Loss Of BRCA1 Research Articles

53BP1 loss rescues embryonic lethality but not genomic instability of BRCA1 total knockout mice

BRCA1 is critical for DNA double-strand break (DSB) repair by homologous recombination (HR). BRCA1 deficient mice are embryonic lethal. Previous studies have shown that 53BP1 knockout (KO) rescues embryonic lethality of BRCA1 hypomorphic mutant mice by restoring HR. Here, we show that 53BP1 KO can partially rescue embryonic lethality of BRCA1 total KO mice, but HR is not restored in BRCA1-53BP1 double knockout (DKO) mice. As a result, BRCA1-53BP1 DKO cells are extremely sensitive to PARP inhibitors (PARPi). In addition to HR deficiency, BRCA1-53BP1 DKO cells have elevated microhomology-mediated end joining (MMEJ) activity and G2/M cell cycle checkpoint defects, causing severe genomic instability in these cells. Interestingly, BRCA1-53BP1 DKO mice rapidly develop thymic lymphoma that is 100% penetrant, which is not observed in any BRCA1 mutant mice rescued by 53BP1 KO. Taken together, our study reveals that 53BP1 KO can partially rescue embryonic lethality caused by complete BRCA1 loss without rescuing HR-related defects. This finding suggests that loss of 53BP1 can support the development of cancers with silenced BRCA1 expression without causing PARPi resistance.

Genomic characteristics associated with clinical activity of rucaparib in patients (pts) with BRCA1 or BRCA2 (BRCA)-mutated metastatic castration-resistant prostate cancer (mCRPC).

178 Background: The phase 2 TRITON2 study (NCT02952534) is evaluating the PARP inhibitor rucaparib in pts with mCRPC and a deleterious germline or somatic alteration in BRCA ( BRCA pts) or 1 of 13 other DNA damage repair (DDR) genes. Methods: Eligible pts had progressed on 1–2 lines of androgen receptor-directed therapy and 1 taxane-based chemotherapy. Deleterious DDR gene alterations were identified from central testing of plasma and/or tissue samples by Foundation Medicine or from local testing. Results: As of Jul 2, 2019 (visit cutoff), 98 BRCA pts had received rucaparib; median (range) duration of follow-up was 13.0 (4.1–25.8) mo. Confirmed objective response rate (ORR) in BRCA pts (with investigator-assessed measurable disease) and PSA response rates (in pts with and without measurable disease) are shown in the Table. A biallelic alteration was observed in 81.4% (35/43) of BRCA pts for whom zygosity could be determined, and pts with homozygous BRCA loss (20.4%; 20/98) demonstrated high ORR (66.7% [95% CI, 38.4-88.2]; 10/15) and PSA response rates (75.0% [95% CI, 50.9-91.3]; 15/20). The safety profile in BRCA pts was consistent with the overall TRITON2 pt population. Conclusions: Results from TRITON2 showed antitumor activity with rucaparib in pts with mCRPC and a deleterious BRCA alteration. Responses were observed in patients with germline or somatic alterations in BRCA1 or BRCA2. The activity of rucaparib in these and additional genomic subgroups (e.g., based on zygosity, plasma genomic testing, or co-occurring alterations) will be reported. Clinical trial information: NCT02952534. [Table: see text]

Abstract P1-10-15: BRCA1 alterations with additional defects in DNA damage repair genes may confer chemoresistance to BRCA-like breast cancers treated with neoadjuvant chemotherapy

Abstract The BRCA-like phenotype is a feature that some sporadic breast cancers share with those occurring in germinal BRCA1- or BRCA2-mutation carriers. Since tumors with the phenotype have defects in the DNA damage repair pathway, which may increase sensitivity to drugs such as DNA cross-linking agents and PARP inhibitors, a method to identify this phenotype is important. The prediction of chemoresistance, which frequently develops in these tumors, is also crucial for improving therapy. We examined genomic aberrations and BRCA1 promoter methylation in tumors of 73 breast cancer (20 HR-/HER2- and 53 HR+/HER2-) patients, who underwent diagnostic core-needle biopsy and received neoadjuvant chemotherapy with anthracycline, cyclophosphamide, and taxane, using SNP array CGH and quantitative PCR. They had a tumor size of T1, T2, T3, or T4, a lymph node status of N0, N1, N2, or N3, and no distant metastasis (M0) at diagnosis, and were enrolled as subjects in this study. The methylation and/or loss or UPD of BRCA1 (termed BRCA1 alterations) and the loss or UPD of BRCA2 (termed BRCA2 alterations) were detected in 27 (37%) and 21 (29%), respectively, out of 73 tumors. Tumors with BRCA1 or BRCA2 alterations were associated with a higher number of genomic aberrations (P<0.001 and P<0.001) and higher percentage of TP53 alterations (the loss or UPD and/or mutations) than those without (P<0.001 and P<0.001). Overall survival (OS) rates were similar between patients with or without BRCA1 or BRCA2 alterations. However, when 27 patients with BRCA1-altered tumors were classified into those with or without the loss or UPD of PALB2, PAGR1, RAD51B, FANCM, MLL4 or ERCC1/2 in tumors, patients with additional defects in DNA damage repair genes had worse OS (P=0.037, 0.045, 0.038, 0.044, 0.041, or 0.019) than those without. These defects may confer chemoresistance and predict poor outcomes in patients with BRCA1-altered breast cancer. Citation Format: Mamoru Takada, Yasuhiko Kaneko, Masayuki Haruta, Shigenori Nagai, Kenichi Inoue, Katsunori Toduka, Masafumi Kurozumi, Hiroyuki Takei, Seishi Ogawa, Takeshi Nagashima, Takafumi Sangai, Hiroshi Fujimoto, Junta Sakakibara, Ryotaro Teranaka. BRCA1 alterations with additional defects in DNA damage repair genes may confer chemoresistance to BRCA-like breast cancers treated with neoadjuvant chemotherapy [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-10-15.

