Homologous Recombination Repair Deficiency Research Articles

Homologous recombination repair deficient prostate cancer represents an immunologically distinct subtype

ABSTRACT Homologous recombination repair deficiency (HRD) is observed in 10% of patients with castrate-resistant prostate cancer (PCa). Preliminary data suggest that HRD-PCa might be more responsive to immune checkpoint inhibitors (ICIs). In this study, we compare the tumor immune landscape and peripheral T cell receptor (TCR) repertoire of patients with and without HRD-PCa to gain further insight into the immunogenicity of HRD-PCa. Immunohistochemistry was performed on tumor tissue of 81 patients, including 15 patients with HRD-PCa. Peripheral TCR sequencing was performed in a partially overlapping cohort of 48 patients, including 16 patients with HRD-PCa. HRD patients more frequently had intratumoral CD3+, CD3+CD8−FoxP3− or Foxp3+ TILs above median compared to patients without DNA damage repair alterations (DDRwt; CD3+ and Foxp3+: 77% vs 35%, p = .013; CD3+CD8−FoxP3−: 80% vs 44%, p = .031). No significant difference in CD8+ TILs or PD-L1 expression was observed. In peripheral blood, HRD patients displayed a more diverse TCR repertoire compared to DDRwt patients (p = .014). Additionally, HRD patients shared TCR clusters with low generation probability, suggesting patient-overlapping T cell responses. A pooled analysis of clinical data from 227 patients with molecularly characterized PCa suggested increased efficacy of ICIs in HRD-PCa. In conclusion, patients with HRD-PCa display increased TIL density and an altered peripheral TCR repertoire. Further research into the efficacy of ICIs and the presence of shared neoantigens in HRD-PCa is warranted.

Homologous recombination repair deficiency (HRD) testing in newly diagnosed advanced-stage epithelial ovarian cancer: A Belgian expert opinion.

Ovarian cancer (OC) has a poor prognosis as most patients present with non-specific symptoms and the disease is mostly diagnosed at advanced stages. Approximately 90% of cases are classified as epithelial OC (EOC), a category comprising histologically and molecularly distinct tumours. Identifying reliable biomarkers and employing personalised therapies in OC subgroups is crucial for battling the disease. EOCs are often characterised by homologous recombination repair deficiency (HRD), frequently caused by inactivation of the breast cancer susceptibility (BRCA) genes. These findings have led to the development of poly- (adenosine diphosphate [ADP])- ribose polymerase inhibitors (PARPi), which are synthetically lethal to HRD tumour cells. Both patients with HRD and non-HRD tumours can benefit from PARPi therapy in the recurrent setting. Moreover, recent phase III trials in patients with newly diagnosed advanced-stage OC have demonstrated greater clinical benefit from PARPi in treating HRD than non-HRD tumours. These findings offer new opportunities for the use of PARPi as maintenance therapy after first-line chemotherapy based on the presence of HRD. In the current article, we provide recommendations for HRD testing and treatment of patients with newly diagnosed advanced-stage EOC.

The impact of rare germline variants on human somatic mutation processes

Somatic mutations are an inevitable component of ageing and the most important cause of cancer. The rates and types of somatic mutation vary across individuals, but relatively few inherited influences on mutation processes are known. We perform a gene-based rare variant association study with diverse mutational processes, using human cancer genomes from over 11,000 individuals of European ancestry. By combining burden and variance tests, we identify 207 associations involving 15 somatic mutational phenotypes and 42 genes that replicated in an independent data set at a false discovery rate of 1%. We associate rare inherited deleterious variants in genes such as MSH3, EXO1, SETD2, and MTOR with two phenotypically different forms of DNA mismatch repair deficiency, and variants in genes such as EXO1, PAXIP1, RIF1, and WRN with deficiency in homologous recombination repair. In addition, we identify associations with other mutational processes, such as APEX1 with APOBEC-signature mutagenesis. Many of the genes interact with each other and with known mutator genes within cellular sub-networks. Considered collectively, damaging variants in the identified genes are prevalent in the population. We suggest that rare germline variation in diverse genes commonly impacts mutational processes in somatic cells.

Synthetic Lethality Targeting Polθ.

Research studies regarding synthetic lethality (SL) in human cells are primarily motivated by the potential of this phenomenon to be an effective, but at the same time, safe to the patient’s anti-cancer chemotherapy. Among the factors that are targets for the induction of the synthetic lethality effect, those involved in DNA repair seem to be the most relevant. Specifically, when mutation in one of the canonical DNA double-strand break (DSB) repair pathways occurs, which is a frequent event in cancer cells, the alternative pathways may be a promising target for the elimination of abnormal cells. Currently, inhibiting RAD52 and/or PARP1 in the tumor cells that are deficient in the canonical repair pathways has been the potential target for inducing the effect of synthetic lethality. Unfortunately, the development of resistance to commonly used PARP1 inhibitors (PARPi) represents the greatest obstacle to working out a successful treatment protocol. DNA polymerase theta (Polθ), encoded by the POLQ gene, plays a key role in an alternative DSB repair pathway—theta-mediated end joining (TMEJ). Thus, it is a promising target in the treatment of tumors harboring deficiencies in homologous recombination repair (HRR), where its inhibition can induce SL. In this review, the authors discuss the current state of knowledge on Polθ as a potential target for synthetic lethality-based anticancer therapies.

