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The genomics of primary prostate cancer differ from those of metastatic castration-resistant prostate cancer (mCRPC). We studied genomic aberrations in primary prostate cancer biopsies from patients who developed mCRPC, also studying matching, same-patient, diagnostic, and mCRPC biopsies following treatment. We profiled 470 treatment-naive prostate cancer diagnostic biopsies and, for 61 cases, mCRPC biopsies, using targeted and low-pass whole-genome sequencing (n = 52). Descriptive statistics were used to summarize mutation and copy number profile. Prevalence was compared using Fisher’s exact test. Survival correlations were studied using log-rank test. TP53 (27%) and PTEN (12%) and DDR gene defects (BRCA2 7%; CDK12 5%; ATM 4%) were commonly detected. TP53, BRCA2, and CDK12 mutations were markedly more common than described in the TCGA cohort. Patients with RB1 loss in the primary tumor had a worse prognosis. Among 61 men with matched hormone-naive and mCRPC biopsies, differences were identified in AR, TP53, RB1, and PI3K/AKT mutational status between same-patient samples. In conclusion, the genomics of diagnostic prostatic biopsies acquired from men who develop mCRPC differ from those of the nonlethal primary prostatic cancers. RB1/TP53/AR aberrations are enriched in later stages, but the prevalence of DDR defects in diagnostic samples is similar to mCRPC.

Background Loss of ataxia telangiectasia mutated (ATM), a key protein regulating DNA repair signaling, has been suggested to increase sensitivity to DNA damaging agents. We conducted a study analyzing the loss of ATM protein expression in colorectal cancer and correlated this with clinical outcomes. Materials and Methods The clinical outcomes data and tumor samples from metastatic colorectal cancer patients referred to the Royal Marsden Hospital Drug Development Unit (United Kingdom) from 2012 to 2016 and providing consent for a molecular characterization study were analyzed. Immunohistochemistry (IHC) slides were assessed by a pathologist for nuclear staining intensity of ATM and semiquantitatively scored. ATM loss was defined as a nuclear H-score of ≤ 10. Results Of 223 colorectal cancer samples, ATM IHC loss was identified in 17 (8%). ATM loss was independent of the RAS and RAF mutational status. ATM loss was associated with superior overall survival after first-line oxaliplatin-based therapy (49 vs. 32 months; hazard ratio [HR], 2.52) but not with irinotecan-based therapy (24 vs. 33 months; HR, 0.72). ATM loss was not prognostic for survival from the diagnosis (50 vs. 44 months; HR, 1.43). Conclusion ATM could be considered a biomarker for the development of novel DNA repair targeting agents and treatment of colorectal cancer.

Pròstata; PARP; Biomarcadors Próstata; PARP; Biomarcadores Prostate; PARP; biomarkers PARP inhibitors are approved for treating advanced prostate cancers (APCs) 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 immunohistochemical 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 BRCA deletion. Biallelic, but not mono-allelic, PALB2 deleterious alterations were associated with clinical benefit. In the ATM cohort, loss of ATM protein by immunohistochemistry associated with better outcome. RAD51 foci loss identified tumors with biallelic BRCA and PALB2 alteration 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 immunohistochemical expression associated with clinical benefit. TOPARP is an investigator-initiated trial; we are grateful for the support and funding from AstraZeneca, and for the study grants from Cancer Research UK (CRUK/11/029; C12540/A12829; C12540/A13230; C12540/A20447). ICR-CTSU also receives program grant funding from Cancer Research UK (Grant number: C1491/A15955, C1491/A25351). We acknowledge research funding for this work from Cancer Research UK, Prostate Cancer UK, the Movember Foundation through the London Movember Centre of Excellence (CEO13_2-002), the Prostate Cancer Foundation, and the UK Department of Health through an Experimental Cancer Medicine Centre (ECMC) grant. Professor Johann de Bono is a National Institute for Health Research (NIHR) Senior Investigator. The views expressed in this article are those of the author(s) and not necessarily those of the NHS, the NIHR, or the Department of Health; research at the Royal Marsden Hospital is supported by a Biomedical Research Centre grant. Pasquale Rescigno was supported by a PCF Young Investigator Award 19YOUN19. The authors affiliated to VHIO acknowledge funding from “La Caixa” Foundation and European Institute of Innovation and Technology/Horizon 2020 (LCF/TR/CC19/52470003), Fundacion FERO and Fundacion Cellex. J. Mateo, A. Llop-Guevara and V. Serra received grants from Fundacion Cientifica AECC (LABAE16020PORTT) and an ERAPERMED2019-215. J. Mateo gratefully acknowledges funding from the European Union’s Horizon 2020 research and innovation program (Marie Skłodowska-Curie grant 837900), Instituto de Salud Carlos III (Grant PI18/01384), Fundación AECC, CRIS Cancer Foundation and the US Department of Defense CDMRP (Impact Award PC170510P1). S. Arce-Gallego Research. Downloaded from cancerdiscovery.aacrjournals.org on July 1, 2021. © 2021 American Association for Cancer Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on May 27, 2021; DOI: 10.1158/2159-8290.CD-21-0007 3 and V. Serra were supported by Instituto de Salud Carlos III (FI19/00280, CPII19/00033).