Abstract P4-05-07: Whole-exome and RNA-sequencing analyses of acinic cell carcinomas of the breast

Abstract Introduction: Acinic cell carcinoma of the breast (ACC) is an exceedingly rare histologic form of triple-negative breast cancer (TNBC) with a generally good clinical behavior. Despite its unique histology, targeted sequencing analysis of ACC has failed to identify recurrent genetic alterations other than those found in common forms of TNBC. Here, we subjected three breast ACCs to whole-exome and RNA-sequencing, seeking to define whether they would be underpinned by a pathognomonic genetic alteration that would distinguish them from common forms of TNBC. Materials and methods: Three breast ACCs were subjected to whole-exome sequencing (WES) and RNA-sequencing. Somatic mutations, copy number alterations, mutational signatures and fusion genes were determined using a validated bioinformatics pipeline. We also estimated DNA-based measures of genomic instability including mutational signatures (using SigMA), large-scale state transition (LST) and telomeric allelic imbalance (NtAI). Results: No oncogenic in-frame fusion transcript was identified in the breast ACCs analyzed. ACCs displayed a median of 173 (range, 92-230) non-synonymous somatic mutations as defined by WES. We detected clonal TP53 hotspot mutations associated with loss of heterozygosity (LOH) of the wild-type allele in two ACCs (ACC1 and ACC18). In contrast, the TP53 wild-type ACC (ACC12) harbored a pathogenic MLH1 germline mutation (p.Glu227_Ser295del) and a clonal hotspot mutation in CTNNB1 (p.Lys335Ile). No cancer gene other than TP53 was found to be mutated in >1 case. Copy number analysis revealed that ACC18 harbored a BRCA1 homozygous deletion and focal amplification of 20p12.3 encompassing PCNA. Moreover, the TP53-mutant cases, ACC1 and ACC18, displayed complex copy number profiles, with multiple gains and losses and focal high-level amplifications. Consistent with the BRCA1 homozygous deletion, ACC18 displayed genomic features of homologous recombination DNA repair deficiency (HRD), such as a dominant mutational signature 3, a high LST score (24), a high NtAI score (23) and a high number of ‘small deletions’ >5bps. In contrast, and consistent with its DNA mismatch repair (MMR) deficiency, ACC12 had a simple genome without detectable copy number alterations, displayed a dominant mutational signature 6 associated with defective MMR, microsatellite instability (MSI) as determined by MSIsensor and loss of MLH1 expression by immunohistochemical analysis. Conclusions: Our findings lend further support to the notion that ACCs of the breast are genetically heterogeneous and display genomic features overlapping with those of common forms of TNBCs. Although no pathognomonic genetic alterations were detected in these cases, our data suggest that at least some ACCs of the breast may arise in the setting of HRD or MSI through distinct molecular mechanisms. Our data also suggest the possibility that both BRCA1 and TP53 loss of function may not be sufficient for a TNBC to display high-grade features and that inactivation of these two genes may not necessarily result in the development of high-grade TNBC. Citation Format: Francisco Beca, Simon SK Lee, Fresia Pareja, Arnaud Da Cruz Paula, Pier Selenica, Lorenzo Ferrando, Hannah Y Wen, Hong Zhang, Elena Guerini-Rocco, Emad A Rakha, Britta Weigelt, Jorge S Reis-Filho. Whole-exome and RNA-sequencing analyses of acinic cell carcinomas of the breast [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-05-07.

BRCA2 and RB1 loss responds to PARP inhibitors.

BRCA2 and RB1 loss responds to PARP inhibitors.

BRCA1 protects cardiac microvascular endothelial cells against irradiation by regulating p21-mediated cell cycle arrest

AimsMicrovascular endothelial cell dysfunction is a leading cause of radiation-induced heart disease (RIHD). BRCA1 plays an important role in DNA damage repair. The study aims to explore the effect of BRCA1 in endothelial cells involved in RIHD. Materials and methodsBRCA1 and p21 expression were detected in human umbilical vein endothelial cells (HUVECs) and in mouse heart tissue after irradiation exposure. The effects of BRCA1 on cell proliferation, cell cycle and radiosensitivity were determined in HUVECs with overexpression and knockdown of BRCA1. A mouse model of RIHD was established. Heart damage was detected in C57BL/6J mice and endothelial cell specific knockout BRCA1 mice (EC-BRCA1−/−). Key findingsBRCA1 and p21 expression was significantly increased both in vitro and vivo response to irradiation. BRCA1 overexpression in endothelial cells enhanced cell growth and G1/S phase arrest, and the opposite results were observed in BRCA1 knockdown endothelial cells. BRCA1 downregulated endothelial cell cycle-related genes cyclin A, cyclin D1, cyclin E and p-Rb through increasing p21 expression, and HUVECs with BRCA1 gene knockdown were more sensitive to radiation. In vivo, a decrease in cardiac microvascular density, as well as cardiomyocyte hypoxia and apoptosis were observed in a time-dependent manner. EC-BRCA1−/− mice were more prone to severe RIHD than EC-BRCA1+/− mice after 16Gy radiation exposure due to endothelial dysfunction caused by loss of BRCA1, and p21 was declined in EC-BRCA1−/− mice heart. SignificanceThese findings indicate that BRCA1 plays a protective role in RIHD by regulating endothelial cell cycle arrest mediated by p21 signal.

