Concordance in KRAS and BRAF mutations in endoscopic biopsy samples and resection specimens of colorectal adenocarcinoma

Abstract

Background

KRAS testing is mandatory if anti-EGFR therapy is considered in patients with metastatic colorectal cancer (CRC). In addition, BRAF mutations seem to be an important negative prognostic factor. The aim of this study is to establish the concordance of KRAS and BRAF mutational status in paired biopsy and resection specimens of primary CRC using several analytic methods.

Methods

DNA was extracted from paraffin blocks of 126 CRC patients. KRAS codon 12/13 and BRAF V600E mutational status was assessed using high resolution melting (HRM), direct sequencing (DS) of the HRM polymerase chain reaction (PCR) product. In addition, the Therascreen Amplification Refractory Mutation System (ARMS)-Scorpion KRAS assay and BRAF pyrosequencing were employed; both assays claim to require less tumour cells in comparison with DS.

Results

KRAS and BRAF were found to be mutually exclusive. Mutation frequencies were 33.9% for KRAS, and for BRAF 19.0%, respectively. Concordance of KRAS mutational status between biopsy and resection specimens was 97.4% (ARMS), 98.4% (DS) and 99.2% (HRM), respectively. For BRAF concordance was 98.4% (Pyro, DS) and 99.2% (HRM).

Conclusions

KRAS and BRAF mutational status of endoscopic biopsies and resection specimens of CRC showed a >95% concordance. Endoscopic biopsies can be confidently used for molecular analysis.

Introduction

The demand for KRAS mutational analysis as a predictive marker has increased rapidly. Prior to treatment with epidermal growth factor receptor (EGFR) inhibitors in colorectal cancer (CRC), KRAS testing has become mandatory in the European Union1, 2 and is recommended in the United States.³ In addition, BRAF mutations are emerging as a very strong negative prognostic factor in CRC.⁴

KRAS is a member of the RAS proteins which are small GTPases that act as molecular switches. KRAS binds to Guanosine triphosphate (GTP) in the active state and has an intrinsic enzymatic activity which cleaves the terminal phosphate of the nucleotide converting it to Guanosine diphosphate (GDP).5, 6 Upon conversion of GTP to GDP, KRAS is turned off.5, 7 The KRAS protein has an important role in Ras/MAPK signalling in which GTP-bound KRAS regulates a variety of cellular processes, including proliferation, differentiation and apoptosis.6, 8 In CRC, codon 12 and 13 of the KRAS gene are mutated in 35% of cases,⁹ transforming the intrinsic GTPase activity of the protein in the constitutively active conformation.¹⁰

BRAF, a member of the Raf kinase family of serine/threonine-specific protein kinases, is a cytosolic protein kinase and is activated by membrane-bound RAS. Mutated BRAF activates a signalling pathway, which causes cell proliferation and inhibits apoptosis.¹⁰ The most common mutation is a single glutamic acid for valine substitution at codon 600 causing the V600E point mutation,⁷ the BRAF mutation frequency is 11%.⁹ Earlier, Roth and colleagues¹¹ described the mutual exclusivity of BRAF and KRAS mutations.

The activating mutations in KRAS and BRAF induce constitutive Ras/MAPK signalling, which cannot be suppressed by EGFR inhibition. Due to prevent undesirable side-effects of the EGFR antibodies and to suppress excessive treatment costs KRAS mutation analysis prior to anti-EGFR therapy is indicated.¹² In addition, BRAF mutational analysis yields prognostic information.¹¹

Currently the most commonly used method for KRAS and BRAF mutation analysis is direct sequencing (DS). Nevertheless, this method requires high tumour cell percentages and good quality material. Resection specimens normally show large amounts of tumour cells. Biopsies generally consist of small tumour foci that might not meet the requirements for direct sequencing. An alternative is high-resolution-melting (HRM) analysis. High resolution melting (HRM) is advocated to be a rapid and cheap ‘pre-screen’ method, and can distinguish wild type from mutated genes based on subtle differences in temperature-depended denaturation (‘melting’) of double strand DNA fragments. This method requires considerably lower tumour cell percentages compared to DS but is unable to define the exact nature of a mutation when non-wild type melting curves are found. Other methods allowing low tumour cell percentages are pyrosequencing and a specific Amplification Refractory Mutation System (ARMS)-Scorpion PCR assay.

To date, no systematic study has been performed to ascertain the validity of KRAS and BRAF mutational analysis of endoscopic biopsy material in comparison to resection specimens.

The aim of our study was to establish the concordance of KRAS and BRAF mutational status between paired endoscopic biopsy and resection specimens in an unselected group of CRC patients. Three molecular assessment methods were used, e.g. DS and HRM for both genes, and in addition DxS ARMS-Scorpion Therascreen for KRAS and Qiagen Therascreen Pyro kit for BRAF.