Investigation of BRCAness associated miRNA-gene axes in breast cancer: cell-free miR-182-5p as a potential expression signature of BRCAness

The concept of the ‘BRCAness’ phenotype implies the properties that some sporadic breast cancers (BC) share with BRCA1/2-mutation carriers with hereditary BC. Breast tumors with BRCAness have deficiencies in homologous recombination repair (HRR), like BRCA1/2-mutation carriers, and consequently could benefit from poly-(ADP)-ribose polymerase (PARP) inhibitors and DNA-damaging chemotherapy. Triple-negative breast cancers (TNBC) show a higher frequency of BRCAness than the other BC subtypes. Therefore, looking for BRCAness-related biomarkers could improve personalized management of TNBC patients. microRNAs (miRNAs) play a pivotal role in onco-transcriptomic profiles of tumor cells besides their suitable features as molecular biomarkers. The current study aims to evaluate the expression level of some critical miRNAs-mRNA axes in HRR pathway in tumors and plasma samples from BC patients. The expression levels of three multi-target miRNAs, including miR-182-5p, miR-146a-5p, and miR-498, as well as six downstream HRR-related protein-coding genes, have been investigated in the breast tumors and paired adjacent normal tissues by Real-time PCR. In the next step, based on the results derived from the previous step, we examined the level of cell-free miR-182-5p in the blood plasma samples from the patients. Our results highlight the difference between TNBC and non-TNBC tumor subgroups regarding the dysregulation of the key miRNA/mRNA axes involved in the HRR pathway. Also, for the first time, we show that the level of cell-free miR-182-5p in plasma samples from BC patients could be a clue for screening BC patients eligible for receiving PARP inhibitors through a personalized manner. Altogether, some sporadic BC patients, especially sporadic TNBC, have epigenetically dysregulated HRR pathway that could be identified and benefit from BRCAness-specific therapeutic agents.

CRISPR/Cas9‑induced saturated mutagenesis identifies Rad51 haplotype as a marker of PARP inhibitor sensitivity in breast cancer.

Breast cancer treatment with poly(ADP-ribose)polymerase (PARP) inhibitors is currently limited to cells defective in the homologous recombination repair (HRR) pathway. The chemical inhibition of many HRR deficiency genes may sensitize cancer cells to PARP inhibitors. In the present study, Rad51, a central player in the HRR pathway, was selected to explore additional low variation and highly representative markers for PARP inhibitor activity. A CRISPR/Cas9-based saturated mutation approach for the Rad51 WALKER domain was used to evaluate the sensitivity of the PARP inhibitor olaparib. Five amino acid mutation sites were identified in olaparib-resistant cells. Two Rad51 haplotypes were assembled from the mutations, and may represent useful pharmacogenomic markers of PARP inhibitor sensitivity.

Abstract 805: RECAP (REpair CAPacity) identifies a subset of breast cancers unable to form RAD51 foci which are undetected by DNA-based BRCAness tests

Abstract Background: Germline BRCA1/2 mutation status is currently used as predictive biomarker for response to Poly-(ADP-Ribose)-Polymerase (PARP) inhibitors in breast cancer (BC) patients. However, non-germline BRCA1/2 mutated tumors and homologous recombination repair deficient (HRD) tumors with unknown etiology are probably also PARP inhibitor-sensitive. Various HRD biomarkers have been proposed and their clinical validity and utility are under investigation. We recently described the functional REpair CAPacity (RECAP) test as a real-time method to select HRD tumors based on their inability to form RAD51 foci. Here, we investigated whether this functional test defines a similar group of HRD tumors as DNA-based tests for primary and metastatic BCs. Materials and Methods: A cohort (n=71; 52 primary and 19 metastatic BCs) enriched for HRD tumors was selected based on the RECAP test, consisting of 26 RECAP-HRD (37%), 9 RECAP-HR intermediate (HRi; 13%) and 36 RECAP-HR proficient (HRP; 51%) tumors. Whole genome sequencing (WGS) was carried out on matched germline and tumor DNA samples for 38 primary BCs and the 19 metastatic lesions, for which material was available. Three samples were excluded due to a low purity score (<0.2), thus WGS data was present for 54 tumors. The tumors were subjected to DNA-based HRD tests: CHORD and BRCA1/2-like classifier algorithms. Concordance between different HRD tests was reached when a tumor was classified as HRD/HRi or HRP by both tests. Results: The RECAP test identified all bi-allelic BRCA deficient samples (n=15) in this cohort as HRD/HRi. Within the remaining non-BRCA RECAP-HRD/HRi samples, there were no pathogenic mutations in other relevant genes that could explain the HRD status of these tumors. In the RECAP-HR proficient tumors no bi-allelic BRCA1/2 deficiencies were identified. The RECAP status partially correlated with the DNA-based HRD test outcomes using the HRDetect, CHORD and BRCA1/2-like classifier algorithms (70% concordance for RECAP-CHORD and RECAP-BRCA1/2-like classifier, and 66% for CHORD-BRCA1/2-like classifier). However, RECAP identified additional samples unable to form RAD51 foci. The discordance among the tests were evenly distributed among the primary and metastatic BCs. Conclusions: The difference between RECAP and other HRD tests in BC patients is relatively large (30-34% discordance), implying that RECAP may be identifying deficiencies in other components of DNA repair which could also result in PARP inhibitor sensitivity. Therefore, direct comparison of these HRD tests in clinical trials will be required to evaluate the optimal predictive test for clinical decision making. Citation Format: Titia G. Meijer, Luan Nguyen, Arne van Hoeck, Marjolijn M. Ladan, Nicole S. Verkaik, Kirsten Ruigrok-Ritstier, Carolien H. van Deurzen, Harmen J. van de Werken, Esther H. Lips, Sabine C. Linn, Yasin Memari, Helen Davies, Serena Nik-Zainal, Roland Kanaar, John W. Martens, Edwin Cuppen, Agnes Jager, Dik C. van Gent. RECAP (REpair CAPacity) identifies a subset of breast cancers unable to form RAD51 foci which are undetected by DNA-based BRCAness tests [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 805.

