Systematic Analysis of Homologous Recombination Deficiency Testing in Ovarian Cancer-Development of Recommendations for Optimal Assay Performance.

Homologous recombination deficiency (HRD) assays are an important element of personalized oncology in ovarian carcinomas, but the optimal tissue requirements for these complex molecular assays remain unclear. As a result, a considerable percentage of assays are not successful, leading to suboptimal diagnoses for these patients. In this study, we have systematically analyzed tumor and tissue parameters for HRD analysis in a large cohort of real-world cancer samples. The aim of this study is to give recommendations for pathologists and gynecologic oncologists for selection of tissue samples to maximize the success rate of HRD analyses. Tumor samples from 2702 patients were sent to the Institute of Pathology of the Philipps-University Marburg between October 2020 and September 2022, of which 2654 were analyzed using the Myriad MyChoice HRD+ CDx assay. A total of 2396 of 2654 samples (90.3%) were successfully tested, of which 984 of 2396 (41.1%) were HRD positive and 1412 (58.9%) were HRD negative. Three hundred sixty-three of 2396 samples (15.2%) were BRCA1/2-mutated; 27 samples had a BRCA1/2 mutation and a genomic instability score (GIS) < 42. Twenty-two samples (0.9%) failed GIS measurement but displayed a BRCA1/2 mutation. BRCA1/2-mutated samples showed significantly (P < .0001) higher GIS values than those with a wild-type BRCA1/2 status. Tumor cell content, tumor area, and histology significantly (P < .0001) affected the probability of successfully analyzing a sample. Based on a systematic analysis of tumor cell content and tumor area, we recommend selecting patient high-grade serous ovarian cancer samples that display a tumor cell content ≥30% and a tumor area ≥0.5 cm2 (based on their hematoxylin and eosin) for HRD testing to allow for optimal chances of a successful analysis and conclusive results. Considering histologic and sample conditions, success rates of up to 98% can be achieved. Our comprehensive evaluation contributes to further standardization of recommendations on HRD testing in ovarian cancer, which will have a large impact on personalized therapeutic strategies in this highly aggressive tumor type.

Tumour-based Mutational Profiles Predict Visceral Metastasis Outcome and Early Death in Prostate Cancer Patients.

BACKGROUND: Visceral metastases are known to occur in advanced prostate cancer, usually when the tumour is resistant to androgen deprivation and, have worse outcomes regardless of therapies. OBJECTIVE: To analyse genomic alterations in tumour samples according to their lymphatic, bone, and visceral metastatic stages and overall survival. DESIGN, SETTING, AND PARTICIPANTS: We selected 200 patients with metastatic prostate cancer. Genomic profiling of 111 genes and molecular signatures (homologous recombination deficiency [HRD], microsatellite instability, and tumour burden mutation) was performed with the MyChoice test (Myriad Genetics, Inc, Salt Lake City, UT, USA). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The association between genomic profiles and visceral metastatic evolution was evaluated using logistic regression. Kaplan-Meier and Cox proportional hazard analyses were used for analyses of early death. RESULTS AND LIMITATIONS: A total of 173 (87%) genomic profiles were obtained. Eighty-four (49%) patients died during the follow-up period (median duration = 76 mo). TP53 was the most frequently mutated gene, followed by FANC genes, including BRCA2, and those of the Wnt-pathway (APC/CTNNB1). TP53 gene mutations were more frequent in patients of European (42%) than in those of African (16%) ancestry. An HRD score of >25 was predictive of FANC gene mutations. The mutational status of TP53 (p < 0.001) and APC (p = 0.002) genes were significantly associated with the risk of visceral metastases. The mutational status of CTNNB1 (p = 0.001), TP53 (p = 0.015), BRCA2 (p = 0.027), and FANC (p = 0.005) genes were significantly associated with an earlier age at death. The limitations are the retrospective study design based on a selection of genes and the low frequency of certain molecular events. CONCLUSIONS: Mutations in the TP53 gene and genes (APC/CTNNB1) related to the Wnt pathway are associated with metastatic visceral dissemination and early death. These genomic alterations could be considered as markers to identify prostate cancer patients at a high risk of life-threatening disease who might benefit from more intensified treatment or new targeted therapies. PATIENT SUMMARY: In this report, we evaluated the relationships between genomic profiles (gene mutations and molecular signatures) of tumour samples from patients with metastatic prostate cancer and early death. We found that mutations of specific genes, notably TP53 and APC/CTNNB1 related to the Wnt pathway, are associated with visceral metastatic progression and an earlier age at death.

Concordance between an FDA-approved companion diagnostic and an alternative assay kit for assessing homologous recombination deficiency in ovarian cancer.