Background Better blood tests to elucidate the behaviour of metastatic castration-resistant prostate cancer (mCRPC) are urgently needed to drive therapeutic decisions. Plasma cell-free DNA (cfDNA) comprises normal and circulating tumour DNA (ctDNA). Low-pass whole-genome sequencing (lpWGS) of ctDNA can provide information on mCRPC behaviour. Objective To validate and clinically qualify plasma lpWGS for mCRPC. Design, setting, and participants Plasma lpWGS data were obtained for mCRPC patients consenting to optional substudies of two prospective phase 3 trials (FIRSTANA and PROSELICA). In FIRSTANA, chemotherapy-naïve patients were randomised to treatment with docetaxel (75 mg/m2) or cabazitaxel (20 or 25 mg/m2). In PROSELICA, patients previously treated with docetaxel were randomised to 20 or 25 mg/m2 cabazitaxel. lpWGS data were generated from 540 samples from 188 mCRPC patients acquired at four different time points (screening, cycle 1, cycle 4, and end of study). Outcome measurements and statistical analysis lpWGS data for ctDNA were evaluated for prognostic, response, and tumour genomic measures. Associations with response and survival data were determined for tumour fraction. Genomic biomarkers including large-scale transition (LST) scores were explored in the context of prior treatments. Results and limitations Plasma tumour fraction was prognostic for overall survival in univariable and stratified multivariable analyses (hazard ratio 1.75, 95% confidence interval 1.08–2.85; p = 0.024) and offered added value compared to existing biomarkers (C index 0.722 vs 0.709; p = 0.021). Longitudinal changes were associated with drug response. PROSELICA samples were enriched for LSTs (p = 0.029) indicating genomic instability, and this enrichment was associated with prior abiraterone and enzalutamide treatment but not taxane or radiation therapy. Higher LSTs were correlated with losses of RB1/RNASEH2B, independent of BRCA2 loss. Conclusions Plasma lpWGS of ctDNA describes CRPC behaviour, providing prognostic and response data of clinical relevance. The added prognostic value of the ctDNA fraction over established biomarkers should be studied further. Patient summary We studied tumour DNA in blood samples from patients with prostate cancer. We found that levels of tumour DNA in blood were indicative of disease prognosis, and that changes after treatment could be detected. We also observed a “genetic scar” in the results that was associated with certain previous treatments. This test allows an assessment of tumour activity that can complement existing tests, offer insights into drug response, and detect clinically relevant genetic changes., Take Home Message Tumour fraction, independent from cell-free DNA concentration and other clinical variables, is prognostic, with low-pass whole-genome sequencing profiles detecting abiraterone/enzalutamide-induced emerging genomic instability that is likely to be acquired as a result of tumour development.

Inhibició de l’ATR; Resposta de danys a l’ADN; Càncer de pròstata Inhibición de ATR; Respuesta al daño del ADN; Cancer de prostata ATR inhibition; DNA damage response; Prostate cancer 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. We gratefully acknowledge research funding for this work from Cancer Research UK, Prostate Cancer UK, the Movember Foundation through the London Movember Centre of Excellence ( CEO13_2-002 ), the Prostate Cancer Foundation (including Young Investigator Awards to Joaquin Mateo, Pasquale Rescigno, and Adam Sharp), Stand Up To Cancer, and the UK Department of Health through an Experimental Cancer Medicine Centre grant. Professor Johann de Bono is a National Institute for Health Research (NIHR) Senior Investigator; research at the Royal Marsden Hospital is supported by a Biomedical Research Centre grant. Part of this work was also funded by a Deparment of Defense CDMRP Impact Award (W81XWH-18-1-0756) to Joaquin Mateo and by Instituto de Salud Carlos III through Grant FI19/00280 to Sara Arce-Gallego, Grant CP19/00170 to Nicolás Herranz, and Grant PI18/01384 to Joaquin Mateo. The authors affiliated to VHIO acknowledge the “la Caixa” Foundation ( ID 100010434 ) for funding under agreement LCF/PR/PR17/51120011 and funding from Fundacion FERO and Moventia . This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement 837900 . The funding organisations had no role in the design, conduction or data analysis of this project, neither in the manuscript preparation.

