Simultaneous identification of 36 mutations in KRAS codons 61and 146, BRAF, NRAS, and PIK3CAin a single reaction by multiplex assay kit

Hideaki Bando,Katsuya Tsuchihara,Eiji Shinozaki,Kentaro Yamazaki,Satoshi Fujii,Kensei Yamaguchi,Takayuki Yoshino,Tomohiro Nishina,Atsushi Ohtsu,Satoshi Yuki,Shinya Kajiura,Takeharu Yamanaka

doi:10.1186/1471-2407-13-405

Abstract

BackgroundRetrospective analyses in the West suggest that mutations in KRAS codons 61 and 146, BRAF, NRAS, and PIK3CA are negative predictive factors for cetuximab treatment in colorectal cancer patients. We developed a novel multiplex kit detecting 36 mutations in KRAS codons 61 and 146, BRAF, NRAS, and PIK3CA using Luminex (xMAP) assay in a single reaction.MethodsTumor samples and clinical data from Asian colorectal cancer patients treated with cetuximab were collected. We investigated KRAS, BRAF, NRAS, and PIK3CA mutations using both the multiplex kit and direct sequencing methods, and evaluated the concordance between the 2 methods. Objective response, progression-free survival (PFS), and overall survival (OS) were also evaluated according to mutational status.ResultsIn total, 82 of 83 samples (78 surgically resected specimens and 5 biopsy specimens) were analyzed using both methods. All multiplex assays were performed using 50 ng of template DNA. The concordance rate between the methods was 100%. Overall, 49 (59.8%) patients had all wild-type tumors, 21 (25.6%) had tumors harboring KRAS codon 12 or 13 mutations, and 12 (14.6%) had tumors harboring KRAS codon 61, KRAS codon 146, BRAF, NRAS, or PIK3CA mutations. The response rates in these patient groups were 38.8%, 4.8%, and 0%, respectively. Median PFS in these groups was 6.1 months (95% confidence interval (CI): 3.1–9.2), 2.7 months (1.2–4.2), and 1.6 months (1.5–1.7); median OS was 13.8 months (9.2–18.4), 8.2 months (5.7–10.7), and 6.3 months (1.3–11.3), respectively. Statistically significant differences in both PFS and OS were found between patients with all wild-type tumors and those with KRAS codon 61, KRAS codon 146, BRAF, NRAS, or PIK3CA mutations (PFS: 95% CI, 0.11–0.44; P < 0.0001; OS: 95% CI, 0.15–0.61; P < 0.0001).ConclusionsOur newly developed multiplex kit is practical and feasible for investigation of a range of sample types. Moreover, mutations in KRAS codon 61, KRAS codon 146, BRAF, NRAS, or PIK3CA detected in Asian patients were not predictive of clinical benefits from cetuximab treatment, similar to the result obtained in European studies.

Highlights

Retrospective analyses in the West suggest that mutations in KRAS codons 61 and 146, BRAF, NRAS, and PIK3CA are negative predictive factors for cetuximab treatment in colorectal cancer patients
Recent retrospective studies have revealed that mutations in KRAS codons 61 and 146, BRAF, NRAS, and PIK3CA are related to resistance to antiEGFR antibodies [8,9,10,11,12,13]
Patients who met all of the following inclusion criteria were retrospectively included in the analyses: (1) age ≥20 years; (2) histologically confirmed adenocarcinoma of the colon or rectum; (3) presence of unresectable metastatic disease; (4) baseline computed tomography (CT) performed within 28 days of initial cetuximab administration; (5) initial CT evaluation performed within 3 months of initial cetuximab administration; (6) previously documented as refractory or intolerant to fluoropyrimidines, oxaliplatin, and irinotecan; (7) Eastern Cooperative Oncology Group performance status score ≤2; and (8) adequate hematological, hepatic, and renal functions

Summary

Introduction

Retrospective analyses in the West suggest that mutations in KRAS codons 61 and 146, BRAF, NRAS, and PIK3CA are negative predictive factors for cetuximab treatment in colorectal cancer patients. In Japan, a KRAS mutation assay kit based on the ARMS–scorpion method that detects seven frequently observed mutations in KRAS codons 12 and 13 (TheraScreen® K-RAS Mutation Kit; QIAGEN) was first approved for in vitro diagnostic use, and a kit using Luminex (xMAP) assay (MEBGEN KRAS Mutation Detection Kit, MBL) followed [14,15]. We recently developed another Luminexbased research-use kit, GENOSEARCH Mu-PACK, which simultaneously detects 36 mutations in KRAS codons 61 and 146, BRAF, NRAS, and PIK3CA. In addition to the hitherto approved KRAS codon 12 and 13 mutation kit, the multiplex kit identifies mutations by a single tube reaction using 50 ng of template DNA from formalin-fixed paraffin-embedded (FFPE) specimens

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