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BACKGROUNDClinical trials have suggested antitumor activity from PARP inhibition beyond homologous recombination deficiency (HRD). RNASEH2B loss is unrelated to HRD and preclinically sensitizes to PARP inhibition. The current study reports on RNASEH2B protein loss in advanced prostate cancer and its association with RB1 protein loss, clinical outcome, and clonal dynamics during treatment with PARP inhibition in a prospective clinical trial.METHODSWhole tumor biopsies from multiple cohorts of patients with advanced prostate cancer were interrogated using whole-exome sequencing (WES), RNA-Seq (bulk and single nucleus), and IHC for RNASEH2B and RB1. Biopsies from patients treated with olaparib in the TOPARP-A and TOPARP-B clinical trials were used to evaluate RNASEH2B clonal selection during olaparib treatment.RESULTSShallow codeletion of RNASEH2B and adjacent RB1 - colocated at chromosome 13q14 - was common, deep codeletion infrequent, and gene loss associated with lower mRNA expression. In castration-resistant prostate cancer (CRPC) biopsies, RNASEH2B and RB1 mRNA expression correlated, but single nucleus RNA-Seq indicated discordant loss of expression. IHC studies showed that loss of the 2 proteins often occurred independently, arguably due to stochastic second allele loss. Pre- and posttreatment metastatic CRPC (mCRPC) biopsy studies from BRCA1/2 WT tumors, treated on the TOPARP phase II trial, indicated that olaparib eradicated RNASEH2B-loss tumor subclones.CONCLUSIONPARP inhibition may benefit men suffering from mCRPC by eradicating tumor subclones with RNASEH2B loss.TRIAL REGISTRATIONClinicaltrials.gov NCT01682772.FUNDINGAstraZeneca; Cancer Research UK; Medical Research Council; Cancer Research UK; Prostate Cancer UK; Movember Foundation; Prostate Cancer Foundation.

It has been recognized for decades that ERBB signaling is important in prostate cancer, but targeting ERBB receptors as a therapeutic strategy for prostate cancer has been ineffective clinically. However, we show here that membranous HER3 protein is commonly highly expressed in lethal prostate cancer, associating with reduced time to castration resistance (CR) and survival. Multiplex immunofluorescence indicated that the HER3 ligand NRG1 is detectable primarily in tumor-infiltrating myelomonocytic cells in human prostate cancer; this observation was confirmed using single-cell RNA sequencing of human prostate cancer biopsies and murine transgenic prostate cancer models. In castration-resistant prostate cancer (CRPC) patient-derived xenograft organoids with high HER3 expression as well as mouse prostate cancer organoids, recombinant NRG1 enhanced proliferation and survival. Supernatant from murine bone marrow-derived macrophages and myeloid-derived suppressor cells promoted murine prostate cancer organoid growth in vitro , which could be reversed by a neutralizing anti-NRG1 antibody and ERBB inhibition. Targeting HER3, especially with the HER3-directed antibody-drug conjugate U3-1402, exhibited antitumor activity against HER3-expressing prostate cancer. Overall, these data indicate that HER3 is commonly overexpressed in lethal prostate cancer and can be activated by NRG1 secreted by myelomonocytic cells in the tumor microenvironment, supporting HER3-targeted therapeutic strategies for treating HER3-expressing advanced CRPC. SIGNIFICANCE: HER3 is an actionable target in prostate cancer, especially with anti-HER3 immunoconjugates, and targeting HER3 warrants clinical evaluation in prospective trials., (©2021 The Authors; Published by the American Association for Cancer Research.)

PARP inhibitors are approved for treating advanced prostate cancers (APC) with various defective DNA repair genes; however, further studies to clinically qualify predictive biomarkers are warranted. Herein we analyzed TOPARP-B phase II clinical trial samples, evaluating whole-exome and low-pass whole-genome sequencing and IHC and IF assays evaluating ATM and RAD51 foci (testing homologous recombination repair function). BRCA1/2 germline and somatic pathogenic mutations associated with similar benefit from olaparib; greater benefit was observed with homozygous BRCA2 deletion. Biallelic, but not monoallelic, PALB2 deleterious alterations were associated with clinical benefit. In the ATM cohort, loss of ATM protein by IHC was associated with a better outcome. RAD51 foci loss identified tumors with biallelic BRCA and PALB2 alterations while most ATM - and CDK12 -altered APCs had higher RAD51 foci levels. Overall, APCs with homozygous BRCA2 deletion are exceptional responders; PALB2 biallelic loss and loss of ATM IHC expression associated with clinical benefit. SIGNIFICANCE: Not all APCs with DNA repair defects derive similar benefit from PARP inhibition. Most benefit was seen among patients with BRCA2 homozygous deletions, biallelic loss of PALB2 , and loss of ATM protein. Loss of RAD51 foci, evaluating homologous recombination repair function, was found primarily in tumors with biallelic BRCA1/2 and PALB2 alterations. This article is highlighted in the In This Issue feature, p. 2659 ., (©2021 The Authors; Published by the American Association for Cancer Research.)