A gene-expression-based test can outperform bap1 and p16 analyses in the differential diagnosis of pleural mesothelial proliferations.

The demonstration of tissue invasion by histology is an essential criterion for the differential diagnosis of benign and malignant mesothelial proliferations. When tissue invasion cannot be identified, the use of ancillary tests is sometimes necessary. Among investigated markers, the loss of BRCA1 associated protein 1 (BAP1) protein expression and the homozygous deletion of p16 have shown 100% specificity in separating benign and malignant mesothelial lesions. However, beyond the excellent specificity of these two markers, their low sensitivity limits their clinical utility. In this context, a previous study developed and tested a novel tool for use in the differential diagnosis of malignant pleural mesothelioma (MPM) using the NanoString System and a classification algorithm. In the current study, the performance of gene classifiers were compared using BAP1 and p16 testing. p16 FISH and BAP1 immunohistochemistry were performed on the same series of 34 epithelioid MPM and 20 benign pleural lesions, which were previously analyzed by the system. The diagnostic performance of p16, BAP1 and our classification models were compared using ROC analysis. It was observed that BAP1 loss and p16 deletion were highly specific for MPM, since they were not detected in benign lesions. However, their AUC values were not completely satisfying (BAP1: 0.8235; p16: 0.7647) particularly due to their low sensitivities. As expected, combining BAP1 and p16 tests increased the diagnostic sensitivity, thus improving the AUC (0.8824). In the same series of cases, our MPM tool outperformed BAP1 and p16 tests using the 22 and 40-gene classification models (AUC 22-gene model: 0.9996; AUC 40-gene model: 0.9990). In conclusion, the present gene-expression-based classification exhibited great potential and further validation is required to support these findings in a prospective fashion, in order to provide a solid alternative for pleural proliferation diagnosis.

BRCA1 Deficiency Upregulates NNMT, Which Reprograms Metabolism and Sensitizes Ovarian Cancer Cells to Mitochondrial Metabolic Targeting Agents.

BRCA1 plays a key role in homologous recombination (HR) DNA repair. Accordingly, changes that downregulate BRCA1, including BRCA1 mutations and reduced BRCA1 transcription, due to promoter hypermethylation or loss of the BRCA1 transcriptional regulator CDK12, disrupt HR in multiple cancers. In addition, BRCA1 has also been implicated in the regulation of metabolism. Here, we show that reducing BRCA1 expression, either by CDK12 or BRCA1 depletion, led to metabolic reprogramming of ovarian cancer cells, causing decreased mitochondrial respiration and reduced ATP levels. BRCA1 depletion drove this reprogramming by upregulating nicotinamide N-methyltransferase (NNMT). Notably, the metabolic alterations caused by BRCA1 depletion and NNMT upregulation sensitized ovarian cancer cells to agents that inhibit mitochondrial metabolism (VLX600 and tigecycline) and to agents that inhibit glucose import (WZB117). These observations suggest that inhibition of energy metabolism may be a potential strategy to selectively target BRCA1-deficient high-grade serous ovarian cancer, which is characterized by frequent BRCA1 loss and NNMT overexpression. SIGNIFICANCE: Loss of BRCA1 reprograms metabolism, creating a therapeutically targetable vulnerability in ovarian cancer.

Abstract KN01: Dissecting antitumor immune response in ovarian cancer

Abstract In epithelial ovarian cancer (EOC), intratumoral or intraepithelial tumor infiltrating lymphocytes (TILs), i.e. T cells specifically infiltrating tumor islets, occur in approximately half of the patients and correlate with longer survival. As with many other solid tumors, despite promising results in mouse models, response of EOC to PD-1 blockade has been quite limited in the clinic, but the mechanisms underlying therapeutic failures in immunoreactive human tumors harboring TILs are not well understood. Our work has sought to answer the following key questions: a) whether ovarian TILs are tumor-specific; b) if so, what are the relevant antigens recognized by TILs; c) whether the PD-1/PD-L1 pathway is relevant; d) if so, what are the mechanisms underlying response to PD-1 blockade; and finally e) what mechanisms govern TIL recruitment and engraftment. The data presented will show that there is indeed a spontaneous and tumor-specific T-cell response in a proportion of EOC, accurately heralded by intraepithelial CD8+ TILs. Tumor-specific antigens will be discussed. Our work also shows that the PD-1/PD-L1 is a central immune checkpoint pathway in these tumors. However, only a fraction of such tumors with ieTILs respond to PD-1. We dissected the mechanisms underlying response to PD-1 blockade and therapeutic approaches to enhance response to PD-1 in human and mouse preclinical models. Therapeutic approaches to achieve effective immunotherapy in ovarian cancer will be discussed, including combination checkpoint blockade, adoptive T cell therapy and new rational combinations. Finally, successful T-cell engraftment in ovarian cancer requires tumor cell-derived CCL5 and is amplified by myeloid cell-secreted IFNg-inducible CXCL9, dependent on activation of tumor-infiltrating lymphocytes (TILs). Spontaneous, epigenetically-mediated loss or stable knockdown of ccl5 leads to CD8+ T-cell loss and immune desertification in the mouse, while forced ccl5 expression prevents loss of cxcl9 and TILs, and leads to attenuated tumor growth. Accordingly, CCL5-CXCL9 co-expression reveals immunoreactive tumors with prolonged survival and response to PD-1 blockade. The impact of homologous recombination and BRCA loss on the establishment of immunoreactive tumors will be also discussed. Citation Format: George Coukos. Dissecting antitumor immune response in ovarian cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr KN01. doi:10.1158/1535-7163.TARG-19-KN01

