a pooled result.
T91400 788178-788306 Sentence denotes Hybrid Capture 2 is a signal amplification nucleic acid hybridization assay that utilizes microplate chemiluminescent detection.
T41332 788307-788338 Sentence denotes It targets high risk HPV types.
T93191 788339-788399 Sentence denotes DNA sample hybridize with a specific HPV RNA probe cocktail.
T37051 788400-788498 Sentence denotes The RNA:DNA hybrids are captured onto the surface of a microplate coated with specific antibodies.
T39139 788499-788663 Sentence denotes Once Immobilized the hybrids react with alkaline phosphatase conjugated specific antibodies, and then detected with a chemiluminescent substrate that emits a light.
T34262 788664-788778 Sentence denotes The results of 25 patients run using Genomica microarray and other platforms were compared using the EP evaluator.
T42427 788779-788787 Sentence denotes Results:
T11435 788788-788947 Sentence denotes GeneXpert assigned two cases as « HPV18/45 » and other high risk types, whereas Genomica specified them differently as « 51/83 » and « 31/51/66 » respectively.
T69983 788948-789056 Sentence denotes As for the other patients there was no difference between the methods with a correlation coefficient R=0.92.
T47258 789057-789068 Sentence denotes Conclusion:
T24678 789069-789251 Sentence denotes The Genomica microarray, Hybrid Capture 2, GeneXpert and the nucleic acid hybridization assay are 4 acceptable assays that can be used for the detection of HPV in cervical specimens.
T20783 789252-789265 Sentence denotes Introduction:
T8388 789266-789389 Sentence denotes As a clinical core laboratory, streamlined chemistries and processes are essential to provide efficient and timely results.
T79705 789390-789465 Sentence denotes Standardized chemistries across assays allow for easier reagent management.
T94635 789466-789642 Sentence denotes Likewise, standardized processes reduce the number of workflows that must be supported in laboratory developed software and liquid handler methods necessary to process samples.
T19755 789643-789651 Sentence denotes Methods:
T31872 789652-789797 Sentence denotes Two existing TruSeq Nano (Illumina, San Diego, CA) library preparation processes supporting three assays were consolidated into one new workflow.
T39960 789798-789893 Sentence denotes Reagent volumes were standardized and validated across assays utilizing the existing workflows.
T6888 789894-790058 Sentence denotes Additionally, custom designed universal adapters and 10 base-pair (bp) index tags were validated to allow for future reagent standardization with other chemistries.
T30415 790059-790366 Sentence denotes Updates were made to the in-house developed LIS (Laboratory Information System), to write a driver file for Biomek FX P Liquid Handlers (Beckman Coulter, Indianapolis, IN) containing variables and transfer instructions for each process step, reducing the number of necessary robot methods from 11 down to 3.
T97220 790367-790375 Sentence denotes Results:
T58317 790376-790481 Sentence denotes Standardized reagent volumes produced concordant variant call results compared to the existing workflows.
T8757 790482-790626 Sentence denotes Batching of samples into single library preparation runs can now occur, allowing more efficient use of liquid handlers and technologist efforts.
T31494 790627-790813 Sentence denotes Combination of sample processing from the two workflows results in 8 hours of saved bench time per batched run, and opens up the use of a liquid handler for other work in the laboratory.
T95021 790814-790965 Sentence denotes TruSeq Nano library preparation reagent kits come with 24 index tags, limiting the amount of samples per pool on an Illumina HiSeq or MiSeq instrument.
T95710 790966-791128 Sentence denotes Of the custom designed 10 bp index tags, 96 individual oligos were validated, resulting in a four-fold increase of the maximum number of samples allowed per pool.
T34277 791129-791321 Sentence denotes LIS updates to write Biomek driver files allowed for the reduction in the amount of methods to manage, and will also reduce the inadvertent selection of the wrong method during run processing.
T81786 791322-791334 Sentence denotes Conclusions:
T49301 791335-791496 Sentence denotes Development of streamlined chemistries and processes are necessary in a core laboratory to efficiently manage the growing number of NGS assays under development.
T50824 791497-791650 Sentence denotes Having established core processes allows for faster run processing by sample batching, and overall process and reagent management is less time consuming.
T7469 791651-791757 Sentence denotes The development and implementation of custom reagents are a powerful strategy to use in a core laboratory.
T6786 791758-792034 Sentence denotes Custom adapters and 10 bp indexes will be utilized by other library preparation methods in the laboratory, thus eliminating issues associated with particular kits and also allowing sample pooling between library preparation methods currently using different length index tags.
T26951 792035-792037 Sentence denotes B.
T58846 792038-792045 Sentence denotes Wei, M.
T58084 792046-792058 Sentence denotes Kibukawa, J.
T56626 792059-792067 Sentence denotes Kang, M.
T75391 792068-792078 Sentence denotes Marton, D.
T9909 792079-792117 Sentence denotes Levitan Merck & Co., Inc., Rahway, NJ.
T56322 792118-792319 Sentence denotes Introduction: KEYNOTE-051 is a clinical trial studying Pembrolizumab (MK-3475) in pediatric patients with advanced melanoma or advanced, relapsed, or refractory PD-L1-positive solid tumors or lymphoma.
T46822 792320-792498 Sentence denotes One of the exploratory objectives is to perform mutation analysis of 8 genes that may be associated with pediatric tumors and potentially portend differential treatment response.
T46795 792499-792507 Sentence denotes Methods:
T82784 792508-792710 Sentence denotes To maximize amplifiable DNA extracted from the often limited pediatric tumor specimens, we selected and optimized Roche High Pure RNA Paraffin method for FFPE DNA isolation over 3 other commercial kits.
T28919 792711-792882 Sentence denotes We utilized NEBNext FFPE DNA Repair Mix to remove false positive calls in poor quality DNA caused by cytosine deamination, and matrix effect such as melanin contamination.
T91931 792883-793044 Sentence denotes A custom hotspot mutation panel based on Ion AmpliSeq targeted sequencing was designed to increase coverage for two challenging RB1 amplicons at 20 ng DNA input.
T2870 793045-793113 Sentence denotes For assay validation, we examined precision and analytical accuracy.
T35203 793114-793309 Sentence denotes We performed inter-run comparison starting from library preparation for precision evaluation with DNA extracted from 35 commercial pediatric tumor specimens, with 2 to 3 specimens per tumor type.
T26513 793310-793529 Sentence denotes To evaluate sensitivity and positive predictive value (PPV) for accuracy, we utilized reference material with known mutations (AcroMetrix Oncology Hotspot Control and HDx FFPE Quantitative Multiplex Reference Standard).
T92787 793530-793636 Sentence denotes We also compared the NGS results of FFPE DNA against digital PCR (dPCR) or commercial FoundationOne panel.
T7259 793637-793645 Sentence denotes Results:
T11905 793646-793841 Sentence denotes Panel-specific false positives such as indels caused by specific homopolymers and base substitutions caused by certain non-specific PCR priming were identified and eliminated to improve accuracy.
T10639 793842-794213 Sentence denotes We achieved high sensitivity and PPV (>0.98) for the AcroMetrix and HDx controls and high specificity (>0.99) for 9 HapMap DNA at 3% variant allele frequency (VAF) cutoff. dPCR confirmed the few low VAF (5-10%) somatic mutations detected in pediatric specimens and the FoundationOne panel confirmed somatic mutation calls of 4 FFPE DNA extracted from adult tumor samples.
T63486 794214-794383 Sentence denotes Although precision is excellent (100% concordance) for half of the pediatric FFPE DNA at 5% VAF cutoff, there were some low VAF false positive calls in poor quality DNA.
T14238 794384-794512 Sentence denotes To reduce the false positive calls, we decided to use consensus calls from two independent NGS libraries for mutation detection.
T76282 794513-794575 Sentence denotes Such practice greatly improved precision for all FFPE samples.
T70170 794576-794667 Sentence denotes Doubling DNA input to 40 ng removed false positives in 3 of the 7 most challenging samples.
T55995 794668-794680 Sentence denotes Conclusions:
T22296 794681-794906 Sentence denotes A robust and sensitive NGS assay combined with efficient FFPE DNA extraction and repair methods was developed and validated to study mutations that may be associated with pediatric tumors and/or treatment response to MK-3475.
T33487 794907-794939 Sentence denotes School of Medicine, Lebanon, NH.
T76479 794940-795077 Sentence denotes Introduction: DNA quantification is an important and necessary step for most DNA analyses, particularly next-generation sequencing (NGS).
T74814 795078-795184 Sentence denotes Amplicon-based library preparation offers the powerful option of sequencing only a targeted pool of genes.
T5312 795185-795364 Sentence denotes FFPE-preserved tissue presents a technical challenge to reproducible DNA extraction of sufficient quality for NGS due to chemical modification of the DNA during tissue processing.
T43299 795365-795480 Sentence denotes Standard DNA isolation procedures often result in low DNA yield or poor performance in downstream PCR applications.
T5703 795481-795639 Sentence denotes As the CGAT laboratory processes many variable FFPE tissues, the DNA concentrations can greatly vary, introducing the need for a broad range detection method.
T56022 795640-795756 Sentence denotes The scarcity of some samples requires an accurate DNA concentration while using as little of the sample as possible.
T97190 795757-795879 Sentence denotes The aim of this study is to determine which DNA quantification method allows for the most successful library construction.
T26605 795880-795888 Sentence denotes Methods:
T87686 795889-796084 Sentence denotes Genomic DNA was extracted from 60 FFPE tissues from patients using a DNA Extraction Column kit (QIAamp DNA FFPE Kit; QIAGEN) in the automated QIACube according to the manufacturer's instructions.
T69949 796085-796153 Sentence denotes The tissue included melanoma, glioma, lung, colon and breast tumors.
T17650 796154-796398 Sentence denotes The DNA concentrations were determined spectrophotometrically (Nanodrop), fluorometrically (PicoGreen, Qubit HS and Qubit Broad Range) and by amplification of a short, conserved region in the human genome (KAPA hgDNA Quantification and QC Kit).
T39023 796399-796464 Sentence denotes Each sample was used with the five methods of DNA quantification.
T91194 796465-796612 Sentence denotes Libraries were generated using Life Technology's Ion AmpliSeq Cancer Hotspot Panel v2 and quantified using the Ion Library TaqMan Quantitation Kit.
T71020 796613-796621 Sentence denotes Results:
T31059 796622-796714 Sentence denotes The Nanodrop consistently overestimated the DNA concentration compared to the other methods.
T14240 796715-796873 Sentence denotes The three methods using DNA binding dyes gave similar DNA concentrations with a good correlation between the library quantification and the DNA concentration.
T59709 796874-796972 Sentence denotes It is imperative that the determined concentration is not outside the range of the standard curve.
T64101 796973-797212 Sentence denotes The qPCR-based method gave in most cases similar DNA quantification as the DNA binding method, with the advantage of also measuring DNA quality, reflecting the ability of PCR amplification, necessary for amplicon-based library preparation.
T95869 797213-797306 Sentence denotes This method showed the best correlation between DNA concentration and library quantification.
T72597 797307-797473 Sentence denotes Appropriate dilution of the samples is necessary to obtain accurate DNA concentration, requiring the need to have an estimated DNA concentration range of the samples.
T71664 797474-797485 Sentence denotes Conclusion:
T71354 797486-797670 Sentence denotes For NGS purposes, the Qubit/High Sensitivity in junction with the KAPA hgDNA Quantification and QC Kit gave optimal results to obtain the expected quantity and high quality of library.
T541 797671-797733 Sentence denotes jmd.amjpathol.org ■ The Journal of Molecular Diagnostics TT14.
T49602 797734-797866 Sentence denotes Evaluation of the Qiagen GeneReader NGS System for the Detection and Reporting of Clinically Actionable Mutations in Solid Tumors S.
T3629 797867-797869 Sentence denotes A.
T79206 797870-797882 Sentence denotes Turner, H.S.
T50048 797883-797908 Sentence denotes Jung, F.B. de Abreu, J.D.
T91060 797909-797923 Sentence denotes Peterson, G.J.
T43294 797924-798041 Sentence denotes Tsongalis Dartmouth Hitchcock Medical Center, Norris Cotton Cancer Center and Geisel School of Medicine, Lebanon, NH.
T66160 798042-798055 Sentence denotes Introduction:
T61023 798056-798203 Sentence denotes The identification of actionable mutations has resulted in novel treatments and improved outcomes for various solid and hematological malignancies.
T86974 798204-798381 Sentence denotes Although the use of next-generation sequencing (NGS) in the clinical laboratory has continued to expand, there is a growing need for integrated wet bench and analytic pipelines.
T1050 798382-798652 Sentence denotes The Qiagen GeneReader NGS System and the Qiagen Actionable Tumor Panel (ATP) has been designed as such, from semi-automated FFPE extraction to integrated bioinformatics and clinical insight, potentially allowing for wider clinical adoption of NGS solid tumor sequencing.
T56291 798653-798799 Sentence denotes Here, we compared the Qiagen GeneReader System (ATP) with our current clinical pipeline using the IonTorrent PGM (Ampliseq Tumor Hotspot Panelv2).
T20868 798800-798808 Sentence denotes Methods:
T14058 798809-798985 Sentence denotes Fifty (31 unique, 19 duplicate) FFPE samples from lung, colon, melanoma, and GIST tumors were extracted, sequenced, and analyzed on the IonTorrent PGM and the Qiagen GR system.
T40581 798986-799183 Sentence denotes The clinical pipeline for the Ampliseq Cancer Hotspot Panelv2 (50-gene) has been published and includes 500X coverage for 10 samples on a 318 Chip with a custom GoldenHelix bioinformatics pipeline.
T35047 799184-799382 Sentence denotes Sample preparation and sequencing on the GeneReader NGS system was performed according to the manufacturer's instructions using the hotspot Actionable Tumor Panel (12-gene) and 10 samples/ flowcell.
T99741 799383-799578 Sentence denotes Bioinformatics and clinical interpretation were performed using the included Qiagen Clinical Insight-Analyze (QCI-A) software package and Qiagen Clinical Insight-Interpret (QCI-I) online service.
T12586 799579-799587 Sentence denotes Results:
T56805 799588-799697 Sentence denotes The stock analysis for the Qiagen panel focuses on the detection of 773 unique variant positions in 12 genes.
T46159 799698-799798 Sentence denotes While additional bases are sequenced, this comparison was limited to clinically actionable variants.
T10026 799799-799913 Sentence denotes Of the 50 samples only one failed to meet established QCI-A quality metrics and was removed from further analysis.
T64055 799914-800063 Sentence denotes We noted high concordance (100%, 30/30 unique samples) across the actionable variants reported by QCI-I in comparison to those identified on the PGM.
T66489 800064-800156 Sentence denotes We also noted high reproducibility of the Qiagen GR System within duplicate samples (19/19).
T43086 800157-800302 Sentence denotes Greater than 99% of variant positions were covered at over 500X with KRAS p.A146 covered at >200 but <500X in 12 samples across 4 of 5 flowcells.
T75263 800303-800315 Sentence denotes Conclusions:
T46260 800316-800497 Sentence denotes The Qiagen GeneReader System is a fully-integrated NGS platform that offers the ability to identify a number of clinically relevant variants for clinical molecular oncology testing.
T27612 800498-800790 Sentence denotes The inclusion of a bioinformatics and clinical interpretation pipeline provides the user with up-to-date clinical information required in making clinical observations without the need for establishing additional in-house knowledge bases and maintaining additional analytics software packages.
T15911 800791-800804 Sentence denotes Introduction:
T32941 800805-800974 Sentence denotes Liquid biopsies are easier to collect and potentially offer a more comprehensive picture of the mutational landscape for more advanced solid tumors than tissue biopsies.
T12797 800975-801147 Sentence denotes However, the abundance of overall cell-free DNA (cfDNA) in these samples is generally low, ranging from less than 1ng to 30ng/mL of cfDNA in blood from healthy individuals.
T94576 801148-801340 Sentence denotes Unfortunately, this material is generally low abundance and cancer cell derived DNA (ctDNA) contributes a very small percentage to the overall material, except in more advanced disease states.
T52238 801341-801410 Sentence denotes Additionally, ctDNA is typically highly fragmented down to 100-300bp.
T26891 801411-801611 Sentence denotes Therefore, NGS-based assays to detect variants in ctDNA must be sensitive enough to detect low-frequency mutations (allele fractions <2%) from low mass inputs (10-100ng) of highly fragmented material.
T77117 801612-801620 Sentence denotes Methods:
T21298 801621-801784 Sentence denotes We modified our Anchored Multiplex PCR (AMP) method for DNA library construction to specifically enrich for low molecular weight ctDNA over larger cfDNA fragments.
T73217 801785-801936 Sentence denotes AMP is a target enrichment strategy for NGS that ligates molecular barcoded adapters to DNA fragments to generate random start sites for multiplex PCR.
T89168 801937-802059 Sentence denotes AMP is well suited to amplify small ctDNA fragments, as it only requires one intact primer-binding site within a fragment.
T64430 802060-802154 Sentence denotes Additionally, our method allows for capture of target regions from both strands independently.
T71030 802155-802320 Sentence denotes Through the use of the molecular barcoded adapters and a specific library purification step, we are able to precisely quantify the number of sequenced DNA molecules.
T68657 802321-802529 Sentence denotes Our ctDNA assay contains primers to enable NGS-based coverage of >3.4kb of many of the most common oncogenic driver mutations, acquired cancer drug resistance mutations, as well as full exon coverage of TP53.
T37271 802530-802538 Sentence denotes Results:
T84447 802539-802672 Sentence denotes Here, we describe our AMP-based ctDNA library preparation method, highlighting the use and advantages of molecular barcoded adapters.
T51468 802673-802831 Sentence denotes Molecular barcodes are used to unambiguously identify PCR duplicates, correct for PCR or sequencer-derived sequence errors, and flag run-to-run contamination.
T4369 802832-803054 Sentence denotes Using a set of reference ctDNA standards, we demonstrate that these benefits of molecular barcodes enabled confident variant detection at allele frequencies ~0.5% with as little as 50ng of input DNA and less than 4M reads.
T62627 803055-803178 Sentence denotes Furthermore, our ctDNA profiling assay demonstrated a similar power to call variants in ctDNA derived from liquid biopsies.
T82659 803179-803191 Sentence denotes Conclusions:
T13203 803192-803382 Sentence denotes We demonstrate that our modified AMP-based library construction method for ctDNA mutational profiling is a powerful tool for sensitive detection of variants in model ctDNA reference samples.
T82097 803383-803490 Sentence denotes The advantages observed with reference materials will likely be applicable to clinically derived specimens.
T64056 803491-803496 Sentence denotes TT16.
T38674 803497-803615 Sentence denotes Detection and Quantitation of Circulating Tumor BRAF Mutant DNA Using the BioRad Digital Droplet PCR (ddPCR) Method D.
T34304 803616-803631 Sentence denotes Milosevic, M.B.
T8550 803632-803643 Sentence denotes Campion, N.
T97483 803644-803661 Sentence denotes Vidal Folch, J.R.
T98944 803662-803673 Sentence denotes Mills, K.C.
T59110 803674-803687 Sentence denotes Halling, M.C.
T23600 803688-803697 Sentence denotes Liu, E.W.
T14406 803698-803713 Sentence denotes Highsmith, B.R.
T43530 803714-803724 Sentence denotes Kipp, J.S.
T71926 803725-803735 Sentence denotes Voss, E.L.
T94948 803736-803749 Sentence denotes Enninga, L.A.
T7204 803750-803766 Sentence denotes Kottschade, S.N.
T92870 803767-803781 Sentence denotes Markovic, S.K.
T47798 803782-803830 Sentence denotes Grebe Mayo Clinic and Foundation, Rochester, MN.
T4639 803831-803844 Sentence denotes Introduction:
T42657 803845-803953 Sentence denotes Defining BRAF status of melanoma patients is critical for targeted treatment decisions with BRAF inhibitors.
T99885 803954-804055 Sentence denotes Liquid biopsies provide a less invasive alternative to tissue biopsy for theranostics and monitoring.
T51287 804056-804212 Sentence denotes In this study we evaluated the performance characteristics of a quantitative multiplex assay for V600E and V600K assay using BioRad's ddPCR instrumentation.
T50477 804213-804221 Sentence denotes Methods:
T74144 804222-804354 Sentence denotes Blood samples were collected in Streck Cell-Free DNA BCT tubes from 20 healthy individuals and 47 patients with metastatic melanoma.
T7400 804355-804435 Sentence denotes DNA was extracted from plasma using Qiagen's QIAmp Circulating Nucleic Acid kit.
T26929 804436-804564 Sentence denotes Simultaneous quantitation of the wild-type and mutant BRAF V600E/K was performed using AutoDG and QX200 multi well plate system.
T27611 804565-804573 Sentence denotes Results:
T91502 804574-804802 Sentence denotes Intra-and inter-assay precision were assessed using 3 pools of BRAF V600E and V600K mutation-positive cell lines and varying percentages of mutant versus wild-type DNA (1% to 10% mutant in a background of wild-type sheared DNA).
T50718 804803-804915 Sentence denotes There was 100% concordance with expected results for the fractional abundance, with a copy number CV of 4.3-18%.
T27271 804916-805049 Sentence denotes Analytical sensitivity experiments showed a limit of detection of 1% mutant in 3ng of DNA input and 0.1% mutant in 30ng of DNA input.
T90639 805050-805155 Sentence denotes Serial dilutions of both V600E and V600K mutants were linear down to 1% mutant in 5ng of total DNA input.
T66031 805156-805290 Sentence denotes Detection specificity, assessed by testing 20 normal plasma DNA samples showed only 1 false positive droplet with the multiplex assay.
T20898 805291-805412 Sentence denotes No mutations were found in 27 patient plasma samples with negative BRAF tumor tissue testing (100% clinical specificity).
T7947 805413-805651 Sentence denotes Out of 20 patients with positive BRAF tumor tissue testing for V600E or K mutations, 9 patients were found to have the identical mutation in plasma (47% clinical sensitivity) whereas 11 patients were found to have wildtype BRAF in plasma.
T95960 805652-805767 Sentence denotes This could be attributed to delayed time between biopsy and blood sampling or low overall tumor burden.Conclusions:
T97149 805768-805869 Sentence denotes We developed a quantitative multiplex BRAF V600E/K assay for detection of circulating BRAF mutations.
T39076 805870-805967 Sentence denotes It is a fast, reliable and accurate method with a total analytical time of approximately 3 hours.
T16069 805968-806266 Sentence denotes Our findings indicate that monitoring melanoma patients' plasma for BRAF V600E/K with the BioRad ddPCR system could be an effective strategy for the detection of BRAF mutations and could allow early detection of disease recurrence which might obviate the need for BRAF analysis of tissue specimens.
T49040 806267-806360 Sentence denotes Additionally, this testing might play a role in assessing patient response to drug treatment.
T98946 806361-806374 Sentence denotes Introduction:
T93297 806375-806540 Sentence denotes As multiple next-generation sequencing (NGS) platforms become part of mainstream clinical testing, regulatory agencies require monitoring of various quality metrics.
T67013 806541-806653 Sentence denotes One important metric is the reproducibility of variant allele frequencies detected in control samples over time.
T15458 806654-806768 Sentence denotes We hereby describe our experience with inter-run and inter-platform control variant frequency as a quality metric.
T70521 806769-806777 Sentence denotes Methods:
T62080 806778-806964 Sentence denotes We performed retrospective analysis of sequencing data using the Ion Ampliseq Cancer Hotspot Panel V2 (Life Technologies) obtained from 6 different control samples over the past 2 years.
T27243 806965-807153 Sentence denotes Each control was sequenced on a MiSeq (Illumina, San Diego, CA) as the primary analysis method and on the Ion Torrent PGM (ThermoFisher Scientific, Waltham, MA) as the confirmatory method.
T90019 807154-807296 Sentence denotes The mutant allele frequencies from all expected variants from each control were compared run to run and between the MiSeq and PGM instruments.
T60812 807297-807304 Sentence denotes Result:
T12934 807305-807458 Sentence denotes Variant frequencies across the six different controls showed excellent correlation between the MiSeq and Ion Torrent sequencers with an R² value of 0.98.
T54069 807459-807718 Sentence denotes When comparing the run to run variant frequencies of the 104 variants across all controls, the MiSeq had an average standard deviation of 0.9% and coefficient of variation (CV) of 3.2%, whereas the PGM had an average standard deviation of 1.7% and CV of 7.0%.
