title: Clinical Validation of a Multiplex Kit for RAS Mutations in Colorectal Cancer: Results of the RASKET (RAS KEy Testing) Prospective, Multicenter Study.
abstract: 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.
  
  
  RESULTS
  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%).
  
  
  CONCLUSIONS
  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).
  
  
  BACKGROUND
  Medical & Biological Laboratories Co., Ltd. (MBL). MBL had roles in study design, data collection, data analysis, and writing of the report for the study.
authors.affiliation: Department of Gastroenterology and Gastrointestinal Oncology, National Cancer Center Hospital East, Chiba, Japan.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Yoshino
authors.affiliation: Department of Clinical Oncology, Aichi Cancer Center Hospital, Aichi, Japan.
authors.forename: Kei
authors.initials: K
authors.lastname: Muro
authors.affiliation: Division of Gastroenterology, Saitama Cancer Center, Saitama, Japan.
authors.forename: Kensei
authors.initials: K
authors.lastname: Yamaguchi
authors.affiliation: Department of Gastrointestinal Medical Oncology, National Hospital Organization Shikoku Cancer Center, Ehime, Japan.
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Nishina
authors.affiliation: Division of Gastroenterology, Chiba Cancer Center, Chiba, Japan.
authors.forename: Tadamichi
authors.initials: T
authors.lastname: Denda
authors.affiliation: Department of Frontier Science for Cancer and Chemotherapy, Osaka University Graduate School of Medicine, Osaka, Japan.
authors.forename: Toshihiro
authors.initials: T
authors.lastname: Kudo
authors.affiliation: Division of Translational Research, Exploratory Oncology Research & Clinical Trial Center, National Cancer Center, Chiba, Japan.
authors.forename: Wataru
authors.initials: W
authors.lastname: Okamoto
authors.affiliation: Department of Clinical Oncology, Aichi Cancer Center Hospital, Aichi, Japan.
authors.forename: Hiroya
authors.initials: H
authors.lastname: Taniguchi
authors.affiliation: Division of Molecular Diagnosis and Cancer Prevention, Saitama Cancer Center, Saitama, Japan.
authors.forename: Kiwamu
authors.initials: K
authors.lastname: Akagi
authors.affiliation: Department of Gastrointestinal Medical Oncology, National Hospital Organization Shikoku Cancer Center, Ehime, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Kajiwara
authors.affiliation: Clinical Trial Promotion Department, Chiba Cancer Center, Chiba, Japan.
authors.forename: Shuichi
authors.initials: S
authors.lastname: Hironaka
authors.affiliation: Department of Frontier Science for Cancer and Chemotherapy, Osaka University Graduate School of Medicine, Osaka, Japan.
authors.forename: Taroh
authors.initials: T
authors.lastname: Satoh
mesh_terms: D015179:Colorectal Neoplasms / Q000235:genetics*; D004252:DNA Mutational Analysis; D023381:Endpoint Determination; D005091:Exons / Q000235:genetics; D060005:Genotyping Techniques; D006801:Humans; D009154:Mutation / Q000235:genetics*; D059645:Mutation Rate; D011446:Prospective Studies; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics*; D011933:Reagent Kits, Diagnostic; D012015:Reference Standards
chemical_list: C117307:KRAS protein, human; D011933:Reagent Kits, Diagnostic; D016283:Proto-Oncogene Proteins p21(ras)
keywords: Anti-EGFR antibody treatment; Biomarker; Colorectal cancer; In vitro diagnostics; RAS mutation; RASKET study