Synthetic Lethal Interaction between Apto-253 and IDH1/2 Mutations

APTO-253 is a small molecule with a novel mechanism of action and potent antiproliferative activity against cell lines derived from a wide range of human malignancies. A Phase 1 study of APTO-253 was completed in patients with advanced solid tumors. It was well tolerated and produced evidence of antitumor activity in patients but did not cause myelosuppression even at the maximum tested dose. A phase 1a/b trial of APTO-253 in patients with relapsed/refractory acute leukemias (including AML) and high-risk MDS (NCT02267863) is currently underway. We previously reported that APTO-253 was converted intracellularly to a complex containing one molecule of iron and three molecules of APTO-253 [Fe(253)3]. Both APTO-253 and Fe(253)3 stabilized DNA quadruplex (G4) structures in the Myc promotor region to reduce the expression of Myc (Figure 1). Stabilization of G4 structures has been reported to stall replication forks and produce DNA strand breaks. Treatment of cancer cells with APTO-253 produced time and concentration-dependent γH2AX foci formation and DNA double strand breaks that have to be repaired by homologous recombination. Loss of either BRCA1 or BRCA2 function in multiple isogenic paired cell lines resulted in hypersensitivity to APTO-253 whose magnitude was similar to the effects of PARP inhibitors, olaparib. Thus, APTO-253 is a member of the limited repertoire of drugs which can exploit defects in homologous recombination and is of particular interest because it does not produce myelosuppression. The goal of this project was to identify synthetic lethal interactions in addition to BRCA1/2 deficiency that can guide combination drug studies. The normal function of the isocitrate dehydrogenase (IDH) enzymes is to catalyze the conversion of isocitrate to α-ketoglutarate (αKG) in the citric acid cycle. IDH1 mutations occur in more than 70% of low-grade gliomas, 20% of high-grade glioblastomas and with a frequency of about 10% in AML, cholangiocarcinomas, melanomas and chondrosarcomas. Additionally, mutations are also identified in IDH2 in about 4% of gliomas and 10% of AMLs. IDH1/2 alterations are heterozygous missense mutations that confer a neomorphic activity on the encoded enzyme such that they covert αKG to (R)-2-hydroxyglutarate [(R)-2HG]. (R)-2HG is an oncometabolite with pleiotropic effects on cell biology including chromatin methylation and cellular differentiation. It was reported that IDH1/2 mutations induce a homologous recombination defect that renders tumor cells exquisitely sensitive to PARP inhibitors. We used 2 pairs of isogenic IDH1 wild type and mutant cell lines to test the hypothesis that cells carrying IDH1/2 mutations are hypersensitive to APTO-253. Induction of the expression of IDH1 R132H in THP-1 AML cells increased the 2-HG concentration ~150-fold whereas induction of wild-type IDH1 expression increased it only 2-fold (doi: 10.1038/nm.3788). THP-1 cells expressing the mutant IDH1 R132H were hypersensitive the treatment of APTO-253 compared to cells expressing the wild-type IDH1 (Figure 2). Comparison of wild-type and isogenic IDH1 R132H HCT116 cells disclosed a ~100-fold higher level of (R)-2-HG in the mutant cells (doi: 10.1126/scitranslmed.aal2463), and an increase in sensitivity to the cytotoxic effect of APTO-253 (Figure 3). These results are consistent with the observation that both APTO-253 and increased levels of 2-HG cause DNA damage that depends on homologous recombination for repair and that this favors a synthetic lethal interaction. We surmise that the ability of APTO-253 to markedly reduce the expression of Myc further contributes to the favorable interaction. This data further supports the conclusion that defects in homologous DNA repair can enhance the potency of APTO-253 and suggest the use of this non-myelosuppressive drug in both IDH1/2 mutant AML and solid tumors. Disclosures Zhang: Aptose Biosciences, Inc: Employment. Rice:Aptose Biosciences, Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Howell:Aptose Biosciences, Inc: Consultancy, Research Funding.

The Indenoisoquinoline TOP1 Inhibitors Selectively Target Homologous Recombination-Deficient and Schlafen 11-Positive Cancer Cells and Synergize with Olaparib.

