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Background: Individuals with germline BRCA1 and BRCA2 pathogenic variants (BRCA carriers) are at high risk of developing high grade serous ovarian carcinoma (HGSC). HGSC is predominantly driven by TP53 mutations, but mutations in this gene are also commonly found in non-cancerous tissue as a feature of normal human aging. We hypothesized that HGSC predisposition in BRCA carriers may be related to increased TP53 somatic evolution, which could be detectable by ultra-deep sequencing of TP53 mutations in gynecological liquid biopsies., Methods: Duplex sequencing was used to identify TP53 mutations with high sensitivity in peritoneal washes and cervical liquid-based cytology (LBC) collected at surgery from 60 individuals including BRCA1 and BRCA2 carriers, and non-carriers. TP53 mutation pathogenicity was compared across groups and with TP53 cancer mutations., Results: TP53 mutations were more abundant in cervical LBC than in peritoneal washes but increased with age in both sample types. In peritoneal washes, but not in cervical LBC, pathogenic TP53 mutation burden was increased in BRCA1 carriers compared to non-carriers, independently of age. Five individuals shared identical pathogenic TP53 mutations in peritoneal washes and cervical LBC, but not in blood., Conclusions: Ultra-deep sequencing of TP53 mutations in peritoneal washes collected at surgery reveals increased burden of pathogenic TP53 mutations in BRCA1 carriers. This excess of pathogenic TP53 mutations might be linked to the elevated risk of HGSC in these individuals. In some patients, concordant TP53 mutations were found in peritoneal washes and cervical LBCs, but the cell of origin remains unknown and deserves further investigation., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: RAR is a consultant and equity holder at TwinStrand Biosciences Inc. and an equity holder at NanoString Technologies Inc. RAR is named inventor on patents owned by the University of Washington and licensed to TwinStrand Biosciences Inc. RAR received research funding from a joint research grant with TwinStrand Biosciences Inc. and Ovartec GmbH., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Somatic TP53 mutations are prevalent in normal tissue but little is known about their association with cancer risk. Cervical liquid-based cytology (LBC), commonly known as Pap test, provides an accessible gynecological sample to test the value of TP53 somatic mutations as a biomarker for high-grade serous ovarian cancer (HGSC), a cancer type mostly driven by TP53 mutations. We used ultra-deep duplex sequencing to analyze TP53 mutations in LBC and blood samples from 70 individuals (30 with and 40 without HGSC) undergoing gynecologic surgery, 30 carrying BRCA1 or BRCA2 germline pathogenic variants (BRCApv). Only 30% of the tumor mutations were found in LBC samples. However, TP53 pathogenic mutations were identified in nearly all LBC and blood samples, with only 5.4% of mutations in LBC (20/368) also found in the corresponding blood sample. TP53 mutations were more abundant in LBC than in blood and increased with age in both sample types. BRCApv carriers with HGSC had more TP53 clonal expansions in LBC than BRCApv carriers without cancer. Our results show that, while not useful for direct cancer detection, LBC samples capture TP53 mutation burden in the gynecological tract, presenting potential value for cancer risk assessment in individuals at higher hereditary risk for ovarian cancer., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

The multi-cohort phase 2 trial NCT02203513 was designed to evaluate the clinical activity of the CHK1 inhibitor (CHK1i) prexasertib in patients with breast or ovarian cancer. Here we report the activity of CHK1i in platinum-resistant high-grade serous ovarian carcinoma (HGSOC) with measurable and biopsiable disease (cohort 5), or without biopsiable disease (cohort 6). The primary endpoint was objective response rate (ORR). Secondary outcomes were safety and progression-free survival (PFS). 