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

<p>(<b>a</b>) Sanger sequencing; (<b>b</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.</p

Automated Universal BRAF State Detection within the Activation Segment in Skin Metastases by Pyrosequencing-Based Assay U-BRAF^V600

<div><p>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 <i>braf</i>. 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.</p> </div

BRAF mutations within activation segment in exon 15 in cutaneous melanoma metastases.

<p>different samples of the same tumor are specified by 1, 2 etc., different tumors of the same patient specified by A, B etc.; age in years, f = female, m = male;</p>1<p>wt – wild type, mt - mutant.</p>2<p>“+” Mutation Detected, “–” Mutation Not Detected (cobas® 4800 report).</p

Recognition patterns for 36 BRAF mutations by U-BRAF^V600 assay.

1<p>wt – wild type, mt – mutant; I – intensity value of correspondent nucleotide dispensation. A-peak reduction factor 0.9 should be taken into consideration.</p>2<p>Catalogue of Somatic Mutations in Cancer (COSMIC) database, version 62 (Wellcome Trust Sanger Institute).</p

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

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

Low-abundance BRAF mutations.

<p><b>a</b>) Pyrogram of cloned wild-type BRAF. Red arrow indicates the reduction of peak intensity values; <b>b</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; <b>c</b>) 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>b</b>).</p

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

<p>*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.</p

Dependence of the predicted probability of <i>BRAF</i> mutation according to the four most influential variables; main effects model.

<p>Individual variable effects for each compartment were standardized for the effects of the other three compartments as shown by the vertical dashed red lines. The main effects model allows estimation of the <i>BRAF</i> mutation probability for each combination of levels of the four variables: as indicated by the vertical dashed red lines, the estimated <i>BRAF</i> mutation probability for a 60-year-old patient at the Heidelberg Center with melanoma subtype SSM and UV exposure was 39.6% (95% confidence interval: 23.1–58.8). The vertical blue lines represent the 95%-confidence intervals, plotted for each level of categorical covariates. In the case of continuous variables, such as age, a 95% confidence interval was plotted (gray). ALM, acral lentiginous melanoma; LMM, lentigo maligna melanoma; NM, nodular melanoma; SSM, superficial spreading melanoma; UV, ultraviolet.</p

Patient characteristics and <i>BRAF</i> mutation status.

<p>Patient characteristics and <i>BRAF</i> mutation status.</p

Distribution of patient age at time of sampling.

<p>Distribution of patient age at time of sampling.</p