Irinotecan and topotecan are used to treat a variety of different cancers. However, they have limitations, including chemical instability and severe side effects. To overcome these limitations, we developed the clinical indenoisoquinolines: LMP400 (indotecan), LMP776 (indimitecan), and LMP744. The purpose of the study is to build the molecular rationale for phase II clinical trials. CellMinerCDB (http://discover.nci.nih.gov/cellminercdb) was used to mine the cancer cell lines genomic databases. The causality of Schlafen11 (SLFN11) was validated in isogenic cell lines. Because topoisomerase I (TOP1)-mediated replication DNA damage is repaired by homologous recombination (HR), we tested the "synthetic lethality" of HR-deficient (HRD) cells. Survival and cell-cycle alterations were performed after drug treatments in isogenic DT40, DLD1, and OVCAR cell lines with BRCA1, BRCA2, or PALB2 deficiencies and in organoids cultured from prostate cancer patient-derived xenografts with BRCA2 loss. We also used an ovarian orthotopic allograft model with BRCA1 loss to validate the efficacy of LMP400 and olaparib combination. CellMinerCDB reveals that SLFN11, which kills cells undergoing replicative stress, is a dominant drug determinant to the clinical indenoisoquinolines. In addition, BRCA1-, BRCA2-, and PALB2-deficient cells were hypersensitive to the indenoisoquinolines. All 3 clinical indenoisoquinolines were also synergistic with olaparib, especially in the HRD cells. The synergy between LMP400 and olaparib was confirmed in the orthotopic allograft model harboring BRCA1 loss. Our results provide a rationale for molecularly designed clinical trials with the indenoisoquinolines as single agents and in combination with PARP inhibitors in HRD cancers expressing SLFN11.

MA23.03 BAP1 Loss Induces Genome Instability Through BRCA1-Dependent and Independent Mechanisms in Mesothelioma

BRCA1 associated protein 1 (BAP1) is a tumor suppressor that is the most frequently mutated in the majority of mesotheliomas. We have previously reported that loss of BRCA1 expression in mesothelioma is a common event, and mediates resistance to spindle checkpoint activator vinorelbine, a drug with relevance to treatment of mesothelioma. However, the loss of BRCA1 is unknown in mesothelioma. The aim of this study is to determine the functional relationship between BAP1 and BRCA1 and examine their role in vinorelbine resistance in mesothelioma cells. We conducted functional genetic analysis of BAP1 and BRCA1 in two MPM cell lines, MSTO and H2452, the latter carrying an inactivating A95D mutation in the UCH domain of BAP1. BAP1 knockdown was achieved by siRNA transfection, while BRCA1 knockdown was achieved by doxycycline induction of an integrated shRNA. Patient samples were processed for BAP1 and BRCA1 immunohistochemistry from MEDUSA cohort. Loss of BAP1 expression led to reduced expression of BRCA1, whereas knockdown of BRCA1 did not affect BAP1 expression. Treatment with the proteasome inhibitor, MG132, restored BRCA1 expression in the absence of BAP1 indicating that BAP1 contributes to post-translational stabilization of BRCA1 protein and the stabilization of BRCA1 by BAP1 is independent of its de-ubiquitination activity. Knockdown of BAP1 induced SAC deficiency and vinorelbine resistance concurrent with reduced expression of BRCA1 and the SAC component, MAD2L1. We also identified a positive and significant correlation between BAP1 and BRCA1 expressionin patient samples. Our data demonstrates that BAP1 regulates BRCA1 expression through regulating its protein stability. Our findings suggest that BAP1 inactivation dysregulates the spindle assembly checkpoint via BRCA1 dependent and independent mechanisms, conferring resistance to vinorelbine. As such BAP1 may have potential as a predictive biomarker for spindle poisons, to underpin chemotherapy stratification. A hypothesis that would be tested in a multicentre randomised phase II VIM trial.

1876PD - Immunogenicity of BRCA1-deficient ovarian cancers is driven through DNA sensing and is augmented by PARP inhibition

BackgroundBRCA1 is an essential gene of the homologous recombination repair pathway. Ovarian cancers with BRCA1 mutations represent about 20% of HGSOC and are characterized by loss-of-function TP53 mutations, copy number alterations, chromosomal instability, high neoantigen loads and increased infiltration of intraepithelial CD8 tumor-infiltrating lymphocytes. MethodsTo address this hypothesis, we investigated the effects of BRCA1 loss on tumor immunogenicity in human and mouse ovarian cancer BRCA1 isogenic cell lines. We further studied tumor immunogenicity and immune recognition in situ in human ovarian carcinomas and in vivo using syngeneic transplantable Tp53-/-BrcaWT and Tp53-/-Brca1-/- mouse models. ResultsWe propose that DNA damage induced by BRCA1 loss could be a tumor-autonomous inflammatory mechanism. Our hypothesis was corroborated by studies in human and mouse isogenic ovarian cancer cell lines which revealed that BRCA1 deficiency leads to increased cytoplasmic translocation of nuclear DNA, increased DNA sensing, induction of proinflammatory cytokines and T cell recruiting chemokines and increased tumor CD8 T cell infiltration. PARP inhibition exacerbated type I IFN responses in BRCA1-deficient ovarian cancer cell lines and simultaneously increased surface expression of PDL1. Increased DNA damage, as measured by γH2AX tumor staining, was also detected in situ in human ovarian cancers with BRCA1 mutations. Importantly, we detected tumor expression of pSTAT1 confirming a type I IFN activation in tumors with DNA damage. Both DNA damage and pSTAT1 activation correlated with higher TIL infiltration and better overall survival. Our results translated in mouse models of ovarian cancer where Trp53-/-Brca1-/- but not Trp53-/-Brca1WT tumors presented with biomarkers of DNA damage, type I IFN pathway activation and responded to a therapeutic combination of PARP inhibitor Olaparib with dual checkpoint blockade antibodies. ConclusionsOur results provide a mechanistic link between loss of BRCA1 and induction of tumor-driven inflammatory and immunogenic responses in ovarian cancer that was mediated by tumor-cell autonomous type I IFN signaling, which translated to increased immune surveillance by CD8 T cells. Legal entity responsible for the studyGeorge Coukos. FundingThis study was supported by the Ludwig Institute for Cancer Research and grants P50 CA083638 SPORE in Ovarian Cancer, the Emma Mouschamp Foundation, the Porphyrogenis Fundation and by a Stand Up To Cancer-Ovarian Cancer Research Fund Alliance-National Ovarian Cancer Coalition Dream Team Translational Cancer Research Grant (SU2C-AACRDT16- 15, to EMS). Stand Up To Cancer is a division of the Entertainment 2 Industry Foundation. Research grants are administered by the American Association for Cancer Research, the Scientific Partner of SU2C. DisclosureAll authors have declared no conflicts of interest.