OBJECTIVE: Authors evaluated the performance of a commercially available next-generation sequencing assay kit; this was based on genomic content from Illumina's TruSight™ Oncology 500 research assay that identifies BRCA variants and proprietary algorithms licensed from Myriad and, with additional genomic content, measures the homologous recombination deficiency (HRD) genomic instability score (GIS) in tumor tissue (TSO 500 HRD assay). METHODS: Data from the TSO 500 HRD assay were compared with data from the Myriad MyChoice®CDx PLUS assay (Myriad assay). Prevalence rates for overall HRD status and BRCA mutations (a deleterious or suspected deleterious BRCA1 or BRCA2 mutation or both) and assay agreement rates for HRD GIS and BRCA analysis were assessed in ovarian tumor samples. Pearson correlations of the continuous HRD GIS and analytic sensitivity and specificity were evaluated. RESULTS: The prevalence of overall HRD positivity was 51.2% (TSO 500 HRD assay) versus 49.2% (Myriad assay) and the prevalence of BRCA mutations was 27.6% (TSO 500 HRD assay) versus 25.5% (Myriad assay). After post-processing optimization, concordance of the HRD GIS was 0.980 in all samples and 0.976 in the non-BRCA mutation cohort; the area under the receiver operating characteristic curve was 0.995 and 0.992, respectively. CONCLUSIONS: Comparison between the Illumina and Myriad assays showed that overall HRD status, the individual components of BRCA analysis, and HRD GIS detection results were highly concordant (>93%), suggesting the TSO 500 HRD assay will approach the analytical accuracy of the FDA-approved Myriad assay.

Identifying homologous recombination deficiency in breast cancer: genomic instability score distributions differ among breast cancer subtypes.

PURPOSE: A 3-biomarker homologous recombination deficiency (HRD) score is a key component of a currently FDA-approved companion diagnostic assay to identify HRD in patients with ovarian cancer using a threshold score of ≥ 42, though recent studies have explored the utility of a lower threshold (GIS ≥ 33). The present study evaluated whether the ovarian cancer thresholds may also be appropriate for major breast cancer subtypes by comparing the genomic instability score (GIS) distributions of BRCA1/2-deficient estrogen receptor-positive breast cancer (ER + BC) and triple-negative breast cancer (TNBC) to the GIS distribution of BRCA1/2-deficient ovarian cancer. METHODS: Ovarian cancer and breast cancer (ER + BC and TNBC) tumors from ten study cohorts were sequenced to identify pathogenic BRCA1/2 mutations, and GIS was calculated using a previously described algorithm. Pathologic complete response (pCR) to platinum therapy was evaluated in a subset of TNBC samples. For TNBC, a threshold was set and threshold validity was assessed relative to clinical outcomes. RESULTS: A total of 560 ovarian cancer, 805 ER + BC, and 443 TNBC tumors were included. Compared to ovarian cancer, the GIS distribution of BRCA1/2-deficient samples was shifted lower for ER + BC (p = 0.015), but not TNBC (p = 0.35). In the subset of TNBC samples, univariable logistic regression models revealed that GIS status using thresholds of ≥ 42 and ≥ 33 were significant predictors of response to platinum therapy. CONCLUSIONS: This study demonstrated that the GIS thresholds used for ovarian cancer may also be appropriate for TNBC, but not ER + BC. GIS thresholds in TNBC were validated using clinical response data to platinum therapy.

The landscape of BRCA1 and BRCA2 large rearrangements in an international cohort of over 20 000 ovarian tumors identified using next-generation sequencing.

BACKGROUND: Approximately half of ovarian tumors have defects within the homologous recombination repair pathway. Tumors carrying pathogenic variants (PVs) in BRCA1/BRCA2 are more likely to respond to poly-ADP ribose polymerase (PARP) inhibitor treatment. Large rearrangements (LRs) are a challenging class of variants to identify and characterize in tumor specimens and may therefore be underreported. This study describes the prevalence of pathogenic BRCA1/BRCA2 LRs in ovarian tumors and discusses the importance of their identification using a comprehensive testing strategy. METHODS: Sequencing and LR analyses of BRCA1/BRCA2 were conducted in 20 692 ovarian tumors received between March 18, 2016 and February 14, 2023 for MyChoice CDx testing. MyChoice CDx uses NGS dosage analysis to detect LRs in BRCA1/BRCA2 genes using dense tiling throughout the coding regions and limited flanking regions. RESULTS: Of the 2217 PVs detected, 6.3% (N = 140) were LRs. Overall, 0.67% of tumors analyzed carried a pathogenic LR. The majority of detected LRs were deletions (89.3%), followed by complex LRs (5.7%), duplications (4.3%), and retroelement insertions (0.7%). Notably, 25% of detected LRs encompassed a single or partial single exon. This study identified 84 unique LRs, 2 samples each carried 2 unique LRs in the same gene. We identified 17 LRs that occurred in multiple samples, some of which were specific to certain ancestries. Several cases presented here illustrate the intricacies involved in characterizing LRs, particularly when multiple events occur within the same gene. CONCLUSIONS: Over 6% of PVs detected in the ovarian tumors analyzed were LRs. It is imperative for laboratories to utilize testing methodologies that will accurately detect LRs at a single exon resolution to optimize the identification of patients who may benefit from PARP inhibitor treatment.

