Automated universal BRAF state detection within the activation segment in skin metastases by pyrosequencing-based assay U-BRAF(V600)

Abstract

Malignant melanoma is a highly-aggressive type of malignancy with considerable metastatic potential and frequent resistance to cytotoxic agents. BRAF mutant protein was recently recognized as therapeutic target in metastatic melanoma. We present a newly-developed U-BRAF(V600) approach - a universal pyrosequencing-based assay for mutation detection within activation segment in exon 15 of human braf. We identified 5 different BRAF mutations in a single assay analyzing 75 different formalin-fixed paraffin-embedded (FFPE) samples of cutaneous melanoma metastases from 29 patients. We found BRAF mutations in 21 of 29 metastases. All mutant variants were quantitatively detectable by the newly-developed U-BRAF(V600) assay. These results were confirmed by ultra-deep-sequencing validation ((~)60,000-fold coverage). In contrast to all other BRAF state detection methods, the U-BRAF(V600) assay is capable of automated quantitative identification of at least 36 previously-published BRAF mutations. Under the precaution of a minimum of 3% mutated cells in front of a background of wild type cells, U-BRAFV600 assay design completely excludes false wild-type results. The corresponding algorithm for classification of BRAF-mutated variants is provided. The single-reaction assay and data analysis automation makes our approach suitable for the assessment of large clinical sample sizes. Therefore, we suggest U-BRAF(V600) assay as a most powerful sequencing-based diagnostic tool to automatically identify BRAF state as a prerequisite to targeted therapy.

Figure 1. BRAF mutation analysis by Sanger sequencing and pyrosequencing-based assay U-BRAF^V600.

(a) Sanger sequencing; (b) pyrosequencing-based assay U-BRAF^V600. “+” indicates the positive peaks of the dispensation nucleotides within recognition patterns of U-BRAF^V600 assay. mt – mutant; wt – wild-type. Recognition patterns are shown in black boxes.

Figure 2. Low-abundance BRAF mutations.

a) Pyrogram of cloned wild-type BRAF. Red arrow indicates the reduction of peak intensity values; b) pyrograms of cloned BRAF mutants. Red asterisks indicate the dispensation nucleotide’s peaks, which are characteristic for corresponding BRAF mutant in low-copy-number analysis; c) pyrograms of premixed BRAF mutants with wild type. Red arrows indicate the tendency of peak-pairs’ difference included in low-copy-number analysis. Red asterisks indicate the peaks with the contribution of correspondent mutant nucleotides shown in (b).

Figure 3. Dispensation order for 5 mutated BRAF variants detected by U-BRAF^V600 assay.

*A5 = Awt +3Amt. Recognition patters are indicated in black boxes, individual mutation features are marked in grey boxes dispensation order’s nucleotides, which are involved into mt:wt ratio, are bolded.

Figure 4. Algorithm for automated BRAF state classification of U-BRAF^V600 pyrosequencing data analysis.

Reduction factors for both A-peak and dispensation steps should be taken into consideration calculating individual peak intensities.