SUMMARY OF SAFETY AND EFFECTIVENESS DATA (SSED)I.GENERAL INFORMATIONDevice Generic Name:Germline Gene Mutation TestDevice Trade Name:BRACAnalysis CDx Device Procode:PJGApplicant’s Name and Address:Myriad Genetic Laboratories, Inc.320 Wakara WaySalt Lake City, UT 84108Date(s) of Panel Recommendation:NonePremarket Approval Application (PMA) Number:P140020Date of FDA Notice of Approval:December 19, 2014Priority Review: Granted priority review status on October 15, 2014, becauseBRACAnalysis CDx addresses an unmet medical need, as there is no approvedalternative and as demonstrated by significant clinically meaningful advantage.II.INDICATIONS FOR USEBRACAnalysis CDx is an in vitro diagnostic device intended for the qualitativedetection and classification of variants in the protein coding regions and intron/exonboundaries of the BRCA1 and BRCA2 genes using genomic DNA obtained from wholeblood specimens collected in EDTA. Single nucleotide variants and small insertions anddeletions (indels) are identified by polymerase chain reaction (PCR) and Sangersequencing. Large deletions and duplications in BRCA1 and BRCA2 are detected usingmultiplex PCR. Results of the test are used as an aid in identifying ovarian cancerpatients with deleterious or suspected deleterious germline BRCA variants eligible fortreatment with Lynparza (olaparib). This assay is for professional use only and is to beperformed only at Myriad Genetic Laboratories, a single laboratory site located at 320Wakara Way, Salt Lake City, UT 84108.III.CONTRAINDICATIONSPatients who have undergone a previous allogeneic bone marrow transplant should not betested with the BRACAnalysis CDx .IV.WARNINGS AND PRECAUTIONSThe warnings and precautions can be found in the BRACAnalysis CDx labeling.
V.DEVICE DESCRIPTIONBRACAnalysis CDx is an in vitro diagnostic device performed in a single laboratory,Myriad Genetic Laboratories, Inc. (Myriad), in Salt Lake City, UT. The test includes asample collection kit, which is sent to ordering laboratories and healthcare providers.The collection kit contains the following components: Monoject Blood Collection Tube (part no. 8881-311743); Test Request Form; Instructions for Sample Collection and Mailing; Technical Information Summary.BRACAnalysis CDx consists of the following two assays to detect sequence variantsand large rearrangements in the BRCA1 and BRCA2 genes: BRACAnalysis CDx Sanger Sequencing; BRACAnalysis CDx Large Rearrangement Test (BART CDx).All reportable variants, including deleterious and suspected deleterious mutations, areconfirmed by repeat analysis with the BRACAnalysis CDx Sanger Sequencing test orthe BART CDx test, or by confirmatory testing. Approximately 98% and 99% of allvariants detected by the BRACAnalysis CDx Sanger Sequencing test and the BART CDx tests, respectively, are confirmed by repeat testing; the remaining variants (about2% and 1%, respectively) require confirmatory analysis by the following tests: Alternate Primer Sequencing (APS); Confirmatory PCR Analysis (CPA).BRACAnalysis CDx is intended to be performed with serial number-controlledinstruments, as indicated in the table below.Instruments for Use with BRACAnalysis CDx InstrumentSerial Number(s)QIASymphony SP14957Tecan Freedom Evo 1501310005128; 1402003965Tecan Infinite F200 Pro Platereader 13090019875344 003700; 5344 009645;5344 010536; 5344 010537;5344 010530; 5344 010552;5344 010572; 5344 026026;5344 010613; 5344 010521;5344 012323; 5344 009646;6324CH818083; 6324CH718080;MasterCycler EP & MasterCycler6324CK418379; 6324CH018081;Pro 384 & 96 well6324CK118382; 6324CK318376;6324CH418082; 6324CH618076;6324CK818381; 6324CK018387;6324CK518383; 6324CK218385;6324AR315529; 6324AR715518;6324AR915513; 6324AR015528;6324AR415521; 6324AR815510;
ABI 3730xlE-Gel iBaseE-Gel Imaging System InGenius3E-Gel Safe Imager Real-timeTransilluminator6321CP8188051408-038; 16112-019;18127-015; 25193-005;24189-002; 24180-009;25193-003; 1412-0272113127IG3/121912093074Blood Collection and DNA ExtractionPeripheral whole blood ( 7 mL) is collected in a blood collection tube containing EDTA,and then the sample is mailed to Myriad at ambient temperature. At Myriad, the bloodsample is accessioned, aliquoted, and then processed using an automated DNA extractionprocess. One aliquot (1 mL) per sample is loaded onto the QIASymphony SP instrument,which is configured for silica-based isolation and purification of genomic DNA usingQIAGEN Software v4.0.1. When the extraction run is complete, the DNA is suspendedin 185 µL TE buffer. The DNA is then quantified and normalized using an automatedrobotic platform (Tecan Freedom EVO 150 with Tecan