Cisplatin with veliparib or placebo in metastatic triple-negative breast cancer and BRCA mutation-associated breast cancer (S1416): a randomised, double-blind, placebo-controlled, phase 2 trial.

BACKGROUND: Poly(ADP-ribose) polymerase (PARP) inhibitors are effective in germline BRCA1 or BRCA2 (BRCA1/2) mutation-associated metastatic breast cancer. However, studies evaluating PARP inhibitors plus platinum-based chemotherapy in germline BRCA1/2-wildtype triple-negative breast cancer are scarce. A large proportion of germline BRCA1/2-wildtype triple-negative breast cancer shows homologous recombination deficiency (HRD), resulting in a BRCA-like phenotype that might render sensitivity to PARP inhibitors. The S1416 trial assessed the efficacy of cisplatin combined with the PARP inhibitor veliparib in three predefined groups of metastatic breast cancer: germline BRCA1/2-mutated, BRCA-like, and non-BRCA-like. METHODS: S1416 was a randomised, double-blind, placebo-controlled, phase 2 trial conducted at 154 community and academic clinical sites across the USA. Eligible patients aged 18 years or older had metastatic or recurrent triple-negative breast cancer or germline BRCA1/2-associated metastatic or recurrent breast cancer, an Eastern Cooperative Oncology Group performance status of 0-2, and had received up to one line of chemotherapy for metastatic disease. Patients were randomly assigned (1:1) via the National Clinical Trials Network open interactive system with dynamic balancing on number of previous cytotoxic regimens for metastatic disease to receive intravenous cisplatin (75 mg/m2, day 1) combined with either veliparib or matching placebo (300 mg orally twice a day, days 1-14) on a 21-day cycle. Investigators, patients, and the sponsors were masked to treatment assignment; the study statisticians were unmasked. Central testing after ran domisation classified patients as having mutated or wildtype germline BRCA1/2. A biomarker panel established a priori was used to classify patients with wildtype germline BRCA1/2 into BRCA-like and non-BRCA-like phenotype groups, with BRCA-like status based on at least one of the biomarkers: genomic instability score (≥42), somatic BRCA1/2 mutations, BRCA1 promoter methylation, or non-BRCA1/2 homologous recombination repair germline mutations. The primary endpoint was investigator-assessed progression-free survival, analysed separately for the three predefined biomarker groups with a prespecified α value for each analysis. Efficacy analyses were done by intention to treat and included all eligible patients. Safety analyses of toxicities attributed to treatment included all patients who received at least one dose of veliparib or placebo. The study is ongoing and registered with ClinicalTrials.gov, NCT02595905. FINDINGS: Between July 7, 2016, and June 15, 2019, 335 patients were enrolled and randomly assigned. 320 patients (n=162 to cisplatin plus veliparib, all women; and n=158 to cisplatin plus placebo, 157 women and one man) were eligible for efficacy evaluation. 247 patients were classified into the three biomarker groups: germline BRCA1/2-mutated (n=37), BRCA-like (n=101), and non-BRCA-like (n=109). 73 patients could not be classified due to missing biomarker information. Median follow-up was 11·1 months (IQR 5·6-20·8). In the germline BRCA1/2-mutated group, median progression-free survival was 6·2 months (95% CI 2·3-9·2) in the cisplatin plus veliparib group and 6·4 months (4·3-8·2) in the cisplatin plus placebo group (HR 0·79 [95% CI 0·38-1·67]; log-rank p=0·54). In the BRCA-like group, median progression-free survival was 5·9 months (95% CI 4·3-7·8) in the cisplatin plus veliparib group versus 4·2 months (2·3-5·0) in the cisplatin plus placebo group (HR 0·57 [95% CI 0·37-0·88]; p=0·010). In the non-BRCA-like group, median progression-free survival was 4·0 months (95% CI 2·5-4·7) in the cisplatin plus veliparib group versus 3·0 months (2·2-4·4) in the cisplatin plus placebo group (HR 0·89 [95% CI 0·60-1·33]; p=0·57). The most common grade 3 or worse adverse events attributed to treatment were neutropenia (71 [46%] of 155 patients in the cisplatin plus veliparib group vs 29 [20%] of 147 in the cisplatin plus placebo group), leukopenia (42 [27%] vs 11 [7%]), anaemia (35 [23%] vs 12 [8%]), and thrombocytopenia (29 [19%] vs four [3%]). Serious adverse events attributed to treatment occurred in 48 (31%) patients in the cisplatin plus veliparib group and 53 (36%) patients in the cisplatin plus placebo group. Treatment-related adverse events led to death in one patient in the cisplatin plus veliparib group (sepsis) and one patient in the cisplatin plus placebo group (acute kidney injury due to cisplatin plus heart failure from previous doxorubicin exposure). INTERPRETATION: The addition of veliparib to cisplatin significantly improved progression-free survival in patients with BRCA-like metastatic triple-negative breast cancer, but not in patients with non-BRCA-like metastatic breast cancer. PARP inhibitors combined with platinum-based chemotherapy should be explored further in BRCA-like triple-negative breast cancer. FUNDING: National Cancer Institute and National Institute of General Medical Sciences (US National Institutes of Health); AbbVie; Myriad Genetics; the Biomarker, Imaging, and Quality of Life Studies Funding Program (awarded by the National Cancer Institute); and The University of Kansas Cancer Center.

