Presents significant progress in melanoma therapy: patients’ treatment with 166518-60-1 vemurafenib resulted in complete or partial tumor regression in the majority of patients with BRAFV600E-positive metastatic melanoma [8]. Current report presents a U-BRAFV600 approach that enables automated BRAF mutation detection within the activation segment in exon 15 by a single pyrosequencing-based assay.Methods Terlipressin Ethics StatementThe study was approved by the Institutional Review Board of the Heidelberg University Hospital, Germany, and all patients signed written informed consent at time of initial clinical investigation.U-BRAFV600 State DetectionTable 1. BRAF mutations within activation segment in exon 15 in cutaneous melanoma metastases.Case 1 2 3 4 5 6 7 8Sample A,B A,B A,B,C 
A,B A A,B,C A A,B,C,D,E A BAge/Sex 53/f 47/f 40/m 79/m 55/m 69/m 80/m 53/f 87/fSanger sequencing V600E V600E V600E V600E ??V600E V600E ?Pyrosequencing mt:wt ratio in 1 24; 25 25; 26 16; 20; 20 53; 59 ??33 36; 18; 26; 25; 35 ?Deep-Sequencing mt:wt ratio in 1 21; 21 19; 22 9; 15; 14 52; 60 ??33 35; 14; 20; 22; 35 2V600E ?cobas2 + + +?10 11 12 13 14A,C,B A,B,C A A,B,C,D A,B,C,D,E A B80/m 82/f 83/m 56/m 57/m 74/fV600E ???VKS600_602.DT ?56; 62; 45 ???33; 23; 37; 24; 35 9V600E ?55; 62; 43 1; 1; 1V600E ??33; 22; 38; 22; 37 4V600E ?11; 17 7 16 24 5V600E 14 7; 6; 6; 5 31; 36 44; 43 ?9; 3V600E 11 ?2; 2V600E ??????16A,B A B65/m 52/mV600E ?V600K21; 24 10 17 28 11V600E 22 17; 13; 13; 11 27; 34 39; 39 ?20; 9V600E 16 ?18 19A A D A,B,C,E30/m 75/f 66/mV600E ?V600E ?21 22 23 24 25A,B A,B A A,B A A,B,C,D,E,F G,H73/m 37/m 71/f 52/m 54/f 66/mV600E2 V600K ??V600E ?27 28A,B,C,D,E A,B A,B54/m 78/f 44/mV600K V600E V600E; K601I49; 43; 47; 42; 56 21; 26 61;49; 45; 46; 47; 61 9; 12 61;+?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; 1 wt ?wild type, mt – mutant. 2 “+” Mutation Detected, “?’ Mutation Not Detected (cobasH 4800 report). doi:10.1371/journal.pone.0059221.tFFPE Tissue Samples and Cell LinesFormalin-fixed paraffin embedded (FFPE) tissue cutaneous metastasis 
samples were examined 23727046 in this study. Diagnoses were independently established and controlled in each tumoral sample according to histopathological standards by two experienced dermopathologists (P.H., co-author, and Wolfgang Hartschuh, Department of Dermatology, University of Heidelberg). A549 cells and wild-type HeLa cell lines were purchased from the ATCC (American Type Culture Collection).DNA Extraction and PyrosequencingFor the analysis of tumor samples, haemoltoxylin- and eosinstained slides were reviewed by an experienced pathologist (P.H.,co-author) to ensure sufficient viable tumor content (60?0 tumor cells). Total genomic DNA was extracted from seven 10 mm-thick unstained sections of FFPE tissue blocks according to manufacturer’s instructions, using an automated DNA Extractor (QiasymphonyTM, Qiagen). To avoid cross-contamination, a new disposable microtome blade was used for each FFPE tissue block. In addition, knife holder and anti-roll plate was wiped down with 100 ethanol in between each block. The total DNA was eluted in 50 ml and immediately stored at 220uC for later use. The eluted DNA was quantified using a Qubit dsDNA HS Assay (Invitrogen). For pyrosequencing assay, the region of human braf spanning mutation sites within the activation segment in exon 15 wasU-BRAFV600 State DetectionFigure 1. BRAF