Abstract 1515: Shannon entropy of mutational signatures predict sensitivity of signature detection in targeted sequencing

Abstract Mutational processes can cause driver mutations which are a proximal cause of tumorigenesis. COSMIC identifies signatures of biological and environmental events that drive mutational processes and can be identified by examining patterns of somatic mutations. We describe the complexity of the COSMIC mutational signatures and their impact. We introduce Shannon entropy as a metric to measure complexity of trinucleotide distributions, thus capturing a previously ignored dimension of mutational signatures that may predict their identification. Shannon entropy derives from the rich predictive mathematics of information theory. We calculated entropy for all the COSMIC mutation signatures and found that SBS3, homologous recombination repair deficiency (HRD) signature, had highest entropy of 6.326, while SBS2 signature, activation of AID/APOBEC cytidine deaminases, had the lowest entropy of 1.758.We discovered that high entropy signatures (such as HRD SBS3) require more somatic mutations to be reliably detected than do low entropy signatures, important when planning signature detection using targeted panels that have smaller genomic footprint. We demonstrate this correlation with two approaches i) by simulation using MutSigSim, a tool we developed for simulating mutational profiles given the number of mutations, and ii) by using real datasets. Simulation was performed for high, medium, and low entropy signatures. We used cosine similarity of 0.7 as the threshold for detection. For signatures with entropy <2, a signature can be detected 90% of the time with 10 or more mutations. Similarly, signatures with entropies between 2-3, 3-5 and >5 can be detected 90% of the time with 30, 40, and 100 mutations, respectively. Mutation signatures of 1500 samples from a pan solid tumor cohort sequenced with Oncomine Comprehensive Assay Plus or Oncomine TMB were used to confirm these conclusions. We saw that signatures with higher entropy were difficult to detect at cosine similarity threshold of 0.7 in samples with a smaller number of mutations. In a cohort of samples with MSI-high, we detected mismatch repair (MMR) signatures with low to medium entropy with high specificity and good sensitivity. Furthermore, in a cohort of Ovarian cancer samples, it was difficult to detect the high entropy HRD signature at 0.7 threshold when the number of somatic mutations was not high. We show that ability to detect mutation signatures using targeted panels is directly correlated to the complexity of signature and number of mutations. For research use only. Not for use in diagnostic procedures. Citation Format: Ajithavalli Chellappan, Chintan Vora, Jagannath Patro, Shilpa Nair, Rushikesh S. Kanap, Fiona C. Hyland. Shannon entropy of mutational signatures predict sensitivity of signature detection in targeted sequencing [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 1515.

Abstract 42: High-throughput next-generation sequencing research solutions for detection of oncology variants, gene fusion events, and key oncology endpoints

Abstract Introduction: The Ion Torrent Genexus System has redefined the genomic profiling paradigm as the first fully-integrated, next-generation sequencing (NGS) research platform to provide an automated sample-to-report workflow with results in a single day. With a purification instrument, an enhanced chip architecture, and downstream reporting, the Genexus System provides a convenient solution to enable oncology research. Here we highlight the oncology research applications and high-throughput NGS capabilities of the Genexus System with Oncomine Comprehensive Assay Plus (OCA Plus), an oncology research panel that can detect variants, fusions, and evaluate key oncology research endpoints. We demonstrate the ability of OCA Plus on Genexus to evaluate tumor mutational burden (TMB), microsatellite instability (MSI), loss of heterozygosity (LOH), and homologous recombination repair deficiency (HRD). Methods: The high-throughput capabilities of the Genexus System enable it to support large oncology research panels such as OCA Plus, which comprises over 13,000 amplicons. The extensive per sample coverage allows for comprehensive DNA and RNA genomic profiling of relevant cancer biomarkers in over 500 genes including detection of over 1,300 fusion isoforms. We utilized high-molecular weight and FFPE samples, reference controls, and orthogonally tested research samples to evaluate DNA variant calling, RNA fusion calling, and key oncology research endpoints, including MSI, LOH, TMB, and HRD. Results: Commercially sourced reference control and research samples were sequenced using OCA Plus on the Genexus System. Sequencing data metrics showed ≥24 million reads per sample, with four samples supported per run. The high-throughput capacity of the Genexus chip architecture results in >95% of amplicons achieving a minimum of 500X coverage with an average coverage uniformity of ≥95% when evaluated across all >13,000 amplicons. Variant calling was assessed using the AcroMetrix Oncology Hotspot Control which has 328 hotspot variants covered by OCA Plus. Variant calling performance showed a hotspot and de novo variant sensitivity and PPV >95%. MSI score and status were assessed using known MSI-High and microsatellite stable (MSS) research samples and FFPE samples of interest. Results show high concordance with data from OCA Plus on GeneStudio. Conclusions: The increased throughput of the Genexus System combined with minimal touchpoints and a rapid turnaround time enables comprehensive genomic profiling for research assays such as OCA Plus where an increased number of sequencing reads leads to greater sensitivity for detecting rare variants and low-level fusion transcripts. Further, accurate characterization of key oncology research endpoints, such as TMB, MSI, LOH, and HRD, allow the Genexus System to accelerate research in the field of oncology. Citation Format: Kayla Zochowski, Dinesh Cyanam, Geoffrey Lowman, Jennifer Kilzer, Sameh El-Difrawy, Yan Zhu, Tanaya Puranik, Alex Phan, Derek Wong, Edith Kwong, Coleen Nemes, Iris Casuga, Frances Chan, Eun Ji Kim, Jianjun Guo, Vinay Mittal, Emily Norris, Shrutii Sarda, Mohit Gupta, Fan Shen, Steven Roman, Gabriel Vargas, Ying Jin, Annie Kraltcheva, Paul Williams, Amneet Gulati, Justin Rigby, Christopher Hoff, Richard Meade, Elaine Wong-Ho, Andrew Wong, Jamsheed Ghadiri, David Garcia-Viramontes, Scott Myrand, Seth Sadis. High-throughput next-generation sequencing research solutions for detection of oncology variants, gene fusion events, and key oncology endpoints [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 42.

