Variant Detection Rates in an MPT Series
We previously reported a retrospective series of MPT individuals (defined as having two primary tumors before 60 years of age) referred to a UK clinical genetics service without pre-assessment and observed that 20.7% (44/212) were found to have a molecular diagnosis upon routine targeted molecular genetic testing, including BRCA1 and BRCA2 testing, mismatch-repair gene analysis, or other single-gene testing (APC [MIM: 611731], MUTYH [MIM: 604933], PTEN, TP53 [MIM: 191170], and RB1 [MIM: 614041]).31
In the current study, we addressed whether comprehensive genetic analysis in pre-assessed individuals with MPTs might increase the diagnostic yield over routine targeted testing. Thus, we analyzed 460 MPT-affected individuals who had previously undergone routine genetic assessment and/or molecular testing (but without a molecular diagnosis) by using WGS for variants in 83 CPGs and identified a P/LP variant in 67/440 (15.2%) probands (incorporating SNVs, indels, and SVs), including those affected by moderate- and high-risk CPGs.
Because the MPT cohort reported here was mostly ascertained from UK genetics centers (and was similar to the previous retrospective cohort that did not have a known genetic cause), we estimate (by assuming that WGS would detect variants identified by routine targeted sequencing approaches) that comprehensive genetic analysis in a genetics-center-referred series of individuals with MPTs (and no prior genetic testing) would detect a P/LP variant in around a third of individuals (20.7% + 12.1% [estimated under the assumption of a diagnostic yield of 15.2% in the 79.3% of individuals without a variant in routine testing] = 32.8%). The estimated proportion of individuals with a P/LP variant and a typical tumor would be ∼27.5% (20.7% [all of those with variants detected by targeted analysis had a typical tumor] + [79.3% × 8.6% = 6.8%]). Therefore, in individuals seen in a genetic clinic, the presence of MPTs (two tumors before 60 years of age or three before 70 years of age) could be taken as an indication for considering genetic testing.
The estimates for diagnostic yield are approximate and would be influenced by ascertainment processes but do suggest that a comprehensive testing for CPG variants significantly increases the detection of P/LP variants over the targeted testing that has been routinely employed in most genetics centers.
Most MPT-affected individuals (38/67 [56.7%] and 38/440 [8.6%] of all pre-assessed probands tested in the current study) with a P/LP variant had been diagnosed with a tumor type characteristically associated with variants in the relevant CPG, findings that have the greatest clinical utility. In, addition, a further 8/440 (1.8%) had a VUS and a previous diagnosis of a characteristic tumor. Such VUSs might eventually be reclassified as LP variants with further investigations (e.g., tumor studies or functional analysis) or additional clinical information (e.g., segregation analysis). However, interpretation of segregation data should be cautious in cancer-predisposition syndromes because of incomplete penetrance and a higher probability of phenocopies. Tumor studies for loss of heterozygosity do not provide absolute confirmation or exclusion of pathogenicity, and together these considerations reinforce the importance of data-sharing initiatives such as ClinVar.20
A major influence on the number and pattern of variants detected in a study such as this is the tumor phenotypes occurring in the cohort, which in this case reflect both the population incidence and the patterns of referral for genetic assessment and investigation. Compared with MPT-affected individuals in cancer registries, our series is enriched with combinations such as breast-ovary (4.4% versus 1.9%) and breast-colorectal (5.5% versus 2.8%), most likely reflecting common cancers with a significant hereditary component and for which genetic testing has been routinely available for a number of years. Many of these cancers are sex specific, most likely contributing to the uneven sex distribution in this series. Some combination types making up >1% of MPT combinations, e.g., breast-thyroid (3.6% in MPT data), are not observed frequently (<1%) in the population-based cohort used here, which could be accounted for by referral prompted by suspicion of germline PTEN variants.
Breast cancer accounted for almost a quarter of tumors in our series, and most genes in which deleterious variants were detected are breast CPGs, many of which are not routinely tested in the UK. Pathogenic variants in ATM and CHEK2 are associated with moderate risks,32, 33 and these genes had not been tested by the referring center in any of the individuals with P/LP variants. Six probands had pathogenic variants in PALB2, a gene initially thought to confer moderate risk34 but subsequently reported to have a penetrance somewhere between that of moderate- and high-risk genes such as BRCA1 and BRCA2.35
Genes can remain uninvestigated by clinicians not only because of uncertainty surrounding risks but also because of recency of discovery. A number of CPGs in which variants were identified, such as MAX and FH, have been relatively recently described as causing pheochromocytoma and paraganglioma. The appearance of these variants in this analysis most likely reflects a lack of availability of testing at the time of consultation and subsequent referral for inclusion in the study. Molecular genetic testing has been available for other genes such as MLH1 and PTEN for a greater period of time, but some individuals appeared not to have fulfilled the clinical testing criteria applied in the referring center. TP53 is a further well-established CPG that is associated with diverse and multiple cancers and has clear clinical testing criteria that are often not fulfilled. Despite this, no pathogenic variants were detected. Germline TP53-variant-related phenotypes (including rare and/or early-onset cancers) are more clearly identifiable clinically and are less likely to appear in cohorts such as ours without specific ascertainment for them. Consistent with this are mutation detection rates of ∼4% in individuals with earlier-onset (≤30 years) breast cancer36 and ∼17% in MPT-affected individuals who were referred for germline TP53 testing and who generally fulfilled criteria for that investigation, had tumors characteristic of Li Fraumeni syndrome, and had an average age at diagnosis (of a first primary tumor) before 30 years.2
Although we report the application of WGS to an adult MPT series, other studies have used agnostic NGS strategies in cohorts with single-site cancer. The detection rate of pathogenic variants in these analyses could be influenced by the assay used, the variant filtering and assessment applied, and the nature of the series in terms of both phenotype and ascertainment. The application of a 76-gene panel to ∼1,000 cancer-affected adults referred for germline genetic testing and ACMG-guideline-based assessment of the resulting variants showed a 17.5% rate,37 whereas tumor-normal sequencing of a similarly sized series with advanced cancer from the same center (regardless of genetic testing referral) reported an equivalent figure of 12.6%.38 The genes containing the most frequent pathogenic variants in both studies were similar to those in the current study (BRCA1, BRCA2, CHEK2, and ATM), but the detection rates were lower than our estimate of around a third of newly referred MPT-affected individuals, most likely reflecting a greater likelihood of a germline pathogenic variant in both genetics referrals and in MPT-affected individuals. Studies of WGS and/or WES applied to unselected pediatric cancer series have also shown pathogenic-variant detection rates close to 10% but a contrasting range of affected genes, suggesting that TP53 and genes associated with embryonal tumors play a far greater role.39, 40, 41