Background: Deleterious ATM alterations are found in metastatic prostate cancer (PC); PARP inhibition has antitumour activity against this subset, but only some ATM loss PCs respond., Objective: To characterise ATM-deficient lethal PC and to study synthetic lethal therapeutic strategies for this subset., Design, Setting, and Participants: We studied advanced PC biopsies using validated immunohistochemical (IHC) and next-generation sequencing (NGS) assays. In vitro cell line models modified using CRISPR-Cas9 to impair ATM function were generated and used in drug-sensitivity and functional assays, with validation in a patient-derived model., Outcome Measurements and Statistical Analysis: ATM expression by IHC was correlated with clinical outcome using Kaplan-Meier curves and log-rank test; sensitivity to different drug combinations was assessed in the preclinical models., Results and Limitations: Overall, we detected ATM IHC loss in 68/631 (11%) PC patients in at least one biopsy, with synchronous and metachronous intrapatient heterogeneity; 46/71 (65%) biopsies with ATM loss had ATM mutations or deletions by NGS. ATM IHC loss was not associated with worse outcome from advanced disease, but ATM loss was associated with increased genomic instability (NtAI:number of subchromosomal regions with allelic imbalance extending to the telomere, p = 0.005; large-scale transitions, p = 0.05). In vitro, ATM loss PC models were sensitive to ATR inhibition, but had variable sensitivity to PARP inhibition; superior antitumour activity was seen with combined PARP and ATR inhibition in these models., Conclusions: ATM loss in PC is not always detected by targeted NGS, associates with genomic instability, and is most sensitive to combined ATR and PARP inhibition., Patient Summary: Of aggressive prostate cancers, 10% lose the DNA repair gene ATM; this loss may identify a distinct prostate cancer subtype that is most sensitive to the combination of oral drugs targeting PARP and ATR., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Purpose: Cyclin-dependent kinase 12 (CDK12) aberrations have been reported as a biomarker of response to immunotherapy for metastatic castration-resistant prostate cancer (mCRPC). Herein, we characterize CDK12-mutated mCRPC, presenting clinical, genomic, and tumor-infiltrating lymphocyte (TIL) data., Experimental Design: Patients with mCRPC consented to the molecular analyses of diagnostic and mCRPC biopsies. Genomic analyses involved targeted next-generation (MiSeq; Illumina) and exome sequencing (NovaSeq; Illumina). TILs were assessed by validated immunocytochemistry coupled with deep learning-based artificial intelligence analyses including multiplex immunofluorescence assays for CD4, CD8, and FOXP3 evaluating TIL subsets. The control group comprised a randomly selected mCRPC cohort with sequencing and clinical data available., Results: Biopsies from 913 patients underwent targeted sequencing between February 2015 and October 2019. Forty-three patients (4.7%) had tumors with CDK12 alterations. CDK12-altered cancers had distinctive features, with some revealing high chromosomal break numbers in exome sequencing. Biallelic CDK12-aberrant mCRPCs had shorter overall survival from diagnosis than controls [5.1 years (95% confidence interval (CI), 4.0-7.9) vs. 6.4 years (95% CI, 5.7-7.8); hazard ratio (HR), 1.65 (95% CI, 1.07-2.53); P = 0.02]. Median intratumoral CD3 + cell density was higher in CDK12 cancers, although this was not statistically significant (203.7 vs. 86.7 cells/mm 2 ; P = 0.07). This infiltrate primarily comprised of CD4 + FOXP3 - cells (50.5 vs. 6.2 cells/mm 2 ; P < 0.0001), where high counts tended to be associated with worse survival from diagnosis (HR, 1.64; 95% CI, 0.95-2.84; P = 0.077) in the overall population., Conclusions: CDK12-altered mCRPCs have worse prognosis, with these tumors surprisingly being primarily enriched for CD4 + FOXP3 - cells that seem to associate with worse outcome and may be immunosuppressive. See related commentary by Lotan and Antonarakis, p. 380 ., (©2020 American Association for Cancer Research.)