Association of Genomic Instability Score, Tumor Mutational Burden, and Tumor-Infiltrating Lymphocytes as Biomarkers in Uterine Serous Carcinoma.

Background: Uterine serous carcinomas represent 10% of uterine carcinomas but account for nearly 40% of deaths from the disease. Improved molecular characterization of these tumors is instrumental in guiding targeted treatment and improving outcomes. This study assessed the genomic instability score (GIS), tumor mutational burden (TMB), and tumor-infiltrating lymphocytes (TILs) in patients with USC. Methods: A retrospective cohort study evaluated patients with USC following staging surgery. The GIS and TMB were determined from archived specimens. We evaluated the tumoral expression of CD3, CD4, CD8, FOXP3, and CD68 using immunohistochemistry. T-tests were used to assess associations of TILs with the GIS. Results: We evaluated 53 patients with USC. The median GIS was 31 (range: 0−52) and a higher GIS was not associated with progression-free (PFS) or overall survival (OS). The median TMB was 1.35 mt/Mb; patients with TMB > 1.35 mt/Mb had improved PFS and OS (p = 0.005; p = 0.002, respectively). Tumors with increased CD3+ and CD4+ immune cells had a higher mean GIS (p = 0.013, p = 0.002). Conclusions: TMB > 1.35 mt/Mb was associated with improved survival in USC patients, whereas the GIS was not. Lower TMB thresholds may provide prognostic value for less immunogenic tumors such as USC. In this limited cohort, we observed that increased TIL populations were correlated with a higher GIS.

Homologous Recombination Deficiency as an Ovarian Cancer Biomarker in a Real-World Cohort: Validation of Decentralized Genomic Profiling.

The diagnostic evaluation of homologous recombination deficiency (HRD) is central to define targeted therapy strategies for patients with ovarian carcinoma. We evaluated HRD in 514 ovarian carcinoma samples by next-generation sequencing of DNA libraries, including BRCA1/BRCA2 and 26,523 single-nucleotide polymorphisms using the standardized Myriad HRD assay, with the predefined cut point of ≥42 for a positive genomic instability score (GIS). All samples were measured in the central Myriad laboratory and in an academic molecular pathology laboratory. A positive GIS was detected in 196 (38.1%) of tumors, whereas 318 (61.9%) were GIS negative. Combining GIS and BRCA mutations, a total of 200 (38.9%) of the 514 tumors were HRD positive. A positive GIS was significantly associated with high-grade serous histology (P < 0.000001), grade 3 tumors (P = 0.001), and patient age <60 years (P = 0.0003). The concordance between both laboratories for the GIS status was 96.9% (P < 0.000001), with a sensitivity of 94.6% and a specificity of 98.4%. Concordance for HRD status was 97.1% (499 of 514 tumors). The percentage of HRD-positive tumors in our real-life cohort was similar to the proportion observed in the recently published PAOLA-1 trial, with high concordance between central and local laboratories. Our results support introduction of the standardized HRD assay in academic molecular pathology laboratories, thus broadening access to personalized oncology strategies for patients with ovarian cancer worldwide.

Homologous Recombination Deficiency: Concepts, Definitions, and Assays.

BACKGROUND: Homologous recombination deficiency (HRD) is a phenotype that is characterized by the inability of a cell to effectively repair DNA double-strand breaks using the homologous recombination repair (HRR) pathway. Loss-of-function genes involved in this pathway can sensitize tumors to poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) inhibitors and platinum-based chemotherapy, which target the destruction of cancer cells by working in concert with HRD through synthetic lethality. However, to identify patients with these tumors, it is vital to understand how to best measure homologous repair (HR) status and to characterize the level of alignment in these measurements across different diagnostic platforms. A key current challenge is that there is no standardized method to define, measure, and report HR status using diagnostics in the clinical setting. METHODS: Friends of Cancer Research convened a consortium of project partners from key healthcare sectors to address concerns about the lack of consistency in the way HRD is defined and methods for measuring HR status. RESULTS: This publication provides findings from the group's discussions that identified opportunities to align the definition of HRD and the parameters that contribute to the determination of HR status. The consortium proposed recommendations and best practices to benefit the broader cancer community. CONCLUSION: Overall, this publication provides additional perspectives for scientist, physician, laboratory, and patient communities to contextualize the definition of HRD and various platforms that are used to measure HRD in tumors.

Concordance of BRCA mutation detection in tumor versus blood, and frequency of bi-allelic loss of BRCA in tumors from patients in the phase III SOLO2 trial.

