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Purpose: The optimal application of maintenance PARP inhibitor therapy for ovarian cancer requires accessible, robust, and rapid testing of homologous recombination deficiency (HRD). However, in many countries, access to HRD testing is problematic and the failure rate is high. We developed an academic HRD test to support treatment decision-making., Experimental Design: Genomic Instability Scar (GIScar) was developed through targeted sequencing of a 127-gene panel to determine HRD status. GIScar was trained from a noninterventional study with 250 prospectively collected ovarian tumor samples. GIScar was validated on 469 DNA tumor samples from the PAOLA-1 trial evaluating maintenance olaparib for newly diagnosed ovarian cancer, and its predictive value was compared with Myriad Genetics MyChoice (MGMC)., Results: GIScar showed significant correlation with MGMC HRD classification (kappa statistics: 0.780). From PAOLA-1 samples, more HRD-positive tumors were identified by GIScar (258) than MGMC (242), with a lower proportion of inconclusive results (1% vs. 9%, respectively). The HRs for progression-free survival (PFS) with olaparib versus placebo were 0.45 [95% confidence interval (CI), 0.33-0.62] in GIScar-identified HRD-positive BRCA-mutated tumors, 0.50 (95% CI, 0.31-0.80) in HRD-positive BRCA-wild-type tumors, and 1.02 (95% CI, 0.74-1.40) in HRD-negative tumors. Tumors identified as HRD positive by GIScar but HRD negative by MGMC had better PFS with olaparib (HR, 0.23; 95% CI, 0.07-0.72)., Conclusions: GIScar is a valuable diagnostic tool, reliably detecting HRD and predicting sensitivity to olaparib for ovarian cancer. GIScar showed high analytic concordance with MGMC test and fewer inconclusive results. GIScar is easily implemented into diagnostic laboratories with a rapid turnaround., (©2023 The Authors; Published by the American Association for Cancer Research.)

Background: PAOLA1 is a phase III study assessing olaparib maintenance therapy in advanced high-grade ovarian carcinoma patients responding to first-line platinum-taxane-based chemotherapy plus bevacizumab as standard of care. Randomization was stratified by treatment outcome and tumor BRCA1/2 status (tBRCA) at screening., Methods: tBRCA was tested on formalin-fixed, paraffin-embedded tumor blocks on 5 French platforms using 2 next-generation sequencing methods based either on hybrid capture or amplicon technology. One of the exploratory objectives was to assess the concordance between germline (gBRCA) and tBRCA testing in French patients. gBRCA testing was performed on blood samples on the same platforms., Results: From May 2015 to July 2017, tBRCA tests were performed for 1176 screened patients. Only 52 (4.4%) tumor samples were noncontributive. The median interval between reception of the tumor sample and availability of the tBRCA status result was 37 days (range = 8-260). A pathogenic variant was reported in 27.1% tumor samples (319 of 1176 screened patients). tBRCA and gBRCA testing were performed for 451 French patients with negative results for both tests in 306 patients (67.8%) and positive results for both tests in 85 patients (18.8%). Only 1 large genomic rearrangement of BRCA1 was detected, exclusively in the blood sample. Interestingly, tBRCA testing revealed 6.4% of pathogenic variant (29 of 451) not detected by gBRCA testing., Conclusions: tBRCA testing is an appropriate tool with an acceptable turnaround time for clinical practice and a low failure rate, ensuring reliable identification of patients likely to benefit from poly(ADP-ribose) polymerase inhibitor therapy., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Interpretation of variants of unknown significance (VUS) is a major challenge for laboratories performing molecular diagnosis of hereditary breast and ovarian cancer (HBOC), especially considering that many genes are now known to be involved in this syndrome. One important way these VUS can have a functional impact is through their effects on RNA splicing. Here we present a custom RNA-Seq assay plus bioinformatics and biostatistics pipeline to analyse specifically alternative and abnormal splicing junctions in 11 targeted HBOC genes. Our pipeline identified 14 new alternative splices in BRCA1 and BRCA2 in addition to detecting the majority of known alternative spliced transcripts therein. We provide here the first global splicing pattern analysis for the other nine genes, which will enable a comprehensive interpretation of splicing defects caused by VUS in HBOC. Previously known splicing alterations were consistently detected, occasionally with a more complex splicing pattern than expected. We also found that splicing in the 11 genes is similar in blood and breast tissue, supporting the utility and simplicity of blood splicing assays. Our pipeline is ready to be integrated into standard molecular diagnosis for HBOC, but it could equally be adapted for an integrative analysis of any multigene disorder.

To optimize the molecular diagnosis of hereditary breast and ovarian cancer (HBOC), we developed a next-generation sequencing (NGS)-based screening based on the capture of a panel of genes involved, or suspected to be involved in HBOC, on pooling of indexed DNA and on paired-end sequencing in an Illumina GAIIx platform, followed by confirmation by Sanger sequencing or MLPA/QMPSF. The bioinformatic pipeline included CASAVA, NextGENe, CNVseq and Alamut-HT. We validated this procedure by the analysis of 59 patients' DNAs harbouring SNVs, indels or large genomic rearrangements of BRCA1 or BRCA2. We also conducted a blind study in 168 patients comparing NGS versus Sanger sequencing or MLPA analyses of BRCA1 and BRCA2. All mutations detected by conventional procedures were detected by NGS. We then screened, using three different versions of the capture set, a large series of 708 consecutive patients. We detected in these patients 69 germline deleterious alterations within BRCA1 and BRCA2, and 4 TP53 mutations in 468 patients also tested for this gene. We also found 36 variations inducing either a premature codon stop or a splicing defect among other genes: 5/708 in CHEK2, 3/708 in RAD51C, 1/708 in RAD50, 7/708 in PALB2, 3/708 in MRE11A, 5/708 in ATM, 3/708 in NBS1, 1/708 in CDH1, 3/468 in MSH2, 2/468 in PMS2, 1/708 in BARD1, 1/468 in PMS1 and 1/468 in MLH3. These results demonstrate the efficiency of NGS in performing molecular diagnosis of HBOC. Detection of mutations within other genes than BRCA1 and BRCA2 highlights the genetic heterogeneity of HBOC.