Open Access Highly Accessed Technical advance

Simultaneous identification of 36 mutations in KRAS codons 61and 146, BRAF, NRAS, and PIK3CA in a single reaction by multiplex assay kit

Hideaki Bando1, Takayuki Yoshino1*, Eiji Shinozaki2, Tomohiro Nishina3, Kentaro Yamazaki4, Kensei Yamaguchi5, Satoshi Yuki6, Shinya Kajiura7, Satoshi Fujii8, Takeharu Yamanaka9, Katsuya Tsuchihara9 and Atsushi Ohtsu19

Author Affiliations

1 Department of Gastroenterology and Gastrointestinal Oncology, National Cancer Center Hospital East, 6-5-1 Kashiwanoha, Kashiwa, Chiba 277-8577, Japan

2 Cancer Institute Hospital of Japanese Foundation for Cancer Research, Tokyo, Japan

3 National Hospital Organization Shikoku Cancer Center, Ehime, Japan

4 Division of Gastrointestinal Oncology, Shizuoka Cancer Center, Shizuoka, Japan

5 Division of Gastroenterology, Saitama Cancer Center, Saitama, Japan

6 Department of Gastroenterology, Hokkaido University Graduate School of Medicine, Hokkaido, Japan

7 The Third Department of Internal Medicine, University of Toyama, Toyama, Japan

8 Pathology Division, Research Center for Innovative Oncology, National Cancer Center Hospital East, Chiba, Japan

9 Exploratory Oncology Research & Clinical Trial Center, National Cancer Center, Chiba, Japan

For all author emails, please log on.

BMC Cancer 2013, 13:405  doi:10.1186/1471-2407-13-405

Published: 3 September 2013



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. 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.


Tumor 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.


In 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).


Our 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.

Luminex assay; KRAS; BRAF; NRAS; PIK3CA; Epidermal growth factor