OBJECTIVE: Maintenance olaparib provided a progression-free survival benefit in the phase III SOLO2 trial (NCT01874353) in patients with platinum-sensitive relapsed ovarian cancer and a BRCA mutation (BRCAm). However, questions remain regarding tumor versus germline BRCA testing and the impact of heterozygous versus bi-allelic loss of BRCA1 or BRCA2 in the tumor. METHODS: Blood and tumor samples were analyzed. A concordance analysis of germline BRCAm status (BRACAnalysis® CLIA test) and tumor BRCAm status (myChoice® CDx test) was conducted (Myriad Genetic Laboratories, Inc.). Bi-allelic loss of BRCA1 and BRCA2 and a genomic instability score (GIS) (myChoice® CDx test) were also determined. RESULTS: 289 of 295 enrolled patients had a germline BRCAm confirmed centrally and tumor BRCAm status was evaluable in 241 patients. There was 98% and 100% concordance between tumor and germline testing for BRCA1m and BRCA2m, respectively, with discordance found in four cases. Of 210 tumor samples evaluable for BRCA zygosity, 100% of germline BRCA1-mutated tumors (n = 144) and 98% of germline BRCA2-mutated tumors (n = 66) had bi-allelic loss of BRCA. One patient with a heterozygous BRCA2m had a GIS of 53, was progression free for 911 days and remained on olaparib at data cut-off. CONCLUSIONS: Very high concordance was demonstrated between tumor and germline BRCA testing, supporting wider implementation of tumor BRCA testing in ovarian cancer. Near 100% rates of bi-allelic loss of BRCA in platinum-sensitive relapsed ovarian tumors suggest routine testing for BRCA zygosity is not required in this population and reflects BRCA loss being a driver of tumorigenesis.

Clinical significance of homologous recombination deficiency score testing in endometrial Cancer.

BACKGROUND: Homologous recombination deficiency (HRD) score is related to chemotherapy response in some cancers, but its role in endometrial cancer in not known. We determined frequency and clinical significance of alterations in the HR pathway in endometrial cancer. METHODS: 253 endometrioid endometrial adenocarcinoma (EEA) samples from two independent cohorts (discovery and replication) were tested for HRD score using the Myriad HRD assay, microsatellite instability (MSI) and tumor mutation burden (TMB) using a next generation sequencing assay. HRD scores were also generated on endometrial cancer cell lines and in vivo response to olaparib was assessed. RESULTS: ROC curves were employed to determine optimal cutoffs of HRD in relation to survival impact in endometrial cancer and a cutoff of HRD ≥ 4 was suggested for DFS using the discovery cohort. Patients from two independent cohorts with HRD score ≥ 4 trended toward worse survival as compared to those with HRD score < 4. Both cohorts were further separated into four groups according to molecular subtypes (TMB positive; MSI positive; HRD positive; all others). When grouped by molecular subtype, there was a significant difference between groups using an HRD ≥4 cutoff in the initial (p = 0.0024) and replication (p = 0.042) cohorts. The Hec1a model (HRD score = 19) was highly sensitive to olaparib in in vitro and in vivo experiments. CONCLUSIONS: High HRD score was associated with worse DFS in our patient cohort. These findings suggest that HRD score may have clinical utility in patients with advanced or recurrent endometrial cancer.

Homologous recombination deficiency (HRD) score in germline BRCA2- versus ATM-altered prostate cancer.

The homologous recombination deficiency (HRD) score integrates three DNA-based measures of genomic instability, and has been understudied in prostate cancer. Given the recent FDA approval of two PARP inhibitors for prostate cancer, HRD score analysis could help to refine treatment selection. We assessed HRD score (defined as the sum of loss-of-heterozygosity, telomeric allelic imbalance, and large-scale state transitions) in three cohorts of primary prostate cancer, including a Johns Hopkins University (JHU) cohort with germline mutations in BRCA2, ATM, or CHEK2 (n = 64), the TCGA cohort (n = 391), and the PROGENE cohort (n = 102). In the JHU cohort, tumors with germline BRCA2 mutations had higher HRD scores (median = 27) than those with germline ATM or CHEK2 mutations (median = 16.5 [p = 0.029] and 9 [p < 0.001], respectively). For TCGA tumors without underlying HR pathway mutations, the median HRD score was 11, significantly lower than ovarian carcinoma lacking BRCA1/2 mutations (median = 28). In the absence of HR gene mutations, the median HRD score was unexpectedly higher among prostate cancers with TP53 mutations versus those without (17 vs. 11; p = 0.015); this finding was confirmed in the PROGENE cohort (24 vs. 16; p = 0.001). Finally, among eight BRCA2-altered patients who received olaparib, progression-free survival trended longer in those with HRD scores above versus below the median (14.9 vs. 9.9 months). We conclude that HRD scores are low in primary prostate cancer and higher in cases with germline BRCA2 or somatic TP53 mutations. Germline BRCA2-altered cases have significantly higher HRD scores than germline ATM-altered or CHEK2-altered cases, consistent with the lower efficacy of PARP inhibitors among the latter.

Prevalence of tumor BRCA1 and BRCA2 dysfunction in unselected patients with ovarian cancer.

