Clinical validation of the next-generation sequencing-based Extended RAS Panel assay using metastatic colorectal cancer patient samples from the phase 3 PRIME study.

PURPOSE: To validate a next-generation sequencing (NGS)-based companion diagnostic using the MiSeqDx® sequencing instrument to simultaneously detect 56 RAS mutations in DNA extracted from formalin-fixed paraffin-embedded metastatic colorectal cancer (mCRC) tumor samples from the PRIME study. The test's ability to identify patients with mCRC likely to benefit from panitumumab treatment was assessed. METHODS: Samples from PRIME, which compared first-line panitumumab + FOLFOX4 with FOLFOX4, were processed according to predefined criteria using a multiplex assay that included input DNA qualification, library preparation, sequencing, and the bioinformatics reporting pipeline. NGS mutational analysis of KRAS and NRAS exons 2, 3, and 4 was performed and compared with Sanger sequencing. RESULTS: In 441 samples, positive percent agreement of the Extended RAS Panel with Sanger sequencing was 98.7% and negative percent agreement was 97.6%. For clinical validation (n = 528), progression-free survival (PFS) and overall survival (OS) were compared between patients with RAS mutations (RAS Positive) and those without (RAS Negative). Panitumumab + FOLFOX4 improved PFS in RAS Negative patients (P = 0.02). Quantitative interaction testing indicated the treatment effect (measured by the hazard ratio of panitumumab + FOLFOX4 versus FOLFOX4) differed for RAS Negative versus RAS Positive for PFS (P = 0.0038) and OS (P = 0.0323). CONCLUSIONS: NGS allows for broad, rapid, highly specific analyses of genomic regions. These results support use of the Extended RAS Panel as a companion diagnostic for selecting patients for panitumumab, and utilization is consistent with recent clinical guidelines regarding mCRC RAS testing. Overall, approximately 13% more patients were detected with the Extended RAS Panel versus KRAS exon 2 alone. CLINICAL TRIAL REGISTRY IDENTIFIER: NCT00364013 (ClinicalTrials.gov).