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Automated Universal BRAF State Detection within the Activation Segment in Skin Metastases by Pyrosequencing-Based Assay U-BRAFV600.doc

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Automated Universal BRAF State Detection within the Activation Segment in Skin Metastases by Pyrosequencing-Based Assay U-BRAFV600

AutomatedUniversalBRAFStateDetectionwithinthe ActivationSegmentinSkinMetastasesby Pyrosequencing-BasedAssayU-BRAFV600 AlexanderSkorokhod1*,PeterHelmbold1,BenediktBrors2,PeterSchirmacher3,AlexanderEnk1, RolandPenzel3 1DepartmentofDermatology,HeidelbergUniversityHospital,Heidelberg,Germany,2DepartmentofTheoreticalBioinformatics,GermanCancerResearchCenter(DKFZ), Heidelberg,Germany,3InstituteofPathology,HeidelbergUniversityHospital,Heidelberg,Germany Abstract Malignant melanoma is a highly-aggressive type of malignancy with considerable metastatic potential and frequent resistancetocytotoxicagents.BRAFmutantproteinwasrecentlyrecognizedastherapeutictargetinmetastaticmelanoma. We present a newly-developed U-BRAFV600 approach – a universal pyrosequencing-based assay for mutation detection withinactivationsegmentinexon15ofhumanbraf.Weidentified5differentBRAFmutationsinasingleassayanalyzing75 differentformalin-fixedparaffin-embedded(FFPE)samplesofcutaneousmelanomametastasesfrom29patients.Wefound BRAF mutations in 21 of 29 metastases. All mutant variants were quantitatively detectable by the newly-developed U- BRAFV600assay.Theseresultswereconfirmedbyultra-deep-sequencingvalidation( 60,000-foldcoverage).Incontrasttoall otherBRAFstatedetectionmethods,theU-BRAFV600assayiscapableofautomatedquantitativeidentificationofatleast36 previously-publishedBRAFmutations.Undertheprecautionofaminimumof3%mutatedcellsinfrontofabackgroundof 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-BRAFV600 assay as a most powerfulsequencing-baseddiagnostictooltoautomaticallyidentifyBRAFstateasaprerequisitetotargetedtherapy. , Citation:SkorokhodA,HelmboldP,BrorsB,SchirmacherP,EnkA,etal.(2013)AutomatedUniversalBRAFStateDete