Objective: The therapeutic benefits of poly(ADP-ribose) polymerase inhibitors highlight the need to evaluate BRCA1/2 defects in tubal/ovarian cancer (OC). We sought to determine the pattern and disease characteristics associated with tumor BRCA1/2 mutations and BRCA1 methylation in women with OC. Methods: We obtained 111 OC specimens from 2 university hospitals and assessed BRCA1/2 mutations and BRCA1 methylation in tumor DNA. The frequency and pattern of BRCA1/2 defects were examined. Associations between patient/disease characteristics and BRCA1/2 defects were ascertained (Fisher's exact test). Platinum-free interval (PFI), progression-free survival (PFS), and overall survival (OS) based on the underlying BRCA1/2 defect were determined (Kaplan-Meier analysis [log-rank test]). Results: We observed a BRCA1/2 dysfunction rate of 40% (28/70) in high-grade serous tubal/ovarian cancer (HGSC), including 14.3% BRCA1 methylation (n=10), 7.1% BRCA1 mutation (n=5), and 18.6% BRCA2 mutation (n=13). Defects in BRCA1/2 genes were associated with stage III/IV HGSC (BRCA1 methylation: P=0.005 [stage III/IV] and P=0.004 [HGSC]; BRCA1/2 mutation: P=0.03 [stage III/IV] and P<0.001 [HGSC]). Patients with BRCA1/2-mutated cancers showed improved OS (hazard ratio [HR], 0.65; 95% confidence interval [CI], 0.43-0.99; P=0.045) and a trend toward improved PFI (HR, 0.48; 95% CI, 0.22-1.06; P=0.07) and PFS (HR, 0.72; 95% CI, 0.51-1.03; P=0.07). No survival differences were observed between BRCA1-methylated and BRCA1/2 wild-type non-BRCA1-methylated cancers. Conclusion: We observed a high tumor BRCA1/2 dysfunction rate in HGSC with a unique predominance of BRCA2 over BRCA1 mutations. While BRCA1/2 mutations conferred survival benefits in OC, no such association was observed with BRCA1 methylation.

Genomic and Clinicopathologic Characterization of ATM-deficient Prostate Cancer.

PURPOSE: The ATM (ataxia telangiectasia mutated) gene is mutated in a subset of prostate cancers, and ATM mutation may confer specific therapeutic vulnerabilities, although ATM-deficient prostate cancers have not been well-characterized. EXPERIMENTAL DESIGN: We genetically validated a clinical grade IHC assay to detect ATM protein loss and examined the frequency of ATM loss among tumors with pathogenic germline ATM mutations and genetically unselected primary prostate carcinomas using tissue microarrays (TMAs). Immunostaining results were correlated with targeted somatic genomic sequencing and clinical outcomes. RESULTS: ATM protein loss was found in 13% (7/52) of primary Gleason pattern 5 cancers with available sequencing data and was 100% sensitive for biallelic ATM inactivation. In a separate cohort with pathogenic germline ATM mutations, 74% (14/19) had ATM protein loss of which 70% (7/10) of evaluable cases had genomic evidence of biallelic inactivation, compared with zero of four of cases with intact ATM expression. By TMA screening, ATM loss was identified in 3% (25/831) of evaluable primary tumors, more commonly in grade group 5 (17/181; 9%) compared with all other grades (8/650; 1%; P < 0.0001). Of those with available sequencing, 80% (4/5) with homogeneous ATM protein loss and 50% (6/12) with heterogeneous ATM protein loss had detectable pathogenic ATM alterations. In surgically treated patients, ATM loss was not significantly associated with clinical outcomes in random-effects Cox models after adjusting for clinicopathologic variables. CONCLUSIONS: ATM loss is enriched among high-grade prostate cancers. Optimal evaluation of ATM status requires both genomic and IHC studies and will guide development of molecularly targeted therapies.

BRCA1 Promoter Methylation and Clinical Outcomes in Ovarian Cancer: An Individual Patient Data Meta-Analysis.

BACKGROUND: BRCA1 methylation has been associated with homologous recombination deficiency, a biomarker of platinum sensitivity. Studies evaluating BRCA1-methylated tubal and ovarian cancer (OC) do not consistently support improved survival following platinum chemotherapy. We examine the characteristics of BRCA1-methylated OC in a meta-analysis of individual participant data. METHODS: Data of 2636 participants across 15 studies were analyzed. BRCA1-methylated tumors were defined according to their original study. Associations between BRCA1 methylation and clinicopathological characteristics were evaluated. The effects of methylation on overall survival (OS) and progression-free survival (PFS) were examined using mixed-effects models. All statistical tests were 2-sided. RESULTS: 430 (16.3%) tumors were BRCA1-methylated. BRCA1 methylation was associated with younger age and advanced-stage, high-grade serous OC. There were no survival differences between BRCA1-methylated and non-BRCA1-methylated OC (median PFS = 20.0 vs 18.5 months, hazard ratio [HR] = 1.01, 95% CI = 0.87 to 1.16; P = .98; median OS = 46.6 vs 48.0 months, HR = 1.02, 95% CI = 0.87 to 1.18; P = .96). Where BRCA1/2 mutations were evaluated (n = 1248), BRCA1 methylation displayed no survival advantage over BRCA1/2-intact (BRCA1/2 wild-type non-BRCA1-methylated) OC. Studies used different methods to define BRCA1 methylation. Where BRCA1 methylation was determined using methylation-specific polymerase chain reaction and gel electrophoresis (n = 834), it was associated with improved survival (PFS: HR = 0.80, 95% CI = 0.66 to 0.97; P = .02; OS: HR = 0.80, 95% CI = 0.63 to 1.00; P = .05) on mixed-effects modeling. CONCLUSION: BRCA1-methylated OC displays similar clinicopathological features to BRCA1-mutated OC but is not associated with survival. Heterogeneity within BRCA1 methylation assays influences associations. Refining these assays may better identify cases with silenced BRCA1 function and improved patient outcomes.