T22790 807719-807912 Sentence denotes HRAS:c.81T>C and TP53:c.215C>G showed the highest variability with an average standard deviation of 1.1% and 0.8% respectively for the MiSeq, and 4.1% and 4.6% respectively for the Ion Torrent.
T95440 807913-808102 Sentence denotes This difference is likely due to the variants being in a homopolymer region and the nature of the Ion Torrent sequencer has more difficulty distinguishing individual bases in these regions.
T68749 808103-808114 Sentence denotes Conclusion:
T35860 808115-808248 Sentence denotes Overall, the MiSeq produces consistent variant frequencies that were slightly better than the Ion Torrent PGM over the 2 year period.
T24115 808249-808347 Sentence denotes In addition, there was excellent correlation of the variant frequencies between the MiSeq and PGM.
T42577 808348-808526 Sentence denotes These results suggest that these sequencing instruments produce reproducible and robust variant allele frequency calls across many different runs over an extended period of time.
T24821 808527-808529 Sentence denotes M.
T59695 808530-808550 Sentence denotes Vaglio Tessitore, A.
T13939 808551-808562 Sentence denotes Sottini, F.
T69660 808563-808573 Sentence denotes Serana, C.
T21466 808574-808585 Sentence denotes Ghidini, A.
T41732 808586-808595 Sentence denotes Sacco, L.
T79075 808596-808651 Sentence denotes Imberti ASST Spedali Civili di Brescia, Brescia, Italy.
T23612 808652-808665 Sentence denotes Introduction:
T12019 808666-808862 Sentence denotes Dried blood (DB) samples spotted on filter paper are a convenient alternative to fresh blood specimens when collection and shipment of biological materials are difficult or used for large surveys.
T30539 808863-809062 Sentence denotes However, the low volume of total DB could be a limiting factor in quantitative assays especially when a large representation of rare targets (i.e., a few copies/μL) is required like, for example, for
T4735 809063-809151 Sentence denotes The Journal of Molecular Diagnostics ■ jmd.amjpathol.org TRECs and KRECs quantification.
T39260 809152-809323 Sentence denotes The aim of this study was to validate if FLOQSwabs (COPAN, Brescia, Italy) can be used as an alternative for storing larger dry blood volumes for an adequate DNA recovery.
T83989 809324-809332 Sentence denotes Methods:
T34827 809333-809437 Sentence denotes Healthy donor blood samples, collected into EDTA tubes, were absorbed for 10 to 60 seconds on FLOQSwabs.
T41575 809438-809631 Sentence denotes Swabs were immediately placed back into their plastic tubes if equipped with active drying system or, alternatively, left to dry for 2 hours or overnight, and then stored back into their tubes.
T8962 809632-809720 Sentence denotes Blood was also placed on COPAN NUCLEIC-CARDS TM or directly subjected to DNA extraction.
T24720 809721-809977 Sentence denotes DNA from FLOQSwabs was extracted using an in-house protocol (cell lysis with ATL buffer, protein digestion overnight with proteinase K at 56°C, protein precipitation with ammonium acetate 7.5 M; DNA precipitated with isopropanol and resuspended in TE pH8).
T77748 809978-810106 Sentence denotes DNA from 300 μL of blood and from six 2-mm punch discs of DB (corresponding to 8.4 μL) was extracted using the Gentra blood kit.
T83529 810107-810175 Sentence denotes DNA concentration was estimated with the Nanodrop spectrophotometer.
T5995 810176-810322 Sentence denotes Percentage of recovery was obtained after calculating the expected DNA using the white blood cell count and an average DNA content of 6.6 pg/cell.
T49842 810323-810351 Sentence denotes Data were analyzed by ANOVA.
T30054 810352-810444 Sentence denotes Results: FLOQSwabs immersed in blood achieved full absorption and reached a plateau in 10 s.
T63758 810445-810725 Sentence denotes The average volume of absorbed blood, which was indirectly estimated by quantifying tube weight prior swab introduction and after to to 132 ng to -CARDS percentage of recovery (i.e., proportion of extracted over expected DNA) did not significantly differ in FLOQSwabs 69% (95% CI:
T70472 810726-810806 Sentence denotes 60% to 78%) compared to cards 74% (62% to 85%) and blood 60% (49% to 70%, p=ns).
T50173 810807-810891 Sentence denotes Intra-and inter-assay CV for swabs, calculated in 26 replicates, were 13.1% (95% CI:
T2556 810892-810947 Sentence denotes 9.9% to 19.4%) and 13.6% (5.7% to 36.7%), respectively.
T40255 810948-811049 Sentence denotes Conclusions: DB collected and stored on FLOQSwabs is a highly reliable and inexpensive source of DNA.
T25054 811050-811241 Sentence denotes This can be a valid alternative to cards when more DNA is needed to detect rare targets together with a practical solution for shipping and storing blood samples for TRECs and KRECs analysis.
T4991 811242-811255 Sentence denotes Introduction:
T39794 811256-811389 Sentence denotes Detecting mutations is becoming important for both predicting disease progression and drug responses to treatment of cancer patients.
T54236 811390-811587 Sentence denotes Current mutation detection methods for cancer diagnosis are mainly based on the invasive sampling technique such as a tissue biopsy, but some patients may not available for this invasive procedure.
T20698 811588-811676 Sentence denotes Therefore, circulating tumor DNA (ctDNA) would be a good alternative for those patients.
T9400 811677-811799 Sentence denotes However, testing methods for tissue biopsy sample are not applicable to ctDNA samples due to relatively lower sensitivity.
T3498 811800-811893 Sentence denotes A highly sensitive assay method is required for detecting mutations in liquid biopsy samples.
T28058 811894-811902 Sentence denotes Methods:
T95732 811903-812016 Sentence denotes We have developed a highly sensitive and simple method to detect somatic mutation from ctDNA in patient's plasma.
T69948 812017-812137 Sentence denotes This new real-time PCR-based testing method (PANAMutyper) has maximized unique properties of peptide nucleic acid (PNA).
T48767 812138-812214 Sentence denotes It contains a PNA clamp and PNA detection probes in the each reaction tubes.
T60880 812215-812344 Sentence denotes An optimized PNA clamp can tightly bind to only wild-type DNA sequences, and then suppress amplification during the PCR reaction.
T4311 812345-812538 Sentence denotes Meanwhile, a PNA detection probe that conjugated with a fluorescent dye and a quencher, can detect a specific target mutant-type DNA and each mutation can be genotyped by melting peak analysis.
T56215 812539-812662 Sentence denotes PANAMutyper is able to detect 29 different mutations in exon 2, 3 and 4 of KRAS gene with detection limits as low as 0.01%.
T51345 812663-812768 Sentence denotes Results: KRAS G12D, G12V, and G13D mutations are the most abundant mutations in human colorectal cancers.
T58002 812769-812850 Sentence denotes These mutations were tested to demonstrate performance of our new testing method.
T20623 812851-813041 Sentence denotes Each standard DNA was mixed with gDNA from HeLa cells (which have the wild-type KRAS gene) at the decreasing ratios, 1:1, 1:10, 1:10 2 , 1:10 3 , 1:10 4 , 2:10 4 , and 1:10 5 , respectively.
T67466 813042-813134 Sentence denotes The results showed 0.005% sensitivity in G12D, G12V and 0.01% sensitivity in G13D mutations.
T29820 813135-813238 Sentence denotes Also, we conducted spiking test using G12D and G12V standard DNA whether it is valid in real condition.
T43790 813239-813322 Sentence denotes Each DNA mixed with human plasma to 0.5, 5, 50 copies per microliter concentration.
T26582 813323-813372 Sentence denotes And then we extracted ctDNA from prepared sample.
T58324 813373-813433 Sentence denotes As a result, all of standard DNA was detected at 0.5 copy/ .
T60089 813434-813554 Sentence denotes Theses result suggest that our new method is highsensitive and applicable to ctDNA derived from patient's plasma sample.
T35498 813555-813567 Sentence denotes Conclusions:
T23749 813568-813695 Sentence denotes Therefore, PANAMutyper can be used in various clinical areas including personalized medicine and monitoring acquired mutations.
T46683 813696-813700 Sentence denotes J.D.
T56509 813701-813711 Sentence denotes Haimes, J.
T98516 813712-813722 Sentence denotes Covino, N.
T71027 813723-813732 Sentence denotes Manoj, E.
T93417 813733-813744 Sentence denotes Baravik, L.
T21970 813745-813756 Sentence denotes Johnson, L.
T2431 813757-813768 Sentence denotes Griffin, J.
T35812 813769-813780 Sentence denotes Stahl, B.P.
T26370 813781-813791 Sentence denotes Culver, B.
T2079 813792-813827 Sentence denotes Kudlow ArcherDX, Inc., Boulder, CO.
T8488 813828-813841 Sentence denotes Introduction:
T45065 813842-813973 Sentence denotes Copy number variants (CNVs) are oncogenic drivers, and impact more of the cancer genome than all other types of mutations combined.
T90358 813974-814102 Sentence denotes Genomic analysis using next-generation sequencing (NGS) has shown promise as a method to detect CNVs from clinical sample types.
T14951 814103-814370 Sentence denotes However, archival storage of formalin-fixed, paraffin-embedded (FFPE) specimens severely damages DNA by inducing proteinnucleic acid crosslinking, base modifications and strand cleavage, which results in poor sequencing coverage and limited CNV detection sensitivity.
T96774 814371-814496 Sentence denotes Therefore, NGSbased detection of low-level CNVs (<3-fold) and CNVs in samples with low tumor cellularity remains challenging.
T97760 814497-814673 Sentence denotes Anchored Multiplex PCR (AMP) is a target enrichment strategy for NGS that enables digital read counting in low-quality samples, resulting in enhanced CNV detection sensitivity.
T54885 814674-814682 Sentence denotes Methods:
T99204 814683-814878 Sentence denotes Based on AMP chemistry, we developed Archer VariantPlex targeted DNA enrichment assays for NGS to detect both low-and high-level CNVs from FFPE specimens and other lowinput clinical sample types.
T48463 814879-815009 Sentence denotes We used the VariantPlex Solid Tumor kit, which contains AMP primers designed to detect CNVs for 43 cancer-associated genes by NGS.
T82880 815010-815172 Sentence denotes To rapidly screen samples prior to library preparation and sequencing, we also developed the Archer PreSeq DNA QC Assay to determine the integrity of genomic DNA.
T14659 815173-815181 Sentence denotes Results:
T73230 815182-815323 Sentence denotes We examined over 150 FFPE tumor samples for genomic DNA integrity and CNV detection using the PreSeq and VariantPlex assays for targeted NGS.
T18851 815324-815520 Sentence denotes Our data show that NGS-based detection sensitivity is driven primarily by the integrity of the input genomic DNA, determined by the PreSeq Assay, which further predicts the limit of CNV detection.
T28124 815521-815703 Sentence denotes Using optimal input amounts of genomic DNA, we detected CNVs as low as 2-fold in FFPE samples and in samples with as low as 3% tumor cellularity with the VariantPlex Solid Tumor kit.
T3631 815704-815716 Sentence denotes Conclusions:
T12994 815717-815903 Sentence denotes These results demonstrate that Archer VariantPlex assays enable sensitive NGS-based detection of low-level CNVs from low-input clinical samples and in samples with low tumor cellularity.
T53341 815904-816076 Sentence denotes Furthermore, the PreSeq DNA QC Assay allows for the adjustment of DNA input to provide adequate genomic copies into the NGS workflow and maximize CNV detection sensitivity.
T81524 816077-816090 Sentence denotes Introduction:
T51997 816091-816201 Sentence denotes Short Tandem Repeat assay is a major method for monitoring chimerism status after bone marrow transplantation.
T65163 816202-816327 Sentence denotes We prepared a limit of detection (LOD) control by mixing 5% genomic DNA of one male with 95% of that from one female subject.
T67525 816328-816381 Sentence denotes LOD is tested in each run along with patient samples.
T53012 816382-816462 Sentence denotes 262 runs from past 2 years were analyzed with our laboratory developed software.
T48410 816463-816543 Sentence denotes Data indicates the difference among the markers is much greater than among runs.
T45014 816544-816631 Sentence denotes Result suggests an improvement in the donor percentage calculation algorithm is needed.
T54489 816632-816640 Sentence denotes Methods:
T77302 816641-816674 Sentence denotes PowerPlex 16 HS System (Promega).
T37136 816675-816778 Sentence denotes Genomic DNA from one male and one female subject were mixed by different ratio (2% to 98% of male DNA).
T72216 816779-816877 Sentence denotes LOD control is made by mixing 5% male as donor and 95% female as recipient, and used in every run.
T36438 816878-816946 Sentence denotes Laboratory developed software used to calculate percentage of donor.
T54841 816947-816955 Sentence denotes Results:
T35062 816956-816973 Sentence denotes 1) Makers tested:
T49668 816974-817207 Sentence denotes 16 markers are tested by the reagent kit, 14 of them are informative for the DNA mixture (D3S1358, D21S11, D18S51, PentaE, D5S818, D7S820, D16S539, CSF1PO, PentaD, AMEL, vWA, D8S1179, TPOX, FGA), 2 are noninformative (TH01, D13S317).
T58955 817208-817336 Sentence denotes 2) Reliability of the assay: a) Linearity test of mixed DNA samples (2% to 98%, 9 test points) shows good linearity of the test.
T61590 817337-817482 Sentence denotes Observed value vs. expected value is x=1.0024y, R 2 = 0.9952. b) Five % mixed DNA is used as LOD and 262 runs were conducted since June 17, 2014.
T86877 817483-817540 Sentence denotes 100% of the tests show positive result close to 5% donor.
T89457 817541-817654 Sentence denotes Average percent calculated with all informative markers (14) is 6.89% (stdev = 1.30%, Max = 10.93%, Min = 3.19%).
T31732 817655-817699 Sentence denotes 3) Variation is significant between markers:
T67178 817700-817842 Sentence denotes Single factor ANOVA shows the variation among markers is much greater than among different runs (F = 44.039, F critical = 1.729, p= 1.7E-105).
T97887 817843-817876 Sentence denotes 4) Markers performed differently:
T310 817877-817942 Sentence denotes Some markers show larger variant range around median than others.
T27131 817943-817955 Sentence denotes Conclusions:
T4783 817956-818047 Sentence denotes 1) PowerPlex 16 HS System shows good linearity of percentage value in the reportable range.
T40250 818048-818129 Sentence denotes 2) Average percent of donor of LOD control sample is close to the expected value.
T1285 818130-818287 Sentence denotes 3) Variation among markers is much greater than among runs, suggests the variation may be due to the nature of each marker, rather than effects between runs.
T13772 818288-818357 Sentence denotes Some markers are more vulnerable to effects between runs than others.
T11591 818358-818603 Sentence denotes 4) Improvement of the algorithm for calculating percentage is needed and in process, which will utilize the fact that each marker is amplified differently by adding a weight to each marker and set individual control matrix range for each marker.
T93762 818604-818608 Sentence denotes I.S.
T19289 818609-818618 Sentence denotes Haque, C.
T46894 818619-818632 Sentence denotes Haverty, J.D.
T70965 818633-818647 Sentence denotes Goldberg, E.A.
T98257 818648-818687 Sentence denotes Evans Counsyl, South San Francisco, CA.
T60093 818688-818701 Sentence denotes Introduction:
T37657 818702-818873 Sentence denotes Although non-invasive prenatal screening (NIPS) for fetal aneuploidy has high sensitivity and specificity, prevalence varies significantly by maternal and gestational age.
T71031 818874-819000 Sentence denotes Variable prevalence affects the probability that a positive test indicates an affected fetus (positive predictive value, PPV).
T84312 819001-819208 Sentence denotes Although ACOG and SMFM direct laboratories to report PPV individualized to the particular patient, no previous work has addressed how uncertain PPV calculations are or to what precision PPV can be estimated.
T47020 819209-819217 Sentence denotes Methods:
T58400 819218-819312 Sentence denotes We introduce a statistical framework to estimate the confidence interval (CI) around NIPS PPV.
T40547 819313-819518 Sentence denotes We estimate the uncertainty in NIPS analytical performance by fitting beta distributions to the 95% CIs on test sensitivity (SENS) and specificity (SPEC) from large meta-analyses (Taylor-Phillips S, et al.
T20719 819519-819526 Sentence denotes 2016) .
T50562 819527-819645 Sentence denotes We sample the posterior distribution of aneuploidy prevalence by using original population data (Hecht CA and Hook HB.
T49735 819646-819672 Sentence denotes 1994; Snijders RJM, et al.
T99645 819673-819702 Sentence denotes 1999; Morris JK and Savva GM.
T63521 819703-819725 Sentence denotes 2008; Savva GM, et al.
T78887 819726-819814 Sentence denotes 2010) to sample from beta distributions or bootstrap Kaplan-Meier curves as appropriate.
T33856 819815-819913 Sentence denotes We estimate the portion of the CI arising from each source by holding individual parameters fixed.
T37430 819914-820009 Sentence denotes With this method, we evaluate the 95% CI of NIPS PPV for trisomies 13, 18, and 21 (T13/18/21) .
T50911 820010-820172 Sentence denotes Sampling all parameters shows that CI breadth mostly varies by maternal age (MA): for example, the CI width for T21 is +/-11% at MA=20yr down to +/-4% at MA=40yr.
T96909 820173-820371 Sentence denotes Low mean PPV for T13 and T18 leads to asymmetric CIs: the T13 PPV CI at MA=20yr and gestational age (GA) 12 weeks has a lower bound 13% below the mean of 20.8% but an upper bound 23% above the mean.
T295 820372-820540 Sentence denotes All CIs are dominated by SPEC uncertainty: at MA=20yr, the T21 CI is +/-10.5% when sampling only over SPEC, +/-4% using only prevalence, and +/-0.1% sampling only SENS.
T43409 820541-820636 Sentence denotes We repeated the analysis using SENS and SPEC from a smaller (N=3,322) study (Porreco RP, et al.
T63621 820637-820644 Sentence denotes 2014) .
T10255 820645-820843 Sentence denotes As expected, at MA=20yr GA=12wk the mean T21 PPVs using parameters from the meta-analysis or the smaller study are similar, but the CI using the meta-analysis is much tighter (+/-12.6% vs +/-32.9%).
T14298 820844-820856 Sentence denotes Conclusions:
T25658 820857-821178 Sentence denotes Our results show that larger meta-analyses of NIPS specificity could narrow the PPV confidence interval two-fold for T21 or fourfold for T13 and T18 (motivating outcome data sharing among laboratories), but that additional gestational prevalence data will then be needed to further improve the precision of PPV estimates.
T63902 821179-821253 Sentence denotes The utility of providing PPV is well-justified even with the estimated CI.
T32439 821254-821401 Sentence denotes This work further demonstrates the need to use large meta-analyses to establish sensitivity and specificity rather than smaller individual studies.
T82448 821402-821404 Sentence denotes A.
T48532 821405-821418 Sentence denotes Robertson, X.
T10212 821419-821429 Sentence denotes Wang, G.M.
T60354 821430-821441 Sentence denotes Gould, J.R.
T81770 821442-821453 Sentence denotes Maguire, H.
T54465 821454-821462 Sentence denotes Kang, I.
T53648 821463-821474 Sentence denotes Haque, E.A.
T69288 821475-821514 Sentence denotes Evans Counsyl, South San Francisco, CA.
T14153 821515-821528 Sentence denotes Introduction:
T18467 821529-821634 Sentence denotes Historically, variant genotyping tests have been validated by concordance with known reference materials.
T20881 821635-821835 Sentence denotes However, for NGS-panel tests detecting any variant in the genes of interest, this strategy is not effective as positive controls for variants that are difficult to detect may not be readily available.
T42208 821836-822039 Sentence denotes Although large numbers of SNPs in genes of interest are available from reference materials, indels (especially large indels) and CNVs are quite sparse or unavailable for most clinically relevant targets.
T53690 822040-822196 Sentence denotes Thus to comprehensively assess the performance of an entire region of interest, in silico methods must be used to supplement reference material comparisons.
T49328 822197-822286 Sentence denotes We present two novel in silico techniques for validating the capability to call variants:
T14963 822287-822452 Sentence denotes 1) simulations to assess the ability to detect copy number variants (CNVs) and 2) a genomic reference editor to assess the ability to detect large or complex indels.
T90274 822453-822461 Sentence denotes Methods:
T92637 822462-822626 Sentence denotes The first technique, simulating CNVs, is designed to simulate realistic data with a particular CNV using a model based on the experimental performance of the assay.
T45891 822627-822724 Sentence denotes Then the simulated data can be analyzed via a bioinformatic pipeline to assess assay performance.
T6179 822725-822870 Sentence denotes The second technique, reference editing, takes advantage of the fact that sequence variants are expressed as a deviation from a reference genome.
T82363 822871-823090 Sentence denotes One makes a modification to the sequence of the reference genome used for variant calling, runs a negative control sample through the full assay and expects to call the inverse of the modification made to the reference.
T39438 823091-823290 Sentence denotes For example, it is possible to insert a 40bp sequence into the reference and re-run the bioinformatics pipeline on a negative sample to determine if the pipeline can detect a simulated 40bp deletion.
T73126 823291-823299 Sentence denotes Results:
T50619 823300-823502 Sentence denotes Using reference editing, we find that off-the-shelf NGS analysis toolchains typically fail to detect large (50bp to 150bp) deletions, and designed a specific variant caller designed to address this gap.
T37437 823503-823644 Sentence denotes As a demonstration, we applied both reference editing and CNV simulation to the development of a NGS assay (Counsyl Inherited Cancer Screen).
T5194 823645-823801 Sentence denotes In particular, CNV simulation led to a refinement of the initial assay design from the baseline design suggested by sequencing reference biological samples.
T12497 823802-823949 Sentence denotes This allowed us to maintain >99.7% CNV sensitivity while reducing our sample retest rate and improving our sensitivity in identified noisy regions.
T69087 823950-823962 Sentence denotes Conclusions:
T78815 823963-824207 Sentence denotes In cases where informative biological samples are not always available, these tools are valuable for validating and improving the quality of a clinical NGS pipeline to provide accurate results to patients and better inform medical management. .
T76372 824208-824351 Sentence denotes Each genotype-specific PNA probe, which is conjugated with a fluorescent dye and a quencher, is used as a reporter in a real-time PCR reaction.
T55421 824352-824456 Sentence denotes PNA probe has the length shorter than the DNA probe and Tm for each single mismatch is a big difference.
T66293 824457-824567 Sentence denotes Due to the above features, PNA probe is useful for multiplex-PCR to detect the target with many variant genes.
T46092 824568-824576 Sentence denotes Results:
T3000 824577-824661 Sentence denotes Test was performed to obtain DNA from 58 samples cultured in a MacConkey agar plate.
T10297 824662-824771 Sentence denotes After a comparison with the culture results, showed a concordance of 93.1% in 54 samples of 58 samples match.
T76584 824772-824885 Sentence denotes For mismatch four samples confirmed through sequencing PCR, the result of the PANA RealTyper CRE kit was correct.
T17812 824886-825010 Sentence denotes In addition, this was also found to be capable of detecting strain obtained from trypticase soy broth (TSB) cultured sample.
T45707 825011-825140 Sentence denotes Conclusion: PANA RealTyper CRE kit is easily and -lactamase gene, and it is expected to help in the inhospital infection control.
T90689 825141-825143 Sentence denotes B.
T48501 825144-825150 Sentence denotes Wu, A.
T10526 825151-825163 Sentence denotes McCarthy, E.
T13028 825164-825173 Sentence denotes Craig, W.
T50128 825174-825185 Sentence denotes Tahaney, A.
T25356 825186-825197 Sentence denotes Edmunds, S.
T37254 825198-825249 Sentence denotes Brahmasandra NeuMoDx Molecular Inc., Ann Arbor, MI.
T9199 825250-825263 Sentence denotes Introduction:
T5941 825264-825405 Sentence denotes Widespread use of nucleic acid based testing (NAT) for diagnostics has resulted in the routine use of PCR in molecular diagnostic (MDx) labs.
T1312 825406-825527 Sentence denotes Reagents for such PCR assays have typically required the need for cold or frozen storage to maintain long term viability.
T80592 825528-825771 Sentence denotes The absolute need for cold temperature storage offers substantial challenges to the implementation of NAT in resource limited settings as well as complicates the development of next-generation MDx instrumentation with onboard reagents storage.