Abstract CT201: Ceralasertib and olaparib in the treatment of homologous recombination repair (HRR)-deficient platinum-sensitive ovarian cancer after progression on PARP inhibitors

Abstract Background: The majority of BRCA-mutant or HRR-deficient ovarian cancers are initially sensitive to PARP inhibitors (PARPis) but eventually develop resistance. The most commonly reported PARPi resistance mechanisms are (1) reversal of HRR deficiency through (a) reversion mutations in BRCA or other HRR genes (eg, RAD51C/D) or (b) DNA end resection rewiring with loss of 53BP1, REV7, or Shieldin complex components; or (2) stabilization of stalled replication forks through loss of PTIP or EZH2, preventing nuclease recruitment. The only confirmed PARPi resistance mechanism in the clinic to date is reversion mutations. Combined ATR and PARP inhibition is an attractive strategy that may overcome PARPi resistance, as has been shown in PARPi-resistant cell lines and patient-derived xenograft models (Yazinski et al Genes Dev 2017; Murai et al Oncotarget 2016; Kim et al Nat Commun 2020; data on file, AstraZeneca). The PARPi olaparib 300 mg twice daily (BID) continuously in combination with the ATR inhibitor ceralasertib 160 mg daily on days 1-7 of a 28-day cycle has demonstrated promising activity in the CAPRI study in patients with ovarian cancer who had progressed on PARPis (Wethington et al J Clin Oncol 2021). However, preclinical models of PARPi resistance suggest that a higher dose or duration of ceralasertib with a lower dose of olaparib may be more beneficial (data on file, AstraZeneca). Methods: D5330C00004 (NCT02264678) is a multicenter, modular, phase 1 study assessing ceralasertib in combination with other anti-cancer agents in adults with advanced solid malignancies. In Module 2, a lower dose of olaparib (150 mg BID continuous) and a longer duration of ceralasertib (80 mg BID, days 1-14 of a 28-day cycle) is now being explored in 2 expansion cohorts in patients with platinum-sensitive, high-grade, serous or endometrioid ovarian, fallopian tube, or primary peritoneal cancer with deleterious or suspected deleterious germline or somatic BRCA or RAD51C/D mutations, or HRR deficiency by Myriad MyChoice® or FoundationOne® CDx (F1CDx) assays. Patients will have progressed on prior PARPis after ≥6 months in the maintenance or treatment setting (≥12 months after first-line maintenance). Cohort 1 is enrolling ~30 patients immediately after progression on a PARPi; Cohort 2 is recruiting ~30 PARPi-treated patients after intervening platinum-based chemotherapy. Study endpoints include safety, tumor response by RECIST 1.1, and duration of response. Exploratory analyses include genomic and functional analysis of HRR restoration in tumor and circulating tumor DNA, and analysis of replication fork stability in organoid cultures established from mandatory fresh tumor biopsy samples. Recruitment began in July 2021 with the first patient dosed in August 2021. Citation Format: Geoffrey I. Shapiro, Bristi Basu, Anthony El-Khoueiry, Sophie Postel-Vinay, Seock-Ah Im, Sun Young Rha, Claire F. Friedman, Antoine Italiano, Yong Man Kim, Myong Cheol Lim, Rene Roux, Joyce F. Liu, Elhan Sanai, Simon A. Smith, Claire Smith, Sarah El Farhi, Alan Lau, Natalia Lukashchuk, Emma Dean, Matthew G. Krebs. Ceralasertib and olaparib in the treatment of homologous recombination repair (HRR)-deficient platinum-sensitive ovarian cancer after progression on 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 CT201.