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/mm2; P = 0.07). This infiltrate primarily comprised of CD4+FOXP3− cells (50.5 vs. 6.2 cells/mm2; 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

Prostate cancer; Olaparib; Gene aberrations Càncer de pròstata; Olaparib; Aberracions de gens Cáncer de próstata; Olaparib; Aberraciones de genes Background: Metastatic castration-resistant prostate cancer is enriched in DNA damage response (DDR) gene aberrations. The TOPARP-B trial aims to prospectively validate the association between DDR gene aberrations and response to olaparib in metastatic castration-resistant prostate cancer. Methods: In this open-label, investigator-initiated, randomised phase 2 trial following a selection (or pick-the-winner) design, we recruited participants from 17 UK hospitals. Men aged 18 years or older with progressing metastatic castration-resistant prostate cancer previously treated with one or two taxane chemotherapy regimens and with an Eastern Cooperative Oncology Group performance status of 2 or less had tumour biopsies tested with targeted sequencing. Patients with DDR gene aberrations were randomly assigned (1:1) by a computer-generated minimisation method, with balancing for circulating tumour cell count at screening, to receive 400 mg or 300 mg olaparib twice daily, given continuously in 4-week cycles until disease progression or unacceptable toxicity. Neither participants nor investigators were masked to dose allocation. The primary endpoint of confirmed response was defined as a composite of all patients presenting with any of the following outcomes: radiological objective response (as assessed by Response Evaluation Criteria in Solid Tumors 1.1), a decrease in prostate-specific antigen (PSA) of 50% or more (PSA50) from baseline, or conversion of circulating tumour cell count (from ≥5 cells per 7·5 mL blood at baseline to

Background: PARP inhibitors (PARPi) are approved for the treatment of metastatic prostate cancer (mPC) associated to various DNA damage repair (DDR) gene mutations; but clinical benefit differs among patients. Biomarkers of homologous recombination repair (HRR) deficiency may help refine patient stratification for a more precise therapy selection. We report an exploratory analysis from the TOPARP-B phase II clinical trial of olaparib in mPC (NCT01682772), investigating the predictive value of an HRR function assay detecting RAD51 foci by immunofluorescence in tumor biopsies. Design: We analyzed formalin-fixed paraffin-embedded (FFPE) primary or metastatic biopsies from mPC patients treated with olaparib in the clinical trial. We evaluated baseline HRR function based on detection of RAD51 and γH2AX foci in geminin-positive tumor cells by immunofluorescence (IF). All samples were scored by two trained readers blinded to genomic and clinical data. Samples were considered HRR deficient (HRD) when RAD51 scores were low, pre-defined as 40%). The intra-class correlation score (ICC) between the two blinded readers was 0.88. Overall, 22 of 52 (42%) cases were considered as HRD based on low RAD51 scores. Response rate (based on the composite RECIST/PSA/CTC trial criteria) was 15/22 (68%) vs 7/30 (23%) for patients with low vs high RAD51 scores (p=0.001). Patients with low RAD51 scores also had longer rPFS (median 9.3 vs 2.9 months p=0.002) and overall survival (median 17.4 vs 9.5 months, p=0.05) from initiation of olaparib. All 16/16 cases with BRCA1/2 alterations were identified as RAD51 low. For patients with PALB2 mutations, 2/2 patients with biallelic loss showed RAD51 low scores and responded to olaparib, whereas 2/2 patients with monoallelic PALB2 mutations showed RAD51 high scores and did not respond to olaparib. Mutations in ATM and CDK12 did not associate with low RAD51. Indeed, 10/11 ATM-mutated and 8/10 CDK12-mutated tumors presented high RAD51 scores; RECIST/PSA responses were observed in two patients with ATM mutations and high RAD51 scores. Conclusion: A RAD51-based IF assay performed on FFPE biopsies can detect prostate cancers with deficient HRR function, including BRCA1/2 and biallelic PALB2 mutated cases, and may be useful for patient stratification for PARP inhibitor treatment in prostate cancer. Further validation of the assay in larger cohorts is warranted. Citation Format: Sara Arce-Gallego, Alba Llop-Guevara, Suzanne Carreira, Nuria Porta, Roberta Fasani, Diletta Bianchini, George Seed, Pasquale Rescigno, Alec Paschalis, Claudia Bertan, Chloe Baker, Jane Goodall, Susana Miranda, Ruth Riisnaes, Ines Figueiredo, Ana Ferreira, Rita Pereira, Bora Gurel, Daniel Nava Rodrigues, Wei Yuan, Jan Rekowski, Emma Hall, Violeta Serra, Johann S. de Bono, Joaquin Mateo. A homologous recombination repair (HRR) functional assay to stratify patients with metastatic prostate cancer for PARP inhibitor treatment in the TOPARP-B clinical trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT161.

