KRAS and BRAF mutation analysis on paired biopsy and resection specimens of patients with colorectal cancer: Molecular analysis using high-resolution melting, direct sequencing, KRAS ARMS-scorpion PCR and BRAF pyrosequencing.

Abstract

10609 Background: KRAS testing is mandatory if anti-EGFR therapy for metastatic colorectal cancer (CRC) is considered. In addition, BRAF might prove to be as important for successful treatment with panitumumab or cetuximab. To date, molecular analysis is preferably performed on resection material; however, in metastatic disease a biopsy might be the only available tissue. The aim of this study is to analyze KRAS and BRAF mutations in paired biopsy and resection specimens of the primary tumor to explore if biopsy material is sufficient to define the mutational status. Methods: DNA was extracted from a group of 126 unselected CRC patients following macro dissection of formalin-fixed paraffin-embedded (FFPE) blocks using the QIAmp DNA FFPE Tissue kit. KRAS and BRAF mutation was assessed using high-resolution melting (HRM) and direct sequencing. Additionally the Therascreen ARMS-scorpion KRAS assay (DxS, Manchester, UK) and BRAF Pyrosequencing (Therascreen BRAF Pyro Kit, Qiagen, Venlo, N.L.) were employed as well; both assays claim to require less tumor cells in comparison with direct sequencing. Results: Six KRAS samples were not conclusive in the HRM, one in the sequence and one in the ARMS-scorpion method and are excluded. BRAF showed no exclusions. BRAF and KRAS were found to be mutually exclusive. Mutation frequencies for KRAS codon 12, 13 and BRAF V600E were 33.9% and 19.0%, respectively. Conclusions: Biopsies and resection specimens of CRC patients showed very high KRAS and BRAF concordance. Biopsies of the primary tumor and possibly metastasis, representatives of the primary tumor, can thus be used for molecular analysis. Number of mutated and wild-type samples found for each method. Percentage concordance between biopsy and resection without the nondetectable samples. KRAS-HRM KRAS-Seq ARMS pcr BRAF-HRM BRAF-Seq Pyroseq Biopsy mutated (n) 40 38 38 22 21 22 Resection mutated (n) 40 40 40 23 23 24 Biopsy wild type (n) 85 87 88 104 105 104 Resection wild type (n) 81 86 85 103 103 102 Concordance (%) 99.2 98.4 97.6 99.2 98.4 98.4