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

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

The different image processing steps and the graph generation steps.

<p>(<b>a</b>) original image of the DAPI-channel; (<b>b</b>) image after shading correction and noise removal; (<b>c</b>) result of the watershed segmentation, the segmented cells are highlighted by green contour; (<b>d</b>) the image after removal of single cells; (<b>e</b>) showing the cells which were connected via the graph generation step in the same color (cells marked with the same color belong to the same sub-graph); (<b>f</b>) cell graph representation of the cells. The red dots are the nodes which represent the cells, the black lines are the edges between them.</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

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

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

Additional file 2: Table S2. of Chromosome 9p copy number gains involving PD-L1 are associated with a specific proliferation and immune-modulating gene expression program active across major cancer types

Enrichment analysis of GO categories in the 75-gene list. (XLSX 240 kb

Accuracy of the watershed cell segmentation.

<p>Accuracy of the watershed cell segmentation.</p

A Flowchart showing the single steps of our methodology.

<p>After obtaining the images, pre-processing steps enhance the image quality and watershed segmentation for the subsequent segmentation is applied. Accordingly the cell graphs are generated and features are computerized. The last step uses a SVM to classify the graphs as either tumor or stroma.</p