Association of Tumor-Infiltrating Lymphocytes with Homologous Recombination Deficiency and BRCA1/2 Status in Patients with Early Triple-Negative Breast Cancer: A Pooled Analysis.

PURPOSE: Patients with triple-negative breast cancer (TNBC) with homologous recombination deficient tumors achieve significantly higher pathologic complete response (pCR) rates when treated with neoadjuvant platinum-based therapy. Tumor-infiltrating lymphocytes (TIL) are prognostic and predictive of chemotherapy benefit in early stage TNBC. The relationship between TILs, BRCA1/2 mutation status, and homologous recombination deficiency (HRD) status in TNBC remains unclear. EXPERIMENTAL DESIGN: We performed a pooled analysis of five phase II studies that included patients with TNBC treated with neoadjuvant platinum-based chemotherapy to evaluate the association of TILs with HRD status (Myriad Genetics) and tumor BRCA1/2 mutation status. Furthermore, the relationship between pathologic response assessed using the residual cancer burden (RCB) index and HRD status with adjustment for TILs was evaluated. RESULTS: Among 161 patients, stromal TIL (sTIL) density was not significantly associated with HRD status (P = 0.107) or tumor BRCA1/2 mutation status (P = 0.391). In multivariate analyses, sTIL density [OR, 1.23; 95% confidence interval (CI), 0.94-1.61; P = 0.139] was not associated with pCR, but was associated with RCB 0/I status (OR 1.62; 95% CI, 1.20-2.28; P = 0.001). HRD was significantly associated with both pCR (OR 12.09; 95% CI, 4.11-44.29; P = 7.82 × 10-7) and RCB 0/I (OR 10.22; 95% CI, 4.11-28.75; P = 1.09 × 10-7) in these models. CONCLUSIONS: In patients with TNBC treated with neoadjuvant platinum-based therapy, TIL density was not significantly associated with either tumor BRCA1/2 mutation status or HRD status. In this pooled analysis, HRD and sTIL density were independently associated with treatment response, with HRD status being the strongest predictor.

Characterisation of homologous recombination deficiency in paired primary and recurrent high-grade serous ovarian cancer.

BACKGROUND: Homologous recombination deficiency (HRD) is shown to predict response to DNA-damaging therapies in patients with high-grade serous ovarian cancer (HGSOC); however, changes in HRD during progression remains unknown. METHODS: HRD scores were evaluated in paired primary and/or recurrent HGSOC samples (N = 107) from 54 patients with adjuvant platinum-based chemotherapy. BRCA1/2 mutation, BRCA1 methylation, loss of heterozygosity (LOH), and HRD scores were characterised using tumour DNA-based next-generation sequencing assays. RESULTS: Among 50 evaluable pairs (N = 100 samples), high intra-patient correlation in HRD score was observed (r2 = 0.93). BRCA1/2 mutations, BRCA1/2 LOH, and HRD were maintained between primary and recurrent samples, except for one pair in which a BRCA1 reversion mutation was identified in the recurrent sample. Despite the reversion, both samples were classified as having high HRD scores ( ≥ 42). All samples with BRCA1/2 mutations exhibited high HRD scores; however, high HRD scores were more prevalent than BRCA1/2 mutations (55% vs. 30%, respectively). CONCLUSION: Markers of HRD were maintained between the primary and recurrent samples, regardless of other genomic changes that occurred during recurrence. HRD score/markers in primary tumours may be valuable and adequate for selection of platinum-based therapy and/or poly-ADP-ribose-polymerase (PARP) inhibitors in recurrent HGSOC.

Candidate biomarkers of PARP inhibitor sensitivity in ovarian cancer beyond the BRCA genes.