T44795 825772-825882 Sentence denotes Development of ambient stable NAT reagents is expected to significantly alleviate these traditional drawbacks.
T69139 825883-826037 Sentence denotes Traditionally, lyophilization or freeze-drying has been the method of choice for producing room temperature stable active reagents such as Taq polymerase.
T8043 826038-826208 Sentence denotes However, lyophilization requires expensive specialized equipment & reagents, is time intensive, and is practically impossible to implement in a continuous mode operation.
T82129 826209-826387 Sentence denotes NeuMoDx has developed a simple, cost effective, high-throughput, and rapid drying procedure capable of producing highly effective NAT reagents using a conventional conveyer oven.
T74408 826388-826396 Sentence denotes Methods:
T13229 826397-826500 Sentence denotes Three types of molecular diagnostic reagents were dried using unique formulations of select excipients:
T71387 826501-826647 Sentence denotes 1) Extraction reagents including magnetic particles with a nucleic acid affinity matrix, lytic enzymes, and encapsulated DNA/RNA process controls.
T12724 826648-826688 Sentence denotes 2) PCR reagents, and 3) RT-PCR reagents.
T72874 826689-826826 Sentence denotes Magnetic particle based DNA/RNA isolation was performed in a variety of clinical matrices using these ambient stable extraction reagents.
T49766 826827-826968 Sentence denotes At the end of the isolation step, the eluted nucleic acids were used directly to reconstitute dried PCR or RT-PCR mix and qPCR was performed.
T27142 826969-827116 Sentence denotes The efficacy of these dried reagents were evaluated by assessing critical analytical performance metrics such as limit of detection, linearity etc.
T66200 827117-827257 Sentence denotes Accelerated aging studies were also performed with all three dried reagent formulations to assess the long term stability of these reagents.
T54860 827258-827266 Sentence denotes Results:
T349 827267-827438 Sentence denotes The NeuMoDx ambient temperature stable reagents performed equivalently or better than the wet controls based on PCR Ct values as well as increase in fluorescent intensity.
T73697 827439-827698 Sentence denotes Although multiple excipient formulations were found to provide Ct values comparable to wet controls, only select combinations of the excipients were capable of producing sigmoidal amplification curves with fluorescence increase comparable to the wet controls.
T22010 827699-827712 Sentence denotes Introduction:
T15447 827713-827888 Sentence denotes Formalin-fixed, paraffin-embedded (FFPE) tissue samples are valuable resources for retrospective and prospective molecular analysis in both the clinical and research settings.
T36032 827889-828093 Sentence denotes Extraction of nucleic acid from FFPE samples is a critical initial step in any molecular analysis; however, many commonly used extraction methods produce inconsistent results in both quantity and quality.
T63197 828094-828193 Sentence denotes There are two major types of nucleic acid extraction methods, column-based and magnetic bead-based.
T10187 828194-828288 Sentence denotes Magnetic bead-based purification is more amenable to automation and high-throughput processes.
T23550 828289-828472 Sentence denotes As part of overall lab automation efforts, we decided to explore other commercial kits for high-throughput sample processing, and compared to our previous column-based extraction kit.
T47711 828473-828527 Sentence denotes Our laboratory assessed several methods of extraction.
T42616 828528-828596 Sentence denotes The following characteristics of each kit and method were evaluated:
T30414 828597-828888 Sentence denotes 1) availability of automated protocol, or ease of development of such a protocol; 2) ability to simultaneously extract DNA and RNA in separate eluates within a single prep; 3) flexible range for sample input amount requirement; 4) minimal upfront manual preparation time; 5) reproducibility.
T94676 828889-828897 Sentence denotes Methods:
T61857 828898-828934 Sentence denotes Three extraction kits were assessed:
T89491 828935-829054 Sentence denotes Thermo Fisher Scientific MagMAX FFPE DNA/RNA Ultra kit, Qiagen AllPrep FFPE DNA/RNA, and Covaris truXTRAC FFPE DNA/RNA.
T4976 829055-829271 Sentence denotes Other commercially available extraction kits on the market were not included in the assessment because the end-product was total nucleic acid which required the addition of DNase or RNase to obtain separate analytes.
T68329 829272-829346 Sentence denotes We assessed each kit's performance with the requirement listed previously.
T53921 829347-829470 Sentence denotes FFPE samples were tested in duplicate or triplicate with Qiagen AllPrep FFPE DNA/RNA and the MagMAX FFPE DNA/RNA Ultra kit.
T72734 829471-829479 Sentence denotes Results:
T92782 829480-829680 Sentence denotes Although Covaris truXTRAC FFPE DNA/RNA gave sufficient yield, the upfront manual preparation time was longer than the other kits, making this protocol unsuitable for high-throughput sample processing.
T52067 829681-829756 Sentence denotes Inter-and intra-technician testing was performed and results were compared.
T72082 829757-829938 Sentence denotes Extracted DNA and RNA were assessed on the Nanodrop (UV spectrophotometry), Qubit (fluorescence-based quantitation), QuantStudio (qPCR), and Bioanalyzer (capillary electrophoresis).
T20980 829939-829951 Sentence denotes Conclusions:
T17385 829952-830064 Sentence denotes Both Qiagen and MagMAX produced comparable and acceptable quality metrics across all tests for both DNA and RNA.
T29667 830065-830258 Sentence denotes However, for high-throughput sample processing, the MagMAX FFPE DNA/RNA Ultra kit's protocol has met all of our necessary requirements and can be integrated with liquid handling for automation.
T4027 830259-830261 Sentence denotes H.
T72459 830262-830271 Sentence denotes Leong, E.
T88100 830272-830285 Sentence denotes Schreiber, S.
T32685 830286-830297 Sentence denotes Berosik, S.
T60052 830298-830306 Sentence denotes Chen, W.
T71000 830307-830317 Sentence denotes George, A.
T70276 830318-830330 Sentence denotes Gerstner, J.
T251 830331-830340 Sentence denotes Marks, S.
T62966 830341-830401 Sentence denotes Schneider Thermo Fisher Scientific, South San Francisco, CA.
T44986 830402-830415 Sentence denotes Introduction:
T75364 830416-830514 Sentence denotes Detecting minor genetic variants has become essential to cancer and infectious disease management.
T8392 830515-830709 Sentence denotes Many have turned to next-generation sequencing to fill this need given the common perception that Sanger sequencing can reliably detect genetic variants at allelic proportions no lower than 25%.
T49987 830710-830848 Sentence denotes We discovered a way to reduce this reference-based detection limit to 5% and have demonstrated detection at even lower allele frequencies.
T55682 830849-830857 Sentence denotes Methods:
T72550 830858-830905 Sentence denotes The discovery is a two-part software algorithm.
T95447 830906-830981 Sentence denotes The first part minimizes the noise that underlies Sanger sequencing traces.
T27930 830982-831054 Sentence denotes The second part detects variants, if any, in the noise-minimized traces.
T35277 831055-831177 Sentence denotes For noise minimization, a model of the noise is made from reference traces and subtracted from the traces of the unknowns.
T90785 831178-831377 Sentence denotes For variant detection, information from the noise-minimized traces of the forward and reverse sequencing reactions is combined to distinguish spurious peaks from those truly associated with variants.
T74438 831378-831537 Sentence denotes The performance of the algorithm was measured on samples from 22 amplicons involving eight different genes: TP53, KRAS, BRAF, EGFR, FLT3, RB1, CDH1, and ERBB2.
T68687 831538-831613 Sentence denotes Many of these were extracted from formalin-fixed, paraffinembedded samples.
T49899 831614-831775 Sentence denotes Some were commercially available reference standards, others were quantified using the RNase-P quantitative polymerase chain reaction assay and serially diluted.
T21971 831776-831819 Sentence denotes Allelic proportions spanned 0.6125% to 50%.
T50336 831820-831987 Sentence denotes These samples were amplified, sequenced, and pre-processed using standard protocols and tools for fluorescent dye terminator Sanger sequencing from Applied Biosystems.
T17965 831988-831996 Sentence denotes Results:
T83588 831997-832239 Sentence denotes A sensitivity of 95.9% and specificity of 99.8% was observed for data at 5% allele frequency; there were 785 variants and 229623 non-variant base positions spanning 704 quadruplets (forward and reverse for both reference and unknown samples).
T62505 832240-832422 Sentence denotes A sensitivity of 98.8% and specificity of 99.8% was observed for data at 10% allele frequency; there were 503 variants and 163037 non-variant base positions spanning 454 quadruplets.
T2258 832423-832605 Sentence denotes Although we have been able to detect variants at allele frequencies 0.6125%, 1%, 1.25%, 2%, and 2.5%, the algorithm did not reach 95% sensitivity and 99% specificity at these levels.
T1416 832606-832618 Sentence denotes Conclusions:
T73454 832619-832760 Sentence denotes It should now be possible to achieve a reference-based limit of detection of 5% allelic proportion with standard Sanger sequencing protocols.
T42557 832761-832985 Sentence denotes Existing protocols for visually reviewing the results can also be used and are enhanced because the algorithm generates results in the form of familiar electropherograms for which the noise has been substantially diminished.
T90503 832986-833174 Sentence denotes These two features of the algorithm may give Sanger sequencing performance and/or economic advantages in some molecular diagnostic applications that require finding minor genetic variants.
T81998 833175-833331 Sentence denotes However, sensitivity and specificity of these separating technologies remain suboptimal, which hinder their widespread application to the clinical practice.
T71362 833332-833637 Sentence denotes In this study, alternating current (AC) potential modulated microfluidic strategy coupled with amperometry was developed to separate and detect small numbers of CTC from patient blood, through the decoration of the CTC surface with an anticancer drug molecule within a short period of time (<400 seconds).
T34072 833638-833646 Sentence denotes Methods:
T35707 833647-833767 Sentence denotes Blood samples were collected from various cancer patients and were blind-tested using the proposed separation technique.
T25686 833768-833920 Sentence denotes The separated cells were collected from the channel and observed under the fluorescent microscope to confirm the presence of cancer cells in the sample.
T85228 833921-834196 Sentence denotes The carbon channel walls were modified with an organic conducting polymer followed by chemical bonding of lipid molecules which provide an affinity towards drug molecules adsorbed on the CTC and allow them to move much slower than the other cells in the microfluidic channel.
T69932 834197-834344 Sentence denotes The separation process was augmented with AC potential and frequency modulations and the overall size of the CTC as compared to normal blood cells.
T7095 834345-834353 Sentence denotes Results:
T64420 834354-834464 Sentence denotes The blood samples with cancer cells showed a peak corresponding to the oxidation of daunomycin (DNM) molecule.
T73325 834465-834646 Sentence denotes Among the patient blood samples 9 out of 15 samples showed electrocatalytic responses for the oxidation of DNM at different retention times, indicating the presence of cancer cells.
T59420 834647-834750 Sentence denotes These collected samples showed the presence of cells that were rather large in size as compared to RBC.
T82826 834751-834889 Sentence denotes When compared to the results given by morphological and molecular diagnoses, we could observe that 86.6 % agreeable results were obtained.
T90394 834890-834902 Sentence denotes Conclusions:
T83889 834903-835051 Sentence denotes Current tumor diagnosis depends on a variety of pathological tests, among which tissue biopsy is considered to be the standard diagnostic procedure.
T68993 835052-835208 Sentence denotes With the recent development of various devices to enrich and detect CTC, the noninvasive screening for cancer diagnosis can assist in early stage detection.
T75934 835209-835344 Sentence denotes The potential of these new technical platforms to improve CTC detection related to the treatment stratification warrants further study.
T79468 835345-835358 Sentence denotes Introduction:
T39812 835359-835429 Sentence denotes Buccal swab has been a convenient collection device for genetic tests.
T8746 835430-835534 Sentence denotes Current practice involves a dry swab that collects DNA from the cells on the inside of a person's cheek.
T18319 835535-835772 Sentence denotes Due to different swabbing techniques of individual patients as well as potential interfering substances, a good extraction system is crucial to harvest DNA of high yield and quality to acquire accurate results and to reduce requeue rate.
T64589 835773-835882 Sentence denotes Roche MagNA Pure 96 System is a high throughput instrument that performs automated nucleic acid purification.
T69282 835883-836065 Sentence denotes The purpose of this study is to evaluate the performance of automated DNA extraction from buccal swabs using Roche MagNA Pure 96 System compared to MagMAX DNA Multi-Sample Ultra Kit.
T5573 836066-836074 Sentence denotes Methods:
T88903 836075-836172 Sentence denotes For Epicentre Catch-All Sample Collection Swabs, 60 swabs were collected with 2 swabs per person.
T61909 836173-836268 Sentence denotes One set of swabs were extracted on MagNa Pure 96 System with DNA and Viral NA Small Volume Kit.
T15786 836269-836370 Sentence denotes The other set of 30 swabs from the same person were extracted with MagMAX DNA Multi-Sample Ultra Kit.
T27912 836371-836442 Sentence denotes DNA yield of all extracted DNA were tested by Qubit dsDNA HS Assay Kit.
T40788 836443-836615 Sentence denotes Copy number variation (CNV) of CYP2D6 and single nucleotide polymorphism (SNP) genotyping assays of 17 genes were performed on all 60 samples and the results were compared.
T10792 836616-836624 Sentence denotes Results:
T36319 836625-836824 Sentence denotes Average DNA yield acquired from MagNA Pure 96 System was 18.01±22.80ng/ul, higher than the average yield of 5.91±6.53ng/ul from the swab collected from the same person extracted using the MagMAX kit.
T89340 836825-836955 Sentence denotes The yield from MagNA Pure 96 varies from 1.86ng/ul to 94.4 ng/ul, and the yield from MagMAX kit ranges from 1.4ng/ul to 29.8ng/ul.
T85082 836956-837044 Sentence denotes This variance is largely due to the inconsistent swabbing technique between individuals.
T4863 837045-837157 Sentence denotes Both CNV assay and 47 genotyping assays of 17 genes achieved 100% agreement using DNA from both extraction kits.
T16338 837158-837170 Sentence denotes Conclusions:
T61646 837171-837275 Sentence denotes Roche MagNA Pure 96 is a reliable system to perform efficient DNA extraction from buccal swab specimens.
T37104 837276-837359 Sentence denotes It recovers DNA with a higher yield and of good quality suitable for genetic tests.
T85672 837360-837373 Sentence denotes Introduction:
T33355 837374-837507 Sentence denotes Detecting mutations is becoming important for both predicting disease progression and drug responses to treatment of cancer patients.
T56449 837508-837705 Sentence denotes Current mutation detection methods for cancer diagnosis are mainly based on the invasive sampling technique such as a tissue biopsy, but some patients may not available for this invasive procedure.
T80012 837706-837794 Sentence denotes Therefore, circulating tumor DNA (ctDNA) would be a good alternative for those patients.
T45370 837795-837917 Sentence denotes However, testing methods for tissue biopsy sample are not applicable to ctDNA samples due to relatively lower sensitivity.
T57704 837918-838011 Sentence denotes A highly sensitive assay method is required for detecting mutations in liquid biopsy samples.
T27426 838012-838020 Sentence denotes Methods:
T30467 838021-838134 Sentence denotes We have developed a highly sensitive and simple method to detect somatic mutation from ctDNA in patient's plasma.
T92555 838135-838255 Sentence denotes This new real-time PCR-based testing method (PANAMutyper) has maximized unique properties of peptide nucleic acid (PNA).
T75759 838256-838332 Sentence denotes It contains a PNA clamp and PNA detection probes in the each reaction tubes.
T97078 838333-838462 Sentence denotes An optimized PNA clamp can tightly bind to only wild-type DNA sequences, and then suppress amplification during the PCR reaction.
T83545 838463-838656 Sentence denotes Meanwhile, a PNA detection probe that conjugated with a fluorescent dye and a quencher, can detect a specific target mutant-type DNA and each mutation can be genotyped by melting peak analysis.
T85696 838657-838787 Sentence denotes PANAMutyper is able to detect 47 different mutations in exon 18, 19, 20 and 21 of EGFR gene with detection limits as low as 0.01%.
T35297 838788-838796 Sentence denotes Results:
T6729 838797-838947 Sentence denotes A total of 83 plasma samples from non-small cell lung cancer (NSCLC) patients were tested to demonstrate clinical efficacy of this new testing method.
T97959 838948-839047 Sentence denotes The results showed mutanttype DNAs in 21 patients and wild-type DNA in 62 patient's plasma samples.
T75381 839048-839132 Sentence denotes These results were compared with direct sequencing data from matched tissue samples.
T77588 839133-839146 Sentence denotes Introduction:
T92926 839147-839283 Sentence denotes A substantial degree of information exists in the cancer-related literature, but systematically extracting this data can be challenging.
T48979 839284-839543 Sentence denotes The ability to rapidly query connections between molecular aberrations, relevant therapies, cancer indications, and available clinical trials requires a comprehensive database comprising a succinct view of the extant data, but in an easily retrievable format.
T60767 839544-839552 Sentence denotes Methods:
T80257 839553-839847 Sentence denotes We have built the JAX Clinical Knowledgebase (CKB), a manually curated database that houses data related to molecular variants, relevant therapeutic drugs, cancer indications, and clinical trials, as well as an application that allows one to methodically search for links between data elements.
T1841 839848-840031 Sentence denotes To demonstrate utility of CKB and provide a snapshot of the data available in CKB, a side-by-side analysis was performed between actionable genes in CKB and those genes in cBioPortal.
T75690 840032-840134 Sentence denotes All genes in CKB with actionable variants were queried in cBioPortal and then analyzed for comparison.
T70587 840135-840281 Sentence denotes The top 15 genes in CKB with the most actionable variants were compared to the top 15 genes in cBioPortal with the highest mutational frequencies.
T79437 840282-840418 Sentence denotes Additional analyses within CKB were executed to illustrate the landscape of available treatment approaches for the top actionable genes.
T59884 840419-840427 Sentence denotes Results:
T7662 840428-840634 Sentence denotes The comparison among the 15 genes with the greatest number of actionable variants in CKB and the top 15 genes in cBioPortal with the highest mutational frequencies demonstrated a concordance of 73% (11/15).
T82377 840635-840804 Sentence denotes In CKB, PTEN and APC comprise the two genes with the largest number of actionable variants while in cBioPortal, TP53 and PIK3CA have the greatest mutational frequencies.
T16982 840805-840944 Sentence denotes The minimal difference between the two databases is primarily due to the frequency at which specific codons in TP53 and PIK3CA are mutated.
T42868 840945-841136 Sentence denotes Within the concordant gene list, PIK3CA demonstrates the highest number of relevant therapeutic drugs, and EGFR represents the gene with the highest number of relevant FDA-approved therapies.
T63066 841137-841149 Sentence denotes Conclusions:
T27721 841150-841337 Sentence denotes The JAX CKB is an inclusive database allowing one to retrieve pertinent information related to specific cancer genes, in a format that can be easily navigated to quickly identify utility.
T83298 841338-841480 Sentence denotes The retrieval analysis performed in this study demonstrates CKB's significant benefit to both the medical and scientific research communities.
T56776 841481-841483 Sentence denotes Y.
T87304 841484-841493 Sentence denotes Cheng, M.
T16169 841494-841537 Sentence denotes Jakubowski Cleveland Clinic, Cleveland, OH.
T80608 841538-841551 Sentence denotes Introduction:
T7993 841552-841623 Sentence denotes Lung cancer is the leading cause of cancer death in both man and women.
T98403 841624-841749 Sentence denotes The identification of critical gene mutations allows better decision making of targeted therapies in managing NSCLC patients.
T52702 841750-841885 Sentence denotes Real-time PCR and nextgeneration sequencing (NGS) have become two popular methods in detecting therapeutic actionable sequence changes.
T87070 841886-842034 Sentence denotes In this study, we explored the accuracy and specificity among NGS platforms and real-time PCR in detecting NSCLC mutations in a clinical laboratory.
T15608 842035-842043 Sentence denotes Methods:
T9649 842044-842192 Sentence denotes Genomic DNA was extracted from NSCLC formalin fixed paraffin embedded tissues and fine needle aspirate specimens collected in PreservCyt or CytoLyt.
T63059 842193-842405 Sentence denotes Targeted genomic regions in 50 oncogenes and tumor suppressor genes were multiplex amplified using Ion AmpliSeq Cancer Hotspot Panel v2 and the Ion AmpliSeq Library Kit 2.0 (ThermoFisher Scientific, Waltham, MA).
T85901 842406-842492 Sentence denotes Selected amplified samples were ligated with Illumina adaptors and sequenced on MiSeq.
T36017 842493-842617 Sentence denotes The resulted fastq files were aligned and analyzed using the NextGENe (SoftGenetics, State College, PA) bioinformatics tool.
T11882 842618-842782 Sentence denotes Only those variants, excluding common single nucleotide polymorphisms, residing in defined mutation hotspots with over 5% allele frequency were examined and scored.
T44544 842783-842928 Sentence denotes The EGFR and KRAS mutation status in the majority of these tumor samples were also determined using real-time PCR and Sanger sequencing approach.
T70619 842929-842937 Sentence denotes Results:
T42716 842938-843077 Sentence denotes Intra-and inter-assay reproducibility were determined by analyzing three specimens that possessed missense and insertion/deletion variants.
T68844 843078-843179 Sentence denotes These specimens were performed in triplicate in the same run or on three separate days, respectively.
T40261 843180-843314 Sentence denotes Inter-tech repeatability was determined by analyzing three specimens that contained missense variants by three separate technologists.
T20529 843315-843359 Sentence denotes All repeatability runs were 100% concordant.
T55005 843360-843443 Sentence denotes Additionally, among 70 previously analyzed specimens, 97% concordance was achieved.
T71173 843444-843515 Sentence denotes The clinical sensitivity and specificity is 97% and 100%, respectively.
T70698 843516-843680 Sentence denotes A multiplex DNA standard (Horizon Dx 200) containing 11 mutations at known allele concentrations, ranging from 1 % to 24.5% allele frequencies was tested six times.
T4100 843681-843786 Sentence denotes Six representative variants with stated allele proportions ranging from 3-25% were consistently detected.
T87967 843787-843799 Sentence denotes Conclusions:
T3841 843800-843915 Sentence denotes In general, the majority of gene mutations were consistently identified among various testing platforms being used.
T62145 843916-844067 Sentence denotes Our data indicate that a small discordance of testing results may be contributed from low mutation allelic frequencies and complex mutation situations.
T2781 844068-844165 Sentence denotes Examples of these cases and the cost effectiveness of applicable diagnostic assays are discussed.
T6457 844166-844179 Sentence denotes Introduction:
T57046 844180-844306 Sentence denotes It is difficult to identify causal agent of respiratory viral infections because of similarity of clinical signs and symptoms.
T71474 844307-844430 Sentence denotes Accurate identification of virus pathogen helps physician with presuming infection route and selecting effective treatment.
T89033 844431-844555 Sentence denotes Recently commercialized DNA probe-based multiplex real-time PCR methods were increased the accuracy of virus identification.
T51219 844556-844750 Sentence denotes However, this DNA probe-based technology required relatively longer probe sequence for target binding and it has its limitation that cannot distinguish sequence variations on target virus genes.
T72241 844751-844759 Sentence denotes Methods:
T19711 844760-844848 Sentence denotes A new peptide nucleic acid (PNA)-assisted melting curve analysis technique is developed.
T2228 844849-844992 Sentence denotes Each genotype-specific PNA probe, which is conjugated with a fluorescent dye and a quencher, is used as a reporter in a real-time PCR reaction.
T32645 844993-845133 Sentence denotes A PNA probe can design relatively shorter binding sequence than its DNA probe, so PNA probe can avoid sequence variation position on a gene.
T10161 845134-845271 Sentence denotes Furthermore, PNA probe showed bigger melting temperature difference than DNA probe when reporter probe bound to a single mismatch target.
T85138 845272-845367 Sentence denotes So, sequence variants on a target gene are easily distinguishable using melting curve analysis.
T85771 845368-845523 Sentence denotes Therefore, PNA-based reporter probe is very useful for multiplex detection in real-time PCR platform to identify a target gene with many sequence variants.
T7658 845524-845532 Sentence denotes Results:
T53348 845533-845631 Sentence denotes We have developed a simple and rapid method for detecting specific virus genes within three hours.