Abstract 1260: Nonclinical evaluation of rucaparib in tumors with mutations in non-BRCA1/2 homologous recombination repair (HRR) genes

Abstract Background: Rucaparib is a PARP inhibitor approved to treat ovarian cancer (OC) and metastatic castration-resistant prostate cancer (mCRPC). Deficiencies in HRR resulting from genetic or epigenetic alterations in BRCA1/2 sensitize tumors to rucaparib through synthetic lethality. Inactivation of other HRR genes may also confer sensitivity to PARP inhibition. Here, we evaluated rucaparib efficacy in preclinical models with genomic or epigenetic alterations in an HRR gene panel. Methods: BRCA1/2 and 14 additional genes were selected for their role in DNA repair and mutation frequency in OC and mCRPC. Small interfering RNA (siRNA) knockdowns were performed in OC (OVCAR-3, OAW28, SK-OV-3) and mCRPC (DU145, PC-3) cell lines to model their inactivation and assess impact on rucaparib sensitivity (IC50) in cell viability assays. Patient-derived xenografts (PDX) harboring deleterious alterations in BRCA1/2, BARD1, BRIP1, NBN, PALB2, RAD51B, RAD51C, or RAD51D, or BRCA1/RAD51C promoter hypermethylation were treated with up to 150 mg/kg rucaparib BID for 25-42 days and tumor growth was monitored. Mutational status was confirmed by next-generation sequencing. Results: In addition to BRCA1/2 knockdown, depletion of BARD1, PALB2, or RAD51 resulted in potent rucaparib activity in OC and mCRPC cells, where the rucaparib IC50 was reduced by >70% vs control in at least 4 of the 5 cell lines tested. Rucaparib also displayed greater potency in cells with siRNA-mediated decreased expression of FANCA, NBN, RAD51C, or RAD54L, where at least 1 cell line had >50% decrease in IC50. Rucaparib treatment in 19/33 HRR-deficient PDX models resulted in significant tumor growth inhibition (TGI). These included BRCA1/2, PALB2, NBN, RAD51B, RAD51C, or RAD51D-mutated and BRCA1/RAD51C-hypermethylated models. Efficacy was strongly associated with biallelic inactivation of BRCA1/2, PALB2, RAD51C, or RAD51D with 87% vs 29% mean TGI for monoallelic BRCA1/2 alterations (P<0.0001). In addition, a single PDX model each for the exploratory genes NBN and RAD51B had 101% and 80% TGI, respectively. No significant difference was observed between PDX models with BRCA1/2 vs non-BRCA1/2 (PALB2, NBN, RAD51B, RAD51C, or RAD51D) biallelic alterations (98% vs 80% mean TGI, respectively; P=0.21). In support of these nonclinical findings, we highlight a patient with RAD51B-mutant mCRPC treated with rucaparib in the TRITON2 trial (NCT02952534) who had confirmed radiographic and PSA responses. Conclusions: Cell lines and PDX models with biallelic alterations in HRR genes other than BRCA1/2, including RAD51B, RAD51C, RAD51D, PALB2, and NBN, are comparably sensitive to rucaparib. Rucaparib is being evaluated in the LODESTAR trial (NCT04171700) in patients with tumors associated with deleterious alterations in BRCA1/2, PALB2, RAD51C, and RAD51D (cohort A) and BARD1, BRIP1, FANCA, NBN, RAD51, and RAD51B (exploratory cohort B). Citation Format: Liliane Robillard, Kevin K. Lin, Andrea Loehr, Tanya Kwan, Rachel Dusek, Andrew D. Simmons, Thomas C. Harding, Brieuc Sautois, Minh Nguyen. Nonclinical evaluation of rucaparib in tumors with mutations in non-BRCA1/2 homologous recombination repair (HRR) genes [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 1260.

Abstract 5394: Uterine leiomyosarcoma with homologous recombination deficiency is highly sensitive to polo-like kinase 4 inhibitor

Abstract INTRODUCTION: Uterine leiomyosarcoma (LMS) is rare and aggressive and without a well-established cancer-driver. Treatment is mainly surgical because most other therapies have limited benefits. Polo-like kinase 4 (PLK4) is a modulator of centriole duplication that is essential for proper cell division. Its inhibition by the PLK4 inhibitor, CFI-400945 resulted in centriole dysregulation, increased in double-strand DNA damage, and ultimately, mitotic catastrophe and cell death. Recent genomic studies have identified a subset of LMS with homologous recombination (HR) repair deficiency. We hypothesize that HR-deficient LMS is highly sensitive to CFI-400945. METHODS: Gene knockouts (KO) were done by CRISPR-Cas9 system. Plasmids encoding the BRCA1- and BRCA2-targeting guide RNA, Cas9 protein, and selection markers were generated and were transfected into SK-UT-1 and SKN followed by selection with antibiotics. The KO and wild-type cell lines were treated with CFI-400945 in vitro. Percentage apoptosis was evaluated by Annexin V-PI staining and cell proliferation was examined by sulforhodamine B assay. To determine whether BRCA1- and BRCA2-KO had any effects on double-strand DNA damage, the γH2AX foci were studied by immunofluorescence staining. RESULTS: CFI-400945 caused a greater degree of apoptosis and less cell viability in the KO than in the wild-type cell lines. In SK-UT-1, quantity of apoptotic cells for BRCA2-KO and BRCA1-KO were 77% and 34%, respectively, versus 27% in the wild-type cell line (p<0.05). Less dramatic findings were observed in SKN with respective percentages of 20% and 25% versus 15% (p<0.05). In SK-UT-1, the mean IC50 for BRCA2-KO and BRCA1-KO were 3.4 nM and 5.5 nM, respectively, versus 7.8 nM in the wild-type cell line (p<0.05). Similar observations were noted in SKN, with respective mean IC50 of 6.5 nM and 7.1 nM versus 10.0 nM (p<0.05). After CFI-400945 treatment, the γH2AX/Hoechst 33342 staining intensity ratio was 0.40 in SK-UT-1 BRAC2-KO and 0.44 in BRCA1-KO, where both were higher than in the corresponding wild-type cell line with 0.29 (p<0.05). Similarly, the intensities in the two SKN KO cell lines were 0.29 and 0.31, respectively, and higher than in the wild-type cell line with 0.19 (p <0.05). CONCLUSIONS: PLK4 inhibitor induced DNA double-strand breaks in LMS and the repair of these breakages was dependent on HR repair. In vitro, LMS with BRCA1 or BRCA2 deficiencies were more sensitive to the effect of PLK4 inhibitor. Citation Format: Tsz Yan Chong, Horace HY Lee, Judy WP Yam, Kin Long Chow, Ho Shing Wong, Ka Yu Tse, Mark R. Bray, Tak Wah Mak, Philip PC Ip. Uterine leiomyosarcoma with homologous recombination deficiency is highly sensitive to polo-like kinase 4 inhibitor [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 5394.

