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

Abstract

Background: 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.

Methods: 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.

Results: 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).

Conclusions: 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.

Figure 1

Associations among KRAS, BRAF, NRAS, and PIK3CA mutations. KRAS codon 12 and 13, KRAS codon 61 and 146, BRAF, and NRAS mutations were mutually exclusive. Only PIK3CA Exon 9 and 20 mutations overlapped KRAS codon 12 and 13 and BRAF mutations.

Figure 2

Kaplan–Meier plots of progression-free survival (PFS) and overall survival (OS) according to KRAS, BRAF, NRAS, and PIK3CA gene status. Figure  2 A. PFS: Median PFS values were 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) among patients with all wild-type tumors (N = 49, blue line), KRAS codon 12 or 13 mutant tumors (N = 21, green line), and KRAS codon 61, KRAS codon 146, BRAF, NRAS, or PIK3CA mutant tumors (N = 12, gray-line), respectively. Differences in PFS values between patients with all wild-type tumors and those with KRAS codon 61, KRAS codon 146, BRAF, NRAS, or PIK3CA mutant tumors were statistically significant (hazard ratio, 0.22; 95% CI, 0.11–0.44; P < 0.0001). Figure  2 B. OS: Median OS values were 13.8 months [95% confidence interval (CI): 9.2–18.4], 8.2 months (5.7–10.7), and 6.3 months (1.3–11.3) among patients with all wild-type tumors (N = 49, blue line), with KRAS codon 12 or 13 mutant tumors (N = 21, green line), and with KRAS codon 61, KRAS codon 146, BRAF, NRAS, or PIK3CA mutations (N = 12, gray-line), respectively. Differences in OS values between patients with all wild-type tumors and those with KRAS codon 61, KRAS codon 146, BRAF, NRAS, or PIK3CA mutant tumors were statistically significant (hazard ratio, 0.30; 95% CI, 0.15–0.61; P < 0.0001).