T37775 845632-845918 Sentence denotes This real-time PCR-based PNA probe-assisted fluorescent melting curve analysis assay (PANA RealTyper RV kit] can detect a total of 25 different virus genotypes (Influenza A, B, PIV1,2,3,4, RSV A, RSV B, AD, CoV-229E, CoV-OC43, CoV-HKU1, CoV-NL63, CoV-MERS, MPV, BoV, PAN-CoV, ENV, RHI).
T72354 845919-846040 Sentence denotes A total of 70 nasopharyngeal flocked swabs samples were tested to demonstrate clinical efficacy of this new assay method.
T64358 846041-846190 Sentence denotes Then, a test result of PNA-based method was compared to a result of DNAbased real-time PCR test that were performed independently by the third party.
T31774 846191-846281 Sentence denotes The results showed 87.1% concordance rate between two test methods (61 out of 70 samples).
T3137 846282-846471 Sentence denotes Meanwhile, nine discordant samples were confirmed by direct sequencing method, and results of sequencing for nine samples were agreed with the results of new PNA-based real-time PCR method.
T48315 846472-846583 Sentence denotes Conclusions: PANA RealTyper RV kit is simple and reliable test method for detecting pandemic respiratory virus.
T7287 846584-846706 Sentence denotes This new method offers a cost-effective and highly sensitive genotyping method that can be used in various clinical areas.
T34283 846707-846765 Sentence denotes GmbH, Hilden, Germany; 3 Medical University Graz, Austria.
T91805 846766-846779 Sentence denotes Introduction:
T29679 846780-846919 Sentence denotes One major goal of cancer clinical research is to establish new prognostic and predictive biomarkers for personalized diagnosis and therapy.
T98569 846920-847102 Sentence denotes Next-Generation Sequencing (NGS) has revolutionized the field of cancer genetic studies by dramatically decreasing costs and time needed for large-scale data generation and analysis.
T88496 847103-847421 Sentence denotes Although NGS has proven to be a very useful tool in research, major hurdles remain for its broad adoption in the clinical research setting: lack of a seamless workflow, ability to handle samples from different sources or pre-analytical treatments, and lack of actionable content to guide the interpretation of results.
T10198 847422-847553 Sentence denotes Here, we apply a complete sample-to-insight NGS workflow, QIAGEN´s GeneReader System, to analyze a diverse set of clinical samples.
T53785 847554-847562 Sentence denotes Methods:
T22525 847563-847709 Sentence denotes Tissue samples from different tumor types (colon or melanoma) were subject to fixation by formalin or the PAXgene system and embedded in paraffin.
T24410 847710-847773 Sentence denotes Donor-matched plasma samples were also analyzed when available.
T14861 847774-847966 Sentence denotes Sample DNA was processed and analyzed accordingly to the GeneReader Workflow: DNA was extracted using the GeneRead FFPE kit and the PreAnalytix PFPE Kit for PAXgene fixed tissue, respectively.
T42881 847967-848036 Sentence denotes Plasma DNA was isolated with the QIAamp Circulating Nucleic Acid Kit.
T61682 848037-848125 Sentence denotes The quality was multiply assessed with the QIAxpert, the QIAxcel and the QuantiMIZE Kit.
T82905 848126-848224 Sentence denotes Targeted amplification of cancer hotspots was performed using the Actionable Insights Tumor Panel.
T40203 848225-848367 Sentence denotes After sequencing with the GeneReader, variants were identified using the QCI-Analyze software and interpreted with the QCI-Interpret software.
T49104 848368-848376 Sentence denotes Results:
T50100 848377-848476 Sentence denotes Despite different preanalytical treatments, all samples showed high quality scores in the QC steps.
T38998 848477-848573 Sentence denotes This was followed by successful amplification of target regions and all the subsequent workflow.
T48897 848574-848715 Sentence denotes For all samples (n=20) more than 99% of the sequence reads were mapped to the human genome (hg19) and > 90% to the regions of interest (ROI).
T73235 848716-848830 Sentence denotes The majority of the samples (19 out of 20) showed more than 95% of the ROI covered with at least 500 single reads.
T51627 848831-849015 Sentence denotes All variants were correctly identified and interpreted, showing the robustness of the GeneReader System and its flexibility and reliability handling different sample types and sources.
T61606 849016-849028 Sentence denotes Conclusions:
T73504 849029-849283 Sentence denotes In clinical research, the development of new prognostic and predictive biomarkers is often dependent on samples from biobanks and prospective cohorts which often suffer from suboptimal conditions such as long storage period and various treatment methods.
T36880 849284-849503 Sentence denotes The data presented show the advantages of a fully integrated sample-to-insight workflow including initial quality control steps and prove the versatility of the GeneReader System for different types of sample materials.
T51166 849504-849526 Sentence denotes For research use only.
T33103 849527-849564 Sentence denotes Not for use in diagnostic procedures.
T75696 849565-849578 Sentence denotes Introduction:
T87407 849579-849700 Sentence denotes Cell-free DNA (cfDNA) controls for next-generation sequencing (NGS) are often derived from patient blood sample remnants.
T25733 849701-849946 Sentence denotes With the expansion of plasma DNA diagnostics, sufficient volumes of patient-derived materials that contain a wide number of target analytes for oncology and non-invasive prenatal screening (NIPS) assays are simply not available to make controls.
T20950 849947-850085 Sentence denotes Therefore we have formulated suitable stabilized biomimetic materials derived from fragmented genomic DNA (gDNA) and synthetic constructs.
T52609 850086-850202 Sentence denotes Stabilization is essential because fragmented DNA is less stable in blood and blood-like matrices than native cfDNA.
T38765 850203-850378 Sentence denotes Several different biomimetic formulations for NIPS aneuploidy controls (Trisomy 13, 18 and 21) were tested for stability by NGS at two different temperatures over nine months.
T48260 850379-850387 Sentence denotes Methods:
T3527 850388-850495 Sentence denotes Encapsulated-stabilized DNA formulations for NIPS were prepared by mixing normal gDNA with aneuploidy gDNA.
T54295 850496-850647 Sentence denotes The mixture was verified by dPCR for copy number and then sheared using Covaris ultrasonic shearing before incorporation into a stabilizing biomimetic.
T7750 850648-850718 Sentence denotes Final samples are blended into modified MatriBase, a synthetic plasma.
T31749 850719-850779 Sentence denotes Samples were stored at 4°C or 42°C for 1, 4, 6 and 9 months.
T74805 850780-850852 Sentence denotes Testing of the samples was performed using a Verinata-derived NGS assay.
T20068 850853-850861 Sentence denotes Results:
T72681 850862-850984 Sentence denotes Stressed stability studies were conducted comparing encapsulated DNA and free DNA in modified MatriBase at 4 °C and 42 °C.
T90414 850985-851155 Sentence denotes The encapsulated DNA samples, using Trisomy 13 reference materials, were found to be unchanged based on NIPS assay performance after 275 days at both temperatures by NGS.
T14733 851156-851232 Sentence denotes Free DNA samples failed library preparation at 47 days at both temperatures.
T88348 851233-851476 Sentence denotes Real-time stability for a Trisomy 21 aneuploidy reference material, a multi-analyte aneuploidy reference material (including Trisomy 13, 18 and 21) and a euploid (an aneuploidy negative control) remained stable for at least six months via NGS.
T65172 851477-851489 Sentence denotes Conclusions:
T65011 851490-851672 Sentence denotes We have demonstrated the ability to combine encapsulate small DNA fragments with synthetic plasma as a general means to prepare biosynthetic reference materials for molecular assays.
T15873 851673-851761 Sentence denotes These materials were found to be viable alternatives to patient samples for NIPS assays.
T74500 851762-851942 Sentence denotes As the biomimetics are readily extractable using commercially available kits, the reference materials can be inserted into existing workflows similar to centrifuged plasma samples.
T560 851943-852116 Sentence denotes Moreover, the protection conferred by the encapsulation creates a stable, refrigerated reference material which will enable improved quality monitoring of diagnostic assays.
T53169 852117-852295 Sentence denotes Recently, exon-based enrichment strategies have been shown to be effective for not only enrichment of transcripts containing known exons, but also depletion of ribosomal content.
T26414 852296-852428 Sentence denotes This study examines how the Agilent Strand Specific RNA Library Prep kit can be used to generate cDNA libraries from FFPE total RNA.
T1990 852429-852549 Sentence denotes Enrichment was then performed on the total cDNA libraries from FFPE RNA using Agilent's V6+UTR exome DNA Enrichment kit.
T76346 852550-852712 Sentence denotes The effectiveness of this approach was examined by comparing the transcriptional data from these FFPE samples to RNASeq data from FF samples from the same source.
T20156 852713-852721 Sentence denotes Methods:
T30132 852722-852787 Sentence denotes Mouse xenografts derived from human breast tumors were collected.
T49130 852788-852906 Sentence denotes Half the material was flash frozen (FF) in OTC matrix and the other half was formalin fixed, paraffin embedded (FFPE).
T22626 852907-852973 Sentence denotes RNA from both FF and FFPE were extracted with standard procedures.
T91492 852974-853071 Sentence denotes All FF and FFPE RNA was pooled to generate the FF Xenografts (PDX) pool as well as FFPE PDX pool.
T75165 853072-853190 Sentence denotes The FFPE PDX pool was further treated to degrade the RNA so that the average size reflected typical archival FFPE RNA.
T86848 853191-853346 Sentence denotes A total of 200ng of FF RNA was processed for RNASeq according to the Agilent protocol, with poly(A) enrichment followed by cDNA synthesis and library prep.
T31292 853347-853602 Sentence denotes For capture only sample processing, 200ng of FF and FFPE RNA were processed with minor modifications (no ribosomal depletion or poly(A) enrichment, modifications to the fragmentation step as well as extending second strand/end repair and A tailing steps).
T17037 853603-853727 Sentence denotes Final libraries were sequenced on an Illumina NextSeq500 flowcell and resulting data were processed as standard RNASeq data.
T68276 853728-853736 Sentence denotes Results:
T28847 853737-853840 Sentence denotes Performing V6+UTR enrichment only on FFPE total RNA library preps was effective in depleting ribosomes.
T864 853841-853954 Sentence denotes In addition, the overall alignment statistics were similar between the FFPE V6+UTR samples and FF Poly(A) RNASeq.
T25002 853955-854083 Sentence denotes Exome captured sample was biased toward exonic reads, with little to no coverage of deep intronic regions or intergenic regions.
T4953 854084-854228 Sentence denotes A Pearson's average correlation coefficient of 0.93 between the FF poly-A enriched samples and FFPE V6+UTR enrichment only samples was observed.
T33613 854229-854362 Sentence denotes Additionally, improved uniformity of reads across transcripts from 5' to 3' was observed for the FFPE V6+UTR enrichment only samples.
T85963 854363-854375 Sentence denotes Conclusions:
T7155 854376-854618 Sentence denotes This data indicate that using the Agilent Strand Specific RNA Library Prep kit on total RNA followed by V6+UTR exome enrichment is effective for performing RNASeq analysis on FFPE samples in the absence of a separate ribosomal depletion step.
T55025 854619-854853 Sentence denotes This, along with increased sensitivity to low expressers and rare transcripts and making FFPE RNA samples candidates for transcriptome sequencing, indicate an effective approach to transcriptional profiling or challenging RNA samples.
T45562 854854-854856 Sentence denotes C.
T26157 854857-854871 Sentence denotes Bissaillon, M.
T12222 854872-854881 Sentence denotes Young, L.
T34705 854882-854892 Sentence denotes Bonomi, A.
T56620 854893-854906 Sentence denotes Tyropolis, K.
T99118 854907-854916 Sentence denotes Lebel, F.
T54450 854917-854956 Sentence denotes Moore Baystate Health, Springfield, MA.
T69711 854957-854970 Sentence denotes Introduction:
T23935 854971-855100 Sentence denotes Herpes simplex viruses (HSV), consisting of Types 1 and 2, are ubiquitous and infectious DNA viruses in the family Herpesviridae.
T61214 855101-855191 Sentence denotes Like most herpesviruses, primary infection with HSV can lead to lifelong latency in cells.
T42650 855192-855281 Sentence denotes Sporadically, they can reactivate and cause outbreaks during which infections are spread.
T99688 855282-855445 Sentence denotes Though there is no cure for latent infection, reactivation rates and decrease of symptom severity can result from treatment with antiviral drugs such as acyclovir.
T89708 855446-855508 Sentence denotes The most common HSV-induced lesions are orolabial and genital.
T51445 855509-855634 Sentence denotes Diagnostic tests for HSV can help distinguish HSV infections from many other conditions that cause similar-appearing lesions.
T485 855635-855749 Sentence denotes It is also important to distinguish Type 1 from Type 2, as the chance of reactivation is much greater with Type 2.
T92131 855750-855872 Sentence denotes The Luminex Aries Two module System (IVD) is a sample to answer system that utilizes real-time PCR performed in cassettes.
T2096 855873-856008 Sentence denotes The cassettes used in this system come preloaded with all the required reagents and internal controls for testing symptomatic patients.
T43518 856009-856017 Sentence denotes Methods:
T64085 856018-856067 Sentence denotes A total of 50 samples were used to test accuracy.
T77595 856068-856217 Sentence denotes A total of 31 residual, de-identified M4 VTM samples were tested as well as 19 Copan UTM samples obtained from Luminex for the purpose of validation.
T82850 856218-856321 Sentence denotes The 50 samples were comprised of 17 HSV 1 positive, 17 HSV 2 positive and 16 negative for both targets.
T8117 856322-856452 Sentence denotes ARIES results obtained from the M4 samples were compared to the Luminex MultiCode RTx HSV 1 & 2 real-time PCR, thermal melt assay.
T96592 856453-856546 Sentence denotes The ARIES results for the Copan UTM were compared to the expected result provided by Luminex.
T743 856547-856744 Sentence denotes The precision of the assay was evaluated by testing one positive HSV 1 sample and one positive HSV 2 sample twice per day for 10 days, between at least two technologists for a total of 40 measures.
T22514 856745-856848 Sentence denotes Five HSV 1 and 5 HSV 2 samples at 2,000 cp/ml were tested to verify the established limit of detection.
T58794 856849-856857 Sentence denotes Results:
T73687 856858-856976 Sentence denotes Correlation samples show 100% (50/50) agreement with expected results for both the Copan UTM and M4 VTM samples types.
T98779 856977-857049 Sentence denotes Precision results show 100% (40/40) concordance for both HSV1 and HSV 2.
T43152 857050-857139 Sentence denotes The limit of detection was verified as 100% (10/10) samples at 2,000 cp/ml were detected.
T8842 857140-857151 Sentence denotes Conclusion:
T24995 857152-857245 Sentence denotes The Luminex Aries system has been shown to provide accurate and precise results for HSV 1 &2.
T57126 857246-857346 Sentence denotes The instrument is easy to use, has a sample to result process, and requires very little maintenance.
T37577 857347-857360 Sentence denotes Introduction:
T81476 857361-857545 Sentence denotes Studies on circulating cell free DNA require 1) exclusion of contaminant cellular DNA, 2) uniform sampling, 3) prevention of degradation, and 4) protection from external contamination.
T53395 857546-857897 Sentence denotes We compared the performance of a novel collection tube with a barrier separating cell-free from cellular material consisting of a photopolymer solidified by UV light to 1) BD EDTA tubes, 2) BD EDTA Plasma separator tubes containing a soft gel separator, and 3) Streck Cell-Free DNA BCT tubes containing a preservative to minimize cellular degradation.
T68451 857898-857906 Sentence denotes Methods:
T6753 857907-858011 Sentence denotes Peripheral blood was collected from 10 normal subjects in duplicate Plasma Separator and Photogel tubes.
T43364 858012-858101 Sentence denotes Following low speed centrifugation, one tube of each type was kept at -80 o C for 7 days.
T39942 858102-858230 Sentence denotes Cell-free DNA was isolated from samples in the remaining tubes and in the frozen samples using a Promega RSC Maxwell instrument.
T6371 858231-858374 Sentence denotes Amounts of amplifiable DNA recovered were tested by digital PCR and amounts of contaminating genomic DNA were tested on an Agilent TapeStation.
T11270 858375-858686 Sentence denotes Peripheral blood from 10 normal subjects was also collected in triplicates in EDTA, Streck and Photogel tubes, centrifuged, and cell-free DNA was extracted either without further manipulation (aliquot #1) or following a second centrifugation at 16,000 x g with or without subsequent treatment with Proteinase K.
T99721 858687-858695 Sentence denotes Results:
T89325 858696-858893 Sentence denotes Differences in the average amount of amplifiable DNA recovered from fresh and frozen Photogel plasma samples were of borderline significance (0.9+/-0.27 and 1.45+/-0.48 ng/μL respectively (p=0.09).
T39616 858894-859130 Sentence denotes Higher yields of amplifiable DNA obtained with plasma separator tubes and significant differences (P=0.01) between fresh and frozen samples (13.70+/-2.82 versus 34.74+/-4.45 μg/μL, P=.01) were accounted for by contaminating genomic DNA.
T46257 859131-859296 Sentence denotes Photogel tubes provided higher yields than Streck tubes for samples processed by low speed centrifugation only (122.3+/-24.8 pg/μL versus 63.1+/-19.1 pg/μL, P=.004).
T53537 859297-859498 Sentence denotes High speed centrifugation proteinase K digestion did not increase recovery from Photogel tubes, but increased recovery from EDTA or Streck tubes to levels similar to those obtained with Photogel tubes.
T94389 859499-859600 Sentence denotes Only trace amounts of genomic DNA contaminants were seen with either EDTA, Streck, or Photogel tubes.
T68598 859601-859613 Sentence denotes Conclusions:
T59440 859614-859788 Sentence denotes Recovery of cell-free DNA using our novel Photogel tube was high, required minimal processing, and the quality was comparable to that of material recovered from Streck tubes.
T79556 859789-860133 Sentence denotes The advantages of the Photogel tube over other commonly used tubes include 1) ability to standardize and preserve cell-free fractions in the primary tube, 2) ability to tolerate vigorous mixing to ensure uniform sampling, minimal processing by untrained personnel, ease of shipping, freezing tolerance, and 3) suitability for long-term storage.
T80228 860134-860174 Sentence denotes Ampliprep/COBAS Taqman HCV Test, v2.0 B.
T81347 860175-860184 Sentence denotes Lemos, C.
T26767 860185-860194 Sentence denotes Labaj, A.
T36027 860195-860208 Sentence denotes Tyropolis, C.
T64606 860209-860223 Sentence denotes Bissaillon, K.
T75890 860224-860233 Sentence denotes Lebel, F.
T51576 860234-860273 Sentence denotes Moore Baystate Health, Springfield, MA.
T57720 860274-860287 Sentence denotes Introduction:
T6352 860288-860420 Sentence denotes The purpose of this study is to verify the use of plasma preparation tubes (PPT) on the COBAS Ampliprep/COBAS Taqman HCV Test, v2.0.
T91887 860421-860521 Sentence denotes Currently, blood collected in EDTA tubes must be spun and the plasma removed within 6 hours of draw.
T69910 860522-860665 Sentence denotes Some collection sites cannot decant the plasma from EDTA tubes and this occasionally results in cancelled tests and recollection of the sample.
T88938 860666-860765 Sentence denotes Plasma preparation tubes need to be spun within 6 hours but do not need to be decanted immediately.
T60835 860766-860982 Sentence denotes The benefits to collecting in PPT tubes over EDTA are limiting aliquoting outside of the laboratory to reduce exposure and potential sample misidentification as well as decreasing the number of recollected specimens.
T63537 860983-860991 Sentence denotes Methods:
T17632 860992-861101 Sentence denotes For this study one PPT tube and two EDTA tubes per patient were collected for Hepatitis C viral load testing.
T89249 861102-861186 Sentence denotes EDTA tubes were spun and plasma separated from the red cells at the collection site.
T80605 861187-861284 Sentence denotes The PPT tubes were spun for 10 minutes at the collection site then transported to the laboratory.
T30759 861285-861408 Sentence denotes Once received in the lab, the PPT tube was briefly spun again and the plasma was removed and stored at -20°C until testing.
T71267 861409-861523 Sentence denotes The EDTA samples and the corresponding PPT sample were then run on the COBAS Ampliprep/COBAS Taqman HCV Test v2.0.
T47784 861524-861692 Sentence denotes The log viral load results from both sample types were recorded and compared using the Bland-Altman xy-scatter plot and regression line, and the Bland-Altman bias plot.
T95481 861693-861822 Sentence denotes Results: jmd.amjpathol.org ■ The Journal of Molecular Diagnostics Matched EDTA and PPT samples were tested on 64 patient samples.
T79583 861823-861881 Sentence denotes Nineteen numerical results by both tube types are plotted.
T28989 861882-861946 Sentence denotes Two outliers were identified and omitted from the data analysis.
T17230 861947-862061 Sentence denotes Linear regression analysis gives the equation y = 1.0365x -0.1813 with a correlation coefficient (R 2 ) of 0.9894.
T32839 862062-862229 Sentence denotes The slope of the line is not statistically different from 1 indicating no proportional bias, and the y intercept is close to zero indicating no constant systemic bias.
T15036 862230-862284 Sentence denotes The R 2 value was 0.9894, indicating good correlation.
T74919 862285-862361 Sentence denotes The mean bias is calculated at 0.12, indicating a systemic bias of 1.3 fold.
T79955 862362-862374 Sentence denotes Conclusions:
T262 862375-862543 Sentence denotes For this study we verified that plasma preparation (PPT) tubes provide results equivalent to EDTA samples utilizing the COBAS Ampliprep/COBAS Taqman for HCV Test, v2.0.
T91976 862544-862703 Sentence denotes Although there were two outliers, the results show that the use of plasma preparation tubes versus EDTA plasma tubes does not show a significant clinical bias.
T33576 862704-862706 Sentence denotes M.
T18051 862707-862721 Sentence denotes Debeljak, M.C.
T74139 862722-862734 Sentence denotes Morrison, E.
T20012 862735-862746 Sentence denotes Mocci, A.P.
T59610 862747-862758 Sentence denotes Klein, J.R.
T79833 862759-862808 Sentence denotes Eshleman Johns Hopkins University, Baltimore, MD.
T96709 862809-862822 Sentence denotes Introduction:
T25459 862823-862945 Sentence denotes Fields of forensics, paternity, and hematopoietic stem cell transplantation (HSCT) testing require human identity testing.
T28845 862946-863068 Sentence denotes The polymorphic nature of short tandem repeats (STRs) makes them most frequently used but they are relatively insensitive.
T45541 863069-863277 Sentence denotes We previously demonstrated that using multiple SNPs on a short amplicon (haplotype counting) could overcome the inherently high error rate of NGS, and was used for ultrasensitive detection of human DNA mixes.
T16116 863278-863390 Sentence denotes In addition, we identified >4,000 additional loci in the human genome that could be used for haplotype counting.
T78728 863391-863522 Sentence denotes Here we present haplotypes of 45 individuals from three different populations (15 individuals/population) at five polymorphic loci.
T79756 863523-863531 Sentence denotes Methods:
T33806 863532-863610 Sentence denotes We designed primers to target five loci (HLA-A, HLA-B, CSMD1, FARP1, and MT4).
T55518 863611-863776 Sentence denotes We then obtained and amplified samples from 15 individuals in three populations used in 1000 Genomes Study (total of 45 individuals; CEU, JPT, YRI) at all five loci.
T87193 863777-863813 Sentence denotes Amplicons were pooled and sequenced.
T22576 863814-863870 Sentence denotes Haplotypes were determined from aligned sequencing data.
T92786 863871-863879 Sentence denotes Results:
T49343 863880-863971 Sentence denotes Comparison of heterozygous loci across populations showed few differences in informativity.
T68552 863972-864125 Sentence denotes Of 45 individuals sequenced, we found 41 to be heterozygous at MT4 (91%), 36 at HLA-B (80%), 34 at FARP1 (76%), 31 at HLA-A (69%), and 30 at CSMD1 (67%).
T87283 864126-864259 Sentence denotes One locus, MT4, was the most informative in all 3 populations with heterozygosity score of 0.89 in YRI, 0.87 in JPT, and 0.81 in CEU.
T78050 864260-864386 Sentence denotes Among these 45 individuals, there were 22 different alleles for MT4, 15 for HLA-A, 10 for HLA-B, 8 for CSMD1, and 7 for FARP1.
