Detection of BRAF, NRAS, KIT, GNAQ, GNA11 and MAP2K1/2 mutations in Russian melanoma patients using LNA PCR clamp and biochip analysis

Abstract

Target inhibitors are used for melanoma treatment, and their effectiveness depends on the tumor genotype. We developed a diagnostic biochip for the detection of 39 clinically relevant somatic mutations in the BRAF, NRAS, KIT, GNAQ, GNA11, MAP2K1 and MAP2K2 genes. We used multiplex locked nucleic acid (LNA) PCR clamp for the preferable amplification of mutated over wild type DNA. The amplified fragments were labeled via the incorporation of fluorescently labeled dUTP during PCR and were hybridized with specific oligonucleotides immobilized on a biochip. This approach could detect 0.5% of mutated DNA in the sample analyzed. The method was validated on 253 clinical samples and six melanoma cell lines. Among 253 melanomas, 129 (51.0%) BRAF, 45 (17.8%) NRAS, 6 (2.4%) KIT, 4 (1.6%) GNAQ, 2 (0.8%) GNA11, 2 (0.8%) MAP2K1 and no MAP2K2 gene mutations were detected by the biochip assay. The results were compared with Sanger sequencing, next generation sequencing and ARMS/Scorpion real-time PCR. The specimens with discordant results were subjected to LNA PCR clamp followed by sequencing. The results of this analysis were predominantly identical to the results obtained by the biochip assay. Infrequently, we identified rare somatic mutations. In the present study we demonstrate that the biochip-based assay can effectively detect somatic mutations in approximately 70% of melanoma patients, who may require specific targeted therapy.

Keywords: biochip; diagnostic tool; melanoma; somatic mutations; targeted therapy.

Figure 1. Biochip spotting scheme

The biochip includes paired probes for the detection of each somatic mutations and corresponding wild-type sequences. Marker spots with Cy5 are located in the corners.

Figure 2. Mutational analysis by biochip

(A) Biochip image and histogram of normalized signal intensity obtained for a sample with a V600K BRAF mutation. The bright spots in the hybridization image correspond to the outstanding bars on the histogram. All gel spots correspond to one analyzed site with common primers, and LNA oligonucleotides are combined into one group. Fluorescent signals from the paired probes are averaged. The fluorescent signal is normalized to the maximum signal in a group of gel spots. The sample contains a V600K BRAF mutation because J_(V600K)>J_(600WT). (B) Biochip images and histograms of normalized signal intensity obtained for a sample with a Q61K NRAS mutation.

Figure 3. Biochip assay sensitivity

The assay sensitivity for BRAF V600E mutation detection was determined by an analysis of serially diluted mutant DNA in the background of WT DNA: 0%, 0.25%, 0.5%, 1%, 5%, 10%, 50% and 100%. Fragment of a biochip images (in the upper part) and the normalized signal intensity (in the lower part) only for the spots corresponding to BRAF 600WT and V600E are present for each dilution.

Figure 4. Detection of rare mutations in the BRAF gene using Sanger sequencing with preliminary enrichment with mutant allele

(A) V600D, c.1799_1800TG>AC, COSM308550; (B) V600V, c.1800G>A, COSM249890; (C) A598V, c.1793C>T, COSM21549; (D) T599_V600insT, c.1797_1798insACA, COSM144982; (E) A598_T599insV, c.1794_1795insGTT, COSM26625.

Figure 5. Frequency of rare BRAF mutations in different age groups of patients

Rare mutations are more common in older patients (P = 0.05).