MA23.07 Loss of Expression of BAP1 and/or MTAP Aids in the Diagnosis of Malignant Mesothelioma Metastatic to Lymph Nodes

Stage and histology are the strongest prognostic parameters in malignant pleural mesothelioma and aid management of patients. However, the distinction between reactive intranodal mesothelial cells and metastatic malignant mesothelioma (MM) can be challenging. Loss of BRCA1 associated protein-1 (BAP1) and/or methylthioadenosine phosphorylase (MTAP) expression has been identified in a subset of MM but not in reactive mesothelial proliferation. We investigated the value of these markers in the distinction between reactive mesothelial cells and metastatic MM in lymph nodes. Surgical files of Mayo Clinic Rochester (1996-2018) were searched for metastatic MM in lymph nodes. All cases and if available corresponding primary MM were reviewed by a thoracic pathologist (ACR) to confirm the diagnosis. Primary MM and lymph nodes were stained with BAP1 (clone C-4) and MTAP (2G4). Absence of nuclear staining of BAP1 and absence of nuclear and cytoplasmic staining of MTAP in essentially all tumor cells was considered as loss of expression. Forty-four patients (25 males, 56.8%) had a median age of 64 years (range, 24-75) at time of surgery. Tissue was available from nodal metastases in all cases, either paired with the primary MM at time of nodal sampling (N=37) or at a different time (N=4) (time between tissue collections, range, 1day- 4 years, respectively), or without paired primary MM (N=3). Thirty-seven pleural, 6 peritoneal and 1 pericardial MM were of epithelioid (N=39) or biphasic (N=5) subtype. Patients underwent extrapleural pneumonectomy (N=17), pleurectomy (N=7), resection (N=9), debulking (N=2), biopsy (N=8), or autopsy (N=1). In nodal metastases, BAP1 and/or MTAP expression was lost in 29 (of 43, 67.4%) cases; specifically, BAP1 expression was lost in 28 (of 44, 63.6%), MTAP was lost in 14 (of 43, 32.6%), and both were lost in 12 (of 43, 27.9%) cases. Agreement in expression/loss of expression of BAP1 and/or MTAP in primary and metastatic MM occurred in all cases. During a median follow up of patients who underwent extrapleural pneumonectomy or pleurectomy (available in N=23) of 14.8 months (range, 1-119) 17 patients died within a median time of 16 months. BAP1 and MTAP immunostains are helpful in the distinction between metastatic MM and reactive mesothelial cells in lymph nodes when one or both markers lost expression in the mesothelial cells. Expression of both markers does not exclude the possibility of metastatic MM.

1879PD - Primary ovarian carcinomas arising in BRCA1 mutation carriers contain a small fraction of BRCA1-proficient cells, which rapidly repopulate tumor mass during neoadjuvant chemotherapy but become outgrown by BRCA1-deficient clones during platinum-free intervals