Purpose: Circulating tumor cells (CTCs) have clinical relevance, but their study has been limited by their low frequency. Experimental Design: We evaluated liquid biopsies by apheresis to increase CTC yield from patients suffering from metastatic prostate cancer, allow precise gene copy-number calls, and study disease heterogeneity. Results: Apheresis was well tolerated and allowed the separation of large numbers of CTCs; the average CTC yield from 7.5 mL of peripheral blood was 167 CTCs, whereas the average CTC yield per apheresis (mean volume: 59.5 mL) was 12,546 CTCs. Purified single CTCs could be isolated from apheresis product by FACS sorting; copy-number aberration (CNA) profiles of 185 single CTCs from 14 patients revealed the genomic landscape of lethal prostate cancer and identified complex intrapatient, intercell, genomic heterogeneity missed on bulk biopsy analyses. Conclusions: Apheresis facilitated the capture of large numbers of CTCs noninvasively with minimal morbidity and allowed the deconvolution of intrapatient heterogeneity and clonal evolution. Clin Cancer Res; 24(22); 5635-44. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Purpose: CHD1 deletions and SPOP mutations frequently cooccur in prostate cancer with lower frequencies reported in castration-resistant prostate cancer (CRPC). We monitored CHD1 expression during disease progression and assessed the molecular and clinical characteristics of CHD1 -deleted/ SPOP -mutated metastatic CRPC (mCRPC). Experimental Design: We identified 89 patients with mCRPC who had hormone-naive and castration-resistant tumor samples available: These were analyzed for CHD1, PTEN, and ERG expression by IHC. SPOP status was determined by targeted next-generation sequencing (NGS). We studied the correlations between these biomarkers and (i) overall survival from diagnosis; (ii) overall survival from CRPC; (iii) duration of abiraterone treatment; and (iv) response to abiraterone. Relationship with outcome was analyzed using Cox regression and log-rank analyses. Results: CHD1 protein loss was detected in 11 (15%) and 13 (17%) of hormone-sensitive prostate cancer (HSPC) and CRPC biopsies, respectively. Comparison of CHD1 expression was feasible in 56 matched, same patient HSPC and CRPC biopsies. CHD1 protein status in HSPC and CRPC correlated in 55 of 56 cases (98%). We identified 22 patients with somatic SPOP mutations, with six of these mutations not reported previously in prostate cancer. SPOP mutations and/or CHD1 loss was associated with a higher response rate to abiraterone (SPOP: OR, 14.50 P = 0.001; CHD1: OR, 7.30, P = 0.08) and a longer time on abiraterone (SPOP: HR, 0.37, P = 0.002, CHD1: HR, 0.50, P = 0.06). Conclusions: SPOP -mutated mCRPCs are strongly enriched for CHD1 loss. These tumors appear highly sensitive to abiraterone treatment. Clin Cancer Res; 24(22); 5585-93. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Background: Understanding the integrated immunogenomic landscape of advanced prostate cancer (APC) could impact stratified treatment selection., Methods: Defective mismatch repair (dMMR) status was determined by either loss of mismatch repair protein expression on IHC or microsatellite instability (MSI) by PCR in 127 APC biopsies from 124 patients (Royal Marsden [RMH] cohort); MSI by targeted panel next-generation sequencing (MSINGS) was then evaluated in the same cohort and in 254 APC samples from the Stand Up To Cancer/Prostate Cancer Foundation (SU2C/PCF). Whole exome sequencing (WES) data from this latter cohort were analyzed for pathogenic MMR gene variants, mutational load, and mutational signatures. Transcriptomic data, available for 168 samples, was also performed., Results: Overall, 8.1% of patients in the RMH cohort had some evidence of dMMR, which associated with decreased overall survival. Higher MSINGS scores associated with dMMR, and these APCs were enriched for higher T cell infiltration and PD-L1 protein expression. Exome MSINGS scores strongly correlated with targeted panel MSINGS scores (r = 0.73, P < 0.0001), and higher MSINGS scores associated with dMMR mutational signatures in APC exomes. dMMR mutational signatures also associated with MMR gene mutations and increased immune cell, immune checkpoint, and T cell-associated transcripts. APC with dMMR mutational signatures overexpressed a variety of immune transcripts, including CD200R1, BTLA, PD-L1, PD-L2, ADORA2A, PIK3CG, and TIGIT., Conclusion: These data could impact immune target selection, combination therapeutic strategy selection, and selection of predictive biomarkers for immunotherapy in APC., Funding: We acknowledge funding support from Movember, Prostate Cancer UK, The Prostate Cancer Foundation, SU2C, and Cancer Research UK.