EVOWare v220.127.116.11) andfluorometer (Tecan Infinite F200 PRO with Magellan v7.0 Software). DNA samplesare stored at 4 C until tested for variants in the BRCA genes.Detection of Sequence VariantsSequence analysis of the BRCA genes is conducted with the BRACAnalysis CDx Sanger Sequencing test. For BRCA1, full sequence determination of approximately 5,400base pairs (bp) comprising 22 coding exons and approximately 750 adjacent bp in thenon-coding intervening (intron) sequences is performed. Exons 1 and 4, which are noncoding, are not analyzed. For BRCA2, full sequence determination of approximately10,200 bp comprising 26 coding exons and approximately 900 adjacent bp in the noncoding intervening sequences is performed. Exon 1 is non-coding, and therefore, is notanalyzed. The intronic regions of BRCA1 and BRCA2 that are analyzed generally do notextend more than 20 bp proximal to the 5’ end and 10 bp distal to the 3’ end of eachexon.The BRACAnalysis CDx Sanger Sequencing test uses primers that define specific basepair sequences to amplify each of the targeted regions by polymerase chain reaction(PCR). Each primer also contains an M13 tail on the 5’end to facilitate the downstreamsequencing reactions. An automated robotic platform (Tecan Freedom EVO 150 withTecan EVOWare v18.104.22.168) is used to add the appropriate primers and DNA samples tothe wells of 384-well PCR plates containing Sanger PCR MasterMix, which consists ofpre-mixed reagents for the amplification reactions. Following inoculation, the PCRplates are centrifuged and then placed onto a thermocycler (Eppendorf MasterCycler EPor MasterCycler Pro 384) for PCR amplification. In total, 35 PCR reactions are carriedout for BRCA1, and 49 PCR reactions for BRCA2.The amplified products are each directly sequenced in the forward and reverse directionsusing fluorescent dye-labeled sequencing primers. Fluorescently-labeled Sanger
sequencing fragments are generated using the Eppendorf MasterCycler thermocyclers,and then purified prior to sequencing on the Applied Biosystems (ABI) 3730xl. Toevaluate the possibility of carryover throughout the procedure, each batch run includestwo negative controls for the BRACAnalysis CDx Sanger Sequencing test: a NoGenomic DNA Control and an M13 F R Negative Control. In each control, no DNAtemplate is added. The No Genomic DNA Control contains all PCR components and aPCR primer pair for one targeted amplicon. The M13 F R Negative Control includes allPCR components in addition to the M13 forward and reverse primer pair. The controlsmust produce the expected results in order to evaluate the data from test samples.Chromatographic tracings of each amplicon are automatically generated with the ABISequence Analysis Software v5.3.1 and Gene Mapper v4.0, and subsequently analyzedusing Myriad’s proprietary Alignment Software v1.7.4 and Mutation Calling Softwarev1.9.6 to identify possible sequence variants. The variant calling software numericallycompares each base of the sequencing traces to consensus wild-type sequences, and anymismatches are flagged as potential variants. The flagged variants may then be routedfor review by trained data analysts. In this case, two independent reviewers visuallyinspect the traces to confirm the variant calls. If the analysts do not agree, the results arereviewed by a supervisor for final determination. All reportable variants areindependently confirmed by repeated PCR amplification of the indicated gene region(s)and subsequent sequencing.Detection of Large RearrangementsBRACAnalysis CDx Large Rearrangement Test (BART CDx) is a multiplex PCRassay intended to detect large genomic rearrangements (e.g., deletions and duplications)across all coding regions, limited flanking intron regions, and the proximal promoterregions of the BRCA1 and BRCA2 genes. DNA is normalized to 2 ng/µL and then addedto the wells of 384-well PCR plates containing pre-mixed reagents using the TecanFreedom EVO 15 automated robotic platform. The PCR plates are centrifuged, andthen amplified products are generated using Eppendorf MasterCycler thermocyclers.During this process, fluorescently labeled primers are incorporated into the amplifiedDNA. In total, 11 multiplex PCR reactions are performed, and on average, there are 12amplicons per multiplex with at least 2 amplicons per exon. The reactions are run induplicate to obtain at least 4 data points for each region.The amplified products are purified using the AMPure magnetic bead system and thenloaded onto the Applied Biosystems (ABI) 3730xl for fragment analysis.Chromatographic traces are generated with the ABI Gene Mapper v4.0 software