49 heavily pretreated patients were enrolled (24 in cohort 5, 25 in cohort 6). Among the 39 RECISTv1.1-evaluable patients, ORR was 33.3% in cohort 5 and 28.6% in cohort 6. Primary endpoint was not evaluable due to early stop of the trial. The median PFS was 4 months in cohort 5 and 6 months in cohort 6. Toxicity was manageable. Translational research was an exploratory endpoint. Potential biomarkers were investigated using pre-treatment fresh biopsies and serial blood samples. Transcriptomic analysis revealed high levels of DNA replication-related genes (POLA1, POLE, GINS3) associated with lack of clinical benefit [defined post-hoc as PFS < 6 months]. Subsequent preclinical experiments demonstrated significant cytotoxicity of POLA1 silencing in combination with CHK1i in platinum-resistant HGSOC cell line models. Therefore, POLA1 expression may be predictive for CHK1i resistance, and the concurrent POLA1 inhibition may improve the efficacy of CHK1i monotherapy in this hard-to-treat population, deserving further investigation., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Poly(ADP-ribose) polymerase inhibitors (PARPis) have changed the treatment paradigm in breast cancer gene ( BRCA )-mutant high-grade serous ovarian carcinoma (HGSC). However, most patients eventually develop resistance to PARPis, highlighting an unmet need for improved therapeutic strategies. Using high-throughput drug screens, we identified ataxia telangiectasia and rad3-related protein/checkpoint kinase 1 (CHK1) pathway inhibitors as cytotoxic and further validated the activity of the CHK1 inhibitor (CHK1i) prexasertib in PARPi-sensitive and -resistant BRCA -mutant HGSC cells and xenograft mouse models. CHK1i monotherapy induced DNA damage, apoptosis, and tumor size reduction. We then conducted a phase 2 study (NCT02203513) of prexasertib in patients with BRCA -mutant HGSC. The treatment was well tolerated but yielded an objective response rate of 6% (1 of 17; one partial response) in patients with previous PARPi treatment. Exploratory biomarker analyses revealed that replication stress and fork stabilization were associated with clinical benefit to CHK1i. In particular, overexpression of Bloom syndrome RecQ helicase ( BLM ) and cyclin E1 ( CCNE1 ) overexpression or copy number gain/amplification were seen in patients who derived durable benefit from CHK1i. BRCA reversion mutation in previously PARPi-treated BRCA -mutant patients was not associated with resistance to CHK1i. Our findings suggest that replication fork-related genes should be further evaluated as biomarkers for CHK1i sensitivity in patients with BRCA -mutant HGSC.

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/m 2 , 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., Competing Interests: Declaration of interests PS reports an S1416 subaward from SWOG (payments made to the institution) as support for the present work; other relationships include grants (payments made to the institution) from Novartis, Bristol Myers Squibb, Merck, and Gilead; royalties from UpToDate; and advisory board participation for Pfizer, Merck, Gilead, Genzyme Corporation, Novartis, AstraZeneca, GSK, and Seattle Genetics. WEB reports a grant from the US National Cancer Institute (payments made to the institution) as support for the present work; other relationships include grants (payments made to the institution) from Merck and AstraZeneca; and data safety monitoring board participation for Frontier Science and the National Eye Institute. JRG reports steering committee membership for Genentech (Roche); advisory board participation for AstraZeneca and Puma Biosciences; consulting fees from Seagen; data safety monitoring board or advisory board participation (no compensation) for Genentech, AstraZeneca, Novartis, and Immunomedics; and a role as Chief Medical Officer for the American Society of Clinical Oncology. SLP reports grants (payments made to the institution) from Pfizer, AstraZeneca, Puma Biosciences, Medivation, Genentech, Eli Lilly, and Bayer HealthCare Pharmaceuticals; faculty or speaker payment from Pfizer; and steering committee membership (no compensation) for Genentech and AbbVie. CKA reports grants from Puma Biosciences, Lilly, Merck, Seattle Genetics, Nektar, Tesaro, G1-Therapeutics, Zion Pharma China, Novartis, Pfizer, AstraZeneca, and Elucida; royalties or licenses from UpToDate and Jones and Bartlett Publishing Company; consulting fees from Genentech, Eisai, Seattle Genetics, AstraZeneca, Novartis, Immunomedics, Elucida, and Athenex; and data safety monitoring board or advisory board participation for Genentech. LG reports grants (payments made to the institution) from Genentech and Merck; advisory board participation for Genentech, Merck, AstraZeneca, Novartis, Syndax, Biovica, GE