Functional RECAP (REpair CAPacity) assay identifies homologous recombination deficiency undetected by DNA-based BRCAness tests

Germline BRCA1/2 mutation status is predictive for response to Poly-[ADP-Ribose]-Polymerase (PARP) inhibitors in breast cancer (BC) patients. However, non-germline BRCA1/2 mutated and homologous recombination repair deficient (HRD) tumors are likely also PARP-inhibitor sensitive. Clinical validity and utility of various HRD biomarkers are under investigation. The REpair CAPacity (RECAP) test is a functional method to select HRD tumors based on their inability to form RAD51 foci. We investigated whether this functional test defines a similar group of HRD tumors as DNA-based tests. An HRD enriched cohort (n = 71; 52 primary and 19 metastatic BCs) selected based on the RECAP test (26 RECAP-HRD; 37%), was subjected to DNA-based HRD tests (i.e., Classifier of HOmologous Recombination Deficiency (CHORD) and BRCA1/2-like classifier). Whole genome sequencing (WGS) was carried out for 38 primary and 19 metastatic BCs. The RECAP test identified all bi-allelic BRCA deficient samples (n = 15) in this cohort. RECAP status partially correlated with DNA-based HRD test outcomes (70% concordance for both RECAP-CHORD and RECAP-BRCA1/2-like classifier). RECAP selected additional samples unable to form RAD51 foci, suggesting that this functional assay identified deficiencies in other DNA repair genes, which could also result in PARP-inhibitor sensitivity. Direct comparison of these HRD tests in clinical trials will be required to evaluate the optimal predictive test for clinical decision making.

BRCA reversion mutations mediated by microhomology-mediated end joining (MMEJ) as a mechanism of resistance to PARP inhibitors in ovarian and breast cancer.

5559 Background: PARP inhibitors exploit synthetic lethality in tumor cells with deficiency in homologous recombination repair (HRR). In line with this, most reported mechanisms of PARP inhibitor resistance restore HRR. Of multiple resistance mechanisms reported preclinically, reversion mutations in BRCA genes are the only confirmed mechanism of resistance to both platinum and PARP inhibitors in patients (pts) to date (Lin K et al. Cancer Discov 2019), with most studies focusing on ovarian cancer. MMEJ is an alternative DNA damage repair pathway, in which DNA polymerase θ (POLθ) has a key role; MMEJ has been suggested to play a role in BRCA reversion mutations (Tobalina L et al. Ann Oncol 2021). Methods: Targeted circulating tumor DNA (ctDNA) sequencing analyzed over 500 plasma samples collected at baseline and at progression to therapy in pts with ovarian or breast cancer and a mutation in BRCA1 and/or BRCA2 (BRCAm) who were treated with olaparib or chemotherapy in one of three Phase II/III clinical studies (LIGHT NCT02983799, SOLO3 NCT02282020, OlympiAD NCT02000622). Only pts with an original pathogenic BRCAm detected in ctDNA were evaluable. BRCA reversion mutations were identified using internal computational framework; DNA sequences surrounding BRCA reversion sites were analyzed for MMEJ signatures. Results: At baseline, in pooled data across treatment arms and across all available samples, BRCA reversion mutations were detected in 4/114 (3.5%) and 6/133 (4.5%) of breast and ovarian cancer pts, respectively, which may have developed on prior platinum therapy. At progression, BRCA reversion mutations were detected in 34/79 (43%) breast cancer pts and in 26/101 (26%) ovarian cancer pts who received olaparib, with at least 2/79 and 4/101 reversions already present at baseline, respectively. At progression, in the chemotherapy arm, BRCA reversion mutations were detected in 3/34 (9%) breast cancer pts and 1/29 (3%) ovarian cancer pts, with 2/34 and 0/29 reversions present at baseline, respectively. Reversion mutations varied in allelic frequency and were either present as single or multiple reversions, suggesting multiple events within the tumor were driving resistance. The location and type of reversion mutations reflected the functional importance of BRCA protein domains. A large proportion of BRCA reversion mutations (47/69 [68%] that were evaluable) were mediated by the MMEJ pathway based on the presence of MMEJ signatures around BRCA reversion sites. Conclusions: We detected BRCA reversion mutations in at least ̃40% of breast and ̃20% of ovarian cancer pts following treatment with olaparib. A large proportion of these reversion mutations are likely to have been mediated by MMEJ repair, suggesting that POLθ inhibitors in combination with platinum or PARP inhibitors might prevent or delay emergence of PARP inhibitor resistance. Clinical trial information: NCT02983799, NCT02282020, NCT02000622.

Biomarker-directed therapy in black and white men with metastatic castration-resistant prostate cancer (mCRPC).

