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).