2. Activation of the Proto-Oncogenes KRAS and BRAF in Colorectal Cancer
Somatic mutations of the KRAS gene are found in approximately 40% of CRC [4]. KRAS is a member of the RAS superfamily, a family of monomeric small-G proteins, and functions as a transmitter of key extracellular signals (e.g., epidermal growth factors) into intracellular signal cascades [5]. Patients with CRC tumors expressing mutant KRAS will not benefit from a targeted therapy against EGFR [6]. Therefore mutation analysis of the KRAS gene is important in clinical practice supporting therapy decision. Although direct sequencing is the gold standard for the detection of KRAS mutations in clinical diagnostics, it remains laborious and is not very sensitive [7]. SNaPshot and reverse hybridization StripAssay were tested as alternatives to direct sequencing for KRAS mutation detection in daily routine. Detection limits of direct sequencing, SNaPshot and StripAssay were 20%, 10% and 1%, respectively [7]. Consequently, the authors concluded that SNaPshot and the StripAssay could both be alternatives to direct sequencing, especially in small laboratories without dedicated equipment. However, the risk of false positives is increased. Another study compared the performance and reagent costs of two new sensitive methods, a peptide nucleic acid (PNA) clamp PCR and amplification refractory mutation system PCR assay, for detection of KRAS mutations. The PNA clamping assay had a higher sensitivity as well as 20 times lower costs compared to ARMS [8]. Therefore, the authors concluded that due to high performance and low costs the PNA clamping assay could be a suitable method for detecting KRAS mutations.
BRAF is a serine-threonine protein kinase and a member of the RAF kinase gene family that acts as a downstream effector of the KRAS gene. Mutations of the BRAF gene, which were found at an early stage of CRC carcinogenesis, occur in 5%–15% of overall CRC [9]. Moreover, BRAF mutations appear to be a valid indicator of poor survival in patients with CRC [10]. Direct sequencing is the gold standard for the detection of BRAF mutations. Nevertheless, Benlloch and colleagues suggest a real-time PCR based test as a suitable alternative to direct sequencing. This test is highly sensitive and specific for detecting V600E mutations and has advantages in cost, time and labor [11]. Recently, the use of digital PCR to detect BRAF mutations was also mentioned [12]. This new technology is an attractive tool due to the formidable accuracy of results, time to results and cost per sample. In a further study, the use of COLD-PCR, a modified PCR protocol that allows preferential amplification of minority alleles from a mixture of wildtype and mutation-containing DNA, improves the detection limit of KRAS and BRAF mutations in CRC without requiring expensive and time-consuming procedures [13].