mutation anal.Presents significant progress in melanoma therapy: patients’ treatment with vemurafenib resulted in complete or partial tumor regression in the majority of patients with BRAFV600E-positive metastatic melanoma [8]. Current report presents a U-BRAFV600 approach that enables automated BRAF mutation detection within the activation segment in exon 15 by a single pyrosequencing-based assay.Methods Ethics StatementThe study was approved by the Institutional Review Board of the Heidelberg University Hospital, Germany, and all patients signed written informed consent at time of initial clinical investigation.U-BRAFV600 State DetectionTable 1. BRAF mutations within activation segment in exon 15 in cutaneous melanoma metastases.Case 1 2 3 4 5 6 7 8Sample A,B A,B A,B,C A,B A A,B,C A A,B,C,D,E A BAge/Sex 53/f 47/f 40/m 79/m 55/m 69/m 80/m 53/f 87/fSanger sequencing V600E V600E V600E V600E ??V600E V600E ?Pyrosequencing mt:wt ratio in 1 24; 25 25; 26 16; 20; 20 53; 59 ??33 36; 18; 26; 25; 35 ?Deep-Sequencing mt:wt ratio in 1 21; 21 19; 22 9; 15; 14 52; 60 ??33 35; 14; 20; 22; 35 2V600E ?cobas2 + + +?10 11 12 13 14A,C,B A,B,C A A,B,C,D A,B,C,D,E A B80/m 82/f 83/m 56/m 57/m 74/fV600E ???VKS600_602.DT ?56; 62; 45 ???33; 23; 37; 24; 35 9V600E ?55; 62; 43 1; 1; 1V600E ??33; 22; 38; 22; 37 4V600E ?11; 17 7 16 24 5V600E 14 7; 6; 6; 5 31; 36 44; 43 ?9; 3V600E 11 ?2; 2V600E ??????16A,B A B65/m 52/mV600E ?V600K21; 24 10 17 28 11V600E 22 17; 13; 13; 11 27; 34 39; 39 ?20; 9V600E 16 ?18 19A A D A,B,C,E30/m 75/f 66/mV600E ?V600E ?21 22 23 24 25A,B A,B A A,B A A,B,C,D,E,F G,H73/m 37/m 71/f 52/m 54/f 66/mV600E2 V600K ??V600E ?27 28A,B,C,D,E A,B A,B54/m 78/f 44/mV600K V600E V600E; K601I49; 43; 47; 42; 56 21; 26 61;49; 45; 46; 47; 61 9; 12 61;+?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; 1 wt ?wild type, mt – mutant. 2 “+” Mutation Detected, “?’ Mutation Not Detected (cobasH 4800 report). doi:10.1371/journal.pone.0059221.tFFPE Tissue Samples and Cell LinesFormalin-fixed paraffin embedded (FFPE) tissue cutaneous metastasis samples were examined 23727046 in this study. Diagnoses were independently established and controlled in each tumoral sample according to histopathological standards by two experienced dermopathologists (P.H., co-author, and Wolfgang Hartschuh, Department of Dermatology, University of Heidelberg). A549 cells and wild-type HeLa cell lines were purchased from the ATCC (American Type Culture Collection).DNA Extraction and PyrosequencingFor the analysis of tumor samples, haemoltoxylin- and eosinstained slides were reviewed by an experienced pathologist (P.H.,co-author) to ensure sufficient viable tumor content (60?0 tumor cells). Total genomic DNA was extracted from seven 10 mm-thick unstained sections of FFPE tissue blocks according to manufacturer’s instructions, using an automated DNA Extractor (QiasymphonyTM, Qiagen). To avoid cross-contamination, a new disposable microtome blade was used for each FFPE tissue block. In addition, knife holder and anti-roll plate was wiped down with 100 ethanol in between each block. The total DNA was eluted in 50 ml and immediately stored at 220uC for later use. The eluted DNA was quantified using a Qubit dsDNA HS Assay (Invitrogen). For pyrosequencing assay, the region of human braf spanning mutation sites within the activation segment in exon 15 wasU-BRAFV600 State DetectionFigure 1. BRAF mutation anal.
