Expanded Low Allele Frequency <i>RAS</i> and <i>BRAF</i> V600E Testing in Metastatic Colorectal Cancer as Predictive Biomarkers for Cetuximab in the Randomized CO.17 Trial.

Abstract

Expanded <i>RAS/BRAF</i> mutations have not been assessed as predictive for single-agent cetuximab in metastatic colorectal cancer (mCRC), and low mutant allele frequency (MAF) mutations are of unclear significance. We aimed to establish cetuximab efficacy in optimally selected patients using highly sensitive beads, emulsion, amplification, and magnetics (BEAMing) analysis, capable of detecting alterations below standard clinical assays. CO.17 trial compared cetuximab versus best supportive care (BSC) in <i>RAS/BRAF</i>-unselected mCRC. We performed <i>RAS/BRAF</i> analysis on microdissected tissue of 242 patients in CO.17 trial using BEAMing for <i>KRAS/NRAS</i> (codons 12/13/59/61/117/146) and <i>BRAF</i> V600E. Patients without BEAMing but with previous Sanger sequencing-detected mutations were included. <i>KRAS, NRAS</i>, and <i>BRAF</i> mutations were present in 53%, 4%, and 3% of tumors, respectively. Cetuximab improved overall survival [OS; HR, 0.51; 95% confidence interval (CI), 0.32-0.81; <i>P</i> = 0.004] and progression-free survival (PFS; HR, 0.25; 95% CI, 0.15-0.41; <i>P</i> < 0.0001) compared with BSC in <i>RAS/BRAF</i> wild-type patients. Cetuximab did not improve OS/PFS for <i>KRAS-, NRAS-</i>, or <i>BRAF-</i>mutated tumors, and tests of interaction confirmed expanded <i>KRAS</i> (<i>P</i> = 0.0002) and <i>NRAS</i> (<i>P</i> = 0.006) as predictive, while <i>BRAF</i> mutations were not (<i>P</i> = 0.089). BEAMing identified 14% more tumors as <i>RAS</i> mutant than Sanger sequencing, and cetuximab lacked activity in these patients. Mutations at MAF < 5% were noted in 6 of 242 patients (2%). One patient with a <i>KRAS</i> A59T mutation (MAF = 2%) responded to cetuximab. More <i>NRAS</i> than <i>KRAS</i> mutations were low MAF (OR, 20.50; 95% CI, 3.88-96.85; <i>P</i> = 0.0038). We establish single-agent cetuximab efficacy in optimally selected patients and show that subclonal <i>RAS/BRAF</i> alterations are uncommon and remain of indeterminate significance.