BACKGROUND: Olaparib (Lynparza™) is a PARP inhibitor approved for advanced BRCA-mutated (BRCAm) ovarian cancer. PARP inhibitors may benefit patients whose tumours are dysfunctional in DNA repair mechanisms unrelated to BRCA1/2. We report exploratory analyses, including the long-term outcome of candidate biomarkers of sensitivity to olaparib in BRCA wild-type (BRCAwt) tumours. METHODS: Tumour samples from an olaparib maintenance monotherapy trial (Study 19, D0810C00019; NCT00753545) were analysed. Analyses included classification of mutations in genes involved in homologous recombination repair (HRR), BRCA1 promoter methylation status, measurement of BRCA1 protein and Myriad HRD score. RESULTS: Patients with BRCAm tumours gained most benefit from olaparib; a similar treatment benefit was also observed in 21/95 patients whose tumours were BRCAwt but had loss-of-function HRR mutations compared to patients with no detectable HRR mutations (58/95). A higher median Myriad MyChoice® HRD score was observed in BRCAm and BRCAwt tumours with BRCA1 methylation. Patients without BRCAm tumours derived benefit from olaparib treatment vs placebo although to a lesser extent than BRCAm patients. CONCLUSIONS: Ovarian cancer patients with tumours harbouring loss-of-function mutations in HRR genes other than BRCA1/2 may constitute a small, molecularly identifiable and clinically relevant population who derive treatment benefit from olaparib similar to patients with BRCAm.

Carboplatin in BRCA1/2-mutated and triple-negative breast cancer BRCAness subgroups: the TNT Trial.

Germline mutations in BRCA1/2 predispose individuals to breast cancer (termed germline-mutated BRCA1/2 breast cancer, gBRCA-BC) by impairing homologous recombination (HR) and causing genomic instability. HR also repairs DNA lesions caused by platinum agents and PARP inhibitors. Triple-negative breast cancers (TNBCs) harbor subpopulations with BRCA1/2 mutations, hypothesized to be especially platinum-sensitive. Cancers in putative 'BRCAness' subgroups-tumors with BRCA1 methylation; low levels of BRCA1 mRNA (BRCA1 mRNA-low); or mutational signatures for HR deficiency and those with basal phenotypes-may also be sensitive to platinum. We assessed the efficacy of carboplatin and another mechanistically distinct therapy, docetaxel, in a phase 3 trial in subjects with unselected advanced TNBC. A prespecified protocol enabled biomarker-treatment interaction analyses in gBRCA-BC and BRCAness subgroups. The primary endpoint was objective response rate (ORR). In the unselected population (376 subjects; 188 carboplatin, 188 docetaxel), carboplatin was not more active than docetaxel (ORR, 31.4% versus 34.0%, respectively; P = 0.66). In contrast, in subjects with gBRCA-BC, carboplatin had double the ORR of docetaxel (68% versus 33%, respectively; biomarker, treatment interaction P = 0.01). Such benefit was not observed for subjects with BRCA1 methylation, BRCA1 mRNA-low tumors or a high score in a Myriad HRD assay. Significant interaction between treatment and the basal-like subtype was driven by high docetaxel response in the nonbasal subgroup. We conclude that patients with advanced TNBC benefit from characterization of BRCA1/2 mutations, but not BRCA1 methylation or Myriad HRD analyses, to inform choices on platinum-based chemotherapy. Additionally, gene expression analysis of basal-like cancers may also influence treatment selection.

Biomarker Assessment of HR Deficiency, Tumor BRCA1/2 Mutations, and CCNE1 Copy Number in Ovarian Cancer: Associations with Clinical Outcome Following Platinum Monotherapy.

The current study evaluated three biomarkers [homologous recombination deficiency (HRD), tumor BRCA1/2 (tBRCA) mutations, and CCNE1 copy-number variation (CNV)] in ovarian tumors from patients enrolled on the SCOTROC4 clinical trial for associations with outcome following carboplatin monotherapy. Ovarian tumors (n = 250), with high-grade serous (HGSOC) subgroup analysis (n = 179) were classified as HRD positive (HRD score ≥42 or tBRCA mutation) and as CCNE1 amplification positive (CCNE1 CNV score >2.4). Seventy-four (30%) tumors were HRD positive, including 34 (14%) with tBRCA mutations. Forty-seven (19%) were CCNE1 amplification positive, all of which were tBRCA wild-type. HRD and tBRCA, but not CCNE1 amplification, were significantly associated with CA125 complete response in the entire cohort (HRD, P = 0.00015; tBRCA P = 0.0096), and the HGSOC subgroup (HRD, P = 0.0016; tBRCA P = 0.032). HRD and lack of CCNE1 amplification were associated with improved progression-free survival (PFS) and overall survival (OS) in the full cohort and HGSOC subgroup (HRD, P = 0.00021; CCNE1 status P = 0.038). HRD remained significant for OS and PFS after adjusting for clinical factors, while CCNE1 status only remained significant for PFS. Patients with HRD-positive tumors had greater PFS and OS benefit from platinum dose intensification than HRD-negative tumors (P = 0.049 and P = 0.035, respectively). An alternative exploratory HRD score threshold (≥33 or tBRCA mutation) was also significantly associated with both PFS and OS in the HGSOC subset.Implications: HRD, tumor BRCA1/2 mutations, and absence of CCNE1 amplification are associated with improved survival of ovarian cancer patients treated with platinum monotherapy and HRD-positive patients may benefit from platinum dose intensification. Mol Cancer Res; 16(7); 1103-11. ©2018 AACR.