T63839 864387-864399 Sentence denotes Conclusions:
T90290 864400-864535 Sentence denotes Ultrasensitive detection of human DNA mixes can be achieved using multiple haplotypes despite the high error rates associated with NGS.
T75939 864536-864607 Sentence denotes Here we validated additional loci that are polymorphic and informative.
T8331 864608-864731 Sentence denotes These loci may be useful for detecting early relapse following bone marrow transplantation and other clinical applications.
T2714 864732-864734 Sentence denotes M.
T89567 864735-864745 Sentence denotes Thomas, T.
T84089 864746-864755 Sentence denotes Zhang, R.
T49850 864756-864772 Sentence denotes Dolatshahi, M.A.
T39136 864773-864783 Sentence denotes Sukhai, S.
T18572 864784-864792 Sentence denotes Garg, M.
T95855 864793-864804 Sentence denotes Misyura, T.
T23120 864805-864815 Sentence denotes Pugh, T.L.
T53145 864816-864828 Sentence denotes Stockley, S.
T61260 864829-864892 Sentence denotes Kamel-Reid University Health Network, Toronto, Ontario, Canada.
T37095 864893-864906 Sentence denotes Introduction:
T72405 864907-865142 Sentence denotes The performance of NGS panels and variant analysis methods need to be assessed within technically challenging regions, e.g., high GC-content and large or complex indels, to identify sub-optimal coverage of clinically relevant variants.
T54214 865143-865341 Sentence denotes This is particularly significant for hematological malignancies, in which clinically important genes are known with larger insertions/deletions (FLT3, CALR) or that are particularly GC-rich (CEBPA).
T34877 865342-865567 Sentence denotes In this study, we assessed the TruSight Myeloid Sequencing Panel (TMSP; Illumina) for gaps in NGS data and developed complementary approaches to ensure the capture of all clinically relevant regions in the above listed genes.
T98236 865568-865576 Sentence denotes Methods:
T33122 865577-865680 Sentence denotes We evaluated the TMSP for performance metrics and ability to detect known variants in actionable genes.
T44907 865681-865870 Sentence denotes Read alignments were reviewed to identify target region depth of coverage for all amplicons, and data quality (quality of mapped reads, read balance, base quality) for insertions/deletions.
T30147 865871-865981 Sentence denotes Samples with known variants were used to assess panel performance in detecting clinically actionable variants.
T90370 865982-865990 Sentence denotes Results:
T62320 865991-866095 Sentence denotes Under-performing amplicons (positions covered <500x in >90% of samples tested) were found in 7/54 genes.
T26746 866096-866273 Sentence denotes Although these low coverage regions did not contain known mutation hotspots, 5 of 6 amplicons of CEBPA (mutated in 8% of AML) consistently failed depth of coverage requirements.
T25635 866274-866426 Sentence denotes Routine analysis for CEBPA in AML cases therefore required a non-NGS (Sanger) complementary assay to ensure detection of CEBPA variants in all patients.
T54595 866427-866526 Sentence denotes We assessed TMSP's ability to detect clinically actionable FLT3 insertions (found in ~25% of AMLs).
T66377 866527-866712 Sentence denotes The default setting on MiSeq reporter (MSR) software for insertion/deletion detection was modified from 25bp to 55bp for this purpose; longer read lengths (2x250 bp) were also selected.
T89604 866713-866954 Sentence denotes 15/31 positive cases, including 4 cases with FLT3 insertion sizes >25bp, up to a maximum size of 33bp were detected by NGS (assay sensitivity = 48%; specificity = 100%), necessitating the use of other assays to detect larger FLT3 insertions.
T10713 866955-867077 Sentence denotes A custom-designed script was developed to detect the recurrent clinically actionable 52 bp deletion in CALR from NGS data.
T6243 867078-867174 Sentence denotes Read data from 387 samples were analyzed; all known positive cases were successfully identified.
T86516 867175-867327 Sentence denotes Conclusions: NGS assays may not detect variants in all clinically relevant regions required for diagnosis or management of specific patient populations.
T5514 867328-867471 Sentence denotes Variants in CEBPA, FLT3 and CALR required supplementation with non-NGS assays or informatics approaches to address deficiencies in performance.
T52169 867472-867670 Sentence denotes Laboratories need to assess low performing regions on panels to prevent false negative results, and should consider the necessary optimization as part of NGS test validation prior to implementation.
T17866 867671-867673 Sentence denotes K.
T49548 867674-867686 Sentence denotes Krishnan, E.
T147 867687-867696 Sentence denotes Yigit, M.
T2919 867697-867707 Sentence denotes Karaca, B.
T47003 867708-867721 Sentence denotes Langhorst, T.
T64017 867722-867735 Sentence denotes Shtatland, D.
T10717 867736-867746 Sentence denotes Munafo, D.
T14278 867747-867760 Sentence denotes Rodriguez, P.
T87064 867761-867768 Sentence denotes Liu, L.
T33716 867769-867778 Sentence denotes Apone, V.
T96423 867779-867795 Sentence denotes Panchapakesa, K.
T52161 867796-867806 Sentence denotes Duggan, C.
T7052 867807-867817 Sentence denotes Sumner, C.
T45277 867818-867829 Sentence denotes Rozzi, F.J.
T82751 867830-867841 Sentence denotes Stewart, L.
T55488 867842-867853 Sentence denotes Mazzola, J.
T44695 867854-867863 Sentence denotes Bybee, D.
T38296 867864-867908 Sentence denotes Rivizzigno New England Biolabs, Ipswich, MA.
T61617 867909-868010 Sentence denotes Introduction: RNA-seq (RNA sequencing) has become the most popular method for transcriptome analysis.
T53151 868011-868164 Sentence denotes It is widely used for gene expression analysis, detection of mutations, fusion transcripts, alternative splicing, and post-transcriptional modifications.
T50151 868165-868340 Sentence denotes Recent improvements in next-generation sequencing technologies (NGS) and sample barcoding strategies allow analysis of multiple samples in parallel in a cost effective manner.
T21434 868341-868495 Sentence denotes As RNA-seq is being rapidly adopted for molecular diagnostics, the quality and reproducibility of library preparation methods are becoming more important.
T29926 868496-868659 Sentence denotes In addition, demand for library preparation methods that support successful NGS library construction from low input RNA or precious clinical samples is increasing.
T11602 868660-868863 Sentence denotes To overcome these challenges, we have developed a strandspecific RNA-seq library preparation method that retains information about which strand of DNA is transcribed from as low as 10 ng total RNA input.
T87649 868864-869103 Sentence denotes Strand specificity is important for the correct annotation of genes, identification of antisense transcripts with potential regulatory roles, and for correct determination of gene expression levels in the presence of antisense transcripts.
T72731 869104-869251 Sentence denotes Methods: poly-A mRNA from Universal Human Reference RNA (10 ng -1000 ng total RNA) was enriched to make libraries using our strand specific method.
T18965 869252-869369 Sentence denotes Libraries were analyzed on an Agilent Bioanalyzer, pooled at equimolar ratio and sequenced on Illumina's Nextseq 500.
T67968 869370-869533 Sentence denotes Paired end reads were mapped to a human reference genome (hg19) using Hisat2 and sequencing metrics were calculated using Picard's RNA-seq Metrics and RSeqC tools.
T45005 869534-869618 Sentence denotes Transcript abundance was measured using Salmon and the Ensembl GRCh38 CDS sequences.
T17603 869619-869627 Sentence denotes Results:
T73776 869628-869775 Sentence denotes Libraries prepared with our streamlined method using inputs that range from 10ng to 100ng show greater than 98% directionality at all input levels.
T2745 869776-869981 Sentence denotes Furthermore, GC content analysis, gene body coverage and gene expression correlation are similar for all inputs tested (10 ng, 100 ng, 1000 ng) even though input amount varies by three orders of magnitude.
T96349 869982-870072 Sentence denotes These consistent results are recapitulated with the spiked-in ERCC controls at all inputs.
T81345 870073-870085 Sentence denotes Conclusions:
T73855 870086-870289 Sentence denotes Our library preparation method is streamlined and can be used for a wide range of input RNA without any major modifications to the protocol, making it a convenient method for RNA-seq library preparation.
T97062 870290-870501 Sentence denotes In addition, it has increased sensitivity and specificity, especially for low-abundance transcripts, reduced PCR duplicates and sequence bias, delivering high quality strand-specific data even for low input RNA.
T34119 870502-870670 Sentence denotes Finally, our method is compatible with both poly A-tail enriched and ribosomal RNA depleted samples, and is amenable to large-scale library construction and automation.
T71606 870671-870684 Sentence denotes Introduction:
T3535 870685-870948 Sentence denotes A (GGGGCC)n hexanucleotide repeat expansion in an intronic region of the C9orf72 gene has been observed in the general population with a frequency of ~1/600 and is present in ~10% of all amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) cases.
T1016 870949-871063 Sentence denotes Fewer than 30 repeats are considered normal whereas pathogenic C9orf72 expansions have 100's to 1000's of repeats.
T90633 871064-871305 Sentence denotes The GCrich repeat poses formidable challenges to routine PCR-based fragment sizing methods, and currently requires analysis using multiple short-range PCR reactions for each sample followed by Southern blot (SB) analysis of expanded samples.
T34044 871306-871478 Sentence denotes Here we describe a two-site evaluation of a single-tube, highly streamlined PCR assay that both detects C9orf72 expansions and sizes expanded alleles with 100's of repeats.
T86580 871479-871487 Sentence denotes Methods:
T72184 871488-871581 Sentence denotes AmplideX PCR reagents were optimized for the amplification of C9orf72 hexanucleotide repeats.
T30252 871582-871701 Sentence denotes Amplicons were sized on a 3500xL or 3130xl Genetic Analyzer (Thermo Fisher) and/or SeaKem LE Plus agarose gels (Lonza).
T15452 871702-871796 Sentence denotes The assay was evaluated at both the University of Pennsylvania (Site 1) and Asuragen (Site 2).
T65434 871797-872023 Sentence denotes Site 1 evaluated a subset of NINDS ALS samples (n=50) and an independent set of ALS and FTD patient-derived from peripheral blood (n=100, 50 of which were expanded), saliva (n=10 expanded), and brain (n=30 expanded) specimens.
T48602 872024-872216 Sentence denotes Site 2 assessed a broader set of NINDS ALS samples (n=119, 72 of which were expanded), including those in common with Site 1, as part of a larger study to analyze the full collection (n=2100).
T29086 872217-872225 Sentence denotes Results:
T35182 872226-872385 Sentence denotes Across both testing sites, repeat numbers were resolved consistent with previous annotations for all normal samples and up to 145 repeats for expanded samples.
T7955 872386-872483 Sentence denotes Fragment sizing was limited only by the resolution limitations of capillary electrophoresis (CE).
T500 872484-872662 Sentence denotes Compatibility with both blood and brain FFPE DNA at 40 ng inputs was demonstrated, and samples with size mosaicism or indels could be identified from the repeat-primed CE traces.
T90048 872663-872747 Sentence denotes Site 2 further demonstrated sensitivity to as little as 1 ng input of cell-line DNA.
T16775 872748-872985 Sentence denotes This site also showed proof-of-concept for agarose gel sizing of PCR amplicons from expansions with up to 800 repeats, which is >10-fold larger than any published report The Journal of Molecular Diagnostics ■ jmd.amjpathol.org using PCR.
T82848 872986-872998 Sentence denotes Conclusions:
T94878 872999-873249 Sentence denotes A multi-site evaluation of a long-read C9orf72 PCR technology revealed both the reliable quantification up to 145 hexanucleotide repeats at high resolution on CE and the identification of repeat expansions irrespective of size, all in a single assay.
T39772 873250-873465 Sentence denotes This innovative, high-throughput PCR has important implications for clinical research and emerging diagnostic, therapeutic, and screening applications for ALS and FTD, and possibly other neurodegenerative disorders.
T98439 873466-873468 Sentence denotes C.
T34446 873469-873487 Sentence denotes Christopherson, P.
T38789 873488-873498 Sentence denotes Sankey, C.
T64378 873499-873507 Sentence denotes Chan, L.
T36356 873508-873520 Sentence denotes Cabiling, E.
T40591 873521-873531 Sentence denotes Caoili, T.
T24841 873532-873540 Sentence denotes Fine, C.
T45996 873541-873551 Sentence denotes Fenech, L.
T48114 873552-873562 Sentence denotes Eubank, P.
T5220 873563-873573 Sentence denotes Parmar, O.
T70182 873574-873588 Sentence denotes Badrenkova, J.
T61486 873589-873599 Sentence denotes Wilbur, R.
T18312 873600-873612 Sentence denotes Woodward, M.
T79434 873613-873623 Sentence denotes Nelles, S.
T87071 873624-873651 Sentence denotes Scott CareDx, Brisbane, CA.
T55847 873652-873665 Sentence denotes Introduction:
T68290 873666-873792 Sentence denotes Circulating cell-free DNA (cfDNA) has been described as a marker to assess allograft rejection in organ transplant recipients.
T57814 873793-873974 Sentence denotes We developed a noninvasive targeted next-generation sequencing (NGS) assay employing 266 SNPs to quantify donor-derived cfDNA levels (dd-cfDNA) in solid organ transplant recipients.
T23378 873975-874103 Sentence denotes Tests such as these are complex multi-step assays that require seamless integration of bench, LIMS and bioinformatics workflows.
T19280 874104-874363 Sentence denotes In addition to validation of the analytical performance, software QA testing, and bioinformatics pipeline validation; it is critical to examine the entire test system as a whole in a process validation, to ensure specimens are accurately tracked and analyzed.
T48126 874364-874530 Sentence denotes Additionally, the potential impact of improper specimen handling should be considered and evaluated within the context of unique effects on the assay being validated.
T510 874531-874539 Sentence denotes Methods:
T62415 874540-874664 Sentence denotes Testing was performed using cfDNA extracted from plasma from healthy volunteers collected in Streck Cell-Free DNA BCT tubes.
T18016 874665-874755 Sentence denotes Mock transplant specimens were created by spiking plasma from one individual into another.
T10915 874756-874904 Sentence denotes Before extracting cfDNA, plasma was mixed to simulate "donor" and "recipient" levels consistent with expected levels in organ transplant recipients.
T14354 874905-875155 Sentence denotes For process validation, commonly encountered accessioning, testing, and reporting scenarios, including repeat testing and demographic parameter changes were created to ensure that specimens could be accurately tracked through the system and reported.
T48280 875156-875247 Sentence denotes Interference testing was performed using purified hemoglobin, bilirubin, and triglycerides.
T38815 875248-875343 Sentence denotes Each substance was individually spiked into plasma and processed following standard procedures.
T28938 875344-875589 Sentence denotes Contamination with recipient cellular DNA present as a consequence of hemolysis during specimen handling was evaluated by spiking the separated recipient plasma with the increasing amounts hemolysate prepared from residual recipient blood cells.
T77816 875590-875598 Sentence denotes Results:
T61728 875599-875776 Sentence denotes Process validation confirmed each step in the process was accurate to final reporting, and identified areas in which future integration and workflow improvements were desirable.
T32276 875777-875936 Sentence denotes Testing showed that mishandled specimens may impact the accurate measurement the %dd-cfDNA due to the release of recipient cellular DNA in hemolyzed specimens.
T63704 875937-876053 Sentence denotes This result was also critical for interpretation of results using interferents generated from human source material.
T14729 876054-876066 Sentence denotes Conclusions:
T43947 876067-876159 Sentence denotes Process validation showed each step was accurate from specimen accession to final reporting.
T60741 876160-876245 Sentence denotes Hemolysis in blood collection tubes may impact accurate measurement of the %dd-cfDNA.
T62337 876246-876409 Sentence denotes A hemolysis rating scale is a useful tool in evaluating specimen quality to identify specimens with high levels of hemolysis that need to be excluded from testing.
T93797 876410-876412 Sentence denotes L.
T50322 876413-876427 Sentence denotes Jackson 1 , A.
T37203 876428-876439 Sentence denotes Wolf 2 , W.
T45887 876440-876451 Sentence denotes Hahn 1 , H.
T10544 876452-876466 Sentence denotes Mellert 1 , H.
T68568 876467-876481 Sentence denotes Mellert 1 , G.
T50870 876482-876553 Sentence denotes Pestano 1 1 Biodesix, Inc., Boulder CO; 2 Qiagen GmbH, Hilden, Germany.
T55728 876554-876567 Sentence denotes Introduction:
T21833 876568-876803 Sentence denotes Approximately 25% of patients with advanced non-small cell lung cancer (NSCLC) are not candidates for tissue biopsies and in some cases where tissue is obtained, it is not always of sufficient quantity or quality for molecular testing.
T86388 876804-876908 Sentence denotes Thus, the detection of circulating nucleic acids has become relevant in the Clinical Laboratory setting.
T7407 876909-876917 Sentence denotes Methods:
T86062 876918-877099 Sentence denotes In this study, we have focused on the optimization of the nucleic extraction step (manual versus automated) for a commercially-available diagnostic test system for NSCLC, GeneStrat.
T66264 877100-877340 Sentence denotes Criteria for evaluation included yield of circulating free (cf) nucleic acids, droplet counts for somatic mutant variants and the counterpart wild-type, as well as percentage minor allelic frequency (%MAF) as measured with validated assays.
T13471 877341-877459 Sentence denotes The specific assays used to evaluate performance were for KRAS G12C, KRAS G12D, EGFR T790M and EGFR del19 (A740-E746).
T86646 877460-877636 Sentence denotes As the reference method for evaluation of the automated QIAsymphony SP we used the previously validated "manual" method developed using the QIAamp Circulating Nucleic Acid Kit.
T98901 877637-877734 Sentence denotes Performance was determined by detection of recovered DNA variants by droplet digital PCR (ddPCR).
T25824 877735-877934 Sentence denotes For our reference and test arms, plasma from four donors with NSCLC and from four parallel processed spiked mixed allelic frequency DNA sample with known %MAF, were extracted and tested by GeneStrat.
T26807 877935-877943 Sentence denotes Results:
T50865 877944-878044 Sentence denotes Three of the clinical samples and all four of the spiked control sample extractions yielded results.
T31317 878045-878150 Sentence denotes Operator error accounted for the loss of one of the clinical samples during the manual extraction method.
T55922 878151-878338 Sentence denotes Overall, we observed that the automated method performed at least equivalently or was slightly superior to the established manual method when evaluating any of the measures in this study.
T4277 878339-878578 Sentence denotes The automated system yielded better results when evaluating the average percent differences for the variants and wildtype counts from both clinical samples (16% and 3%, respectively) and for the analytic samples (3% and 11%, respectively).
T25415 878579-878704 Sentence denotes Overall method variability as evaluated by calculated differences in %MAF between the methods were below 0.01% (0% to 0.01%).
T30053 878705-878717 Sentence denotes Conclusions:
T71245 878718-878911 Sentence denotes Our findings demonstrate that the automated and manual methods of recovering circulating nucleic from plasma perform at least equivalently for the gene read-outs and test system evaluated here.
T73400 878912-879071 Sentence denotes The quality and yield of cf DNA from the automated process was sufficient to meet the previously established criteria for the manual performance of the assays.
T53684 879072-879219 Sentence denotes Evaluations are on-going with the automated systems for larger plasma volumes and increased numbers of samples that could be processed in parallel.
T23093 879220-879224 Sentence denotes M.P.
T50612 879225-879238 Sentence denotes Powers 1 , S.
T77738 879239-879258 Sentence denotes Balakrishnan 2 , R.
T49460 879259-879271 Sentence denotes Calef 2 , P.
T51065 879272-879286 Sentence denotes Hartley 2 , J.
T56749 879287-879300 Sentence denotes Stites 2 , C.
T30596 879301-879313 Sentence denotes Troll 2 , N.
T56716 879314-879329 Sentence denotes Putnam 2 , R.E.
T49810 879330-879424 Sentence denotes Green 2 1 Dovetail Genomics, LLC, San Francisco, CA; 2 Dovetail Genomics, LLC, Santa Cruz, CA.
T33105 879425-879438 Sentence denotes Introduction:
T73291 879439-879641 Sentence denotes Presented herein is a novel technique for the generation of hypermegabase phased sequence data via a pre-extraction in situ DNA library preparation from formalin-fixed, paraffin-embedded (FFPE) samples.
T14459 879642-879773 Sentence denotes Until now, FFPE samples typically yielded sub-500bp DNA fragments confounding the identification of longrange sequence information.
T24001 879774-879940 Sentence denotes Long-range sequence information can only be obtained from FFPE tissue in non-repetitive loci where reads overlap unique breakpoints or other unique sequence features.
T28809 879941-880070 Sentence denotes Because the human genome is >50% repetitive, human FFPE samples have to date not provided adequate long-range genome information.
T71865 880071-880079 Sentence denotes Methods:
T53767 880080-880480 Sentence denotes In this first of its kind proof-of-principle study we: i) remove paraffin from FFPE treated cell lines (GM24149, GM24385; Horizon Discovery, Cambridge, United Kingdon), ii) treat the tissue fragments in situ with restriction endonuclease, iii) fill-in the resulting recessed 3' termini with a tag for purification of ligation fragments, and iv) ligate the resulting ends, creating chimeric molecules.
T55487 880481-880599 Sentence denotes These chimeric molecules preserve the long-range information of the starting molecule due to proximity-based ligation.
T46089 880600-880608 Sentence denotes Results:
T18967 880609-880702 Sentence denotes Sequencing of the ligated library provides data similar to those produced by Hi-C or Chicago.
T25956 880703-880889 Sentence denotes Preliminary assembly with a modified version of our HiRise software demonstrates that phasing and longrange information is preserved from these samples with >90% accuracy up to 1.9 Mbps.
T59970 880890-881001 Sentence denotes Connections greater than 1.9 MBps are also seen, but with an increase in errant connections across chromosomes.
T49294 881002-881014 Sentence denotes Conclusions:
T96612 881015-881347 Sentence denotes This technique provides a powerful new way to extract genome information for archives of tumor samples and other FFPE samples from bio-banks including the discovery of novel structural variation, copy-neutral structural variants, and other long-range sequence information including those within (or mediated by) repetitive elements.
T57760 881348-881406 Sentence denotes Experiments are on-going with FFPE samples from bio-banks.
T12208 881407-881464 Sentence denotes Construction Workflow for Use with Challenging Samples V.
T49336 881465-881481 Sentence denotes Panchapakesa, L.
T28723 881482-881491 Sentence denotes Apone, K.
T68123 881492-881502 Sentence denotes Duggan, P.
T10944 881503-881510 Sentence denotes Liu, T.
T43830 881511-881524 Sentence denotes Shtatland, B.
T38737 881525-881538 Sentence denotes Langhorst, J.
T34795 881539-881550 Sentence denotes Murdoch, C.
T53866 881551-881561 Sentence denotes Sumner, C.
T4156 881562-881571 Sentence denotes Rozzi, K.
T73331 881572-881584 Sentence denotes Krishnan, D.
T69561 881585-881598 Sentence denotes Rodriguez, J.
T44981 881599-881608 Sentence denotes Bybee, D.
T61680 881609-881623 Sentence denotes Rivizzigno, L.
T51043 881624-881635 Sentence denotes Mazzola, F.
T43973 881636-881647 Sentence denotes Stewart, E.
T6163 881648-881661 Sentence denotes Dimalanta, T.
T19424 881662-881701 Sentence denotes Davis New England Biolabs, Ipswich, MA.
T46419 881702-881715 Sentence denotes Introduction:
T44612 881716-881836 Sentence denotes Next-generation sequencing (NGS) plays a vital role in understanding molecular mechanisms involved in disease processes.
T74547 881837-881929 Sentence denotes This knowledge is important to support drug development targeted towards precision medicine.
T89848 881930-882042 Sentence denotes Success on NGS platforms begins with optimal sample preparation, which has become a bottleneck in NGS workflows.
T64525 882043-882165 Sentence denotes We have developed a robust, flexible library preparation method that integrates enzymatic fragmentation into the workflow.
T14135 882166-882328 Sentence denotes This method can be easily automated and is compatible with ultra low input as well as challenging samples such as Formalin Fixed Paraffin Embedded samples (FFPE).
T30417 882329-882336 Sentence denotes Method:
T62881 882337-882558 Sentence denotes Genomic DNA (gDNA) isolated from a variety of sources including FFPE were used to construct Illumina compatible, NGS libraries. gDNA ranging from 100pg to 500ng was fragmented, end repaired and dA-tailed in a single step.