Healthcare, Pfizer, Nanostring, Immunomedics, Aduro, Genomic Health, Amgen, Mylan-Lynx, Eisai, Lilly, Exact Sciences, and Biovica; and data safety monitoring board participation for Daiichi Sankyo. DT reports grants from Novartis, Pfizer, and Polyphor; and consulting fees from AstraZeneca, GSK, Exact Sciences, Gilead, Novartis, Pfizer, Personalis, and Sermonix. UAB-G reports consulting fees from Novartis, Biotheranostics, Taiho, Sanofi, Macrogenics, Seattle Genetics, and Gilead; and payment or honoraria from Seattle Genetics. AKG reports a Masonic Cancer Alliance Partners Advisory Board grant from The University of Kansas Cancer Center and Children's Mercy Kansas City as support for the current work; other relationships include grants (payments made to the institution) from VITRAC Therapeutics and Predicine; scientific advisory board membership for Biovica, Clara Biotech, and Sinochips Diagnostics; a role as Deputy Director for The University of Kansas Cancer Center; a role as co-founder of Sinochips Diagnostics; and future stock options with Clara Biotech. EMS reports a SWOG subcontract from the Biomarker, Imaging, and Quality of Life Studies Funding Program as support for the current work; and data safety monitoring board participation for Novartis. KMT reports homologous recombination deficiency patents, employment, and stock or stock options with Myriad Genetics. LP reports research funding or clinical trial support from Bristol Myers Squibb, Merck, Pfizer, AstraZeneca, and Seagen. DFH reports grants (payments made to the institution) from Merrimack Pharmaceuticals (Parexel), Menarini Silicon Biosystems, Pfizer, AstraZeneca, and Cepheid; royalties or licenses from Janssen Research & Development (Johnson & Johnson; transferred to Menarini Silicon Biosystems); consulting fees from Cepheid, Freenome, Artiman Ventures (Cellworks), Lexent Bio, Epic Sciences, L-Nutra, BioVeca, OncoCyte, Turnstone Biologics, Predictus BioSciences, Guardant, Macrogenics, Xilis, and Exact Sciences; lecture payment from Tempus and Macrogenics; patents under Immunicon Corporation (European Patent Office) and University of Michigan (US Patent Office); a role as chair for the International Breast Cancer Study Group; stock options with Inbiomotion; receipt of equipment, materials, drugs, medical writing, gifts, or other services from Menarini Silicon Biosystems and Cepheid; and other financial or non-financial interests from UpToDate (section editor) and CancerExpertNow. All other authors declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Purpose: Cediranib, a pan-vascular endothelial growth factor receptor inhibitor, suppresses expression of homologous recombination repair (HRR) genes and increases sensitivity to poly-(ADP-ribose) polymerase inhibition in preclinical models. We investigated whether cediranib combined with olaparib improves the clinical outcomes of patients with prostate cancer., Methods: Patients with progressive metastatic castration-resistant prostate cancer (mCRPC) were randomly assigned 1:1 to arm A: cediranib 30 mg once daily plus olaparib 200 mg twice daily or arm B: olaparib 300 mg twice daily alone. The primary end point was radiographic progression-free survival (rPFS) in the intention-to-treat patients. The secondary end points were rPFS in patients with HRR-deficient and HRR-proficient mCRPC., Results: In the intention-to-treat set of 90 patients, median rPFS was 8.5 (95% CI, 5.4 to 12.0) and 4.0 (95% CI, 3.2 to 8.5) months in arms A and B, respectively. Cediranib/olaparib significantly improved rPFS versus olaparib alone (hazard ratio [HR], 0.617; 95% CI, 0.392 to 0.969; P = .0359). Descriptive analyses showed a median rPFS of 10.6 (95% CI, 5.9 to not assessed [NA]) and 3.8 (95% CI, 2.33 to NA) months (HR, 0.64; 95% CI, 0.272 to 1.504) among patients with HRR-deficient mCRPC, and 13.8 (95% CI, 3.3 to NA) and 11.3 (95% CI, 3.8 to NA) months (HR, 0.98; 95% CI, 0.321 to 2.988) among patients with BRCA2 -mutated mCRPC in arms A and B, respectively. The incidence of grades 3-4 adverse events was 61% and 18% in arms A and B, respectively., Conclusion: Cediranib combined with olaparib improved rPFS compared with olaparib alone in men with mCRPC. This combination was associated with an increased incidence of grades 3-4 adverse events. BRCA2 -mutated subgroups treated with olaparib with or without cediranib were associated with a numerically longer median rPFS.

Current screening methods for ovarian cancer (OC) have failed to demonstrate a significant reduction in mortality. Uterine lavage combined with TP53 ultra-deep sequencing for the detection of disseminated OC cells has emerged as a promising tool, but this approach has not been tested for early-stage disease or non-serous histologies. In addition, lavages carry multiple background mutations, the significance of which is poorly understood. Uterine lavage was collected preoperatively in 34 patients undergoing surgery for suspected ovarian malignancy including 14 patients with benign disease and 20 patients with OC (6 non-serous and 14 high grade serous-like (serous)). Ultra-deep duplex sequencing (~3000x) with a panel of common OC genes identified the tumor mutation in 33% of non-serous (all early stage) and in 79% of serous cancers (including four early stage). In addition, all lavages carried multiple somatic mutations (average of 25 mutations per lavage), more than half of which corresponded to common cancer driver mutations. Driver mutations in KRAS, PIK3CA, PTEN, PPP2R1A and ARID1A presented as larger clones than non-driver mutations and with similar frequency in lavages from patients with and without OC, indicating prevalent somatic evolution in all patients. Driver TP53 mutations, however, presented as significantly larger clones and with higher frequency in lavages from individuals with OC, suggesting that TP53 -specific clonal expansions are linked to ovarian cancer development. Our results demonstrate that lavages capture cancer cells, even from early-stage cancers, as well as other clonal expansions and support further exploration of TP53 mutation burden as a potential OC risk factor.

Mutations in homologous recombination (HR) genes, including BRCA1 , BRCA2 , and the RAD51 paralog RAD51C , predispose to tumorigenesis and sensitize cancers to DNA-damaging agents and poly(ADP ribose) polymerase inhibitors. However, ∼800 missense variants of unknown significance have been identified for RAD51C alone, impairing cancer risk assessment and therapeutic strategies. Here, we interrogated >50 RAD51C missense variants, finding that mutations in residues conserved with RAD51 strongly predicted HR deficiency and disrupted interactions with other RAD51 paralogs. A cluster of mutations was identified in and around the Walker A box that led to impairments in HR, interactions with three other RAD51 paralogs, binding to single-stranded DNA, and ATP hydrolysis. We generated structural models of the two RAD51 paralog complexes containing RAD51C, RAD51B-RAD51C-RAD51D-XRCC2 and RAD51C-XRCC3. Together with our functional and biochemical analyses, the structural models predict ATP binding at the interface of RAD51C interactions with other RAD51 paralogs, similar to interactions between monomers in RAD51 filaments, and explain the failure of RAD51C variants in binding multiple paralogs. Ovarian cancer patients with variants in this cluster showed exceptionally long survival, which may be relevant to the reversion potential of the variants. This comprehensive analysis provides a framework for RAD51C variant classification. Importantly, it also provides insight into the functioning of the RAD51 paralog complexes.

Triple-negative breast cancer (TNBC) and ovarian carcinomas (OvCas) with BRCA1 promoter methylation ( BRCA1 meth) respond more poorly to alkylating agents compared to those bearing mutations in BRCA1 and BRCA2 ( BRCA mut). This is a conundrum given the biologically equivalent homologous recombination deficiency (HRD) induced by these genetic and epigenetic BRCA perturbations. We dissected this problem through detailed genomic analyses of TNBC and OvCa cohorts and experimentation with patient-derived xenografts and genetically engineered cell lines. We found that despite identical downstream genomic mutational signatures associated with BRCA1 meth and BRCA mut states, BRCA1 meth uniformly associates with poor outcomes. Exposure of BRCA1 meth TNBCs to platinum chemotherapy, either as clinical treatment of a patient or as experimental in vivo exposure of preclinical patient derived xenografts, resulted in allelic loss of BRCA1 methylation and increased BRCA1 expression and platinum resistance. These data suggest that, unlike BRCA mut cancers, where BRCA loss is a genetically "fixed" deficiency state, BRCA1 meth cancers are highly adaptive to genotoxin exposure and, through reversal of promoter methylation, recover BRCA1 expression and become resistant to therapy. We further found a specific augmented immune transcriptional signal associated with enhanced response to platinum chemotherapy but only in patients with BRCA-proficient cancers. We showed how integrating both this cancer immune signature and the presence of BRCA mutations results in more accurate predictions of patient response when compared to either HRD status or BRCA status alone. This underscores the importance of defining BRCA heterogeneity in optimizing the predictive precision of assigning response probabilities in TNBC and OvCa.

Purpose: PARP inhibitors synergize with topoisomerase inhibitors, and veliparib plus modified (m) FOLFIRI (no 5-FU bolus) had preliminary activity in metastatic pancreatic cancers. This study evaluated the safety and efficacy of second-line treatment with veliparib and mFOLFIRI versus FOLFIRI (control) for metastatic pancreatic cancer., Patients and Methods: This randomized phase II clinical trial led by the SWOG Cancer Research Network enrolled patients between September 1, 2016 and December 13, 2017. The median follow-up was 9 months (IQR 1-27). BRCA1/2 and homologous recombination DNA damage repair (HR-DDR) genetic defects were tested in blood and tumor biopsies. Patients received veliparib 200 mg twice daily, days 1-7 with mFOLFIRI days 3-5, or FOLFIRI in 14-day cycles., Results: After 123 of planned 143 patients were accrued, an interim futility analysis indicated that the veliparib arm was unlikely to be superior to control, and the study was halted. Median overall survival (OS) was 5.4 versus 6.5 months (HR, 1.23; P = 0.28), and median progression-free survival (PFS) was 2.1 versus 2.9 months (HR, 1.39; P = 0.09) with veliparib versus control. Grade 3/4 toxicities were more common with veliparib (69% vs. 58%, P = 0.23). For cancers with HR-DDR defects versus wild-type, median PFS and OS were 7.3 versus 2.5 months ( P = 0.05) and 10.1 versus 5.9 months ( P = 0.17), respectively, with FOLFIRI, and 2.0 versus 2.1 months ( P = 0.62) and 7.4 versus 5.1 months ( P = 0.10), respectively, with veliparib plus mFOLFIRI., Conclusions: Veliparib plus mFOLFIRI did not improve survival for metastatic pancreatic cancer. FOLFIRI should be further studied in pancreatic cancers with HR-DDR defects., (©2021 American Association for Cancer Research.)

Acquired PARP inhibitor (PARPi) resistance in BRCA1 - or BRCA2 -mutant ovarian cancer often results from secondary mutations that restore expression of functional protein. RAD51C is a less commonly studied ovarian cancer susceptibility gene whose promoter is sometimes methylated, leading to homologous recombination (HR) deficiency and PARPi sensitivity. For this study, the PARPi-sensitive patient-derived ovarian cancer xenograft PH039, which lacks HR gene mutations but harbors RAD51C promoter methylation, was selected for PARPi resistance by cyclical niraparib treatment in vivo . PH039 acquired PARPi resistance by the third treatment cycle and grew through subsequent treatment with either niraparib or rucaparib. Transcriptional profiling throughout the course of resistance development showed widespread pathway level changes along with a marked increase in RAD51C mRNA, which reflected loss of RAD51C promoter methylation. Analysis of ovarian cancer samples from the ARIEL2 Part 1 clinical trial of rucaparib monotherapy likewise indicated an association between loss of RAD51C methylation prior to on-study biopsy and limited response. Interestingly, the PARPi resistant PH039 model remained platinum sensitive. Collectively, these results not only indicate that PARPi treatment pressure can reverse RAD51C methylation and restore RAD51C expression, but also provide a model for studying the clinical observation that PARPi and platinum sensitivity are sometimes dissociated., (© The Author(s) 2021. Published by Oxford University Press on behalf of NAR Cancer.)

Mutations in homologous recombination (HR) genes predispose to cancer but also sensitize to chemotherapeutics. Although therapy can initially be effective, cancers frequently cease responding, leading to recurrence and poor prognosis. Here we identify a germline mutation in RAD51C , a critical HR factor and known tumor suppressor, in an ovarian cancer patient with exceptionally long, progression-free survival. The RAD51C-T132P mutation is in a highly conserved residue within the nucleotide-binding site and interferes with single-strand DNA binding of the RAD51 paralog complex RAD51B-RAD51C-RAD51D-XRCC2 and association with another RAD51 paralog XRCC3. These biochemical defects lead to highly defective HR and drug sensitivity in tumor cells, ascribing RAD51C-T132P as a deleterious mutation that was likely causal for tumor formation. Conversely, its position within a critical site suggests that it is refractory to secondary mutations that would restore RAD51C gene function and lead to therapy resistance. A need for a greater understanding of the relationship between mutation position and reversion potential of HR genes is underscored, as it may help predict the effectiveness of therapies in patients with HR-deficient cancers., (© 2021 Sullivan et al.; Published by Cold Spring Harbor Laboratory Press.)

Objectives: Mutations in the TP53 tumor suppressor gene are common in ovarian carcinoma (OC) but their impact on outcomes is controversial. We sought to define the relationship of TP53 mutations to cancer outcomes and their interactions with co-occurrent BRCA1 or BRCA2 (BRCA) mutations, comparing three different TP53 mutation classification schemes., Methods: We performed next generation sequencing on 393 cases of OC prospectively followed for survival. TP53 mutations were classified according to three schemes termed Structural, Functional, and Hotspot. Mutation distribution was compared between cases with and without BRCA mutations. In a subset of 281 cases of high grade serous carcinoma (HGSC), overall survival was compared using Kaplan-Meier curves, logrank testing, and multivariate Cox regression analysis, both stratified and adjusted for BRCA mutation status. Multivariate logistic regression was used to analyze the effects of TP53 mutation type on platinum resistance., Results: TP53 mutations were identified in 76.8% of the total cohort (n = 302/393) and 87.9% of HGSC (n = 247/281). Cases with BRCA mutations demonstrated significantly higher TP53 mutation frequency overall (n = 84/91, 92.3% vs. n = 218/302, 72.2%, p < 0.001). TP53 mutations were not associated with overall survival, even when stratified by BRCA mutation. TP53 mutations were associated with platinum sensitivity, even after adjusting for BRCA mutation status (OR 0.41, p = 0.048). The choice of TP53 mutation classification scheme was not found to alter any significant outcome., Conclusions: BRCA mutations significantly co-occur with TP53 mutations. After adjusting for BRCA mutations, TP53 mutations are associated with platinum sensitivity, and this effect is not dependent on TP53 mutation type., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Purpose: Preclinical studies suggest PARP inhibition (PARPi) induces immunostimulatory micromilieu in ovarian cancer thus complementing activity of immune checkpoint blockade. We conducted a phase II trial of PARPi olaparib and anti-PD-L1 durvalumab and collected paired fresh core biopsies and blood samples to test this hypothesis., Patients and Methods: In a single-center, proof-of-concept phase II study, we enrolled women aged ≥18 with recurrent ovarian cancer. All patients were immune checkpoint inhibitor-naïve and had measurable disease per RECISTv1.1, ECOG performance status 0-2, and adequate organ and marrow function. Patients received olaparib 300 mg twice daily and durvalumab 1,500 mg intravenously every 4 weeks until disease progression, unacceptable toxicity, or withdrawal of consent. Primary endpoint was overall response rate (ORR). Secondary objectives were safety and progression-free survival (PFS). Translational objectives included biomarker evaluation for relationships with clinical response and immunomodulatory effects by treatment., Results: Thirty-five patients with ovarian cancer [median, four prior therapies (IQR, 2-5.5), predominantly platinum-resistant (86%), BRCA wild-type (77%)] received at least one full cycle of treatment. ORR was 14% [5/35; 95% confidence interval (CI), 4.8%-30.3%]. Disease control rate (PR+SD) was 71% (25/35; 95% CI, 53.7%-85.4%). Treatment enhanced IFNγ and CXCL9/CXCL10 expression, systemic IFNγ/TNFα production, and tumor-infiltrating lymphocytes, indicating an immunostimulatory environment. Increased IFNγ production was associated with improved PFS [HR, 0.37 (95% CI, 0.16-0.87), P = 0.023], while elevated VEGFR3 levels were associated with worse PFS (HR, 3.22 (95% CI, 1.23-8.40), P = 0.017]., Conclusions: The PARPi and anti-PD-L1 combination showed modest clinical activity in recurrent ovarian cancer. Our correlative study results suggest immunomodulatory effects by olaparib/durvalumab in patients and indicate that VEGF/VEGFR pathway blockade would be necessary for improved efficacy of the combination., (©2020 American Association for Cancer Research.)

Germline loss-of-function mutations in BRCA1 interacting protein C-terminal helicase 1 (BRIP1) are associated with ovarian carcinoma and may also contribute to breast cancer risk, particularly among patients who develop disease at an early age. Normal BRIP1 activity is required for DNA interstrand cross-link (ICL) repair and is thus central to the maintenance of genome stability. Although pathogenic mutations have been identified in BRIP1 , genetic testing more often reveals missense variants, for which the impact on molecular function and subsequent roles in cancer risk are uncertain. Next-generation sequencing of germline DNA in 2,160 early-onset breast cancer and 1,199 patients with ovarian cancer revealed nearly 2% of patients carry a very rare missense variant (minor allele frequency < 0.0001) in BRIP1 . This is 3-fold higher than the frequency of all rare BRIP1 missense alleles reported in more than 60,000 individuals of the general population ( P < 0.0001, χ 2 test). Using CRISPR-Cas9 gene editing technology and rescue assays, we functionally characterized 20 of these missense variants, focusing on the altered protein's ability to repair ICL damage. A total of 75% of the characterized variants rendered the protein hypomorph or null. In a clinical cohort of >117,000 patients with breast and ovarian cancer who underwent panel testing, the combined OR associated with BRIP1 hypomorph or null missense carriers compared with the general population was 2.30 (95% confidence interval, 1.60-3.30; P < 0.0001). These findings suggest that novel missense variants within the helicase domain of BRIP1 may confer risk for both breast and ovarian cancer and highlight the importance of functional testing for additional variants. SIGNIFICANCE: Functional characterization of rare variants of uncertain significance in BRIP1 revealed that 75% demonstrate loss-of-function activity, suggesting rare missense alleles in BRIP1 confer risk for both breast and ovarian cancer., (©2019 American Association for Cancer Research.)

Objective: Pap tests hold promise as a molecular diagnostic for serous ovarian cancer, but previous studies reported limited sensitivity. Furthermore, the presence of somatic mutations in normal tissue is increasingly recognized as a challenge to the specificity of mutation-based cancer diagnostics. We applied an ultra-deep sequencing method with the goal of improving sensitivity and characterizing the landscape of low-frequency somatic TP53 mutations in Pap tests., Methods: We used CRISPR-DS to deeply sequence (mean Duplex depth ~3000×) the TP53 gene in 30 Pap tests from 21 women without cancer and 9 women with serous ovarian carcinoma with known TP53 driver mutations. Mutations were annotated and compared to those in the TP53 cancer database., Results: The tumor-derived mutation was identified in 3 of 8 Pap tests from women with ovarian cancer and intact tubes. In addition, 221 low-frequency (≲0.001) exonic TP53 mutations were identified in Pap tests from women with ovarian cancer (94 mutations) and without ovarian cancer (127 mutations). Many of these mutations resembled TP53 mutations found in cancer: they impaired protein activity, were predicted to be pathogenic, and clustered in exons 5 to 8 and hotspot codons. Cancer-like mutations were identified in all women but at higher frequency in women with ovarian cancer., Conclusions: Pap tests have low sensitivity for ovarian cancer detection and carry abundant low-frequency TP53 mutations. These mutations are more frequently pathogenic in women with ovarian cancer. Determining whether low-frequency TP53 mutations in normal gynecologic tissues are associated with an increased cancer risk warrants further study., Competing Interests: Declaration of competing interest RAR shares equity in NanoString Technologies Inc. and is the principal investigator on a NIH SBIR R44CA221426 subcontract research agreement with TwinStrand Biosciences Inc., (Copyright © 2019 Elsevier Inc. All rights reserved.)

BRCA1 mutant carcinomas are sensitive to PARP inhibitor (PARPi) therapy; however, resistance arises. BRCA1 BRCT domain mutant proteins do not fold correctly and are subject to proteasomal degradation, resulting in PARPi sensitivity. In this study, we show that cell lines and patient-derived tumors, with highly disruptive BRCT domain mutations, have readily detectable BRCA1 protein expression, and are able to proliferate in the presence of PARPi. Peptide analyses reveal that chemo-resistant cancers contain residues encoded by BRCA1 intron 15. Mechanistically, cancers with BRCT domain mutations harbor BRCA1 gene breakpoints within or adjacent to Alu elements in intron 15; producing partial gene duplications, inversions and translocations, and terminating transcription prior to the mutation-containing BRCT domain. BRCA1 BRCT domain-deficient protein isoforms avoid mutation-induced proteasomal degradation, support homology-dependent DNA repair, and promote PARPi resistance. Taken together, Alu-mediated BRCA1 gene rearrangements are responsible for generating hypomorphic proteins, and may represent a biomarker of PARPi resistance.

A key resistance mechanism to platinum-based chemotherapies and PARP inhibitors in BRCA -mutant cancers is the acquisition of BRCA reversion mutations that restore protein function. To estimate the prevalence of BRCA reversion mutations in high-grade ovarian carcinoma (HGOC), we performed targeted next-generation sequencing of circulating cell-free DNA (cfDNA) extracted from pretreatment and postprogression plasma in patients with deleterious germline or somatic BRCA mutations treated with the PARP inhibitor rucaparib. BRCA reversion mutations were identified in pretreatment cfDNA from 18% (2/11) of platinum-refractory and 13% (5/38) of platinum-resistant cancers, compared with 2% (1/48) of platinum-sensitive cancers ( P = 0.049). Patients without BRCA reversion mutations detected in pretreatment cfDNA had significantly longer rucaparib progression-free survival than those with reversion mutations (median, 9.0 vs. 1.8 months; HR, 0.12; P < 0.0001). To study acquired resistance, we sequenced 78 postprogression cfDNA, identifying eight additional patients with BRCA reversion mutations not found in pretreatment cfDNA. SIGNIFICANCE: BRCA reversion mutations are detected in cfDNA from platinum-resistant or platinum-refractory HGOC and are associated with decreased clinical benefit from rucaparib treatment. Sequencing of cfDNA can detect multiple BRCA reversion mutations, highlighting the ability to capture multiclonal heterogeneity. This article is highlighted in the In This Issue feature, p. 151 ., (©2018 American Association for Cancer Research.)

Nucleotide sequences representing nine genes and five presumptive genetic loci were used to infer phylogenetic relationships among seven Baylisascaris species, including one species with no previously available molecular data. These genes were used to test the species status of B. procyonis and B. columnaris using a coalescent approach. Phylogenetic analysis based on combined analysis of sequence data strongly supported monophyly of the genus and separated the species into two main clades. Clade 1 included B. procyonis , B. columnaris , and B. devosi , species hosted by musteloid carnivores. Clade 2 included B. transfuga and B. schroederi from ursids, B. ailuri, a species from the red panda (a musteloid), and B. tasmaniensis from a marsupial. Within clade 2, geographic isolates of B. transfuga , B. schroederi (from giant panda), and B. ailuri formed a strongly supported clade. In certain analyses (e.g., some single genes), B. tasmaniensis was sister to all other Baylisascaris species rather than sister to the species from ursids and red panda. Using one combination of priors corresponding to moderate population size and shallow genetic divergence, the multispecies coalescent analysis of B. procyonis and B. columnaris yielded moderate support (posterior probability 0.91) for these taxa as separate species. However, other prior combinations yielded weak or no support for delimiting these taxa as separate species. Similarly, tree topologies constrained to represent reciprocal monophyly of B. columnaris and B. procyonis individuals (topologies consistent with separate species) were significantly worse in some cases, but not others, depending on the dataset analyzed. An expanded analysis of SNPs and other genetic markers that were previously suggested to distinguish between individuals of B. procyonis and B. columnaris was made by characterization of additional individual nematodes. The results suggest that many of these SNPs do not represent fixed differences between nematodes derived from raccoon and skunk hosts.