andanalyzed for the presence of wild-type and variant fragments. The fragment data fromseparation of the PCR products are sized using an internal lane standard with fragmentsof known sizes. Myriad’s proprietary large rearrangement analysis software, MiniARTApplication v0.2, compares the relative peak intensities within a sample, and betweensamples run in the same batch, to generate statistical values and a gene dosage scatterplot. Briefly, each exon is represented by a minimum of 4 data points, so data from allamplicons of all samples in the same batch are normalized across the batch and thencombined to yield one data point per sample per exon on the gene dosage scatter plot.Three housekeeping genes are used as additional copy number controls. The traces andscatter plot are reviewed by two independent trained analysts. Any sample flagged
during data review is reprocessed and reanalyzed. Any sample with a potential largerearrangement is reviewed by a trained supervisor to verify the result.In order to verify that the BART CDx test can produce the expected results, two positivecontrols and one negative control are included in each run. The positive controls are twoindependent cell lines with defined BRCA large rearrangements. The negative controlcontains all of the components for the BART CDx PCR reactions, with the exception ofDNA template. If the controls produce the expected results, then the data from the testsamples are assessed.Confirmatory TestingAll reportable variants, including deleterious and suspected deleterious mutations, areconfirmed by additional testing prior to result reporting. Approximately 98% ofreportable sequence variants are confirmed by repeating the BRACAnalysis CDx Sanger Sequencing test for the indicated gene region(s), and about 99% of largerearrangements are confirmed by repeat BART CDx testing. Certain variants thatcannot be confirmed with repeat testing are subject to confirmatory analysis usingAlternate Primer Sequencing (APS) or Confirmatory PCR Analysis (CPA).Approximately 2% of atypical variant results are confirmed using the APS test. APS isbased on the same PCR and Sanger sequencing methods used in the BRACAnalysisCDx Sanger Sequencing test; however, PCR is conducted in 96-well PCR plates. TheAPS test is performed using alternative primer sequences, or primer combinations thatflank the primer binding sites used in the BRACAnalysis CDx Sanger Sequencingassay, to allow for the identification of potential heterozygous base changes at primerbinding sites that may have resulted in inefficient PCR amplification in theBRACAnalysis CDx Sanger Sequencing or BART CDx tests. For example, aheterozygous base change could yield either unequal PCR amplification in theBRACAnalysis CDx Sanger Sequencing test or artifacts - such as apparent single exondeletions - in the BART CDx test. The APS test is therefore used to confirm these typesof rare results from the BRACAnalysis CDx Sanger Sequencing or BART CDx tests.The CPA test is used for follow up testing for about 1% of atypical results, such as singleexon deletions, from the BART CDx test. Sequence-specific primers that span one ormore exons are used to amplify breakpoint-specific genomic regions in a 96-well PCRplate format. The amplified products are evaluated using gel electrophoresis andsubsequent imaging (E-Gel Imaging System InGenius3, Invitrogen E-Gel Safe Imagerand E-Gel iBASE Power System with GENESys Gel Documentation System v22.214.171.124software) and may be verified by sequencing. A size-based analysis of the PCR productsis performed by comparing the PCR products from the sample of interest against wildtype PCR products. The confirmatory assays are generally performed as needed toconfirm the presence of certain variants.Variant ClassificationUpon completion of testing at Myriad, a test report is sent to the ordering physician. Theresults of each test component, along with the classification of the germline variant(s)detected by the BRACAnalysis CDx , are provided. Variant classification is conducted
according to a defined classification process by an in-house committee consisting ofLaboratory Directors, the Chief Medical Officer, representatives from Medical Services,Genetic Counselors, and other trained professionals, including PhD-level scientists,board-certified clinical molecular geneticists, or equivalent. Based on the classificationcriteria, each identified variant is classified into one of five categories. If multiplevariants are detected with different classifications, the overall test interpretation isdetermined by the highest tiered variant classification, as listed below.