T33720 882559-882662 Sentence denotes Adaptor ligation was performed in the same tube followed by a bead based cleanup and PCR amplification.
T10030 882663-882743 Sentence denotes Library quality and concentration were determined using the Agilent Bioanalyzer.
T71095 882744-882919 Sentence denotes Libraries were sequenced on the Illumina platform, reads were aligned to the appropriate reference genome using Bowtie 2 and quality metrics were generated using Picard tools.
T59945 882920-882928 Sentence denotes Results:
T2581 882929-883103 Sentence denotes Libraries constructed using intact gDNA and this novel library preparation method produced substantially higher yields than those generated using mechanically fragmented DNA.
T12372 883104-883277 Sentence denotes The greatest differences were observed with the lowest DNA inputs and most challenging samples, where a 4-5 fold increase in yield was observed with use of the novel method.
T44520 883278-883575 Sentence denotes Sequencing quality of libraries generated with inputs ranging from 100pg to 500ng of intact DNA show no significant difference in coverage uniformity, mappable reads, duplication rates, GC bias metrics and fragment size distribution compared to libraries constructed with mechanically sheared DNA.
T44768 883576-883588 Sentence denotes Conclusions:
T9125 883589-883804 Sentence denotes We have developed an enzymatic DNA fragmentation kit that enables the construction of high quality libraries from a range of sample types and inputs with minimal sequence bias, high yields and low duplication rates.
T40960 883805-884011 Sentence denotes This method eliminates the need for expensive equipment to fragment DNA as well as numerous cleanup and liquid transfer steps thereby reducing the time, cost and errors associated with library construction.
T89142 884012-884120 Sentence denotes These advances will permit greater use and adoption of NGS technologies in clinical and diagnostic settings.
T20517 884121-884183 Sentence denotes jmd.amjpathol.org ■ The Journal of Molecular Diagnostics TT48.
T82486 884184-884275 Sentence denotes Automation of NGS Workflow for Processing of High Sample Volume in a Clinical Laboratory S.
T3746 884276-884290 Sentence denotes Mercurio, A.I.
T56074 884291-884301 Sentence denotes Wald, K.M.
T34222 884302-884316 Sentence denotes Callenberg, S.
T46176 884317-884326 Sentence denotes Roy, Y.E.
T4882 884327-884342 Sentence denotes Nikiforov, M.N.
T72882 884343-884410 Sentence denotes Nikiforova University of Pittsburgh Medical Center, Pittsburgh, PA.
T51343 884411-884424 Sentence denotes Introduction:
T43424 884425-884524 Sentence denotes Next-generation sequencing (NGS) technologies have been successfully used in clinical laboratories.
T57310 884525-884679 Sentence denotes However, processing of a high sample volume is complicated due to lack of automation in multiple steps of NGS sample/library processing and data analysis.
T75626 884680-884840 Sentence denotes We have developed a specialized workflow that streamlines all components of testing to efficiently carry out NGS analysis in a high volume molecular laboratory.
T49183 884841-884849 Sentence denotes Methods:
T74373 884850-885135 Sentence denotes To accommodate a high increase in volume of NGS tests, we have developed a clinical NGS laboratory workflow to integrate multiple components of targeted amplification based NGS analysis, including specimen tracking, sample preparation, wet-bench automation and bioinformatics analysis.
T17883 885136-885246 Sentence denotes From November 2015 to April 2016, we have processed about 5,000 specimens for NGS testing (~10,000 libraries).
T46903 885247-885336 Sentence denotes Sequencing was performed on Ion Torrent PGM and Ion Proton platforms (Life Technologies).
T64926 885337-885345 Sentence denotes Results:
T53621 885346-885453 Sentence denotes The created NGS workflow utilizes automation and high-throughput technologies for each step of NGS testing:
T72096 885454-886000 Sentence denotes 1-nucleic acids isolation is performed using automated extractors (Magnapure Compact, Qiacube), 2-DNA and RNA yields are measured by high-throughput fluorometer (Promega Glomax Discover), 3multiplex PCR for custom targeted AmpliSeq library generation is set-up with the automated pipetter instruments (QIAgility), 4-library preparation, quantitation, normalization, template preparation and Ion Proton chip loading is automated using high-throughput instruments that require minimal user input (Tecan Freedom EVO 100, TapeStation 4200, Ion Chef).
T32010 886001-886216 Sentence denotes In addition, we developed automated bioinformatics pipelines (Variant Explorer, SeqReporter) that allows for fast variant analysis, QC monitoring, specimen tracking, functional variants categorization and reporting.
T57036 886217-886351 Sentence denotes Since implementation of this workflow, the average turn-around time was 8.2 days despite a 2-fold increase in volume in the past year.
T48457 886352-886578 Sentence denotes The workflow reduced hands-on time from 18 hours to 9 hours for library generation to sequencing of each batch of samples and significantly reduced the technical errors and repeat testing from ~5% to under 2% of all libraries.
T81318 886579-886591 Sentence denotes Conclusions:
T22242 886592-886776 Sentence denotes We have developed a special clinical NGS laboratory workflow infrastructure to integrate specimen tracking, sample preparation, wet-bench automation, sequencing analysis and reporting.
T69022 886777-887016 Sentence denotes This type of organization is necessary to facilitate operations in a high-throughput laboratory while maintaining appropriate turn-around time, decreasing hands-on technologist time, and ensuring assay consistency, accuracy and efficiency.
T71212 887017-887030 Sentence denotes Introduction:
T57975 887031-887195 Sentence denotes Standard FISH techniques require hybridization times of 12 hours or greater, thus amounting to turn around times (TAT) of 24 hours or greater for results reporting.
T20216 887196-887343 Sentence denotes To improve the TAT for the FISH tests, the new fast working Vysis IntelliFISH hybridization buffer has been recently developed by Abbott Molecular.
T77093 887344-887567 Sentence denotes Compared to standard LSI hybridization buffer protocols, the new fast working Vysis IntelliFISH hybridization buffer protocol decreases the required hybridization time and has the potential to improve hybridization quality.
T48508 887568-887576 Sentence denotes Methods:
T62074 887577-887693 Sentence denotes The Vysis IntelliFISH hybridization buffer was evaluated in bone marrow and formalin fixed Lymphoma tissues samples:
T61299 887694-887866 Sentence denotes 1) 20 pairs of matched bone marrow samples were probed with Vysis TP53/ CEP17 FISH probe kit using both the standard LSI protocol & Vysis IntelliFISH fast working protocol.
T83542 887867-888045 Sentence denotes 2) 10 pairs of matched formalin fixed Lymphoma tissues samples were probed with Vysis IGH/MYC/CEP8 probe using both the standard LSI protocol & Vysis IntelliFISH buffer protocol.
T6308 888046-888054 Sentence denotes Results:
T61188 888055-888190 Sentence denotes 1) The IntelliFISH hybridization buffer was clearly superior to the standard LSI hybridization buffer in analyzing bone marrow samples.
T13854 888191-888371 Sentence denotes Hybridization time (2 or 3 hours) for the Vysis TP53/ CEP17 probes demonstrated comparable performance in bone marrow samples when using the Vysis IntelliFISH hybridization buffer.
T96596 888372-888559 Sentence denotes 2) The signal intensity and specificity of Vysis IGH/MYC/CEP8 probes in Lymphoma samples was comparable using either the Vysis IntelliFISH hybridization buffer or the standard LSI buffer.
T32717 888560-888572 Sentence denotes Conclusions:
T34340 888573-888746 Sentence denotes The Vysis IntelliFISH hybridization buffer significantly reduces FISH hybridization time and simplifies the workflow of the standard overnight Vysis hybridization protocols.
T30952 888747-888870 Sentence denotes Signal intensity, specificity and signal to noise ratio of FISH probes were comparable to standard hybridization protocols.
T55743 888871-888925 Sentence denotes Regardless of Donor-Recipient Familial Relationship K.
T81982 888926-888938 Sentence denotes Thompson, J.
T29584 888939-888950 Sentence denotes Collins, C.
T54474 888951-888962 Sentence denotes Marchis, J.
T63782 888963-888976 Sentence denotes Sninsky, R.N.
T64356 888977-888989 Sentence denotes Woodward, M.
T66099 888990-889020 Sentence denotes Grskovic CareDx, Brisbane, CA.
T68931 889021-889034 Sentence denotes Introduction:
T20003 889035-889146 Sentence denotes Circulating cell-free DNA (cfDNA) is a novel biomarker for noninvasive assessment of transplanted organ injury.
T45310 889147-889332 Sentence denotes We developed AlloSure, a clinicalgrade NGS assay to measure the level of donor-derived cfDNA (dd-cfDNA) using a panel of 266 SNPs that does not require genotyping of donor or recipient.
T41548 889333-889434 Sentence denotes Previous studies validated the accuracy and linearity in genetically unrelated donor-recipient pairs.
T42406 889435-889591 Sentence denotes Compared to unrelated donor-recipient pairs, related donor-recipient pairs are expected to be more challenging due to the smaller number of variant alleles.
T66653 889592-889710 Sentence denotes In this study, we evaluate the performance of AlloSure in donor-recipient pairs with different degrees of relatedness.
T34250 889711-889816 Sentence denotes Methods: DNA from verified extended reference families from NIGM Biorepository was obtained from Coriell.
T12063 889817-889929 Sentence denotes DNA mixtures mimicking plasma cfDNA from transplant recipients with related and unrelated donors were developed.
T12281 889930-890143 Sentence denotes Twenty-two panels representing 22 'transplant recipients' were created across four families; and included seven unrelated, two grandparent/grandchild, five parent/child, and eight sibling donor-recipient mixtures.
T53910 890144-890241 Sentence denotes Each panel contained 'donor' DNA mixed into 'recipient' DNA at levels ranging from 0.1% to 12.5%.
T28681 890242-890326 Sentence denotes Accuracy was assessed by comparing dd-cfDNA results to trace amount spike-in levels.
T31215 890327-890455 Sentence denotes Limit of Detection (LOD) and Limit of Quantification (LOQ) were determined in comparison to the unrelated donor-recipient pairs.
T55078 890456-890464 Sentence denotes Results:
T22186 890465-890666 Sentence denotes There is a strong correlation between expected and AlloSure-determined values of dd-cfDNA for both related and unrelated donor-recipient pairs (R 2 0.981 and 0.993; RMSE 0.82% and 1.31%, respectively).
T29337 890667-890791 Sentence denotes Panels assembled from different family DNAs and replicate runs of select panels produced reproducible results with CVs <10%.
T53264 890792-890948 Sentence denotes LOD and LOQ are incrementally higher for the closely related 'donor-recipient' pairs compared to unrelated pairs (0.22% vs 0.16% LOD and 0.22% vs 0.2% LOQ).
T91705 890949-890960 Sentence denotes Conclusion:
T40956 890961-891151 Sentence denotes The analytical performance of a clinical-grade NGS dd-cfDNA assay was demonstrated using custom familial reference material panels mimicking both unrelated and related donor-recipient pairs.
T87277 891152-891289 Sentence denotes AlloSure is a sensitive, accurate, and precise non-invasive assay with application for surveillance of solid organ transplant recipients.
T86436 891290-891502 Sentence denotes With the exception of donor-recipient monozygotic twins, this study suggests AlloSure is applicable to single solid organ transplant recipients regardless of the degree of relatedness between donor and recipient.
T48010 891503-891505 Sentence denotes A.
T87945 891506-891521 Sentence denotes Ullius 1,3 , T.
T86148 891522-891533 Sentence denotes Voss 1 , S.
T36521 891534-891547 Sentence denotes Busche 2 , W.
T96673 891548-891562 Sentence denotes Hofmann 2 , D.
T78335 891563-891673 Sentence denotes Groelz 1 1 PreAnalytiX GmbH, Hilden, Germany; 2 LifeCodexx, Konstanz, Germany; 3 Qiagen GmbH, Hilden, Germany.
T48135 891674-891687 Sentence denotes Introduction:
T32512 891688-891805 Sentence denotes Non-invasive prenatal testing (NIPT) uses fetal circulating cell-free DNA (ccfDNA) in the maternal blood circulation.
T33631 891806-892002 Sentence denotes The release of maternal genomic DNA from blood cells during blood collection, transport and storage significantly reduces the sensitivity of the next-generation sequencing (NGS) based NIPT assays.
T91591 892003-892135 Sentence denotes Moreover, the reliable and reproducible extraction of the low abundance and short fetal ccfDNA fragments is technically challenging.
T12621 892136-892452 Sentence denotes Both difficulties are addressed by the PAXgene Blood ccfDNA System, consisting of the PAXgene Blood ccfDNA Tube, a plastic BD Vacutainer blood collection tube with a unique, non-crosslinking chemistry for stabilization of blood cells, and the QIAsymphony PAXgene Blood ccfDNA Kit, an automated ccfDNA extraction kit.
T30713 892453-892588 Sentence denotes The performance of the new system was evaluated in two research studies in comparison to K2EDTA tubes and the Streck Cell-Free DNA BCT.
T87837 892589-892597 Sentence denotes Methods:
T60330 892598-892606 Sentence denotes Study 1:
T15219 892607-893005 Sentence denotes Blood from 20 consented, apparently healthy donors in three replicates was collected into paired PAXgene and K2EDTA tubes and stored at different conditions to simulate sample transport and storage. ccfDNA was extracted from separated plasma using the QIAsymphony kit or the QIAGEN QIAamp Circulating Nucleic Acid Kit and analyzed by a validated quantitative real-time PCR assay targeting 18S rDNA.
T21451 893006-893014 Sentence denotes Study 2:
T2313 893015-893156 Sentence denotes Paired blood samples from 25 pregnant women were collected into PAXgene and Streck tubes, and ccfDNA was extracted using the QIAsymphony kit.
T180 893157-893238 Sentence denotes Fetal ccfDNA fraction was quantified by qPCR with the LifeCodexx QuantYfeX assay.
T13921 893239-893344 Sentence denotes Finally, eluates were analyzed using the NGS-based LifeCodexx PraenaTest assay for chromosomal disorders.
T49162 893345-893353 Sentence denotes Results:
T8551 893354-893362 Sentence denotes Study 1:
T43673 893363-893528 Sentence denotes The relative mean ccfDNA yield from PAXgene tubes was 97% ± 8% in comparison to the paired K2EDTA tubes when plasma was processed immediately after blood collection.
T28007 893529-893735 Sentence denotes Simulation of transport and storage of the PAXgene tubes for up to 7 days at 25°C did not increase yield significantly as compared to PAXgene tubes processed immediately after blood collection (113% ± 34%).
T6421 893736-893891 Sentence denotes Consistent and stable performance has been demonstrated by more than 70 runs using 6 different instruments and more than 1700 samples from over 200 donors.
T68862 893892-893900 Sentence denotes Study 2:
T26863 893901-894033 Sentence denotes Relative fetal ccfDNA fractions in maternal blood in PAXgene and Streck tubes were not significantly different (11.4% versus 12.4%).
T18729 894034-894144 Sentence denotes All samples which passed the internal quality standards produced equivalent results with the PraenaTest assay.
T76781 894145-894157 Sentence denotes Conclusions:
T14047 894158-894390 Sentence denotes The PAXgene Blood ccfDNA System provides consistent performance after 7 days of simulated transport and storage at RT and automated ccfDNA extraction from plasma with minimal variation between replicates and highly reliable results.
T24499 894391-894413 Sentence denotes For research use only.
T84227 894414-894451 Sentence denotes Not for use in diagnostic procedures.
T31688 894452-894465 Sentence denotes Introduction:
T10676 894466-894681 Sentence denotes Detection of single nucleotide variants (SNVs) and insertion/deletions (indels) using next-generation sequencing (NGS) technology is gaining increasing importance in research into cancer development and progression.
T19779 894682-894854 Sentence denotes Tissue biopsies are typically archived as formalin-fixed, paraffin embedded (FFPE) blocks, which preserve tissue morphology and allow long-term storage at room temperature.
T33152 894855-894978 Sentence denotes However, the methods used for fixation significantly damage and compromise the quality of nucleic acids from these samples.
T74671 894979-895078 Sentence denotes Formalin damage can fragment the DNA jmd.amjpathol.org ■ The Journal of Molecular Diagnostics TT56.
T481 895079-895200 Sentence denotes Validation of a Clinical Mate Pair Sequencing Assay to Characterize Apparently Balanced Structural Chromosome Variants Y.
T39687 895201-895208 Sentence denotes Cao, S.
T11459 895209-895219 Sentence denotes Smoley, B.
T2020 895220-895230 Sentence denotes Porath, R.
T64741 895231-895241 Sentence denotes Rowsey, J.
T48558 895242-895252 Sentence denotes Rustin, S.
T49919 895253-895264 Sentence denotes Johnson, G.
T3877 895265-895278 Sentence denotes Vasmatzis, S.
T23253 895279-895290 Sentence denotes Yerneni, J.
T37384 895291-895302 Sentence denotes Blommel, L.
T82774 895303-895315 Sentence denotes Peterson, K.
T82797 895316-895326 Sentence denotes Pearce, H.
T37198 895327-895338 Sentence denotes Kearney, U.
T312 895339-895348 Sentence denotes Aypar, R.
T33637 895349-895360 Sentence denotes Jenkins, W.
T44677 895361-895370 Sentence denotes Sukov, N.
T1909 895371-895406 Sentence denotes Hoppman Mayo Clinic, Rochester, MN.
T37443 895407-895420 Sentence denotes Introduction:
T19119 895421-895657 Sentence denotes Mate Pair sequencing, an emerging powerful technology able to detect structural variation, is able to further characterize the breakpoints of apparently balanced rearrangements and better understand the genetic content of these regions.
T39315 895658-895866 Sentence denotes Here we validate the performance of a laboratory-developed whole genome Mate Pair sequencing assay to characterize apparently balanced rearrangements identified in 75 samples by validated Cytogenetic testing.
T51531 895867-896029 Sentence denotes Methods: DNA samples were library-prepped using the Illumina Nextera Mate Pair library preparation kit and sequenced on the Illumina HiSeq 2500 in rapid run mode.
T90001 896030-896152 Sentence denotes Pooled libraries are hybridized two samples per flow cell and sequenced using 101basepair reads and paired end sequencing.
T13925 896153-896240 Sentence denotes Data is aligned to the GRCh38 reference genome using BIMAv3 (in-house developed tools).
T30244 896241-896340 Sentence denotes Structural variants and their breakpoints are identified using SVAtools (in-house developed tools).
T43700 896341-896349 Sentence denotes Results:
T71706 896350-896496 Sentence denotes Twenty-five abnormal samples were collected from each tissue type, including peripheral blood (PB), bone marrow (BM) and fresh/frozen tissue (FT).
T95663 896497-896642 Sentence denotes To evaluate accuracy, we compared Mate Pair sequencing results to the structural variants previously identified by validated Cytogenetic testing.
T58320 896643-896712 Sentence denotes Mate Pair sequencing successfully detected 71/75 structural variants:
T36782 896713-896755 Sentence denotes 24/25 in PB, 23/25 in BM, and 24/25 in FT.
T76316 896756-896926 Sentence denotes Two abnormalities in BM were not detected probably due to the low level of abnormalities, 15% (3/20 metaphases) and 25% (5/20 metaphases) estimated by chromosome studies.
T20315 896927-897216 Sentence denotes Although, Mate Pair sequencing, with high reproducibility, is able to detect most abnormalities with variable DNA input (as low as 0.25ug) and variable level of mosaicism (as low as 5-10%), the detection level may vary among different variants due to the difference of sequence structures.
T62061 897217-897320 Sentence denotes Mate Pair sequencing also missed one translocation in PB and one uncharacterizable rearrangement in FT.
T41215 897321-897682 Sentence denotes Of note, previous Mate Pair sequencing with high coverage (88x) research setting did detect the constitutional translocation, but only with few data supports due to the repetitive sequence of this region, suggesting the failure of detection is probably due to the limited coverage (36x) in rapid run mode as well as the genomic repetitive nature of this region.
T98152 897683-897695 Sentence denotes Conclusions:
T29472 897696-897773 Sentence denotes Mate Pair sequencing successfully detected 71/75 (94.7%) structural variants:
T41059 897774-897840 Sentence denotes 24/25 (96.0%) in PB, 23/25 (92.0%) in BM, and 24/25 (96.0%) in FT.
T26248 897841-897979 Sentence denotes These results provide evidence supporting the clinical utility of mate pair sequencing to characterize apparently balanced rearrangements.
T26915 897980-898198 Sentence denotes Meanwhile, the failure of detection in 4/75 samples suggest the limitation of this assay: it may not be able to detect low level abnormalities or regions with complex genomic structures such as repetitive DNA sequence.
T11130 898199-898262 Sentence denotes During Cutting-needle Biopsy Guided by Computed Tomography S.I.
T6380 898263-898277 Sentence denotes Meireles, S.S.
T25317 898278-898289 Sentence denotes Koide, C.D.
T5755 898290-898301 Sentence denotes Godoy, R.A.
T1314 898302-898351 Sentence denotes Coudry Hospital Sírio Libanês, São Paulo, Brazil.
T21120 898352-898365 Sentence denotes Introduction:
T91911 898366-898555 Sentence denotes Cutting-needle biopsy guided by computed tomography (CT) is used largely for lung, pleural and mediastinal lesions and provides tissue samples that are suitable for histological evaluation.
T98515 898556-898681 Sentence denotes On-site assessment of tumor adequacy (rapid onsite evaluation) has been performed to maximize precise sampling for diagnosis.
T40335 898682-898829 Sentence denotes Since molecular analysis has a fundamental role to drive therapy in lung cancer, an incremental volume of tumor tissue has been demanded over time.
T78905 898830-899025 Sentence denotes In this study, we investigated the usefulness for molecular analysis of tumor material from rapid touch-prepared glass slides generated for assessment of adequacy during imaging guided procedure.
T68233 899026-899034 Sentence denotes Methods:
T74649 899035-899225 Sentence denotes Panoptic or Thionin stained cells from 26 cases harvested from the slides were submitted to DNA isolation and mutational analysis by next-generation sequencing (NGS) or capillary sequencing.
T33438 899226-899234 Sentence denotes Results:
T26204 899235-899364 Sentence denotes The number of cells in the touch-prepared slides ranged from 200 to 5000 cells and the neoplastic content was between 40% to 90%.
T22941 899365-899468 Sentence denotes There was only one case were the quantity of cells in the imprint was insufficient for further testing.
T37113 899469-899603 Sentence denotes 17 cases were successfully analyzed by NGS and 8 cases were applied in capillary sequencing using as low as 16 ng of DNA per reaction.
T26233 899604-899736 Sentence denotes Mutations were detected in 65.4% of the samples, including KRAS (5 cases), EGFR (6 cases), ERBB2 (1 case) and other genes (4 cases).
T21720 899737-899917 Sentence denotes Mutational status in the touch-prepared specimen was compared with the FFPE matched specimen in 8 cases and demonstrated 100% concordance for both presence and absence of mutation.
T95465 899918-900038 Sentence denotes FFPE material from the remaining 18 cases yielded insufficient number of cells or quantity of DNA for molecular testing.
T29654 900039-900051 Sentence denotes Conclusions:
T77797 900052-900213 Sentence denotes Material obtained from on-site assessment of sample adequacy from cutting-needle CT guided biopsy may represent a valuable specimen source for molecular testing.
T48601 900214-900340 Sentence denotes Additional cases are under evaluation to expand the comparison between touch-prepared glasses slides and matched FFPE samples.
T68867 900341-900343 Sentence denotes J.
T99868 900344-900356 Sentence denotes Eberlein, T.
T98161 900357-900369 Sentence denotes Harrison, I.
T91517 900370-900384 Sentence denotes McKittrick, M.
T99472 900385-900397 Sentence denotes Wemmer, L.M.
T49462 900398-900411 Sentence denotes Griffin, B.P.
T17526 900412-900424 Sentence denotes Culver, L.A.
T12772 900425-900438 Sentence denotes Johnson, B.A.
T14999 900439-900474 Sentence denotes Kudlow ArcherDX, Inc., Boulder, CO.
T4430 900475-900488 Sentence denotes Introduction:
T67920 900489-900639 Sentence denotes The adaptive immune system is involved in various disease conditions including cancer, chronic infection, autoimmune disease and transplant rejection.