BackgroundSomatic loss of the wild-type allele is a key event in the pathogenesis of BRCA1-driven carcinomas. BRCA1 deficiency renders pronounced sensitivity of these tumors to platinum compounds and PARP inhibitors. MethodsWe analyzed BRCA1 loss of heterozygosity (LOH) in serial ovarian cancer (OC) samples, which were obtained from BRCA1 germ-line mutation carriers before neoadjuvant chemotherapy (NACT), at surgery and at disease relapse. Results26/36 (72%) OC had BRCA1 LOH before NACT. 15 (58%) of these 26 tumors showed retention of the normal BRCA1 allele after NACT. The analysis of linked SNPs and FISH assay strongly indicated, that the restoration of BRCA1 function is caused not by the second mutation, but by the selection of pre-existing BRCA1-proficient cells. Tumor relapses were available in 7 patients with BRCA1 LOH in the chemonaive tumor and/or BRCA-proficiency in the residual post-NACT neoplasm; 6 (86%) of these relapses had BRCA1 LOH thus resembling the primary tumor. Secondary open reading frame (ORF) restoring BRCA1 mutation was detected in 1 recurrent OC. Serial samples retained same TP53 mutation during the treatment course. Whole exome sequencing revealed that chemonaive, post-NACT and relapsed tumors had both shared and individual mutations. Conclusions1) BRCA1 LOH is not the first event in the pathogenesis of BRCA1-driven cancer: gain of TP53 mutation probably precedes BRCA1 inactivation in order to prevent apoptosis; 2) Chemonaïve BRCA1-driven tumors contain a small fraction of BRCA1-proficient cells, which rapidly repopulate the tumor lump during the first weeks of therapy; 3) Change of BRCA1 status during NACT may call to reconsider the existing approaches to adjuvant therapy, as the residual post-NACT tumor masses are likely to be platinum-resistant; 4) The balance between BRCA1-deficient and BRCA1-proficient cells is a subject of fluctuations, depending whether therapy is applied or not; 5) Clinical trials on BRCA1-driven OCs need to address the issue of intratumoral heterogeneity of BRCA1 status. Legal entity responsible for the studyThe authors. FundingRussian Science Foundation (grant 19-15-00168). DisclosureAll authors have declared no conflicts of interest.

A Functional Analysis of the Unclassified Pro2767Ser BRCA2 Variant Reveals Its Potential Pathogenicity that Acts by Hampering DNA Binding and Homology-Mediated DNA Repair.

BRCA1 and BRCA2 are the genes most frequently associated with hereditary breast and ovarian cancer (HBOC). They are crucial for the maintenance of genome stability, particularly in the homologous recombination-mediated repair pathway of DNA double-strand breaks (HR-DSBR). Widespread BRCA1/2 next-generation sequencing (NGS) screening has revealed numerous variants of uncertain significance. Assessing the clinical significance of these variants is challenging, particularly regarding the clinical management of patients. Here, we report the functional characterization of the unclassified BRCA2 c.8299C > T variant, identified in a young breast cancer patient during BRCA1/2 NGS screening. This variant causes the change of Proline 2767 to Serine in the DNA binding domain (DBD) of the BRCA2 protein, necessary for the loading of RAD51 on ssDNA during the HR-DSBR. Our in silico analysis and 3D-structure modeling predicted that the p.Pro2767Ser substitution is likely to alter the BRCA2 DBD structure and function. Therefore, to evaluate the functional impact of the p.Pro2767Ser variant, we used a minigene encoding a truncated protein that contains the BRCA2 DBD and the nearby nuclear localization sequence. We found that the ectopically expressed truncated protein carrying the normal DBD, which retains the DNA binding function and lacks the central RAD51 binding domain, interferes with endogenous wild-type BRCA2 mediator functions in the HR-DSBR. We also demonstrated that the BRCA2 Pro2767Ser DBD is unable to compete with endogenous BRCA2 DNA binding, thereby suggesting that the p.Pro2767Ser substitution in the full-length protein causes the functional loss of BRCA2. Consequently, our data suggest that the p.Pro2767Ser variant should be considered pathogenic, thus supporting a revision of the ClinVar interpretation. Moreover, our experimental strategy could be a valid method with which to preliminarily evaluate the pathogenicity of the unclassified BRCA2 germline variants in the DBD and their risk of predisposing to HBOC.

Defining and Modulating 'BRCAness'.

The concept of 'BRCAness' defines the pathogenesis and vulnerability of multiple cancers. The canonical definition of BRCAness is a defect in homologous recombination repair, mimicking BRCA1 or BRCA2 loss. In turn, BRCA-deficient cells utilize error-prone DNA-repair pathways, causing increased genomic instability, which may be responsible for their sensitivity to DNA damaging agents and poly-(ADP)-ribose polymerase inhibitors (PARPis). However, recent work has expanded the mechanistic basis of BRCAness, to include defects in replication fork protection (RFP). Here, we broaden the definition of BRCAness to include RFP and regulatory mechanisms that cause synthetic lethality with PARPis. We highlight these recent discoveries, which include mechanisms of RFP regulation, DNA damage checkpoint proteins, as well as kinases that regulate BRCA1/2 function. Importantly, many of these emerging mechanisms may be targeted for inhibition with small molecule inhibitors, thus inducing BRCAness in a much larger subset of BRCA-proficient tumors, with significant translational potential.

Clinical utility of FoundationOne tissue molecular profiling in men with metastatic prostate cancer

PurposeTargeted inhibitors and immunotherapy have entered the treatment landscape of metastatic prostate cancer. Genomic testing may uncover which patients benefit most from these therapies. We report the clinical utility and benefits of FoundationOne testing in men with advanced prostate cancer. Patients and methodsWe retrospectively identified all men with prostate cancer who received tissue FoundationOne testing at our institution between January 2010 and April 2017. Genomic alterations, treatment selection based on FoundationOne results, and clinical outcomes including response and duration of therapy following matched targeted therapy were analyzed. ResultsA total of 77 men with metastatic prostate cancer were referred for FoundationOne testing; 59 (77%) had sufficient tumor tissue for testing. Of these, 22% (17/77) of men had a targetable mutation and 9% (7/77) of men received matched off-label targeted therapy. Overall, 5% (4/77) of patients derived clinical benefit. One patient with a BRCA2 loss had a complete response on olaparib (>27 months) and 3 patients (ATM substitution, PALB2 frameshift, CDK12 frameshift) had stable disease with olaparib (10.3, 18.7, and 7.8 months, respectively). Three patients (BRCA2 frameshift, PDL1 + PDL2 amplification, PMS2 missense) had progressive disease despite targeted therapy. ConclusionsTissue genomic testing can uncover patients who may benefit from targeted therapies such as poly(adenosine diphosphate-ribose) polymerase inhibitors or immunotherapy. In our limited single institution study, genomic testing led to clinical benefit in 5% of patients. Combined germline and circulating tumor DNA testing may be helpful to identify additional patients suitable for matched genomic therapies.