5013 Background: Black men have been underrepresented in large-scale molecular prostate cancer (PC) surveys, despite having higher PC incidence and mortality. Since molecular profiling to guide the use of targeted agents is increasingly important in mCRPC, we compared precision medicine data and utilization in a cohort of black and white men with mCRPC. Methods: The PROMISE precision medicine database is an academic collaboration to compile clinical and genomic data from men with PC. All patients have had germline and/or somatic genetic testing performed. Eligibility criteria for this analysis included a diagnosis of mCRPC with available race and biomarker data. The primary outcome was the proportion of non-Hispanic black (NHB) and non-Hispanic white (NHW) men with actionable molecular data, defined as the presence of mismatch repair deficiency (MMRd/MSI-H), homologous recombination repair deficiency (HRRd), tumor mutational burden (TMB) ≥ 10 mut/MB, or AR-V7. Secondary outcomes included the proportion of NHB and NHW men with other alterations, the type and timing of genomic testing performed, and the use of targeted therapy. Results: A total of 962 mCRPC patients (21.2% NHB; 78.8% NHW) met inclusion criteria of 1619 in the overall database. Median age (NHB/NHW) was 61/63; 77.5/68.8% had Gleason 8-10; 52.5/56.7% presented with de novo metastatic disease (33.8/29.9% LN, 36.2/32.2% bone and 8.3/6.1% viscera). The median time from diagnosis to first molecular result was 56.3 mo for NHB v 58.7 mo for NHW (p = 0.45). Use of blood-based molecular testing was more common in NHB men (48.7% v 36.4%, p < 0.001). Overall, 32.8% of NHB men harbored actionable molecular data compared to 30.3% of NHW men (Table). MMRd/MSI-H was more common in NHB men (9.1 v 4.9%, p = 0.04). Other than PTEN (12.7/23.8% NHB/NHW, p = 0.0001), no significant differences were seen in the 15 most frequently mutated genes, including TP53, AR, CDK12, RB1, and PIK3CA. Tumor suppressor co-mutations (PTEN/TP53/RB1) were found in 13.1% of NHB and 18.0% NHW (p = 0.13). Delivery of targeted therapy was reported in 19.6% of NHB and 23.7% of NHW men (p = 0.25) after a median of 2 CRPC lines. Median OS from development of mCRPC was 41.5 mo (95% CI, 34.7-51.3) and 44.7 mo (95% CI, 41.1-51.5) for NHB and NHW men, respectively (p = 0.14). Conclusions: In a real-world mCRPC molecular profiling cohort, we found similar overall rates of actionable molecular alterations in NHB and NHW men, but higher rates of MMRd/MSI-H and lower frequency of PTEN alterations in NHB men. We did not find differences in delivery of targeted therapy. [Table: see text]

Tumor immune microenvironment of primary prostate cancer with and without germline mutations in homologous recombination repair genes

BackgroundAberrations in homologous recombination repair (HRR) genes are emerging as important biomarkers for personalized treatment in prostate cancer (PCa). HRR deficiency (HRD) could affect the tumor immune microenvironment (TIME), potentially...

Identification of homologous recombination deficiency (HRD) by RAD51 in a tumor molecular profiling program for precision medicine.

3137 Background: Tumor molecular profiling by panel sequencing helps to identify candidate patients for precision oncology. Mutations in genes of the homologous recombination repair (HRR) pathway confer DNA repair deficiency and sensitivity to DNA damaging agents, such as PARP inhibitors (PARPi) or platinum-based drugs. RAD51 nuclear foci is a biomarker of functional HRR deficiency (HRD). We aimed to incorporate the RAD51 test in an academic molecular profiling program to identify HRD tumors beyond genetic testing. Methods: We included patients with metastatic breast cancer (TNBC, ER+ <60y, 3rd line HER2+ BC), newly diagnosed high-grade epithelial ovarian cancer (HGOC) and metastatic and/or castration-resistant prostate adenocarcinoma (PC) undergoing tumor/germline genetic testing. A customized capture-based targeted sequencing of 450 genes was performed in FFPE tumor samples. RAD51 was manually stained by immunofluorescence and functional HRD was defined as RAD51 score ≤10%. Results: Between January 2021 and January 2022, tumor panel sequencing was performed in 279 tumors. Panel sequencing was informative in 264/279 (95%) samples and the RAD51 test was evaluable in 81/90 (90%). In total, 77 samples were evaluable for both tumor/germline sequencing and RAD51, namely 42 BC, 16 HGOC and 19 PC (Table 1). Functional HRD by RAD51 was observed in 15/77 (20%) cases. Panel sequencing identified 17/77 (22%) cases carrying HRR gene alterations, of which nine were confirmed as HRD by RAD51. In BC, 2/6 tumors with HRD by RAD51 did not carry a germline or tumor mutation in HRR genes. HRD tumor profiling triggered the germline analysis of one hereditary BRCA2 BC patient. Four g BRCA1/2 BC with HRD by RAD51 became HRR proficient (HRP) at PARPi progression. In addition, the RAD51 test identified 2/5 HGOC and 2/4 PC tumors as HRD despite not carrying a tumor HRR gene mutation. Conclusions: RAD51 testing is feasible in an established molecular profiling program and complements gene panel sequencing results by providing evidence of functional HRR status. In particular, the RAD51 test extends the identification of HRD tumors beyond those with HRR gene mutations and captures HRR restoration after PARPi treatment. We aim to expand the analysis of RAD51 to other tumor types.[Table: see text]

The significance of the fusion partner gene genomic neighborhood analysis in translocation‐defined tumors