T28213 900640-900783 Sentence denotes Adaptive immunity is mediated by B and T lymphocytes, which are activated upon antigen binding to antigen receptors expressed on their surface.
T5304 900784-900971 Sentence denotes Therefore, the spectrum of these antigen receptors, or immune repertoire (IR), provides a means to monitor adaptive immune responses to disease, vaccination and therapeutic interventions.
T85222 900972-901120 Sentence denotes Next-generation sequencing (NGS) of antigen receptor genes is a valuable tool in the study of disease states and responses to various interventions.
T74412 901121-901244 Sentence denotes Traditional amplicon-based NGS assays use opposing primers for targeted amplification of rearranged antigen receptor genes.
T80969 901245-901353 Sentence denotes Thus, large primer panels are required to capture the extensive combinatorial diversity exhibited by the IR.
T81234 901354-901515 Sentence denotes Quantification from such assays requires a complex system of synthetic controls to account for differential amplification efficiency across segment combinations.
T30034 901516-901725 Sentence denotes Here, we describe an Anchored Multiplex PCR (AMP)-based NGS assay to analyze the IR, employing a minimal set of gene-specific primers in conjunction with molecular barcodes (MBCs) to reduce amplification bias.
T13775 901726-901875 Sentence denotes Methods: AMP uses MBCs ligated to cDNA ends and gene-specific primers for amplification, enabling immune chain mRNA interrogation from a single side.
T99719 901876-902025 Sentence denotes This eliminates the need for opposing primers that bind within the highly variable V-segment, eliminating clone dropout due to somatic hypermutation.
T66552 902026-902112 Sentence denotes Furthermore, this facilitates CDR3 sequence capture from highly fragmented RNA inputs.
T44328 902113-902364 Sentence denotes We validated the quantitative reproducibility and sensitivity of AMP-based B-and T-cell IR assays using high-quality mRNA isolated from peripheral blood leukocytes and highly fragmented RNA isolated from formalinfixed paraffin-embedded (FFPE) samples.
T9672 902365-902373 Sentence denotes Results:
T39774 902374-902476 Sentence denotes Our data showed high reproducibility between replicates and quantitative clone tracking down to 0.01%.
T27682 902477-902637 Sentence denotes Furthermore, our data indicate that clonal diversity in sequencing data is driven by input quantity, total T-cell number, and, to a lesser degree, mRNA quality.
T54966 902638-902769 Sentence denotes Conclusions: AMP-based NGS with MBC quantification and error-correction is a powerful method to characterize the immune repertoire.
T89355 902770-902876 Sentence denotes This enables sensitive clone tracking and measurement of lymphocyte diversity from fragmented RNA samples.
T95009 902877-902890 Sentence denotes Introduction:
T41334 902891-903133 Sentence denotes Target enrichment of selected exonic regions for deep sequence analysis is a widely used practice for the discovery of novel variants, and identification and phenotypic association of known variants for a wide range of practical applications.
T72578 903134-903464 Sentence denotes Current available strategies for selective enrichment can be characterized as either hybridization-based enrichment, where long synthetic oligonucleotides are used to selectively capture regions of interest, or multiplexed amplicon-based, where pairs of short primer sequences leverage PCR to selectively amplify sequence targets.
T70473 903465-903726 Sentence denotes Although hybridization-based methods have proven to be a tractable approach for large panels scaling to whole exome, the approach presents challenges in a relatively high sample input requirement, longer workflows, and inability to scale to very focused panels.
T27929 903727-904028 Sentence denotes In contrast, multiplexed amplicon approaches have proven valuable for small, highly focused panels, yet suffer from inherent challenges including the inability to scale content, loss of specificity associated with PCR duplication, and difficulties annealing primer pairs to already degraded materials.
T87073 904029-904036 Sentence denotes Method:
T76004 904037-904227 Sentence denotes We have developed a technology that utilizes a novel approach to selectively enrich nucleic acid targets ranging from a single gene to several hundred genes, without sacrificing specificity.
T36882 904228-904353 Sentence denotes Fragmented DNA is rapidly hybridized to biotinylated oligonucleotide baits that define the 3´ end of each target of interest.
T65906 904354-904466 Sentence denotes The bait-target hybrids are bound to streptavidin beads and any 3´ off target sequence is removed enzymatically.
T73140 904467-904611 Sentence denotes The trimmed targets are then converted into Illumina-compatible libraries that include unique molecular identifiers (UMIs) and a sample barcode.
T64285 904612-904773 Sentence denotes A Cancer Hotspot panel has been developed to enable highly specific hybridization-based capture of 190 common cancer targets from 50 genes using this technology.
T44215 904774-904782 Sentence denotes Results:
T14924 904783-904984 Sentence denotes The combination of a short hybridization time with the enzymatic removal of 3´ off-target sequence enabled greater sequencing efficiency relative to conventional hybridization-based enrichment methods.
T94445 904985-905208 Sentence denotes With the Cancer HotSpot panel, 100% of bases were covered to 25% or higher of the mean target coverage (MTC), 98% of bases were covered to 33% or higher of the MTC, and 90% of bases were covered to 50% or higher of the MTC.
T84027 905209-905417 Sentence denotes A high percentage of sequence reads also mapped to targets when challenging sample types such as FFPE and circulating tumor DNA (ctDA) were used, and the panel provided minimized bias across sequence content.
T87004 905418-905430 Sentence denotes Conclusions:
T8231 905431-905654 Sentence denotes This one-day protocol enables the preparation of sequence-ready libraries with high specificity, uniformity, and sensitivity for the discovery and identification of nucleic acid variants, even with challenging sample types.
T18066 905655-905659 Sentence denotes B.J.
T95593 905660-905671 Sentence denotes Dokus, H.B.
T94804 905672-905687 Sentence denotes Steinmetz, G.J.
T54143 905688-905703 Sentence denotes Tsongalis, L.J.
T81642 905704-905757 Sentence denotes Tafe Dartmouth-Hitchcock Medical Center, Lebanon, NH.
T85442 905758-905771 Sentence denotes Introduction:
T30654 905772-905998 Sentence denotes Manual assessment of fluorescence in situ hybridization (FISH) slides is labor intensive, susceptible to interobserver variability and subjectivity and requires technologist and Pathologist review on a fluorescence microscope.
T5979 905999-906135 Sentence denotes We implemented an automated imaging analysis platform into our FISH workflow to alleviate variables associated with manual slide review.
T15280 906136-906144 Sentence denotes Methods:
T39157 906145-906335 Sentence denotes The BioView Allegro Plus (BioView Inc., Billerica, MA) is an automated imaging and analysis system with an 8 slide motorized stage which can scan multiple FISH probe types in a single batch.
T13446 906336-906481 Sentence denotes We validated this system using the PathVysion HER2 probe in FFPE breast and gastro-intestinal tissues (for which the instrument is FDA approved).
T77501 906482-906631 Sentence denotes This system allows for tissue matching of the FISH tissue slide with the H&E slide for the purposes of tumor localization and review of scored cells.
T50810 906632-906712 Sentence denotes The developed workflow includes the following steps performed by a technologist:
T87984 906713-907220 Sentence denotes 1) Scan H&E slide; 2) low resolution scan of FISH slide; 3) tissue matching of FISH and H&E slides to digitally align; 4) select 20 fields of view (FOV) from the regions of tumor previously marked by a Pathologist; 5) scan FOVs at the 60X resolution; 6) review and select 40 cells using the analysis software for classification and scoring using the Solo workstations; 7) second technologist review, formalize the case and electronically assign the case to the Pathologist for final review and verification.
T81653 907221-907229 Sentence denotes Results:
T51635 907230-907426 Sentence denotes Our HER2 FISH validation study included analysis of 50 samples on the Bioview system that have been previously analyzed manually (20 positive, 20 negative and 10 equivocal for HER2 amplification).
T27264 907427-907588 Sentence denotes A significant amount of time and effort was invested into optimizing the operating procedures and establishing a workable protocol before validation could begin.
T61440 907589-907692 Sentence denotes Although there was a significant learning curve, scanned results were comparable to the manual scoring.
T73917 907693-907762 Sentence denotes Harmonized analysis and improved turnaround times were also achieved.
T55010 907763-907775 Sentence denotes Conclusions:
T82100 907776-908085 Sentence denotes Adoption of the BioView Allegro Plus into our Laboratory, although requiring a significant initial investment of time and resources, ultimately allowed for more standardization and automation in the scoring of FISH slides, time savings for the reviewers and provides more accurate correlation with H&E slides.
T78409 908086-908233 Sentence denotes The pathologist can use the Solo web to review the case on a local or remote computer which can also potentially be linked to the laboratory's LIS.
T84069 908234-908399 Sentence denotes With tissue matching, the pathologist can review exactly which cells were selected and scored by the technologist and view the corresponding region on the H&E slide.
T91622 908400-908545 Sentence denotes Both methods require prior knowledge of the fusion partners, have no or limited multiplex capacity, and lack the ability to detect novel fusions.
T47914 908546-908852 Sentence denotes We evaluated the performance of a next-generation sequencing (NGS)-based assay utilizing Anchored Multiplex PCR technology (Archer FusionPlex, Boulder, CO) for detection of gene fusions in RNA isolated from formalin-fixed, paraffin-embedded (FFPE) sarcomas in comparison with FISH and cytogenetic findings.
T25500 908853-908861 Sentence denotes Methods:
T90309 908862-909065 Sentence denotes Total RNA was extracted from 17 cases of microdissected FFPE sarcomas including 1 alveolar rhabdomyosarcoma (ARM), 3 alveolar soft-part sarcoma (ASPS), 5 Ewing sarcoma (EWS), and 8 synovial sarcoma (SS).
T80866 909066-909300 Sentence denotes Sequencing analysis for gene fusions was performed using the Universal RNA Fusion Detection Kit (ArcherDX), the Archer FusionPlex Sarcoma Panel (ArcherDX) and the Ion Torrent personal genomic machine (Life Technologies, Carlsbad, CA).
T84619 909301-909426 Sentence denotes RNA from a EWSR1-FLI1 positive cell line (SK-N-MC) and two negative (RNAs from normal blood and FFPE) controls were included.
T22161 909427-909485 Sentence denotes Data were analyzed using the Archer Analysis Pipeline 4.0.
T12464 909486-909562 Sentence denotes All cases had been tested by FISH (14) and/or conventional cytogenetics (3).
T69461 909563-909571 Sentence denotes Results:
T14000 909572-909824 Sentence denotes Gene fusions with specific breakpoints were detected in 15 tumors: ASPSCR1-TFE3 in 3 cases of ASPS; 3 fusion variants in 5 cases of EWS: EWSR1-FLI (3), EWRS1-ERG (1), and FUS-ERG (1); 2 fusion variants in 7 cases of SS: SS18-SSX1 (3) and SS18-SSX2 (4).
T46078 909825-909876 Sentence denotes No fusion was detected in ARM (1) and 1 case of SS.
T62702 909877-909948 Sentence denotes Fifteen of 17 cases had concordant FISH or cytogenetics findings (88%).
T8596 909949-910122 Sentence denotes Two Ewing sarcomas were FISH-negative using the EWRS1 breakapart probe but were positive for EWRS1-ERG (low level) and FUS-ERG fusion, respectively, by this NGS-based assay.
T90442 910123-910135 Sentence denotes Conclusions:
T75543 910136-910276 Sentence denotes This NGS-based gene fusion detection assay is a reliable platform for simultaneous detection of multiple gene fusions from various sarcomas.
T47186 910277-910333 Sentence denotes The assay works well with RNA isolated from FFPE tissue.
T37997 910334-910408 Sentence denotes It is sensitive and can separate different fusions at a single base level.
T75544 910409-910553 Sentence denotes Using this assay, we were able to identify all the translocations identified by FISH or cytogenetics and found fusions that were missed by FISH.
T41069 910554-910721 Sentence denotes Most importantly, this assay allows discovery of new fusions without prior knowledge of the fusion partners, a unique characteristics that neither FISH nor RT-PCR has.
T15833 910722-910770 Sentence denotes Next-Generation Sequencing-Based Technologies L.
T26931 910771-910779 Sentence denotes Wang, J.
T74441 910780-910788 Sentence denotes Wang, M.
T60234 910789-910796 Sentence denotes Rao, R.
T20834 910797-910807 Sentence denotes Cimera, R.
T80758 910808-910822 Sentence denotes Aryeequaye, L.
T95836 910823-910830 Sentence denotes Cao, R.
T87782 910831-910842 Sentence denotes Benayed, M.
T91259 910843-910854 Sentence denotes Ladanyi, T.
T91899 910855-910867 Sentence denotes Hollmann, K.
T28792 910868-910877 Sentence denotes Busam, M.
T77025 910878-910938 Sentence denotes Hameed Memorial Sloan Kettering Cancer Center, New York, NY.
T42074 910939-910952 Sentence denotes Introduction:
T58313 910953-911173 Sentence denotes Gene/locus-specific fluorescence in situ hybridization (FISH) probes are typically derived from cloned genomic regions presented in BAC (bacterial artificial chromosome) and PAC (P1-derived artificial chromosome) clones.
T68387 911174-911320 Sentence denotes The limitations of these conventional probes are the availability of specific clones and the large size of their genomic inserts (150 to ~300 kb).
T61637 911321-911578 Sentence denotes The use of array-and nextgeneration sequencing-based technologies is expanding our understanding of structural abnormalities at a previously unappreciated resolution, and many of the abnormalities detected are difficult to visualize using conventional FISH.
T63903 911579-911649 Sentence denotes Thus, there is a need to develop high-resolution (<100Kb) FISH probes.
T789 911650-911819 Sentence denotes We present here our experience with oligonucleotide-based FISH (oFISH) probes which are derived from synthesized oligonucleotides targeting specific sequences in genome.
T67338 911820-911828 Sentence denotes Methods:
T33315 911829-912129 Sentence denotes Two cases with BRAFstructural aberrations revealed by high-resolution SNP-array analysis were studied by FISH using a custom BRAF break-apart probe set consisting of a 300kb probe targeting 5' BRAF (exons 1 to 7 plus 5' upstream sequence) and a 60kb probe precisely targeting 3' BRAF (exons 8 to 18).
T24913 912130-912255 Sentence denotes Probes were synthesized using Agilent's oligo-based SureFISH technology and labeled with FITC (5') and Cy3 (3') fluorophores.
T76018 912256-912299 Sentence denotes FISH was performed on FFPE tissue sections.
T75286 912300-912417 Sentence denotes For comparison, conventional FISH for BRAF gene rearrangement was performed using BAC-clone probes (Empire Genomics).
T68369 912418-912426 Sentence denotes Results:
T28394 912427-912650 Sentence denotes High resolution SNP-array analysis revealed intragenic copy number alterations in BRAF in both cases, showing gain of 3' portion of BRAF and loss or no copy number change of 5' BRAF with the change point mapped to intron 8.
T29421 912651-912823 Sentence denotes The boundaries of the gain segments were clearly defined by BRAF intron 8 and exon 18, whereas the 3' flanking sequence of BRAF was not involved in the gain in either case.
T92352 912824-912990 Sentence denotes FFPE FISH analysis using our custom oFISH probes successfully detected BRAF gene rearrangement with signals consistent with the pattern predicted from array analysis.
T67844 912991-913076 Sentence denotes In contrast, FISH using conventional probes failed to illustrate the BRAF alteration.
T17816 913077-913184 Sentence denotes Furthermore, a targeted RNA-seq assay (ArcherDX) identified in-frame BRAF fusion transcripts in both cases.
T38181 913185-913345 Sentence denotes Conclusions: oFISH probes can provide precise control over the sequence to be targeted, allowing for accurate analysis of regions as small as tens of kilobases.
T38846 913346-913698 Sentence denotes The resolution of oFISH is comparable to that of array-based technology in copy number analysis, therefore, can be used to develop complementary FISH assays accordingly. oFISH works well in FFPE tissue, and the implementation of oFISH probes in clinical diagnosis can significantly improve the precision and accuracy of FISH analysis in selected cases.
T80967 913699-913712 Sentence denotes Introduction:
T95015 913713-913889 Sentence denotes Success rate, accuracy and turnaround time of laboratory results being generated using increasingly complex technologies are of paramount importance in patient care and safety.
T75719 913890-914071 Sentence denotes Occupations that deal with risky technologies in high complexity environments (e.g. aviation, nuclear) apply proactive measures to avoid disastrous consequences of potential errors.
T80960 914072-914254 Sentence denotes We describe our proactive adoption of this model and development of strategies to achieve a High Reliability Organization (HRO) status in a clinical molecular diagnostics laboratory.
T8001 914255-914263 Sentence denotes Methods:
T682 914264-914520 Sentence denotes We aligned our ongoing patient safety, quality and process improvement strategies with five HRO traits (see results) and Joint Commission Center for Transforming Healthcare's 3-domain (Leadership, Safety Culture, Robust Process Improvement) maturity model.
T11265 914521-914650 Sentence denotes New initiatives, engagement of staff, and proactive use of tools and monitors were added to achieve the goal of high reliability.
T64642 914651-914698 Sentence denotes Results: HRO trait 1-Sensitivity to operations:
T55433 914699-914830 Sentence denotes An operations command center allowed real-time monitoring of critical operational components (IT, staffing, reagents, instruments).
T27579 914831-914953 Sentence denotes An electronic dashboard allowed monitoring of production line, resources, quality, patient safety and training indicators.
T48141 914954-915039 Sentence denotes Industrial and quality engineering expertise were added to the laboratory operations.
T3518 915040-915108 Sentence denotes HRO trait 2-Reluctance to accept "simple" explanations for problems:
T35004 915109-915229 Sentence denotes Detailed root cause analyses and documentation systems allowed identification and remediation of multi-factorial issues.
T33738 915230-915269 Sentence denotes HRO trait 3-Preoccupation with failure:
T27957 915270-915411 Sentence denotes Systematic evaluation of laboratory processes allowed proactive implementation of preventive measures and detailed "Stop the Line" protocols.
T21781 915412-915447 Sentence denotes HRO trait 4-Deference to expertise:
T48526 915448-915585 Sentence denotes A patient safety team (frontline technologist on rotating membership) performed proactive compliance and patient safety risk assessments.
T8473 915586-915671 Sentence denotes A culture of safety was created through open discussions and active staff engagement.
T56653 915672-915748 Sentence denotes Also, a high level of informatics expertise was embedded throughout the lab.
T24596 915749-915772 Sentence denotes HRO trait 5-Resilience:
T44979 915773-916089 Sentence denotes Highly coordinated team efforts allowed the laboratory to continue offering high quality patient care during significant changes such as the laboratory move to a new location, national clinical trial adoptions, automation, implementations of a new LIS and a new institutional EMR, all within a total span of <1 year.
T8786 916090-916102 Sentence denotes Conclusions:
T27541 916103-916466 Sentence denotes Systematic evaluation of laboratory processes, engagement of staff and proactive implementation of laboratory-wide initiatives facilitate strengthening of operations, creates a culture of patient safety and helps to eliminate the risk of errors thereby providing high quality patient care and moving closer to the goal of becoming a High Reliability Organization.
T78857 916467-916480 Sentence denotes Introduction:
T55994 916481-916660 Sentence denotes Deregulation of the MET receptor tyrosine kinase is associated with aggressive phenotypes in a variety of human cancers, promoting proliferation, invasive growth and angiogenesis.
T20844 916661-916836 Sentence denotes Several types of genetic aberrations can drive MET deregulation, including gene amplification, overexpression, single nucleotide variants (SNVs), exon 14 skipping and fusions.
T12729 916837-916968 Sentence denotes MET is a target of intensive drug development efforts, although the various mutated forms of MET exhibit unique drug sensitivities.
T51948 916969-917086 Sentence denotes Therefore, detection of these mutations has the potential to guide treatments for cancers driven by MET deregulation.
T80217 917087-917212 Sentence denotes Next-generation sequencing (NGS) enables comprehensive detection of all mutation types from whole genomes and transcriptomes.
T80014 917213-917395 Sentence denotes However, low detection sensitivity, high input requirement and high costs render these approaches impractical for routine detection of mutations from low-input clinical sample types.
T28451 917396-917567 Sentence denotes Anchored Multiplex PCR (AMP) is a target enrichment strategy for NGS that, by its scalable and quantitative nature, is well suited to detect all modes of MET deregulation.
T75947 917568-917696 Sentence denotes Methods: AMP-based Archer VariantPlex and FusionPlex library preparation assays detect mutations from DNA and RNA, respectively.
T46539 917697-917842 Sentence denotes We designed AMP probes covering the MET gene to detect copy numbers and SNVs from DNA, and fusions, exon skipping and expression levels from RNA.
T11757 917843-917994 Sentence denotes Results: MET amplifications and resulting overexpression were detected in FFPE samples using the VariantPlex and FusionPlex kits and confirmed by FISH.
T3566 917995-918138 Sentence denotes Exon 14 skipping with concomitant splice site mutations was also detected using the Archer kits and confirmed by RT-PCR in both FFPE and cells.
T5503 918139-918251 Sentence denotes Interestingly, we detected a novel GTF2I:MET gene fusion and a Y1253D activating point mutation in FFPE samples.
T36981 918252-918264 Sentence denotes Conclusions:
T86072 918265-918452 Sentence denotes These results show that AMP-based VariantPlex and FusionPlex Assays enable comprehensive detection of multiple mutation types from low-input clinical sample types, such as FFPE specimens.
T49688 918453-918482 Sentence denotes Suspected B-Cell Clonality Y.
T40881 918483-918492 Sentence denotes Huang, A.
T37233 918493-918505 Sentence denotes Jacobsen, J.
T92467 918506-918520 Sentence denotes Panganiban, K.
T15021 918521-918529 Sentence denotes Hutt, D.
T4082 918530-918539 Sentence denotes Duong, J.
T91254 918540-918551 Sentence denotes Thornes, J.
T90745 918552-918562 Sentence denotes Miller, T.
T71604 918563-918599 Sentence denotes Stenzel Invivoscribe, San Diego, CA.
T73970 918600-918819 Sentence denotes Introduction: PCR-based methods targeting immunoglobulin heavy chain (IGH) frameworks 1, 2, and 3 (FR1, FR2 and FR3) are the current gold standard for the first line clonality testing in suspected B-cell proliferations.
T14427 918820-919037 Sentence denotes Recently, next-generation sequencing (NGS) based approaches for immune receptor geneshave been suggested to improve sensitivity and identify the specific V-J DNA sequence required to track clones in follow up testing.
T77679 919038-919190 Sentence denotes Here we report the development of a comprehensive IGH NGS assay which allows detection of all three FRs, simultaneously, in a single Illumina MiSeq run.
T1436 919191-919199 Sentence denotes Methods:
T38777 919200-919293 Sentence denotes The MiSeq IGH Assay consists of three master mixes, targeting FR1/J, FR2/J and FR3/J regions.
T98062 919294-919427 Sentence denotes The proprietary V and J consensus primers were designed and adapted to enable the PCR products to be sequenced on the MiSeq platform.
T67271 919428-919550 Sentence denotes Each MiSeq IGH FR master mix uses 24 indices, allowing analysis of 22 patient samples plus positive and negative controls.
T7580 919551-919635 Sentence denotes Multiplexed PCR was followed by amplicon purification using the AMPureXP PCR system.
T49157 919636-919737 Sentence denotes Purified equimolar amounts of amplicons from different samples and FRs were pooled to form a library.
T91527 919738-919834 Sentence denotes The harmonized and quantified library was sequenced using the MiSeq v2 Reagent kit (500 cycles).
T21467 919835-920126 Sentence denotes MiSeq output data was analyzed using proprietary bioinformatics software, which can sort the sequences by both index and FR, generate frequency distributions of V-J rearrangements, and determine the somatic hypermutation (SHM) rate of the IGHVregion based on the sequence of FR1/J amplicons.
T16110 920127-920135 Sentence denotes Results:
T89758 920136-920257 Sentence denotes The ability to detect FR1, FR2 and FR3 regions was verified by testing different known V-J rearrangement B cell line DNA.
T67333 920258-920385 Sentence denotes Cell line DNA, serially diluted into tonsil DNA, demonstrated the limit of detection (LoD) at 5% dilution with 50 ng DNA input.
T90481 920386-920544 Sentence denotes The assay achieved good linearity (R 2 > 0.95), and good reproducibility (<25% CV) was demonstrated over 2 operators, 2 MiSeqs and 2 lots of PCR master mixes.
T39080 920545-920817 Sentence denotes Preliminary testing of genomic DNA from peripheral blood (PB), bone marrow (BM) aspirates and formalin-fixed paraffin-embedded tissue (FFPE) demonstrated that the clonality detection missed by one FR could be detected by another FR, thus increasing overall detection rate.
T74765 920818-920830 Sentence denotes Conclusions:
T65444 920831-920976 Sentence denotes A comprehensive IGH NGS assay has been developed for the Illumina MiSeq platform that identifies clonal IGH V-J rearrangements and DNA sequences.
T13591 920977-921164 Sentence denotes The assay has demonstrated that combining FR1, FR2 and FR3 helps to decrease the false-negative rate due to somatic hypermutation in primer binding sites of the involved VH gene segments.
T77575 921165-921178 Sentence denotes Introduction:
T71856 921179-921349 Sentence denotes Accurate detection of low occurrence somatic mutations is paramount when using next-generation sequencing (NGS) based results to direct personalized management of cancer.
T56378 921350-921551 Sentence denotes Between March 2013 and March 2016, the Center for Personalized Diagnostics sequenced 3010 clinical cases on its amplicon based solid tumor panel (Sv1) which covers mutational hot spots across 47 genes.
T70314 921552-921763 Sentence denotes Our potential to include more genes, cover all exons and report variant allele frequencies (AF) of < 5% using low DNA input were limited due to the assay methodology and duplication errors of PCR and sequencing.
T1618 921764-921931 Sentence denotes To address these limitations, we designed our next solid tumor panel (Sv2) using Agilent Haloplex HS methodology that combines molecular barcoding and deep sequencing.
T61238 921932-922089 Sentence denotes We present our data from 48 samples and show that unique molecular identifiers can be used for accurate detection of variants below 5% AF with low DNA input.
T59597 922090-922098 Sentence denotes Methods:
T21081 922099-922211 Sentence denotes The final version of Sv2 captured 99.81% of the 583kbp target region designed to include all exons of 155 genes.
T86005 922212-922309 Sentence denotes We tested 48 DNA samples isolated from FFPE tissues (n=38), blood (n=6) and assay controls (n=4).
T883 922310-922375 Sentence denotes DNA input range was 25ng to 100ng and all DNA were <30% degraded.
T21502 922376-922482 Sentence denotes Expected AF range was 0.05% to 100% for single-nucleotide variants (SNV) and insertion-deletions (indels).
T63112 922483-922573 Sentence denotes Data were analyzed using SureCall analysis software (v3.5.1.46) from Agilent Technologies.
T21901 922574-922694 Sentence denotes Variants were called only with unique reads, while at least three such reads were required to deem a variant to be true.
T21789 922695-922703 Sentence denotes Results:
T22093 922704-922774 Sentence denotes We successfully generated libraries at all DNA input ranges we tested.
T94150 922775-922877 Sentence denotes We observed >75% duplicate molecules when samples were >15% degraded and/or when DNA input was < 35ng.
T8977 922878-923008 Sentence denotes More than 80% of all target regions had mean coverage >200x when sample mean coverage was >500x and duplicate molecules were <60%.
T18090 923009-923053 Sentence denotes On-target specificity was consistently >98%.
T50442 923054-923167 Sentence denotes SNVs with expected AF <5% (n=28), had 100% sensitivity and specificity across all DNA inputs and sequencing runs.
T44915 923168-923276 Sentence denotes Below 1% AF, we had 100% concordance and accurately detected SNVs at 0.5% (n=3); 0.1% (n=7) and 0.05% (n=2).
T14515 923277-923371 Sentence denotes SNVs with AF >5% (n=38), had 100% concordance and on average AF's varied by 4.5% between runs.
T69986 923372-923491 Sentence denotes Sensitivity and specificity of indels and whole gene/exon alterations for all AF ranges are currently being determined.
T90382 923492-923504 Sentence denotes Conclusions:
T44533 923505-923658 Sentence denotes Accurate identification of somatic mutations in tumors by NGS is required for enrollment into mutation-specific treatment modalities and clinical trials.
T90708 923659-923804 Sentence denotes We show that our Sv2 can reliably detect lowlevel SNVs by tagging DNA fragments with more than a million unique molecular identifiers before PCR.
T6512 923805-923958 Sentence denotes This panel will greatly benefit patients whose tumors carry low-level actionable mutations and those who require monitoring for minimal residual disease.
T21340 923959-923964 Sentence denotes TT67.
T2262 923965-924037 Sentence denotes An Automated Cell-free DNA Extraction System for Clinical Diagnostics T.
T25471 924038-924049 Sentence denotes Ivanova, A.
T85629 924050-924057 Sentence denotes Fan, A.
T5189 924058-924065 Sentence denotes Yeo, P.
T6128 924066-924080 Sentence denotes Ariyaratne, G.
T90802 924081-924116 Sentence denotes Michel Vela Research Singapore Pte.
T1835 924117-924133 Sentence denotes Ltd., Singapore.
T48511 924134-924147 Sentence denotes Introduction:
T9838 924148-924313 Sentence denotes Circulating cell-free DNA (cfDNA) has emerged as an important biomarker in cancer diagnostics and non-invasive progression monitoring of various clinical conditions.
T95119 924314-924398 Sentence denotes This has resulted in the development of new in vitro diagnostics (IVD) cfDNA assays.
T71483 924399-924566 Sentence denotes Challenges encountered in these developments related to the efficient extraction of cfDNA from liquid biopsies, often yielding low quantities of highly fragmented DNA.
T83195 924567-924771 Sentence denotes As assays for cfDNA are typically intended to identify genetic variants present at very low allelic frequencies, many of the established detection technologies are driven to the edge of their performance.
T43546 924772-924780 Sentence denotes Methods:
T32530 924781-924924 Sentence denotes We developed a magnetic bead-based cfDNA extraction kit Sentosa SX cfDNA Kit (4x8) and optimized it for use on the Vela Sentosa SX101 platform.
T77928 924925-925089 Sentence denotes Sentosa SX101 is a CE-IVD certified robotic liquid handling system for nucleic acid extraction, PCR set-up and Next-Generation Sequencing (NGS) library preparation.
T99164 925090-925189 Sentence denotes We compared performance of the Sentosa SX cfDNA Kit (4x8) with a column-based cfDNA extraction kit.
T6934 925190-925277 Sentence denotes Integrity of cfDNA extracted by both methods was assessed using ALU repeats qPCR assay.
T41152 925278-925365 Sentence denotes Quality of the extracted cfDNA was tested using an NGSbased Sentosa SQ CRC Panel (4x8).
T79896 925366-925374 Sentence denotes Results:
T12116 925375-925496 Sentence denotes The Sentosa SX cfDNA Kit (4x8) utilizes 4 mL of human plasma as sample input and can process up to eight samples per run.
T34884 925497-925710 Sentence denotes In this pilot study DNA was extracted from plasma samples with 3.0, 1.5 and 0.75 ng spiked-in fragmented HCT116 gDNA (KRASG13D positive) using Sentosa SX cfDNA and column-based cfDNA extraction kits, respectively.
T46583 925711-925779 Sentence denotes Fragment size of HCT116 gDNA was ~170 bp (confirmed by Bioanalyzer).
T10851 925780-925972 Sentence denotes The ALU247/115 ratio for DNA extracted by the Sentosa SX cfDNA kit was 0.19-0.28 and for the columnbased extraction method >0.7 (expected ratio for cfDNA is less than 0.5 and for gDNA is 1.0).
T66417 925973-926114 Sentence denotes Amount and quality of DNA for all samples extracted by both methods was sufficient to prepare NGS libraries using Sentosa SQ CRC Panel (4x8).
T44143 926115-926280 Sentence denotes KRAS G13D mutation was detected in all samples extracted by the Sentosa SX cfDNA kit and no KRAS G13D mutation was detected in any of column-based extracted samples.
T9115 926281-926293 Sentence denotes Conclusions:
T35408 926294-926384 Sentence denotes The Sentosa SX cfDNA Kit (4x8) selectively extracts cfDNA over high molecular weight gDNA.
T24332 926385-926509 Sentence denotes The Sentosa SX cfDNA Kit (4x8) appears as an efficient and reliable solution for cfDNA extraction from human plasma samples.
T53517 926510-926701 Sentence denotes Integration into the Sentosa qPCR-and NGS-based workflows makes the Sentosa SX cfDNA Kit (4x8) a universal in vitro diagnostics tool, which can be used in combination with various IVD assays.
T3274 926702-926715 Sentence denotes Introduction:
T68323 926716-926838 Sentence denotes Plasma cell-free DNA is challenging to analyze due to a high degree of fragmentation and low concentration in circulation.
T91779 926839-926935 Sentence denotes Detection of low level mutations in cfDNA requires highly sensitive molecular detection methods.
T25708 926936-927120 Sentence denotes In this study, we compare the utility of the Bio Rad droplet digital PCR (ddPCR) and MassARRAY System, powered by UltraSEEK chemistry for detecting low level mutations in plasma cfDNA.
T73084 927121-927206 Sentence denotes Methods: cfDNA was extracted from plasma of 25 patients with metastatic solid tumors:
T69428 927207-927474 Sentence denotes 14 adenocarcinomas including 7 colorectal, 3 breast, 2 pancreas, 2 intestinal; 1 oral squamous cell carcinoma, 1 mucoepidermoid carcinoma, 6 melanoma, and 3 astrocytoma/glioblastoma using the QIAamp circulating nucleic acid kit within 2-16 hrs of specimen collection.
T14 927475-927602 Sentence denotes Mutations in primary (n=14) or metastatic (n=11) tumors were tested using the Ampliseq Cancer Hotspot Panel v2 (Thermo Fisher).
T77267 927603-927926 Sentence denotes 10ng cfDNA was used for each assay. cfDNA genotyping was performed for 11 SNPs in IDH1 (R132H), PIK3CA (H1047R, E542K, E545K), KRAS (G12D, G12V, G13D), BRAF (V600E, V600M), NRAS(Q61R, Q61K) genes using ddPCR (Bio Rad) and 12 gene hotspot Ultraseek MassARRAY 1% MAF, all 37 expected calls were detected using the Accel TP53.
T93499 927927-928087 Sentence denotes Further, Accel TP53 assay detected 1 additional nonsense mutation (TP53. c.372C>A p.C124*) at 1.1% MAF (region not covered by TruSeq 53-gene hotspot amplicons).
T33719 928088-928164 Sentence denotes There was a good correlation of MAFs detected by the two assays (R 2 =0.98).
T93144 928165-928252 Sentence denotes The actual hands-on time was ~4.5 hrs (versus ~ 8 hrs on TruSeq 53-gene hotspot assay).
T98074 928253-928342 Sentence denotes Further optimization of the pipeline for improving the limit of detection is in progress.
T15054 928343-928355 Sentence denotes Conclusions:
T10383 928356-928544 Sentence denotes Accel TP53 is a sensitive, high-throughput "single-gene" NGS-based assay for detection of low-level TP53 mutations, suitable for MRD monitoring and early detection of sub-clonal mutations.
T27 928545-928773 Sentence denotes Compared with the routinely used NGS-based multi-gene panels targeting hot-spot regions, the advantages of this assay include efficient resource utilization and comprehensive coverage of the entire coding region at reduced cost.
T5839 928774-928936 Sentence denotes The need to process small solid tumor specimens is increasing as early detection strategies become more effective and less invasive biopsy strategies are adopted.
T7811 928937-929099 Sentence denotes Moreover, the rapidly changing landscape of molecular testing argues that current strategies need to minimize sample input and preserve tissue for future testing.
T83168 929100-929274 Sentence denotes Processing small regions of tumor can be challenging as traditional manual macro-dissection and purification methods include multiple steps during which material can be lost.
T55046 929275-929415 Sentence denotes We evaluated an approach that combines automated tissue dissection with NGS library preparation directly from fragments of dissected tissue.
T75153 929416-929569 Sentence denotes Methods: FFPE tissue dissection was performed using the Roche Automated Tissue Dissection platform (Roche Diagnostics) which is currently in development.
T88015 929570-929764 Sentence denotes For DNA analysis, tissue fragments were processed using FFPE Direct Reagent (Thermo Fisher) and Ion AmpliSeq Cancer Hotspot Panel v2 (CHPv2) libraries were prepared and run on an Ion PGM system.
T2320 929765-929939 Sentence denotes For RNA analysis, tissue fragments were processed using the HTG Oncology Biomarker Panel (OBP) on the HTG EdgeSeq (HTG Molecular) and libraries were run on an Illumina MiSeq.
T6599 929940-929948 Sentence denotes Results:
T30694 929949-930152 Sentence denotes Automated tissue dissection was performed on slides of unstained sections from archived FFPE cases of melanoma, colon adenocarcinoma, pancreatic ductal adenocarcinoma and pancreatic neuroendocrine tumor.
T46084 930153-930227 Sentence denotes For DNA analysis, we dissected areas of tumor tissue from 50 mm2 to 1 mm2.
T6701 930228-930364 Sentence denotes We show that all CHPv2 libraries yielded excellent sequence data and that there was 100% concordance with previously reported mutations.
T88115 930365-930525 Sentence denotes We compared the performance metrics of libraries prepared from 1 mm2 to larger areas of tissue for the same case and found highly correlated amplicon coverages.
T48560 930526-930600 Sentence denotes For RNA analysis, we dissected areas of tumor tissue from 25 mm2 to 1 mm2.
T37326 930601-930691 Sentence denotes The HTG OPB libraries all passed QC metrics and yielded high quality gene expression data.
T83668 930692-930808 Sentence denotes In particular, samples processed from equivalent areas of tumor on adjacent sections yielded highly correlated data.
T63602 930809-930821 Sentence denotes Conclusions:
T34801 930822-931036 Sentence denotes The precision isolation of FFPE tumor provided by automated tissue dissection combined with extraction free DNA/RNA library preparation can be used to minimize material losses and reduce the amount of tissue input.
T6122 931037-931154 Sentence denotes The approach allows the processing of extremely small samples that are otherwise too small for standard NGS analysis.
T77592 931155-931157 Sentence denotes V.
T61648 931158-931170 Sentence denotes Kumar 1 , R.
T88623 931171-931182 Sentence denotes Webb 2 , N.
T36715 931183-931198 Sentence denotes Palomino 3 , E.
T38423 931199-931210 Sentence denotes Fang 1 , C.
T97288 931211-931223 Sentence denotes Perez 2 , J.
T77791 931224-931236 Sentence denotes Punia 1 , K.
T83003 931237-931250 Sentence denotes Fisher 1 , W.
T88644 931251-931265 Sentence denotes Parsons 1 , D.
T66411 931266-931286 Sentence denotes López-Terrada 1 , L.
T75357 931287-931309 Sentence denotes Suarez Ferguson 2 , A.
T72816 931310-931437 Sentence denotes Roy 1 1 Baylor College of Medicine, Houston TX; 2 Texas Children's Hospital, Houston, TX; 3 Texas Tech University, Lubbock, TX.
T67011 931438-931451 Sentence denotes Introduction:
T55917 931452-931548 Sentence denotes Internal tandem duplications (ITD) are a rare class of oncogenic genetic aberrations in cancers.
T37034 931549-931893 Sentence denotes The recent discovery of recurrent C-terminal ITDs in the X-linked BCL-6 co-repressor (BCOR) gene in diverse childhood solid tumors, including clear cell sarcoma of the kidney (CCSK), primitive neuroectodermal tumors of the brain, and soft tissue sarcomas, highlights the emergence of a useful marker for these diagnostically-challenging tumors.
T42792 931894-932102 Sentence denotes Herein, we describe the development and clinical implementation of a DNA-based fluorescent fragment analysis test to detect BCOR ITDs from formalin-fixed and paraffin-embedded (FFPE) and frozen tumor samples.
T90764 932103-932111 Sentence denotes Methods:
T48763 932112-932409 Sentence denotes Genomic DNA extracted from frozen and FFPE specimens of ITD-positive CCSK tumors (true positives, n=9), an ITD-negative CCSK and tumor-adjacent normal kidney specimens (true negatives, n=7), Wilms tumors (true negatives, n=23), and congenital mesoblastic nephromas (true negatives, n=9) were used.
T42394 932410-932586 Sentence denotes DNA was amplified with specific (6-FAM-labeled forward and unlabeled reverse) primers designed to amplify a fragment of the last coding exon of the BCOR gene that harbors ITDs.
T14810 932587-932772 Sentence denotes Fluorescent Fragments were detected using the ABI 3130 Capillary Electrophoresis system, sized with ABI GeneScan-500 LIZ ISS and analyzed with GeneMapper Software 5 (Life Technologies).
T60076 932773-932898 Sentence denotes Limit of detection studies were performed using serial dilutions of an ITD-positive case with known variant allelic fraction.
T15084 932899-932975 Sentence denotes Results were compared with Sanger sequencing and next-generation sequencing.
T14808 932976-932984 Sentence denotes Results:
T11763 932985-933177 Sentence denotes Fragment analysis-based ITD detection was successfully achieved with a total DNA input of >10ng (FFPE) and >1ng (frozen specimens), at an analysis threshold of 100 relative fluorescence units.
T65978 933178-933302 Sentence denotes Three different ITDs (90 to 96 bp) in eight of nine true-positive samples were detected by the fragment analysis assay (8/9:
T68728 933303-933400 Sentence denotes 89% clinical sensitivity), with the single discordant result being in a sample with degraded DNA.
T98181 933401-933511 Sentence denotes Clinical specificity of 100% was observed with all (39/39) true negative samples confirmed to be ITD negative.
T4940 933512-933600 Sentence denotes A high degree of concordance (0.97, Cohen's Kappa) was obtained between the two methods.
T92708 933601-933726 Sentence denotes Allelic ratio estimation using peak heights (ITD/wild-type) revealed a lower limit of detection of 5% tumor/normal DNA ratio.
T95942 933727-933870 Sentence denotes Both repeatability and reproducibility studies demonstrated sizing variation <0.15bp for the wild-type (expected size 287bp) and ITD fragments.
T21430 933871-933883 Sentence denotes Conclusions:
T3897 933884-934089 Sentence denotes The newly-developed fragment analysis test is a highly sensitive and specific clinical diagnostic tool that can be used on minimal archival FFPE specimens for the detection of BCOR ITDs in tumor specimens.
T78826 934090-934230 Sentence denotes Prospective application of this assay in a wide variety of BCOR ITDpositive tumor types would enable the evaluation of its clinical utility.
T88359 934231-934244 Sentence denotes Introduction:
T89192 934245-934406 Sentence denotes A majority of molecular diagnostic tests using formalin-fixed paraffinembedded (FFPE) tissue start from DNA extraction, a labor-intensive and timeconsuming step.
T33945 934407-934709 Sentence denotes To accommodate increasing demands of DNA-based testing on FFPE samples, we sought to decrease the labor and time of the deparaffinization step of extraction, and combine Zymo Research (ZR) Pinpoint isolation with Qiagen spin columns to facilitate automation of the DNA purification step on the QIAcube.
T14709 934710-934980 Sentence denotes Methods: FFPE slides were deparaffinized using a xylene alternative on the BiogenX Xmatrx NANO, followed by isolation of tissue from areas containing tumor cells using the ZR Pinpoint Solution and DNA extraction using ZR Pinpoint >40% Extraction Buffer and Proteinase K.
T26750 934981-935135 Sentence denotes The DNA purification was compared using the Zymo-Spin I columns (Zymo method) to Qiagen MinElute columns (hybrid method), which allowed us to use QIAcube.
T74990 935136-935223 Sentence denotes DNA quantity was determined with Qubit dsDNA BR Assay Kit and Agilent 2200 TapeStation.
T74818 935224-935232 Sentence denotes Results:
T74657 935233-935491 Sentence denotes Both methodologies require fewer unstained slides compared to our previously validated extraction and purification method, which involves scraping tumor from unstained FFPE slides, xylene deparaffinization, lysis, and purification on QIAamp Mini spin column.
T47829 935492-935602 Sentence denotes The ZR Pinpoint Solution allows for precise tissue selection and less tissue loss compared to scraping method.
T5488 935603-935780 Sentence denotes DNA yields were equivalent in both Zymo and hybrid methods, determined by Qubit BR assay and TapeStation (350-900 ng total), and are higher than our traditional scraping method.
T77037 935781-935825 Sentence denotes OD 260/OD280 ratio ranged from 1.85 to 2.00.
T66709 935826-936050 Sentence denotes Combining ZR Pinpoint DNA isolation with Qiagen DNA purification has enabled us to decrease hands-on time to 30 min from 60 min, when the QIAcube is incorporated; and the total extraction time to 6 hours from 18 to 20 hours.
T32639 936051-936124 Sentence denotes This method also allows for a decrease in error and recovery of more DNA.
T37339 936125-936137 Sentence denotes Conclusions:
T72537 936138-936350 Sentence denotes Automated deparaffinization of FFPE slides followed by ZR Pinpoint isolation of tumor tissue and Qiagen's spin column technology results in DNA yields similar to those of using ZR Pinpoint Isolation System alone.
T67949 936351-936470 Sentence denotes Pinpoint Solution allows for more precise tissue sampling and less loss of tissue than the traditional scraping method.
T26547 936471-936605 Sentence denotes Using Qiagen spin columns enables automation of DNA purification on the QIAcube, and results in significantly decreased hands-on time.
T33123 936606-936705 Sentence denotes Alternative deparaffinization eliminates xylene, thereby decreasing chemical exposure and disposal.
T6744 936706-936830 Sentence denotes This semiautomated hybrid method will increase efficiency as well as the quality of the workflow in our clinical laboratory.
T82398 936831-936898 Sentence denotes Rejection of Specimens Previously Judged Inadequate for Analysis S.
T94568 936899-936909 Sentence denotes Rapp, T.C.
T98361 936910-936923 Sentence denotes Greiner, A.M.
T66037 936924-936988 Sentence denotes Cushman-Vokoun University of Nebraska Medical Center, Omaha, NE.
T8967 936989-937002 Sentence denotes Introduction:
T93727 937003-937101 Sentence denotes Manual processing of specimens for Next-generation sequencing (NGS) is complex and time consuming.
T15057 937102-937172 Sentence denotes Automation can allow for better standardization and improved workflow.
T69802 937173-937289 Sentence denotes The Ion Torrent Chef requires 8 specimens be pooled on an Ion 318 chip, thus 16 specimens per 2 chip sequencing run.
T65569 937290-937366 Sentence denotes However, our lab volume is not sufficient to run two Ion 318 chips per week.
T43142 937367-937628 Sentence denotes We describe an alternative NGS automated workflow with combined data from two Ion 316 chips resulting in improved wet-bench turnaround time, reduced technician hands-on time, equivalent data and the ability to adequately analyze previously inadequate specimens.
T4470 937629-937637 Sentence denotes Methods:
T48248 937638-937835 Sentence denotes Twenty clinical specimens were previously sequenced using a manual workflow and the 50 gene Ion AmpliSeq Cancer Hotspot Panel, the Ion One Touch 2 and the Ion Torrent Personal Genome Machine (PGM).
T46832 937836-937992 Sentence denotes Eight additional specimens that previously failed or were excluded from clinical testing due to low quality or quantity DNA were also included in the study.
T64521 937993-938135 Sentence denotes Laboratory validation was then performed using the Ion AmpliSeq Kit for Chef DL8 and the Ion AmpliSeq Cancer Hotspot Panel v2 on the Ion Chef.
T23953 938136-938341 Sentence denotes Combined libraries were prepared in duplicate (8 specimens per Ion 316 chip including control) using the Ion PGM Hi-Q Chef Kit and Ion 316 Chip Kit v2 for clonal amplification, chip loading and sequencing.
T73966 938342-938593 Sentence denotes Aligned data from the same 8 specimens on two 316 chips were combined, using the Torrent Suite Software v5.0.4 with the variantCaller plugin v5.0.4.0, to achieve adequate coverage for detecting somatic mutations, rather than employing an Ion 318 chip.
T38122 938594-938676 Sentence denotes This strategy has not been previously reported in the literature or by the vendor.
T50728 938677-938685 Sentence denotes Results:
T29387 938686-938786 Sentence denotes For specimens with previously known clinical results (n=20), all expected mutations were identified.
T30993 938787-938895 Sentence denotes Additionally the 8 specimens, deemed inadequate for the manual workflow by lab experience, were successfully