Clinical Validation of a Multiplex Kit for RAS Mutations in Colorectal Cancer: Results of the RASKET (RAS KEy Testing) Prospective, Multicenter Study

BACKGROUND: RAS (KRAS and NRAS) testing is required to predict anti-epidermal growth factor receptor (EGFR) treatment efficacy in metastatic colorectal cancer (CRC). Although direct sequencing (DS) with manual microdissection (MMD) is widely used, a diagnostic kit providing rapid detections of RAS mutations would be clinically beneficial. We evaluated the MEBGEN(TM) RASKET KIT (RASKET KIT), a multiplex assay using PCR-reverse sequence specific oligonucleotide and xMAP(®) technology to concurrently detect exon 2, 3, and 4 RAS mutations in a short turnaround time (4.5 h/96-specimens). METHODS: Formalin-fixed paraffin-embedded (FFPE) tissues were obtained from 308 consenting patients with histologically-confirmed CRC at six hospitals in Japan. For the RASKET KIT, we used only 50–100 ng DNA from each FFPE specimen not processed by MMD. The primary endpoint was the concordance rate between RAS mutations identified with the RASKET KIT and two reference assays (DS with MMD and TheraScreen(®) K-RAS Mutation Kit). As the secondary endpoints, we evaluated the concordance rate between DS and the RASKET KIT for RAS mutations in the wild-type KRAS exon 2 population and the genotyping performance of the RASKET KIT compared with DS. FINDINGS: Among 307 analyzable specimens, the reference assays detected 140 (45.6%, 140/307) RAS mutations: 111 KRAS exon 2 and 29 other (minor) RAS mutations. The RASKET KIT detected 143 (46.6%, 143/307) mutations: 114 KRAS exon 2 and 29 minor RAS mutations. The between-method concordance rate was 96.7% (297/307) (95% CI: 94.1–98.4%). Minor RAS mutations were detected in 15.7% (30/191) of the wild-type KRAS exon 2 population (n = 191); the concordance rate was 98.4% (188/191) (95% CI: 95.5–99.7%). The concordance rate of RAS genotyping was 100% (139/139) (95% CI: 97–100%). INTERPRETATION: The RASKET KIT provides rapid and precise detections of RAS mutations and consequently, quicker and more effective anti-EGFR therapy for CRC (Study ID: UMIN000011784). FUNDING: Medical & Biological Laboratories Co., Ltd. (MBL). MBL had roles in study design, data collection, data analysis, and writing of the report for the study.