IntroductionThis study presents a novel molecular parameter potentially co‐defining tumor biology—the total tumor suppressor gene (TSG) count at chromosomal loci harboring genes rearranged in fusion‐defined tumors. It belongs to the family of molecular parameters created using a black‐box approach.MethodIt is based on a public curated Texas TSG database. Its data are regrouped based on individual genes loci using another public database (Genecards). The total TSG count for NTRK (NTRK1; OMIM: 191315; NTRK2; OMIM: 600456; NTRK3; OMIM: 191316), NRG1 (OMIM: 142445), and RET (OMIM: 164761) rearranged tumors in patients treated with a theranostic approach is calculated using the results of recently published studies.ResultsAltogether 138 loci containing at least three TSGs are identified. These include 21 “extremely hot” spots, with 10 to 28 TSGs mapping to a given locus. However, the study falls short of finding a correlation between tumor regression or patient survival and the TSG count owing to a low number of cases meeting the study criteria.ConclusionThe total TSG count alone cannot predict the biology of translocation‐defined tumors. The addition of other parameters, including microsatellite instability (MSI), tumor mutation burden (TMB), homologous recombination repair deficiency (HRD), and copy number heterogeneity (CNH), might be helpful. Thus a multi‐modal data integration is advocated. We believe that large scale studies should evaluate the significance and value of the total TSG count.

Olaparib outcomes in metastatic castration-resistant prostate cancer: First real-world experience in safety and efficacy from the Chinese mainland.

BackgroundOlaparib, a poly (ADP-ribose) polymerase (PARP) inhibitor, has been approved for use in breast cancer susceptibility gene (BRCA)-mutated metastatic castration-resistant prostate cancer (mCPRC) patients. Our aim was to evaluate the adverse events (AEs) and efficacy of Olaparib in the treatment of mCRPC patients from the Chinese mainland.MethodsWe retrospectively included mCPRC patients treated with Olaparib more than for 28 days. Patients with alterations in 15 homologous recombination repair (HRR) genes were defined as the HRRmt group, and the rest were defined as the HRRwt group. The efficacy was analyzed by prostate-specific antigen (PSA) decreased rate and PSA progression-free survival (PFS). The partial response, good response, and high response of PSA were defined as a reduction of between 0% and 50%, greater than 50%, and greater than 90% from baseline.ResultsA total of 43 patients were enrolled in this study, including 26 HRRmt group patients and 17 HRRwt group patients. Two HRRwt patients received additional abiraterone therapy. A partial response, good response, and high response were achieved in 89% (23/26), 59% (15/26), and 15% (4/26) of HRRmt group patients, respectively. In HRRwt group, 59% (10/17), 35% (6/17), and 12% (2/17) of patients met the criteria of partial response, good response, and high response, respectively. Median PFS was 8.0 months in the HRRmt group and 3.0s months in the HRRwt group (HR, 0.61; 95% CI, 0.24–1.14; p = 0.148), respectively. All the 20 patients had AEs during Olaparib treatment. Ten episodes of grade 3 or 4 AEs were observed in four patients. The most common all-grade AEs were fatigue or asthenia (70%), anemia (65%), and decreased appetite (55%).ConclusionsMost of the AEs were tolerated, and Olaparib was effective in mCRPC patients with HRR deficiency. In addition, the underlying mechanism of the efficacy of Olaparib observed in HRRwt group patients remained explored.

Intra-Tumoral CD8+ T-Cell Infiltration and PD-L1 Positivity in Homologous Recombination Deficient Pancreatic Ductal Adenocarcinoma.

The immune contexture of pancreatic ductal adenocarcinoma (PDAC) is generally immunosuppressive. A role for immune checkpoint inhibitors (ICIs) in PDAC has only been demonstrated for the rare and hypermutated mismatch repair (MMR) deficient (MMR-d) subtype. Homologous recombination repair (HR) deficient (HR-d) PDAC is more prevalent and may encompass up to 20% of PDAC. Its genomic instability may promote a T-cell mediated anti-tumor response with therapeutic sensitivity to ICIs. To investigate the immunogenicity of HR-d PDAC, we used multiplex immunohistochemistry (IHC) to compare the density and spatial distribution of CD8+ cytotoxic T-cells, FOXP3+ regulatory T-cells (Tregs), and CD68+ tumor-associated macrophages (TAMs) in HR-d versus HR/MMR-intact PDAC. We also evaluated the IHC positivity of programmed death-ligand 1 (PD-L1) across the subgroups. 192 tumors were evaluated and classified as HR/MMR-intact (n=166), HR-d (n=25) or MMR-d (n=1) based on germline testing and tumor molecular hallmarks. Intra-tumoral CD8+ T-cell infiltration was higher in HR-d versus HR/MMR-intact PDAC (p<0.0001), while CD8+ T-cell densities in the peri-tumoral and stromal regions were similar in both groups. HR-d PDAC also displayed increased intra-tumoral FOXP3+ Tregs (p=0.049) and had a higher CD8+:FOXP3+ ratio (p=0.023). CD68+ TAM expression was similar in HR-d and HR/MMR-intact PDAC. Finally, 6 of the 25 HR-d cases showed a PD-L1 Combined Positive Score of >=1, whereas none of the HR/MMR-intact cases met this threshold (p<0.00001). These results provide immunohistochemical evidence for intra-tumoral CD8+ T-cell enrichment and PD-L1 positivity in HR-d PDAC, suggesting that HR-d PDAC may be amenable to ICI treatment strategies.