Background: Advanced prostate cancer (PC) is invariably fatal and largely insensitive to established immune checkpoint inhibition (ICI). The immune checkpoint, PD-L1 (B7-H1; CD274), is infrequently overexpressed in PC; whilst other B7 family immunomodulatory glycoprotein, B7-H3 (CD276), is overexpressed in many PCs. B7-H3 targeted immunoconjugates are in clinical development. We therefore studied the longitudinal expression of B7-H3 in PC and its associations with advanced PC immunogenomics. Design: We analysed matching, same-patient, formalin-fixed paraffin-embedded (FFPE) metastatic castration resistant PC (mCRPC) (n=98), and treatment-naïve, castration-sensitive PC (CSPC) biopsies (n=43) from patients treated at The Royal Marsden Hospital (UK). Biopsies were analysed by: targeted next-generation sequencing (NGS) for deleterious DNA repair gene alterations; immunohistochemistry (IHC) for ATM, PTEN, B7-H3 and mismatch repair (MMR) proteins using validated antibodies; and multi-color immunofluorescence (IF) assays for T cell surface markers to determine the density of tumour infiltrating T lymphocyte (TIL) subsets. Wilcoxon signed-rank test and Mann-Whitney U test compared B7-H3 expression across matched and unmatched subsets, respectively. Spearman correlation determined associations between continuous variables. Results: Most CRPC and CSPC biopsies had both membranous B7-H3 (mB7-H3) expression (134/141, 95.0%) and cytoplasmic B7-H3 expression (137/141, 97.2%). Analysis of the 43 matched samples sets showed that there was no significant change in mB7-H3 expression as tumours progressed from CSPC to CRPC (median Histoscore [HS] [range]: 130 [5-300] for CSPC vs. 130 [5-290] for CRPC; p=0.6). There was significant interpatient and intratumor heterogeneity in mB7-H3 expression, but a subset of tumour biopsies had very high mB7-H3 expression (HS ≥200: 28/98 [28.6%] CRPC; 14/43 [32.6%] CSPC). mB7-H3 expression strongly associated with the presence of deleterious alterations of genes involved in homologous recombination (HR) repair (p Conclusion: mB7-H3 is highly expressed in advanced PC, with higher expression associating with BRCA2 and ATM loss of function alterations and low intratumor TILs. B7-H3 may be an actionable target for treating this disease subset. Citation Format: Christina Guo, Ines Figueiredo, Bora Gurel, Matues Crespo, Jan Rekowski, Suzanne Carreira, Antje Neeb, Adam Sharp, Maria D. Fenor de la Maza, Pasquale Rescigno, Khobe Chandran, Ana Ferreira, Ruth Riisnaes, Susana Miranda, Rita Pereira, Veronica Gil, George Seed, Claudia Bertan, Chloe Baker, Wei Yuan, Johann S. de Bono. B7-H3 as a therapeutic target in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB035.

