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

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

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

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

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

CT characteristics in pulmonary adenocarcinoma with epidermal growth factor receptor mutation

<div><p>Comprehensively investigate the association of CT morphology and clinical findings of adenocarcinoma with <i>EGFR</i> mutation status. Retrospectively included 282 patients who was pathologically proved as lung adenocarcinoma with known <i>EGFR</i> mutation status (mutations: 138 patients, female: 86, median age: 66 years; wildtype: 144 patients, female: 67, median age: 62 years) and their pre-treatment CT scans were analyzed. CT findings and clinical information were collected. Univariate and multivariable logistic regression analysis were performed. Adjusted for age, gender and smoking history of two groups, significantly more patients with pleural tags, pleural and liver metastases were found in the <i>EGFR</i> mutated group (<i>P</i> = 0.007, 0.004, and 0.043, respectively). Multivariable logistic regression analysis found that the model included age, gender, smoking history, air bronchogram, pleural tags, pleural and liver metastasis had a moderate predictive value for <i>EGFR</i> mutation status (AUC = 0.741, <i>P</i> < .0001). Exon-19 deletion was associated with air bronchogram which adjusted for age, gender and smoking history (<i>P</i> = 0.007, OR: 2.91, 95%CI: 1.25–7.79). The evidence of pleural tags, pleural and liver metastases go along with a higher probability of <i>EGFR</i> mutation in adenocarcinoma patients and air bronchogram is positively associated with Exon-19 deletion mutation.</p></div

Receiver operator characteristic curve for the model which composed by age, gender, smoking history, air bronchogram, pleural tags, pleural and liver metastasis to predict <i>EGFR</i> mutation status.

<p>The area under the curve is 0.741.</p

Statistically significant imaging characteristics comparison between <i>EGFR</i> mutation statuses.

<p>Statistically significant imaging characteristics comparison between <i>EGFR</i> mutation statuses.</p