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

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.

Challenges That Hinder the Translation of Clinical Advances Into Practice: Results From an International Assessment in Colorectal Cancer

“…13 Second, a number of new therapeutic agents have been approved, increasing oncologists' treatment options. 14,15 These new agents include mutation/marker specific drugs that may lack a positive risk benefit ratio on patients not meeting that specific tumor profile.…”

Development of a Luminex-Based Multiplex Assay for Detection of Mutations Conferring Resistance to Echinocandins in Candida glabrata

“…In recent years, microsphere-based technology has become an attractive alternative to DNA sequencing. This technology had been applied for species identification in molds and yeasts (16)(17)(18)(19)(20) and for the identification of single nucleotide polymorphisms (SNPs) (21,22). Here, we describe the adaptation of this technology to identify SNPs in the FKS1 HS1 and FKS2 HS1 domains of C. glabrata which confer in vitro resistance to echinocandins.…”

Current companion diagnostics in advanced colorectal cancer; getting a bigger and better piece of the pie

“…Therascreen uses an allele specific amplification refractory mutation system (ARMS) and incorporates a fluorescent probe (Scorpion) during real-time PCR when a mutation is present in template material. It detects the seven most common mutations in KRAS exon 2 (codon 12 and 13) and has been shown to result in equivalent, if not better sensitivity compared to direct sequencing and HRM, with the ability to detect mutant allele frequencies of 1% (38,49,58,59). The assay identifies the specific mutation which is present in a sample but does not offer extended RAS coverage outside of exon 2 (59).…”

“…Multiplex assays allow assessment of numerous genes within one reaction and have similar or better sensitivity to direct sequencing methods (58,117,118). They are cheaper than direct sequencing and are better able to deal with degraded or poor quality DNA.…”