Background In men with castration-sensitive prostate cancer (CSPC), the HSD3B1 c.1245A>C variant has been reported to be associated with shorter responses to first-line androgen-deprivation therapy (ADT). Here, we evaluated the association between the inherited HSD3B1 c.1245A>C variant and outcomes from metastatic castration-resistant prostate cancer (mCRPC) after first-line treatment with abiraterone (Abi) or enzalutamide (Enza). Patients and methods Patients with mCRPC (n = 266) were enrolled from two centers at the time of starting first-line Abi/Enza. Outcomes after Abi/Enza included best prostate-specific antigen (PSA) response, treatment duration, and overall survival (OS). Outcomes after first-line ADT were determined retrospectively, and included treatment duration and OS. As was prespecified, we compared patients with the homozygous variant HSD3B1 genotype (CC genotype) versus the combined group with the heterozygous (AC) and homozygous wild-type (AA) genotypes. Results Among the 266 patients, 22 (8.3%) were homozygous for the HSD3B1 variant (CC). The CC genotype had no association with PSA response rate; the median Abi/Enza treatment duration was 7.1 months for the CC group and 10.3 months for the AA/AC group (log rank P = 0.34). Patients with the CC genotype had significantly worse OS, with median survival at 23.6 months for the CC group and 30.7 months for the AA/AC group (log rank P = 0.02). In multivariable analysis adjusting for age, Gleason score, PSA, prior chemotherapy, and M1 disease, the association between the CC genotype and OS remained significant (hazard ratio 1.78, 95% confidence interval 1.03–3.07, P = 0.04). Poor outcome after first-line ADT in the CC group was also observed when evaluating retrospective ADT duration data for the same combined cohort. Conclusions In this large two-center study evaluating the HSD3B1 c.1245 genotype and outcomes after first-line Abi/Enza, homozygous variant (CC) HSD3B1 genotype was associated with worse outcomes. Novel therapeutic strategies are needed to enable treatment selection based on this genetic marker.

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.

155 Background: Metastatic castration resistant prostate cancer (mCRPC) is a heterogeneous disease in which molecular stratification is needed to improve clinical outcomes. Targeting programmed cell death protein 1 (PD-1) elicits durable antitumor responses in multiple cancer types. Putative biomarkers predictive of response to anti-PD-1 therapies include programmed death-ligand-1 (PD-L1), tumour mutational burden (TMB), T cell-inflamed 18-gene expression profile (GEP), and DNA repair defects. Objective: To study potential biomarkers of response in mCRPC to anti-PD-1 therapy (PD-L1, GEP, mismatch repair (MMR) protein), other biomarkers of interest including BRCA2, PALB2, CDK12, PTEN, TP53, SOX2, and to determine association with clinical outcomes. Methods: The study population included 100 men with mCRPC treated at the Royal Marsden Hospital with available fresh mCRPC biopsy tissue. All men had received at least one line of therapy with a next generation hormonal agent and one line of taxane-based chemotherapy. Clinical characteristics and outcomes were extracted from medical records. mCRPC biopsies were assayed by whole exome sequencing (WES), targeted next generation sequencing (NGS), RNA sequencing (RNAseq), GEP score (Nanostring), PD-L1 immunohistochemistry (IHC; DAKO 22C3 assay), SOX2 IHC, ATM IHC. Correlations among these biomarkers and clinical outcomes were assessed. Results: The median age of patients was 68.0 years; 46/84 (54.8%) had de novo metastatic disease at diagnosis and 24/100 (24.0%) patients had visceral disease. Median follow-up from mCRPC biopsy was 56.2 months. The prevalence of loss of protein expression by IHC and/or pathogenic mutation by NGS of MMR was 7/100 (7%). Loss of PTEN and ATM by IHC was 29/100 (29%) and 13/100 (13%) respectively. SOX2 expression (defined as expression in >5% of cells) was 27/100 (27%). The prevalence of TP53 mutation was 25/100 (25%); deleterious alterations of BRCA2 was 9/100 (9%), CDK12 3/100 (3%) and PALB2 1/100 (1%). PDL1 and GEP results were available for 70 and 93 samples respectively. PD-L1 was expressed (combined positive score ≥1) in 24 (33%) mCRPC biopsies; 23 (26%) had high GEP scores (> -0.318). PD-L1 and GEP expression were positively correlated (Phi 0.63). No other biomarkers showed strong correlations. Of 5 samples with MMR loss for which PD-L1 was available, 1 (20%) had PD-L1 ≥1; one of the CDK12 samples had PD-L1 ≥1 (33%). 0/6 BRCA2 mutated biopsies expressed PD-L1 (0%). Based on univariate analysis, PD-L1 expression [HR: 1.75 (1.00;3.06), p=0.045], high GEP score [HR: 2.00 (1.18;3.39), p=0.0083] and SOX2 expression [HR: 1.81 (1.12;2.94), p=0.015] were associated with worse overall survival (OS). No other biomarkers associated with OS. Conclusions: PD-L1 IHC expression was detected in 33% of mCRPC patients and associated with high GEP score. Higher PD-L1, GEP, and SOX2 expression were associated with poor prognosis.

