Validation and Implementation of BRCA1/2 Variant Screening in Ovarian Tumor Tissue Marthe M. de Jonge, Dina Ruano, Ronald van Eijk, Nienke van der Stoep,

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Presentation transcript:

Validation and Implementation of BRCA1/2 Variant Screening in Ovarian Tumor Tissue Marthe M. de Jonge, Dina Ruano, Ronald van Eijk, Nienke van der Stoep, Maartje Nielsen, Juul T. Wijnen, Natalja T. ter Haar, Astrid Baalbergen, Monique E.M.M. Bos, Marjolein J. Kagie, Maaike P.G. Vreeswijk, Katja N. Gaarenstroom, Judith R. Kroep, Vincent T.H.B.M. Smit, Tjalling Bosse, Tom van Wezel, Christi J. van Asperen The Journal of Molecular Diagnostics Volume 20, Issue 5, Pages 600-611 (September 2018) DOI: 10.1016/j.jmoldx.2018.05.005 Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Schematic overview of cohort selection. A: Training cohort. Copy number variant-multiplex ligation-dependent probe amplification (CNV-MLPA) was performed only for cases in which no variant was automatically identified via the Ion Torrent specific caller, Torrent Variant Caller version 5.0.2 (Thermo Fisher Scientific). B: Clinical implementation of BRCA1/2 screening on ovarian tumor tissue cohort. Of the 33 cases selected for variants that were potentially more challenging to detect in the training cohort, two had insufficient tumor tissue for analysis. MS, methylation specific; NGS, next-generation sequencing. The Journal of Molecular Diagnostics 2018 20, 600-611DOI: (10.1016/j.jmoldx.2018.05.005) Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Flowchart illustrating the current epithelial ovarian cancer (EOC) BRCA1/2 screening pathway (A) and the proposed EOC BRCA1/2 tumor screening pathway (B). The integration of tumor tissue analysis for BRCA1/2 variants as part of the ovarian cancer patient pathway is more efficient because it avoids referral of most patients when only those women carrying a BRCA1/2 mutation or having a suspected family history are being referred for genetic counseling. Percentages are based on the clinical implementation of BRCA1/2 screening on ovarian tumor tissue cohort. The Journal of Molecular Diagnostics 2018 20, 600-611DOI: (10.1016/j.jmoldx.2018.05.005) Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Supplemental Figure S2 Adjustments in the alignment improved automatic detection of two variants (R32 and R36). A: Initially, the BRCA1 deletion (c.3481_3491delGAAGATACTAG) in sample R32 was not automatically detected by the pipeline, and visual inspection of the reads in Integrative Genomics Viewer was needed for identification. B: Adjustment of the alignment settings resulted in improved alignment of the reads and automatic detection of the deletion. The Journal of Molecular Diagnostics 2018 20, 600-611DOI: (10.1016/j.jmoldx.2018.05.005) Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Supplemental Figure S3 Sequencing artifacts at the border of homopolymer regions of ≥6 bp interfere with variant detection. A: Sample R24 contains a T duplication (BRCA2: NM_000059.3: c.4284dupT) in a stretch of six thymidines. The thymidine duplication is visible in a subset of reads (purple dots; arrows) in the Integrative Genomics Viewer but could not be identified because homopolymer instability caused by the T stretch in the form of a bp deletion (black stripes). B: Sequencing artifacts present at the border of an 8-bp adenine stretch led the software (Ion Torrent specific caller, Torrent Variant Caller 5.0.2) to call a 2-bp deletion (BRCA2: NM_000059.3: c.9099_9100delCT) present in sample R1 as a 3-bp deletion (NM_000059.3: c.9097_9099delACT), which was manually curated. The Journal of Molecular Diagnostics 2018 20, 600-611DOI: (10.1016/j.jmoldx.2018.05.005) Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Supplemental Figure S4 Percentages of BRCA1/2 defects detected in the high-grade ovarian, fallopian tube, and primary peritoneal cancers (HGSCs) of the clinical implementation of BRCA1/2 screening on ovarian tumor tissue (COBRA) cohort compared with The Cancer Genome Atlas (TCGA) cohort. Seven percent (4/54) of gBRCA1 variants, 4% (2/54) of gBRCA2 variants, 7% (4/54) of sBRCA1 variants, 2% (1/54) of sBRCA2 variants, and 16% (8/50) of BRCA1 promoter hypermethylated cases were detected in HGSCs of COBRA cases included in the final analysis. TCGA found 9% (27/316) of gBRCA1 variants, 8% (24/316) of gBRCA2 variants, 3% (10/316) of sBRCA1 variants, 3% (9/316) of sBRCA2 variants, and 11% (56/489) of BRCA1 promoter hypermethylation in HGSCs. Considering the TCGA cohort: One case with both a germline BRCA1 and BRCA2 variant was only counted in the germline BRCA1 mutated group, one case with a simultaneous somatic BRCA1 variant and germline BRCA2 variant was only counted in the germline BRCA2 mutated group, and one case with a simultaneous somatic BRCA2 variant and germline BRCA2 variant was only counted in the germline BRCA2 mutated group. The Journal of Molecular Diagnostics 2018 20, 600-611DOI: (10.1016/j.jmoldx.2018.05.005) Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions