Wild-type BRCA1 Research Articles

Replicative Instability Drives Cancer Progression.

In the past decade, defective DNA repair has been increasingly linked with cancer progression. Human tumors with markers of defective DNA repair and increased replication stress exhibit genomic instability and poor survival rates across tumor types. Seminal studies have demonstrated that genomic instability develops following inactivation of BRCA1, BRCA2, or BRCA-related genes. However, it is recognized that many tumors exhibit genomic instability but lack BRCA inactivation. We sought to identify a pan-cancer mechanism that underpins genomic instability and cancer progression in BRCA-wildtype tumors. Methods: Using multi-omics data from two independent consortia, we analyzed data from dozens of tumor types to identify patient cohorts characterized by poor outcomes, genomic instability, and wildtype BRCA genes. We developed several novel metrics to identify the genetic underpinnings of genomic instability in tumors with wildtype BRCA. Associated clinical data was mined to analyze patient responses to standard of care therapies and potential differences in metastatic dissemination. Results: Systematic analysis of the DNA repair landscape revealed that defective single-strand break repair, translesion synthesis, and non-homologous end-joining effectors drive genomic instability in tumors with wildtype BRCA and BRCA-related genes. Importantly, we find that loss of these effectors promotes replication stress, therapy resistance, and increased primary carcinoma to brain metastasis. Conclusions: Our results have defined a new pan-cancer class of tumors characterized by replicative instability (RIN). RIN is defined by the accumulation of intra-chromosomal, gene-level gain and loss events at replication stress sensitive (RSS) genome sites. We find that RIN accelerates cancer progression by driving copy number alterations and transcriptional program rewiring that promote tumor evolution. Clinically, we find that RIN drives therapy resistance and distant metastases across multiple tumor types.

Tumor Treating Fields (TTFields) Concomitant with PARP Inhibitors for Treatment of Ovarian Cancer Cell Lines

<h3>Purpose/Objective(s)</h3> TTFields are electric fields that disrupt cellular processes critical for cancer cell viability and tumor progression. Recently, TTFields have also been shown to induce a state of BRCAness in various cancer types. PARP inhibitors (PARPi) demonstrate clinical efficacy in patients with solid tumors and deleterious BRCA mutations; however, 3 of 4 patients with ovarian cancer do not harbor BRCA mutations, and thus may experience limited benefit from PARP inhibition. TTFields have shown in vitro and in vivo efficacy in ovarian cancer models. The objective of this study was to examine the effect of TTFields concomitant with PARPi on ovarian cancer cell lines. <h3>Materials/Methods</h3> A2780 (BRCA1/2 wild type) and OVCAR3 (BRCA1 wild type, BRCA2 deep deletion) ovarian carcinoma cells were treated for 72 h with TTFields at an intensity of 1 V/cm RMS and frequency of 200 kHz, alone or with concomitant application of the PARP inhibitors niraparib or olaparib. Efficacy was examined via measurements of cell survival, colony forming ability, and induction of apoptosis. Formation of DNA double strand breaks was examined by fluorescence staining for ɣH2AX. <h3>Results</h3> Application of TTFields or PARPi to A2780 or OVCAR3 cells resulted in cytotoxicity, anti-clonogenic effect, and induction of apoptosis. Concomitant application of TTFields with PARPi displayed synergistic interaction in the BRCA wild type A2780 cells and additively in the BRCA mutant OVCAR-3 cells. Co-application of TTFields and PARPi also resulted in increased levels of DNA double strand breaks in both cell lines relative to each modality alone. <h3>Conclusion</h3> The results support that TTFields induce a state of BRCAness, which together with PARP inhibition can possibly lead to synthetic lethality. Collectively, the data suggest potential benefits for TTFields concomitant application with PARP inhibition for treatment of ovarian cancer even in the absence of background BRCA mutations.

Comparison of Clinical, Pathological, and Prognostic Features in BRCA Mutant and Wild-Type Male Breast Cancer Patients.

Published studies on male breast cancer (MBC) and BRCA mutations are scarce and usually include,a small number of patients. The clinicopathological characteristics of BRCA mutant and wild-type MBC patients were compared in more than forty patients in this study. A retrospective review of MBC patients' clinical and histopathological data was conducted. To compare the patients' characteristics, chi-square test and Fisher's Exact test were utilized. Kaplan-Meier analysis was used to examine the survival analysis. In total 43 cases were reviewed. The average duration of follow-up was 35.8 months. BRCA mutations were found in 11 (25.6%) of the patients. BRCA1 mutations were found in four patients (9.3%), BRCA2 mutations in six patients (14%), and BRCA1 and BRCA2 mutations in one patient (2.3%). The median age at diagnosis was 58 years old, and there was no statistically significant difference between groups (p = 0.7). Tumor location (p = 0.3), human epidermal growth factor receptor 2 overexpression (p = 0.5), estrogen receptor status (p = 0.05), progesterone receptor status (p = 0.6), tumor stage (p = 0.9), lymph node positivity (p = 0.5), tumor histology (p = 0.06), and recurrence status (p = 0.6) were similar between BRCA-wild type and -mutated patients. Overall survival averaged 115.6 months (range: 76.0-155.3), with no statistically significant differences between groups (p = 0.6). This study investigated clinical and pathological characteristics and prognoses of BRCA wild and mutant-type MBC and these were similar in all groups studied.

A phase II study of talazoparib monotherapy in patients with wild-type BRCA1 and BRCA2 with a mutation in other homologous recombination genes

Talazoparib, a PARP inhibitor, is active in germline BRCA1 and BRCA2 (gBRCA1/2)-mutant advanced breast cancer, but its activity beyond gBRCA1/2 is poorly understood. We conducted Talazoparib Beyond BRCA (NCT02401347), an open-label phase II trial, to evaluate talazoparib in patients with pretreated advanced HER2-negative breast cancer (n = 13) or other solid tumors (n = 7) with mutations in homologous recombination (HR) pathway genes other than BRCA1 and BRCA2. In patients with breast cancer, four patients had a Response Evaluation Criteria in Solid Tumors (RECIST) partial response (overall response rate, 31%), and three additional patients had stable disease of ≥6 months (clinical benefit rate, 54%). All patients with germline mutations in PALB2(gPALB2; encoding partner and localizer of BRCA2) had treatment-associated tumor regression. Tumor or plasma circulating tumor DNA (ctDNA) HR deficiency (HRD) scores were correlated with treatment outcomes and were increased in all gPALB2 tumors. In addition, a gPALB2-associated mutational signature was associated with tumor response. Thus, talazoparib has been demonstrated to have efficacy in patients with advanced breast cancer who have gPALB2 mutations, showing activity in the context of HR pathway gene mutations beyond gBRCA1/2.

Methylation Analyses Reveal Promoter Hypermethylation as a Rare Cause of "Second Hit" in Germline BRCA1-Associated Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by the occurrence of pathogenic variants in BRCA1/2 in 5-6% of patients. Biallelic loss of BRCA1/2 enriches for response to platinum agents and poly (ADP-ribose) polymerase 1 inhibitors. There is a dearth of evidence on the mechanism of inactivation of the wild-type BRCA1 allele in PDAC tumors with a germline BRCA1 (gBRCA1) pathogenic or likely pathogenic variant (P/LPV). Herein, we examine promotor hypermethylation as a "second hit" mechanism in patients with gBRCA1-PDAC. We evaluated patients with PDAC who underwent Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) somatic and germline testing from an institutional database. DNA isolated from tumor tissue and matched normal peripheral blood were sequenced by MSK-IMPACT. In patients with gBRCA1-PDAC, we examined the somatic BRCA1 mutation status and promotor methylation status of the tumor BRCA1 allele via a methylation array analysis. In patients with sufficient remaining DNA, a second methylation analysis by pyrosequencing was performed. Of 1012 patients with PDAC, 19 (1.9%) were identified to harbor a gBRCA1 P/LPV. Fifteen patients underwent a methylation array and the mean percentage of BRCA1 promotor methylation was 3.62%. In seven patients in whom sufficient DNA was available, subsequent pyrosequencing confirmed an unmethylated BRCA1 promotor. Loss of heterozygosity was detected in 12 of 19 (63%, 95% confidence interval 38-84) patients, demonstrating loss of heterozygosity is the major molecular mechanism of BRCA1 inactivation in PDAC. Two (10.5%) cases had a somatic BRCA1 mutation. In patients with gBRCA1-P/LPV-PDAC, loss of heterozygosity is the main inactivating mechanism of the wild-type BRCA1 allele in the tumor, and methylation of the BRCA1 promoter is a distinctly uncommon occurrence.

What's beyond BRCA Mutational Status in High Grade Serous Ovarian Cancer? The Impact of Hormone Receptor Expression in a Large BRCA-Profiled Ovarian Cancer Patient Series: A Retrospective Cohort Study.

Simple SummaryOvarian hormones are involved in ovarian cancer pathogenesis. However, few reports have investigated the hormone receptor pattern according to BRCA mutational status. The aim of this single-center, observational, retrospective study was to explore the relationship between hormone receptor status and BRCA1/2 mutation in a cohort of 207 high-grade serous ovarian carcinoma (HGSOC) patients. Interesting differences emerged between BRCA-mutated and BRCA wild-type women, in terms of pattern of receptor expression and its association to the outcome. On the whole, our findings, though needing further validation, extend our understanding of the complex interplay between BRCA1/2 protein and hormone signaling, suggesting new pathways to be exploited in order to develop future personalized therapy.Several studies have explored the prognostic role of hormone receptor status in high-grade serous ovarian cancer (HGSOC) patients. However, few reports have investigated their expression according to BRCA mutational status. The aim of this single-center, observational, retrospective study was to explore the hormone receptor pattern and its potential prognostic role in a cohort of 207 HGSOC women stratified for BRCA mutational status. To this end, ERα, ERβ1, ERβ2, ERβ5, PR, and AR expression were assessed by immunohistochemistry in 135 BRCA-wild type (BRCA-wt) and 72 BRCA1/2 mutation carriers (BRCA-mut). No significant difference emerged in hormone receptor expression between the two sub-samples, except for a significantly lower ERα expression observed in pre-menopausal BRCA1/2-mut as compared to BRCA-wt patients (p = 0.02). None of the examined hormone receptors has revealed a significant prognostic role in the whole sample, apart from the ratio ERα/ERβ5 nuclear, for which higher values disclosed a positive role on the outcome in BRCA-wt subgroup (HR 0.77; CI 0.61–0.96; p = 0.019). Conversely, it negatively affected overall survival in the presence of BRCA1/2-mut (HR 1.41; CI 1.06–1.87; p = 0.020). Finally, higher PR levels were associated with platinum sensitivity in the whole sample (p = 0.019). Our data, though needing further validation, suggest a potential role of oestrogen-mediated pathways in BRCA1/2-associated HGSOC tumorigenesis, thus revealing a possible therapeutic potential for targeting this interaction.

Multicenter Real-World Data of Subsequent Chemotherapy after Progression to PARP Inhibitors in a Maintenance Relapse Setting.

Simple SummarySince the irruption of PARPi in the therapeutic armamentarium for ovarian cancer, concerns regarding post-progression treatment outcomes have emerged, owing to known crossed-resistance mechanisms between PARPi and platinum. In this multicentric retrospective series of ovarian cancer patients, we evaluated chemotherapy results upon progression to maintenance with PARPi in the relapsed setting. We further selected the population of platinum-sensitive patients (according to the classical definition) retreated with platinum (n = 74). In this platinum-sensitive population, overall response rate and survival outcomes of platinum rechallenge after PARPi were similar to historical series of the prePARPi era. However, within this group, analysis according to BRCA status showed that BRCA mutant patients (n = 35) presented higher rates of progression and worse survival outcomes under subsequent platinum than BRCA wild type patients (n = 39), with statistically significant differences. This is the largest real-world data series of ovarian cancer patients treated with platinum rechallenge in the post-PARPi scenario.Background: Despite impressive progression-free survival (PFS) results from PARP inhibitors (PARPi) in ovarian cancer, concerns about their effect on post-progression treatment outcomes have recently arisen, particularly when administered in the relapsed setting. Overlapping mechanisms of resistance between PARPi and platinum have been described, and optimal therapies upon progression to PARPi are unknown. We communicate real-world data (RWD) on outcomes of subsequent chemotherapy upon progression to PARPi used as maintenance in ovarian cancer relapses, particularly focusing on platinum rechallenge, according to BRCA status. Methods: Data from high-grade serous or endometrioid ovarian cancer patients who received subsequent chemotherapy after progression to maintenance PARPi in the relapsed setting, in 16 Catalan hospitals between August 2016 and April 2021, and who were followed-up until July 2021, were included. Endpoints were overall response rate (ORR), and PFS and overall survival (OS) measured from the subsequent chemotherapy starting date. Results: 111 patients were included [46 (41.4%) presented pathological BRCA1/2 mutations, 8 (7.5%) in other homologous recombination-related genes]. Sixty-four patients (57.7%) had received two prior chemotherapy lines, including the one immediately prior to PARPi. PARPi were niraparib (n = 60, 54.1%), olaparib (n = 49, 44.1%), and rucaparib (n = 2, 1.8%). A total of 81 patients remained platinum-sensitive (PS population) after progression to PARPi (when progression-free interval [PFI] was >6 months after the last cycle of prior platinum) [median PFI 12.0 months (interquartile range, IQR, 8.8–17.1)]. Of those, 74 were treated with subsequent platinum regimens, with the following results: ORR of 41.9%, median PFS (mPFS) of 6.6 months (95% CI 6–9.2), and median OS (mOS) of 20.6 months (95% CI 13.6–28.9). Analysis of these 74 patients according to BRCA status showed that PFIs for BRCA mutant and non BRCA-mutant patients were 13.6 [IQR11.2–22.2] and 10.3 [IQR 7.4–14.9] months, respectively (p = 0.010); ORR were 40.0% versus 43.6%, respectively; Rates of progression (as best response) to subsequent platinum were 45.7% versus 17.9%, respectively (p = 0.004); mPFS and mOS were 3.5 (95% CI 2.5–8.6) versus 7.5 months (95% CI 6.5–10.1, p = 0.03), and 16.4 (95% CI 9.3–27.5) versus 24.2 months (95% CI 17.2–NR, p = 0.036), respectively. Conclusion: This is the largest series of real-world data on ovarian cancer patients retreated with platinum in the post-PARPi scenario, separately analyzing BRCA mutant and non-mutant patients, to our knowledge. In our platinum-sensitive population, rechallenge with platinum after progression upon PARPi in the 3rd or later lines for ovarian cancer relapses shows relevant ORR and similar PFS outcomes to historical series of the prePARPi era. However, BRCA mutant patients presented significantly higher rates of progression under subsequent platinum and worse survival outcomes associated with subsequent platinum than non-BRCA-mutant patients.

Dissection of transcriptome dysregulation and immune characterization in women with germline BRCA1 mutation at single-cell resolution

BackgroundHigh-grade serous carcinoma (HGSC) is the most frequent and lethal type of ovarian cancer. It has been proposed that tubal secretory cells are the origin of ovarian HGSC in women with familial BRCA1/2 mutations. However, the molecular changes underlying malignant transformation remain unknown.MethodWe performed single-cell RNA and T cell receptor sequencing of tubal fimbriated ends from 3 BRCA1 germline mutation carriers (BRCA1 carriers) and 3 normal controls with no high-risk history (non-BRCA1 carriers).ResultsExploring the transcriptomes of 19,008 cells, predominantly from BRCA1+ samples, we identified 5 major cell populations in the fallopian tubal mucosae. The secretory cells of BRCA1+ samples had differentially expressed genes involved in tumor growth and regulation, chemokine signaling, and antigen presentation compared to the wild-type BRCA1 controls. There are several novel findings in this study. First, a subset of the fallopian tubal secretory cells from one BRCA1 carrier exhibited an epithelial-to-mesenchymal transition (EMT) phenotype, which was also present in the mucosal fibroblasts. Second, we identified a previously unreported phenotypic split of the EMT secretory cells with distinct evolutionary endpoints. Third, we observed increased clonal expansion among the CD8+ T cell population from BRCA1+ carriers. Among those clonally expanded CD8+ T cells, PD-1 was significantly increased in tubal mucosae of BRCA1+ patients compared with that of normal controls, indicating that T cell exhaustion may occur before the development of any premalignant or malignant lesions.ConclusionThese results indicate that EMT and immune evasion in normal-looking tubal mucosae may represent early events leading to the development of HGSC in women with BRCA1 germline mutation. Our findings provide a probable molecular mechanism explaining why some, but not all, women with BRCA1 germline mutation present with early development and rapid dissemination of HGSC.

CT imaging phenotypes linked to CA125 and HE4 biomarkers are highly predictive in discriminating between hereditary and sporadic ovarian cancer patients.

Hereditary ovarian cancers (HOC) represent about 23% of ovarian cancer (OC) cases: they are most frequently related to germline mutations in the BRCA genes. We aimed to compare CA125/HE4 serum levels and Computed Tomography (CT) features at time of ovarian cancer (OC) diagnosis in two populations: BRCA mutant and BRCA wild-type (WT) OC, and to investigate the relationship between this laboratory and radiological biomarker and BRCA mutation status. This retrospective study included 60 newly diagnosed OC patients with FIGO stage IIIC-IV disease, tested for BRCA1/2 germline mutation status of which preoperative CT scan and serum tumor marker assay were available. The median level of CA125 (708 U/mL) was significantly higher (p < 0.002) in BRCA1/2 mutated patients than in WT patients (176 U/mL), whereas the median level of HE4 (492 pmol/L) was significantly higher (p < 0.002) in WT than in BRCA-mutated patients (252 pmol/L). BRCA mutation carriers showed a higher incidence of bilateral ovarian masses (p = 0.0303) characterized by solid structures (p < 0.00001), higher peritoneal tumor load, macronodular implants >2 cm (p = 0.000099), increased frequency of lymphadenopathies (p = 0.019), and metastasis (p = 0.052) compared to patients with BRCA WT. Tumor markers and CT patterns may help in identifying BRCA mutation status in OC directing patients towards a personalized treatment.

586P BRCA1, BRCA2 and RAD51C somatic RNAseq study in ovarian cancer: A description of physiological and pathogenic splicing patterns

Genomic molecular profiling is becoming a key step in the management of advanced ovarian cancer. Currently, the main indication for tumor analysis is on DNA (BRCA1/2 mutations and HRD status); there is no indication for RNA tumor analysis. The aims of this work were to describe physiological splicing profiles of BRCA1, BRCA2 and RAD51C in ovarian tissue and then search for pathogenic splicing of these genes in patients with high-grade serous papillary adenocarcinomas stage III of FIGO classification with good response to neoadjuvant cisplatin. RNAseq (Agilent SureSelect XT HS/capture-based target enrichment and NextSeq 550 Illumina Sequencer) was performed on RNA extracted from ovarian cell lines OVCAR4 and COV318 and five pre-cisplatin frozen ovarian tumors with wild-type BRCA1 and BRCA2 somatic DNA screening. We obtained the Genomic Instability Score with Myriad approach and performed RNAseq on post-cisplatin FFPE ovarian block. We also checked BRCA1 methylation status by digital PCR before their inclusion to describe physiological splicing patterns, in addition to OVCAR4 and COV318. A pathogenic skip of BRCA1 exon 3 was found in one tumor at a high level, comparing to the normal transcript. The loss of exon 3 caused a truncation of BRCA1 protein at codon 27; no variant was identified on DNA. This splicing event explained the high GIS at 66 and the good clinical response to cisplatin. Exon 3 skipping was tumoral as it was absent on the post-cisplatin residual ovarian block. Another tumor was BRCA1 methylated. There were neither enrichment of alternative nor novel splicing event of BRCA1, BRCA2 and RAD51C, specific of ovarian tissue. Physiological splicing profiles were qualitatively and quantitatively close to those reported in lymphoblastoid cell lines. Unlike BRCA2 and RAD51C, five known reported transcripts of BRCA1 were missing in the ovarian tissue. We provided a complete description of physiological splicing profiles of BRCA1, BRCA2 and RAD51C in the ovarian tissue. We also showed that somatic RNAseq analysis in ovarian cancer may modify the therapeutic prognosis by allowing access to targeted therapies.

590P Ki67 as a predictor of response to PARP inhibitors in platinum sensitive BRCA wild type ovarian cancers: MITO 37 retrospective study

590P Ki67 as a predictor of response to PARP inhibitors in platinum sensitive BRCA wild type ovarian cancers: MITO 37 retrospective study

Aromatase inhibitors and contralateral breast cancer in BRCA mutation carriers.

Deleterious BRCA mutations confer a significant lifetime risk of breast cancer (BC) as well as contralateral BC (CBC) in patients who do not undergo prophylactic mastectomy. Prior reports have suggested that tamoxifen reduces the risk of CBC in BRCA mutation carriers. Whether aromatase inhibitors (AI) have the same effect is unknown. This is a retrospective review of patients diagnosed with non-metastatic ER+ BC between 2004 and 2014 with known BRCA mutation status. Patients were followed from primary diagnosis until CBC diagnosis or death. Median follow-up was 11.5years. Risk of CBC was evaluated as time to event. 935 subjects were included in this analysis, with 53 BRCA1 mutation carriers, and 94 BRCA2 mutation carriers. Median age at diagnosis was 42.7years. Seventy-two percent (676) received tamoxifen and 43% (405) received AI. A total of 66 CBCs occurred, of which 10% (15/147) occurred in BRCA mutation carriers vs 6.5% (51/788) in BRCA wild type. Multivariate analyses indicated that BRCA status and AI use were significantly associated with CBC risk. AI use resulted in a significant reduction in risk of CBC (HR 0.44, p = 0.004) regardless of the BRCA mutation status. Tamoxifen use was not associated with reduced risk of CBC. This is the first report showing that AIs reduce the risk of CBC in BRCA mutation carriers. The potential role of AIs as chemoprevention should be validated in larger independent cohorts.

The disruption of the CCDC6 – PP4 axis induces a BRCAness like phenotype and sensitivity to PARP inhibitors in high-grade serous ovarian carcinoma

BackgroundTreatment with PARP inhibitors (PARPi) is primarily effective against high-grade serous ovarian cancers (HGSOC) with BRCA1/2 mutations or other deficiencies in homologous recombination (HR) repair mechanisms. However, resistance to PARPi frequently develops, mostly as a result of BRCA1/2 reversion mutations. The tumour suppressor CCDC6 is involved in HR repair by regulating the PP4c phosphatase activity on γH2AX. In this work, we reported that in ovarian cancer cells, a physical or functional loss of CCDC6 results synthetic lethal with the PARP-inhibitors drugs, by affecting the HR repair. We also unravelled a role for CCDC6 as predictive marker of PARPi sensitivity in ovarian cancer, and the impact of CCDC6 downregulation in overcoming PARPi resistance in these tumours.MethodsA panel of HGSOC cell lines (either BRCA-wild type or mutant) were treated with PARPi after CCDC6 was attenuated by silencing or by inhibiting USP7, a CCDC6-deubiquitinating enzyme, and the effects on cell survival were assessed. At the cellular and molecular levels, the processes underlying the CCDC6-dependent modification of drugs’ sensitivity were examined. Patient-derived xenografts (PDXs) were immunostained for CCDC6, and the expression of the protein was analysed statistically after digital or visual means.ResultsHGSOC cells acquired PARPi sensitivity after CCDC6 depletion. Notably, CCDC6 downregulation restored the PARPi sensitivity in newly generated or spontaneously resistant cells containing either wild type- or mutant-BRCA2. When in an un-phosphorylated state, the CCDC6 residue threonine 427 is crucial for effective CCDC6-PP4 complex formation and PP4 sequestration, which maintains high γH2AX levels and effective HR. Remarkably, the PP4-dependent control of HR repair is influenced by the CCDC6 constitutively phosphorylated mutant T427D or by the CCDC6 loss, favouring PARPi sensitivity. As a result, the PP4 regulatory component PP4R3α showed to be essential for both the activity of the PP4 complex and the CCDC6 dependent PARPi sensitivity. It's interesting to note that immunohistochemistry revealed an intense CCDC6 protein staining in olaparib-resistant HGSOC cells and PDXs.ConclusionsOur findings suggest that the physical loss or the functional impairment of CCDC6 enhances the PP4c complex activity, which causes BRCAness and PARPi sensitivity in HGSOC cells. Moreover, CCDC6 downregulation might overcome PARPi resistance in HGSOCs, thus supporting the potential of targeting CCDC6 by USP7 inhibitors to tackle PARPi resistance.

MONO-OLA1: A randomized, phase III study of olaparib maintenance monotherapy in patients with BRCA wild-type advanced ovarian cancer following response to first-line platinum-based chemotherapy (334)

Objectives: Results of the SOLO1 study (Moore et al. N Engl J Med 2018) led to the approval of the poly(ADP-ribose) polymerase (PARP) inhibitor olaparib in the USA, the EU, Japan, and China as maintenance treatment in patients with a BRCA1 and/or BRCA2 mutation (BRCAm) following response to first-line (1L) platinum-based chemotherapy (PBC). Maintenance PARP inhibitor, with or without bevacizumab, has been shown to provide benefit to patients with newly diagnosed advanced ovarian cancer (OC) without a BRCAm (González-Martin et al. N Engl J Med 2019; Ray-Coquard et al. N Engl J Med 2019). The MONO-OLA1 study (NCT04884360) assesses the efficacy and safety of olaparib maintenance monotherapy when compared with placebo in patients with BRCA wild-type (BRCAwt) advanced OC following response to 1L PBC. Methods: MONO-OLA1 is a Phase III, randomized, double-blind, placebo-controlled, international study. Eligible patients must have higher risk newly diagnosed BRCAwt advanced OC (FIGO stage III/IV) and have a complete response (CR) or partial response (PR) following 1L PBC. Patients with stage III disease who have complete cytoreduction following primary debulking surgery are not eligible. Patients will be randomly assigned (2:1 target, n=420) to receive either olaparib (300 mg bid) or placebo tablets. Randomization will be stratified by response to 1L PBC (CR and PR), homologous recombination deficiency (HRD) status (positive and negative/unknown), and region (China and other international sites). The number of patients with HRD-negative/unknown status will be capped at approximately 60% of the total patient population. The primary endpoints are investigator-assessed progression-free survival (PFS) by RECIST v1.1 in the BRCAwt HRD-positive and BRCAwt groups. A PFS sensitivity analysis by blinded independent central review will be performed to confirm the primary efficacy findings. The key secondary endpoints are second progression-free survival, overall survival, and safety and tolerability. Other secondary endpoints include time to first subsequent therapy or death, time to second subsequent therapy or death, and health-related quality of life. Exploratory endpoints include translational analyses to identify predictive biomarkers of response to olaparib. Enrollment began in May 2021. Objectives: Results of the SOLO1 study (Moore et al. N Engl J Med 2018) led to the approval of the poly(ADP-ribose) polymerase (PARP) inhibitor olaparib in the USA, the EU, Japan, and China as maintenance treatment in patients with a BRCA1 and/or BRCA2 mutation (BRCAm) following response to first-line (1L) platinum-based chemotherapy (PBC). Maintenance PARP inhibitor, with or without bevacizumab, has been shown to provide benefit to patients with newly diagnosed advanced ovarian cancer (OC) without a BRCAm (González-Martin et al. N Engl J Med 2019; Ray-Coquard et al. N Engl J Med 2019). The MONO-OLA1 study (NCT04884360) assesses the efficacy and safety of olaparib maintenance monotherapy when compared with placebo in patients with BRCA wild-type (BRCAwt) advanced OC following response to 1L PBC. Methods: MONO-OLA1 is a Phase III, randomized, double-blind, placebo-controlled, international study. Eligible patients must have higher risk newly diagnosed BRCAwt advanced OC (FIGO stage III/IV) and have a complete response (CR) or partial response (PR) following 1L PBC. Patients with stage III disease who have complete cytoreduction following primary debulking surgery are not eligible. Patients will be randomly assigned (2:1 target, n=420) to receive either olaparib (300 mg bid) or placebo tablets. Randomization will be stratified by response to 1L PBC (CR and PR), homologous recombination deficiency (HRD) status (positive and negative/unknown), and region (China and other international sites). The number of patients with HRD-negative/unknown status will be capped at approximately 60% of the total patient population. The primary endpoints are investigator-assessed progression-free survival (PFS) by RECIST v1.1 in the BRCAwt HRD-positive and BRCAwt groups. A PFS sensitivity analysis by blinded independent central review will be performed to confirm the primary efficacy findings. The key secondary endpoints are second progression-free survival, overall survival, and safety and tolerability. Other secondary endpoints include time to first subsequent therapy or death, time to second subsequent therapy or death, and health-related quality of life. Exploratory endpoints include translational analyses to identify predictive biomarkers of response to olaparib. Enrollment began in May 2021.

Efficacy of combining tumor treating fields (TTFields) with a PARP inhibitor in ovarian cell lines (300.5)

Objectives: PARP inhibition demonstrates clinical effectiveness in patients with solid tumors and deleterious BRCA mutations. The use of PARP inhibitors is specifically relevant in ovarian cancer, as one of three women with this malignancy harbor BRCA mutations. Tumor Treating Fields (TTFields) are alternating electric fields with antimitotic effects on cancerous cells, which are approved to treat patients with glioblastoma and malignant pleural mesothelioma. TTFields were shown to induce a state of BRCAness in several cancerous cell lines. The objective of this study was to explore the potential of combining TTFields with PARP inhibition for the treatment of ovarian cancer based on in vitro work. Methods: Ovarian carcinoma cells, A2780 (BRCA1/2 wild type) and OVCAR3 (BRCA1 wild type, BRCA2 mutated), were treated for 72 h with TTFields (at an intensity of 1.2 V/cm RMS and frequency of 200 kHz) using the in vitro system. The effectiveness of TTFields in combination with the PARP inhibitor niraparib was examined by measuring cell counts, clonogenic potential, overall effect (multiplicity product of cytotoxicity and clonogenicity), and apoptosis induction. Results: The application of TTFields to A2780 or OVCAR3 cells resulted in about 50% reduction in cell counts. Niraparib displayed a dose-dependent cytotoxic effect on both cell lines. Co-application of niraparib and TTFields resulted in enhanced cytotoxicity compared to the effect of niraparib or TTFields alone. TTFields also elevated the effects of niraparib on the colony-forming ability of the cells, the overall effect, and apoptosis. Conclusions: These results suggest a potential benefit for concomitant treatment of TTFields with niraparib in ovarian cancer. Additional experiments will examine the effect on the BRCA-FANC pathway in ovarian cancer. Objectives: PARP inhibition demonstrates clinical effectiveness in patients with solid tumors and deleterious BRCA mutations. The use of PARP inhibitors is specifically relevant in ovarian cancer, as one of three women with this malignancy harbor BRCA mutations. Tumor Treating Fields (TTFields) are alternating electric fields with antimitotic effects on cancerous cells, which are approved to treat patients with glioblastoma and malignant pleural mesothelioma. TTFields were shown to induce a state of BRCAness in several cancerous cell lines. The objective of this study was to explore the potential of combining TTFields with PARP inhibition for the treatment of ovarian cancer based on in vitro work. Methods: Ovarian carcinoma cells, A2780 (BRCA1/2 wild type) and OVCAR3 (BRCA1 wild type, BRCA2 mutated), were treated for 72 h with TTFields (at an intensity of 1.2 V/cm RMS and frequency of 200 kHz) using the in vitro system. The effectiveness of TTFields in combination with the PARP inhibitor niraparib was examined by measuring cell counts, clonogenic potential, overall effect (multiplicity product of cytotoxicity and clonogenicity), and apoptosis induction. Results: The application of TTFields to A2780 or OVCAR3 cells resulted in about 50% reduction in cell counts. Niraparib displayed a dose-dependent cytotoxic effect on both cell lines. Co-application of niraparib and TTFields resulted in enhanced cytotoxicity compared to the effect of niraparib or TTFields alone. TTFields also elevated the effects of niraparib on the colony-forming ability of the cells, the overall effect, and apoptosis. Conclusions: These results suggest a potential benefit for concomitant treatment of TTFields with niraparib in ovarian cancer. Additional experiments will examine the effect on the BRCA-FANC pathway in ovarian cancer.

Olaparib@human serum albumin nanoparticles as sustained drug-releasing tumour-targeting nanomedicine to inhibit growth and metastasis in the mouse model of triple-negative breast cancer

Poly(ADP-ribose) polymerase inhibitor olaparib demonstrated therapeutic effectiveness in highly metastatic triple-negative breast cancer (TNBC). However, olaparib offers a weak therapeutic response in wild-type BRCA cancers due to the drug’s poor bioavailability. Here, a bioinspired/active-tumour targeted nanoparticles system of human serum albumin with physical entrapment of olaparib was prepared via a low-energy desolvation technique using the crosslinker glutaraldehyde. The developed OLA@HSA NPs were nanosize (∼140 nm), kinetically stable with a low polydispersity (0.3), exhibited olaparib entrapment (EE 76.01 ± 2.53%, DL 6.76 ± 0.22%) and sustained drug release at pH 7.4 with an enhancement of drug release in acidic pH. OLA@HSA NPs decreased the half-maximal inhibitory concentrations (IC50) of olaparib by 1.6-, 1.8-fold in 24 h and 2.2-, 2.4-fold in 48 h for human (MDA-MB 231) and mouse (4T1) TNBC cells, respectively, mediated by their enhanced time-dependent cellular uptake than free olaparib. The OLA@HSA NPs induced concentration-dependent phosphatidylserine (apoptotic marker) externalisation and arrested the cell population in the G2/M phase in both the tested cell lines at a higher level than free olaparib. The NPs formulation increased DNA fragmentation, mitochondrial membrane depolarisation and ROS generation than the free olaparib. The in vivo study conducted using 4T1-Luc tumour-bearing mice demonstrated strong tumour growth inhibitory potential of OLA@HSA NPs by elevating apoptosis ROS generation and reducing the level of the antiproliferative marker, Ki-67. OLA@HSA NPs reduced the occurrence of lung metastasis (formation of metastasis nodules decreased by ∼10-fold). OLA@HSA NPs could be a promising nanomedicine for the TNBC treatment.

Abstract CT113: A phase II study of prexasertib, a cell cycle checkpoint kinase 1 (CHK1) inhibitor, in platinum-resistant recurrent high-grade serous ovarian cancer (HGSOC) with BRCA wild-type (BRCAwt)

Abstract Background: Platinum-resistance is associated with a poor prognosis in HGSOC and has limited treatment options. Inhibition of CHK1 has shown activity in BRCAwt HGSOC. Here, we report the clinical activity and tolerability of the second generation CHK1 inhibitor, prexasertib, in platinum-resistant BRCAwt HGSOC (NCT02203513). Methods: Eligibility included platinum-resistant recurrent HGSOC women without germline or somatic BRCA mutation, ECOG PS 0-2, good end organ function, and measurable disease. The primary endpoint was response rate (RR). Secondary endpoints were progression-free survival (PFS) and safety. Prexasertib was given at 105 mg/m2 IV every 14 days in a 28-day cycle. CT scans were performed every 2 cycles for RECIST v1.1 evaluation, and safety evaluation using CTCAE v4.0 every cycle. CBC was performed on day 8 of cycle 1 for ANC nadir. Pretreatment clinical samples were collected for biomarker analysis including HR deficiency and circulating tumor cells (CTC). Results: Between January 2017 and November 2020, 49 patients (median age 64.1 years [IQR 56.8 - 69.9]) received at least one dose. 12 (24.5%) patients were primary platinum-resistant, and 37 (75.5%) were secondary platinum-resistant. All were heavily pretreated (median 4 prior systemic therapies [IQR 3 - 7]), including bevacizumab (79%), PARP inhibitors (44%), and both (2%). 39 patients were evaluable by RECIST and RR was 30.7%. The median PFS was 5.8 months (range 1.7-26.4 months). The median duration of response among PRs was 5.5 months (range 1-19.8 months). The clinical benefit rate (PR+CR+SD &amp;gt;4 months) was 84.6%. 10 patients were inevaluable due to: withdrawal of consent (n=2), intercurrent illness related to disease (tumor invasion of mainstem bronchus (n=1) or vaginal wall (n=1), small bowel obstruction (n=2), infection (n=2), failure to thrive (n=1), and pneumothorax secondary to thoracentesis (n=1) during cycle 1. The common (&amp;gt;10%) grade 3 or 4 adverse events (AEs) included neutropenia (85%, 42/49) based on the cycle 1 day 8 CBC at the time of ANC nadir, lymphocytopenia (46%, 23/49), thrombocytopenia (40%, 20/49), anemia (30%, 15/49), and febrile neutropenia (12%, 5/49). 83% (41/49) of patients received growth factors support to avoid subsequent treatment delays. There were no deaths on the study. No significant difference of RECIST response or PFS was observed in patients with high (≥2) vs low (&amp;lt;2) CTC at baseline. Conclusions: Prexasertib monotherapy resulted in clinical benefit in subgroups of heavily pretreated BRCAwt platinum-resistant HGSOC. Prexasertib was well tolerated with manageable grade 3/4 AEs. Further studies on predictive biomarkers are ongoing. Citation Format: Grant Zurcher, Ann McCoy, Brooke Solarz, Jay Nair, Min-Jung Lee, Jane B. Trepel, Christina M. Annunziata, Jung-Min Lee. A phase II study of prexasertib, a cell cycle checkpoint kinase 1 (CHK1) inhibitor, in platinum-resistant recurrent high-grade serous ovarian cancer (HGSOC) with BRCA wild-type (BRCAwt) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT113.

Abstract 1765: Discovery and first disclosure of AZD8205, a B7-H4-targeted antibody-drug conjugate utilizing a novel topoisomerase I linker-warhead

Abstract The cell-surface glycoprotein B7-H4 is overexpressed in a range of solid tumors including breast cancer, ovarian serous carcinoma, endometrial carcinoma, and cholangiocarcinoma, yet has limited expression in normal tissue, making it an attractive target for an antibody-drug conjugate (ADC). This presentation describes for the first time the development of AZD8205, a B7-H4 targeted ADC incorporating a novel topoisomerase 1 inhibitor (TOP1i) linker-warhead, AZ’0133 which was designed to exploit the full potential of B7-H4 as an ADC target. Initially, we investigated a series of more than 35 TOP1i compounds as warheads and achieved activity in a clinically relevant nM range. We further optimized the conjugation site and chemistry to reduce the potential for aggregation while maintaining potency, overcoming major synthetic challenges to deliver a robust synthetic route amenable to scale-up. Finally, with a series of optimized linker-warheads, we explored the impact of linker-warhead design on ADC hydrophobicity, stability, efficacy, pharmacokinetics and tolerability culminating in the development of AZD8205. The primary mechanism of action of AZD8205 is intracellular delivery of the TOP1i warhead to B7-H4 positive cells, leading to DNA damage and apoptotic cell death. AZD8205 drove bystander killing of target negative cells in mixed cultures in vitro, which is further supported by robust antitumor activity observed in in vivo studies with patient-derived xenograft (PDX) tumors with heterogeneous target expression, representing multiple tumor indications. In a study of 26 human TNBC PDX tumors, a single IV administration of 3.5 mg/kg AZD8205 provided an overall response rate of 69% (tumor regression of 30% or greater from baseline) and complete responses observed in 9/26 (36%) of models. To understand the biology underlying antitumor response, we conducted a multiparametric analysis including genomics, proteomics and computational pathology and found that deeper antitumor activity was observed in models with elevated B7-H4 expression as well as in models with defects in DNA damage repair (DDR). To further exploit the DNA damage elicited by the TOP1i warhead, we examined combinations of AZD8205 with small molecules, including a novel PARP1 selective inhibitor, in a BRCA wild type MDA-MB-468 model. These data suggest that AZD8205 is a promising therapeutic candidate for the treatment of B7-H4 positive solid tumors. A first in human phase 1 study in patients with advanced solid tumors is currently ongoing (NCT05123482). Citation Format: Krista Kinneer, Niall J. Dickinson, Luke Masterson, Thais Cailleau, Ian Hutchinson, Balakumar Vijayakrishnan, Nazzareno Dimasi, R. James Christie, Mary McFarlane, Kathryn Ball, Arthur Lewis, Sofia Koch, Lee Brown, Yue Huang, Anton I. Rosenbaum, Jiaqi Yuan, Si Mou, Noel R. Monks, Jon Chesebrough, Ravinder Tammali, Judith Anderton, Darrin Sabol, Frances Anne Tosto, Philipp Wortmann, Zachary A. Cooper, Pauline Ryan, John Hood, Carlos Fernandez Teruel, Carlos Serra Traynor, Andy Pike, Michael Davies, Elisabetta Leo, Kimberly Cook, Nadia Luheshi, Philip W. Howard, Puja Sapra. Discovery and first disclosure of AZD8205, a B7-H4-targeted antibody-drug conjugate utilizing a novel topoisomerase I linker-warhead [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1765.

Abstract 2601: Concomitant treatment of ovarian cell lines with Tumor Treating Fields (TTFields) and PARP inhibitors

Abstract Background: PARP inhibitors (PARPi) are an effective treatment option for ovarian cancer patients harboring deleterious BRCA mutations. While such mutations prevail in one third of ovarian cancer patients, most of the other patients gain little survival benefit by the use of PARPi. Tumor Treating Fields (TTFields) are alternating electric fields with antimitotic effects on cancerous cells, also shown to induce a state of BRCAness in several types of cancer cell lines. The current study aimed to explore the potential of combining TTFields with PARP inhibition for the treatment of ovarian cancer in vitro. Methods: Ovarian carcinoma cells A2780 (BRCA1/2 wild type) and OVCAR3 (BRCA1 wild type, BRCA2 deep deletion) were treated with TTFields using the inovitro system. Treatment was applied for 72 h at an intensity of 1.2 V/cm RMS and frequency of 200 kHz. The efficacy of concomitant application of TTFields with the PARP inhibitors niraparib and olaparib was examined via measurements of cell survival, colony forming ability, overall effect (multiplicity product of cytotoxicity and clonogenicity), and apoptosis induction. Results: A2780 or OVCAR3 cells treated with TTFields displayed about 50% reduction in cell counts. Co-application of TTFields with PARPi enhanced the cytotoxic effect compared to that demonstrated by TTFields or PARPi alone. Concomitant application also resulted in reduced clonogenicity, enhanced overall effect, and increased apoptosis. Conclusions: The results suggest potential benefits for TTFields concomitant with PARP inhibiting agents for treatment of ovarian cancer. Citation Format: Antonia Martinez-Conde, Eyal Dor-On, Adi Haber, Moshe Giladi, Uri Weinberg, Yoram Palti. Concomitant treatment of ovarian cell lines with Tumor Treating Fields (TTFields) and PARP inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2601.

Abstract 2608: Pre-clinical evaluation of small molecule CHK2 inhibitor, ART-122 combined with olaparib in BRCA wild type tumors

Abstract Background: PARP inhibitors are widely used for BRCA-mutant cancers, but often tumors become resistant to PARP inhibitors. The resistance develops through various mechanisms, including BRCA1/2 reversion mutation. Several publications have suggested that CHK2 inhibition could help overcome the resistance to PARP inhibitors. We discovered a novel small molecule CHK2 inhibitor, ART-122, and conducted a series of experiments to explore the rationale for combining a PARP inhibitor and ART-122 to treat the BRCA wild-type ovarian and breast tumors. Results: In the LanthaScreen࣪ Kinase assay, ART-122 was shown to have the IC50 values of 0.57 nM and 10.85 nM, for CHK2 and CHK1, respectively. We observed the strong synergy between ART-122 and olaparib in the mouse xenograft model using the MDA-MB-231 cancer cells (BRCA1/2 wild type, triple-negative breast cancer cells). Animals were treated with 50 mg/kg olaparib on days 1-5, 30 mg/kg ART-122 on days 1-2 each week for 3 weeks, or both therapies. The result demonstrated approximately 41% reduction in tumor volume in mice treated with the combination therapy compared to the vehicle group. Animals treated with either of the single-agent therapies showed tumor growth similar to the vehicle group. We did not observe apparent toxicity or significant body weight differences among the animals. Additionally, the synergism between ART-122 and olaparib was confirmed in other cancer cell lines, including several BRCA wild-type and BRCA mutant cells lines such as CaOV3, SK-OV-3, UWB1.289, UWB1.289 with BRCA overexpression, HCC1937, MDA-MB-436, and MCF7. Conclusion: The combination of ART-122 and Olaparib demonstrated a synergistic effect in BRCA wild-type ovarian and breast tumor cell lines. In the mouse xenograft model, the CHK2 inhibitor ART-122 sensitized BRCA1/2 wild-type tumor cells MDA-MB-231 to olaparib treatment. The data provide a strong rationale for the further development of ART-122 as a therapeutic agent. Citation Format: Taegon Baik, Chaeun Park, Young Woo Kang, Danny Joh, Ha-Young Lee, Sojung Park, Supyong Hwang, In Ki Kim, Jong Oh Kim. Pre-clinical evaluation of small molecule CHK2 inhibitor, ART-122 combined with olaparib in BRCA wild type tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2608.