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

PURPOSE: Expanded RAS/BRAF 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. PATIENTS AND METHODS: CO.17 trial compared cetuximab versus best supportive care (BSC) in RAS/BRAF-unselected mCRC. We performed RAS/BRAF analysis on microdissected tissue of 242 patients in CO.17 trial using BEAMing for KRAS/NRAS (codons 12/13/59/61/117/146) and BRAF V600E. Patients without BEAMing but with previous Sanger sequencing-detected mutations were included. RESULTS: KRAS, NRAS, and BRAF 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; P = 0.004] and progression-free survival (PFS; HR, 0.25; 95% CI, 0.15-0.41; P < 0.0001) compared with BSC in RAS/BRAF wild-type patients. Cetuximab did not improve OS/PFS for KRAS-, NRAS-, or BRAF-mutated tumors, and tests of interaction confirmed expanded KRAS (P = 0.0002) and NRAS (P = 0.006) as predictive, while BRAF mutations were not (P = 0.089). BEAMing identified 14% more tumors as RAS 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 KRAS A59T mutation (MAF = 2%) responded to cetuximab. More NRAS than KRAS mutations were low MAF (OR, 20.50; 95% CI, 3.88-96.85; P = 0.0038). CONCLUSIONS: We establish single-agent cetuximab efficacy in optimally selected patients and show that subclonal RAS/BRAF alterations are uncommon and remain of indeterminate significance.

Sensitive molecular testing methods can demonstrate NSCLC driver mutations in malignant pleural effusion despite non-malignant cytology.

Malignant pleural effusion (MPE) may be diagnosed by cytologic evaluation of pleural fluid, though false negative results can occur. Pleural effusions may provide a source of tumour material for genotyping in lung cancer patients. Detection of MPE may be improved through use of highly sensitive molecular techniques. We identified five patients with non-small cell lung cancer (NSCLC) with initial pleural fluid samples that were non-malignant on cytology, but were subsequently clinically confirmed to have MPE. Tumour mutation status was confirmed via routine testing of diagnostic clinical specimens. Cytologically negative pleural fluid cell-block specimens were analysed by amplicon-based parallel sequencing (APS) for somatic mutations commonly detected in NSCLC, and selected cases by improved and complete enrichment CO-amplification at lower denaturation temperature PCR (ICECOLD PCR) for known mutations. Mutations were detected in three out of three (sensitivity 100%) cytologically non-malignant pleural fluids from patients with a known mutation: two patients with known Kirsten rat sarcoma (KRAS) mutation demonstrated the same KRAS mutation in their pleural fluids by APS, both at approximately 2% mutant allele frequency. In one patient with a known KRAS mutation, ICECOLD PCR detected the same KRAS variant at 0.7% frequency. No mutations were detected in patients with wild-type findings from reference samples (specificity 100%). Sensitive DNA sequencing methods can detect cancer-driver mutations in cytologically non-malignant pleural fluid specimens from NSCLC patients with MPE. Our findings demonstrate the feasibility of sensitive molecular diagnostic techniques for improvement of diagnostic assessment of pleural effusions in patients with lung cancer.