Early-phase cancer clinical trials are becoming increasingly accessible for patients with advanced cancer who have exhausted standard treatment options and later phase trial options. Many of these trials mandate research tissue biopsies. Research biopsies have been perceived as ethically fraught due to the perception of potential coercion of vulnerable human subjects. We performed an audit of two years of practice to assess the safety of ultrasound (US)-guided research biopsies, and to look at the yield of a simultaneous tumour next-generation sequencing (NGS) and immunohistochemistry (IHC) molecular characterisation programme. We show that in our institution, US-guided research biopsies were safe, produced adequate tumour content and in a selected subset who underwent in-house NGS sequencing, showed a high rate of actionable mutations with 30% having a Tier 1 variant. Nevertheless, these research biopsies may only provide direct benefit for a minority of patients and we conclude with a reflection on the importance of obtaining truly informed consent.

Background: Methylation is reported to support cancer immune tolerance. We conducted a phase 1 dose-escalation trial [NCT02998567] of combination guadecitabine (G; DNA hypomethylating agent) and pembrolizumab (P) in patients (pts) with advanced cancers. We hypothesized that G can normalize the expression of epigenetically suppressed immune genes, increase interferon producing tumor-infiltrating lymphocytes (TILs), and enhance the anticancer activity of P. Methods: In dose escalation (Es), pts received G (45 mg/m2 or 30 mg/m2, administered SC on days 1-4) with P (200 mg, administered IV starting from cycle 2 onwards) as outpatient Q3W; in expansion (Ex), the RP2D of G (30 mg/m2) with P (200 mg) Q3W was administered. Pre-treatment and on-treatment tumor biopsies were evaluated for PD-L1 expression, tumor infiltrating lymphocytes, gene expression by RNAseq and methylome studies. Longitudinal analyses of peripheral blood CD3, CD4 and CD8 lymphocytes by flow cytometry were performed. Results: Overall, 34 pts (Es, n = 14; Ex, n = 20) were evaluable for safety. The most common treatment-related adverse events (TRAEs) were neutropenia (n = 21), fatigue (n = 6) and thrombocytopenia (n = 3), diarrhea (n = 2). G3+ TRAEs were neutropenia (n = 14), febrile neutropenia (n = 4), raised ALP (n = 1), raised AST (n=1), colitis (n = 1), diarrhoea (n = 1) and lung infection (n = 1). Two DLTs (neutropenia, febrile neutropenia) were reported at G 45mg/m2 with none reported at G 30mg/m2. There were no treatment-related deaths. In total, 28 pts (Es, n = 12; Ex, n = 16) were evaluable for antitumor activity studies (≥2 scans); ORR (CR+PR) and DCR (CR+PR+SD) were 3% and 57%; 10/15 pts with non-small cell lung cancer (13 pts resistant/refractory to PD-1/PD-L1 targeting agents) were evaluable, with a DCR of 80% and 5 pts having DCR > 6 months with 8 pts remaining on study treatment. Overall, 25 paired biopsies were obtained. Using LINE1 sequences to study global methylation, both tumor biopsies and peripheral blood showed reduced methylation post-G treatment. Preliminary data on tumor-infiltrating lymphocytes assessed by multicolor immunofluorescence in 9 paired biopsies showed a numerical increase in median values of T-helper (CD4+FOXP3-) (10.20 to 19.70, p = 0.5469), T-regulatory (CD4+FOXP3+) (5.1 to 6.7, p=0.8438), and T-cytotoxic (CD8+) cell densities (2.7 to 7.4, p=0.6523) . Comparing with matched pre-treatment, on treatment tumor had numerical increases in interferon alpha and gamma response pathway activation in serial biopsy RNAseq analyses but did not reach significance. Conclusions: G plus P resulted in no unexpected toxicities with evidence suggestive of biological and anti-cancer activity. Citation Format: Dionysis Papadatos-Pastos, Abhijit Pal, Melek Akay, Malaka Ameratunga, Sanjena Mithra, Joo-Ern Ang, Sofia Levva, Reece Caldwell, Ruth Riisnaes, Mateus Crespo, Wei Yuan, George Seed, Bora Gurel, Ines Figueiredo, Rita Pereira, Susana Miranda, Anna Ferreira, Suzanne Carreira, Claudia Bertan, Chloe Baker, Ricardo Morilla, Robert Brown, Nahal Masrour, Toby Prout, Anna Zachariou, Alison Turner, Mona Parmar, Mark Van de Velde, Ben Jenkins, Christina Yap, Nina Tunariu, Udai Banerji, Juanita Lopez, Anna Minchom, Johann De Bono. HyPeR: A phase 1, dose escalation and expansion trial of guadecitabine (SGI-110), a second-generation hypomethylating agent in combination with pembrolizumab (MK3475) in patients with refractory solid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT129.