Purpose: Precise detection of copy number aberrations (CNA) from tumor biopsies is critically important to the treatment of metastatic prostate cancer. The use of targeted panel next-generation sequencing (NGS) is inexpensive, high throughput, and easily feasible, allowing single-nucleotide variant calls, but CNA estimation from this remains challenging. Experimental Design: We evaluated CNVkit for CNA identification from amplicon-based targeted NGS in a cohort of 110 fresh castration-resistant prostate cancer biopsies and used capture-based whole-exome sequencing (WES), array comparative genomic hybridization (aCGH), and FISH to explore the viability of this approach. Results: We showed that this method produced highly reproducible CNA results ( r = 0.92), with the use of pooled germline DNA as a coverage reference supporting precise CNA estimation. CNA estimates from targeted NGS were comparable with WES ( r = 0.86) and aCGH ( r = 0.7); for key selected genes ( BRCA2, MYC, PIK3CA, PTEN , and RB1 ), CNA estimation correlated well with WES ( r = 0.91) and aCGH ( r = 0.84) results. The frequency of CNAs in our population was comparable with that previously described (i.e., deep deletions: BRCA2 4.5%; RB1 8.2%; PTEN 15.5%; amplification: AR 45.5%; gain: MYC 31.8%). We also showed, utilizing FISH, that CNA estimation can be impacted by intratumor heterogeneity and demonstrated that tumor microdissection allows NGS to provide more precise CNA estimates. Conclusions: Targeted NGS and CNVkit-based analyses provide a robust, precise, high-throughput, and cost-effective method for CNA estimation for the delivery of more precise patient care. Clin Cancer Res; 23(20); 6070-7. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Biomarkers for more precise patient care are needed in metastatic prostate cancer. We have reported a phase II trial (TOPARP-A) of the PARP inhibitor olaparib in metastatic prostate cancer, demonstrating antitumor activity associating with homologous recombination DNA repair defects. We now report targeted and whole-exome sequencing of serial circulating cell-free DNA (cfDNA) samples collected during this trial. Decreases in cfDNA concentration independently associated with outcome in multivariable analyses (HR for overall survival at week 8: 0.19; 95% CI, 0.06-0.56; P = 0.003). All tumor tissue somatic DNA repair mutations were detectable in cfDNA; allele frequency of somatic mutations decreased selectively in responding patients (χ 2 P < 0.001). At disease progression, following response to olaparib, multiple subclonal aberrations reverting germline and somatic DNA repair mutations ( BRCA2, PALB2 ) back in frame emerged as mechanisms of resistance. These data support the role of liquid biopsies as a predictive, prognostic, response, and resistance biomarker in metastatic prostate cancer. Significance: We report prospectively planned, serial, cfDNA analyses from patients with metastatic prostate cancer treated on an investigator-initiated phase II trial of olaparib. These analyses provide predictive, prognostic, response, and resistance data with "second hit" mutations first detectable at disease progression, suggesting clonal evolution from treatment-selective pressure and platinum resistance. Cancer Discov; 7(9); 1006-17. ©2017 AACR. See related commentary by Domchek, p. 937 See related article by Kondrashova et al., p. 984 See related article by Quigley et al., p. 999 This article is highlighted in the In This Issue feature, p. 920 ., (©2017 American Association for Cancer Research.)

PurposeProstate cancer is the second leading cause of male cancer deaths. Castration-resistant prostate cancer (CRPC) is a lethal stage of the disease that emerges when endocrine therapies are no longer effective at suppressing activity of the androgen receptor (AR) transcription factor. The purpose of this study was to identify genomic mechanisms that contribute to the development and progression of CRPC.Experimental designWe used whole-genome and targeted DNA-sequencing approaches to identify mechanisms underlying CRPC in an aggregate cohort of 272 prostate cancer patients. We analyzed structural rearrangements at the genome-wide level and carried out a detailed structural rearrangement analysis of the AR locus. We used genome engineering to perform experimental modeling of AR gene rearrangements and long-read RNA sequencing to analyze effects on expression of AR and truncated AR variants (AR-V).ResultsAR was among the most frequently rearranged genes in CRPC tumors. AR gene rearrangements promoted expression of diverse AR-V species. AR gene rearrangements occurring in the context of AR amplification correlated with AR overexpression. Cell lines with experimentally derived AR gene rearrangements displayed high expression of tumor-specific AR-Vs and were resistant to endocrine therapies, including the AR antagonist enzalutamide.ConclusionsAR gene rearrangements are an important mechanism of resistance to endocrine therapies in CRPC.