EZH2 Is Overexpressed in BRCA1-like Breast Tumors and Predictive for Sensitivity to High-Dose Platinum-Based Chemotherapy.

BRCA1-deficient breast cancers carry a specific DNA copy-number signature ("BRCA1-like") and are hypersensitive to DNA double-strand break (DSB) inducing compounds. Here, we explored whether (i) EZH2 is overexpressed in human BRCA1-deficient breast tumors and might predict sensitivity to DSB-inducing drugs; (ii) EZH2 inhibition potentiates cisplatin efficacy in Brca1-deficient murine mammary tumors. EZH2 expression was analyzed in 497 breast cancers using IHC or RNA sequencing. We classified 370 tumors by copy-number profiles as BRCA1-like or non-BRCA1-like and examined its association with EZH2 expression. Additionally, we assessed BRCA1 loss through mutation or promoter methylation status and investigated the predictive value of EZH2 expression in a study population of breast cancer patients treated with adjuvant high-dose platinum-based chemotherapy compared with standard anthracycline-based chemotherapy. To explore whether EZH2 inhibition by GSK126 enhances sensitivity to platinum drugs in EZH2-overexpressing breast cancers we used a Brca1-deficient mouse model. The highest EZH2 expression was found in BRCA1-associated tumors harboring a BRCA1 mutation, BRCA1-promoter methylation or were classified as BRCA1 like. We observed a greater benefit from high-dose platinum-based chemotherapy in BRCA1-like and non-BRCA1-like patients with high EZH2 expression. Combined treatment with the EZH2 inhibitor GSK126 and cisplatin decreased cell proliferation and improved survival in Brca1-deficient mice in comparison with single agents. Our findings demonstrate that EZH2 is expressed at significantly higher levels in BRCA1-deficient breast cancers. EZH2 overexpression can identify patients with breast cancer who benefit significantly from intensified DSB-inducing platinum-based chemotherapy independent of BRCA1-like status. EZH2 inhibition improves the antitumor effect of platinum drugs in Brca1-deficient breast tumors in vivo.

Abstract 2228: Comparison of the Epic CTC HRD assay vs. tumor DDR mutations in metastatic castration-resistant prostate cancer (mCRPC) patients (pts)

Abstract Background: PARPi have recently demonstrated clinical activity in a subset of mCRPC pts with defects in homologous recombination (HRD). Predicting PARPi response with HRD gene mutations have struggled to show clinical specificity. Previously, we described a novel circulating tumor cell (CTC) based method (CTC-HRD) that identifies a specific cell morphology associated with genomic scarring [e.g. large scale transitions (LST)] and demonstrated CTC-HRD’s clinical utility in identifying pts likely to respond to PARPi and likely to resist to standard of care (SOC) therapies (Tx), including AR signaling inhibitors. We also observed some discordance between CTC-HRD and DNA damage response (DDR) mutational status from tissue sequencing. Here we compare the prevalence of CTCs, # LSTs/CTC and the % of CTCs with BRCA2 loss in mCRPC patient samples with matched tumor sequencing DDR status. Materials and methods: 302 CTCs from 26 mCRPC pts collected prior to Tx were collected from pts with matched tissue biopsy sequencing. CTCs were identified, assayed for CTC-HRD status, and low pass whole genome sequenced for LSTs and gene copy number alterations (CNA). We compared the prevalence of CTCs, HRD properties of each identified cell including BRCA2 copy loss, # of LSTs/CTC and their prevalence in cells derived from pts vs. CTC-HRD score and tissue DDR mutational status. Results: CTC-HRDDDR Tumor# PtsMedian CTC/mLMedian LST/CTCMedian % CTC BRCA2 Loss per Pt++913.83259.00%+-1010.42347.20%-+72.430.00% CTC-HRD positive pts, independent of tissue DDR status had higher CTC/mL, higher median # LST/CTC and higher percentages of BRCA2 loss in their CTCs vs. those pts who were CTC-HRD negative. Notably, we identified 10 pts that were negative for tissue DDR mutation but were CTC-HRD+ with a high frequency of CTC BRCA2 loss. Conclusions: These results highlight the concordance of the Epic CTC-HRD assay with known markers of HRD and demonstrate differences in CTC-HRD status from tissue DDR mutations which have struggled to achieve the required clinical specificity required for PARPi patient selection. Citation Format: Joseph Schonhoft, Adam Jendrisak, Jerry Lee, Angel Rodriguez, Ramsay Sutton, Yipeng Wang, Ryan Dittamore, Mark Landers. Comparison of the Epic CTC HRD assay vs. tumor DDR mutations in metastatic castration-resistant prostate cancer (mCRPC) patients (pts) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2228.