Background and objective: The rapid development of molecular biology techniques allows for the introduction of real-time polymerase chain reaction (PCR) methods with a limit of mutation detection at 1% in a background of wild-type DNA. Analysis of KRAS mutations in codons 12, 13, and 61, together with analysis of BRAF mutations in codon 600, are predictive biomarkers for anti-epidermal growth factor receptor (EGFR) treatment in colorectal cancer. Our aim was to compare PCR methods for KRAS mutations and BRAF mutation analysis using DNA isolated from tissue samples previously evaluated for presence of tumor cells using a quantitative scale and the percentage of tumor cells (PTC) scale. We addressed the question of whether a low number of tumor cells can be qualified for somatic mutation testing.
Results: Our study showed that PTC as low as 10% was good enough to detect KRAS G12D, G13D, and Q61L mutations in formalin-fixed paraffin-embedded (FFPE) material. Furthermore, our results indicate that up to 20% of colorectal cancer may carry mutations in the KRAS codon 61 and BRAF codon 600, which suggests the value of these mutation analyses because patients carrying them are unlikely to respond to cetuximab or panitumumab. A low level of KRAS somatic mutation detection has not been studied in depth in the context of clinical outcomes in patients; therefore, we compared new PCR methods, (KRBR-RT 50 Entrogen; ViennaLab StripAssay) and re-evaluated KRAS and BRAF status in patients with relapse after targeted therapy.
Conclusions: The importance of molecular results was confirmed by clinical observation of a patient with relapse who had qualified for targeted therapy with KRAS WT status (but was diagnosed by less sensitive single-stranded conformation polymorphisms method). Interestingly, during anti-EGFR treatment, it came to the selection of cells with KRAS G12C mutation which were present from the beginning in the tumor but at a low level (detected by PCR methods) only and led consequently to the metastasis. Taking into consideration the limit of detection, labor time, and assay cost, the real-time PCR method seems to be very promising especially for FFPE material with the PTC below 15%.