Development of an assay for KRAS mutation as a predictive marker for cetuximab in colorectal cancer

Abstract

e15048 Background: The activating KRAS mutation is an important predictive marker for anti-EGFR therapy against colorectal cancer (CRC), although valid assays using pathological specimens have not been yet established. We present a rapid, sensitive assay for KRAS genotyping using small biopsy specimens. Methods: We used Cycleave PCR to detect the KRAS mutation in CRC with a chimeric DNA-RNA-DNA probe labeled with fluorescent dye and quencher, with results obtained within 4 hours. Template DNA was extracted from formalin-fixed, paraffin-embedded specimens, which are surgically resected or biopsied in clinical practice. Results: To evaluate Cycleave PCR accuracy for detecting the KRAS mutation, we compared with results of reverse transcriptase-PCR-coupled direct sequencing (RT-PCR-DS) of metastatic lung tumor specimens from CRC patients. KRAS mutations were present in 40 (43%) of 94 patients, including 28 (30%) and 8 (9%) in codon 12 and codon 13 mutations, respectively. Concordant results between Cycleave PCR and RT-PCR-DS in KRAS codon 12 and 13 were found in 35/36 (97%) and 23/23 (100%), respectively. We also applied this method to surgical specimens in clinical practice. Although 8 from 73 patients (11%) could not be evaluated with Cycleave PCR, corresponding biopsy specimens could be used alternatively. Because biopsy specimens were fixed by formalin for a shorter period, fixation of surgical specimens for longer time may have contributed to PCR failure. Indeed, over- fixation by formalin impaired PCR amplification of KRAS in time-dependently. Furthermore, results of 4 randomly selected biopsy specimens using Cycleave PCR were consistent with those of surgical specimens. Conclusions: Cycleave PCR even using biopsy specimens is accurate, rapid, and useful to detect KRAS mutations in CRC patients. This study also called attention to specimen selection, as over-fixation by formalin may lead to failure of the gene examination due to template DNA fragmentation. This method can be applied to examine BRAF genotyping as well as KRAS. KRAS/BRAF genotyping by Cycleave PCR leads to individualized therapy using cetuximab for CRC patients. No significant financial relationships to disclose.