Background: We, and others, have shown that responses to therapies in metastatic castration-resistant prostate cancer (mCRPC) can be monitored using plasma cell-free DNA (cfDNA), and that this can be both quantitative and qualitative. In this study, we explored the utility of low pass whole genome sequencing (lpWGS) of cfDNA in mCRPC patients treated on two large prospectively collected Phase III trials of taxane chemotherapy, FIRSTANA (NCT01308567) and PROSELICA (NCT01308580). Methods: Plasma samples collated from the FIRSTANA and PROSELICA trials were evaluated. cfDNA was isolated and analysed with lpWGS using the Illumina NovaSeq 6000. Reads were aligned using BWA-MEM and quality controlled using Picard and FASTQC. Whole-genome copy number aberration (CNA) profiles were generated using ichorCNA, which also estimated tumor purity and ploidy. Log-rank tests were used for univariable survival comparisons, and Cox proportional hazard models were used for multivariable survival analysis. Results: Overall, lp-WGS data was generated from 528 samples acquired at three different timepoints (Baseline [SCR and C1], C4 and EOS), representing 188 unique patients. We observed CNA profiles in line with emergent mCRPC subtypes including highly aberrant, large-scale events which have been linked to homologous-repair deficient tumors. We found that CNAs in this cohort matched other published studies with frequent amplifications of AR (~30%), copy-gains of PI3KCA (~45%) and copy-loss of NKX3-1 (~75%), RB1 (~70%) and PTEN (~50%). We did not observe changes in baseline tumor purity or ploidy between the FIRSTANA and PROSELICA cohorts. Following calculation of a large-scale transition (LST) score (measuring CNA breakpoints), we observed that this was significantly elevated (Wilcox test, p=0.0073) in cases that had prior exposure to second line androgen deprivation (abiraterone or enzalutamide). Baseline tumor purity values were associated with several mCRPC clinical variables including ECOG performance status. High lpWGS tumor purity values were univariably associated with poorer overall survival in both FIRSTANA and PROSELICA cohorts (log-rank test p=0.0021 and 0.0009), and were also associated with significantly poorer RPFS and PSAPFS. Tumor purity was also associated (HR 1.66, p=0.036) with survival when assessed using multivariable models alongside other mCRPC clinical variables, including cell-free DNA concentration. Further, longitudinal changes in tumor purity on treatment can be illustrative of drug responses. Conclusions: In our analysis of this prospectively collected clinical trial data, we show mCRPC CNA profiles in cfDNA, and that these CNAs may be induced by prior aggressive treatments. We also found that tumor purity estimation can serve as a robust, competitive indicator of both overall survival and, assessed longitudinally, shows clear association with drug response. We envision that these techniques will enable improved strategies for monitoring mCRPC patients. Citation Format: George Seed, Semini Sumanasuriya, Claudia Bertan, Harry Parr, Gemma Fowler, Rossitza Christova, Lorna Pope, Jane Goodall, Maryou Lambros, Pasquale Rescigno, Penelope Flohr, Suzanne Carreira, Wei Yuan, Mustapha Chadjaa, Sandrine Mace, Johann De Bono. Discovery of genomic correlates and tumor purity as an independent clinical factor of poor outcome in advanced prostate cancer lpWGS CNA data [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-270.

Background: Genomic analyses describing the molecular landscape of primary prostate cancer (PCa) and metastatic castration-resistant prostate cancer (mCRPC) have identified recurrent CDK12 alterations in 2-4% of primary PCa and 4-11% of mCRPC. These aberrations, as well as high lymphocyte tumor infiltration, could be clinically relevant as putative biomarkers of response to immunotherapies. However, there are few studies looking into whether CDK12 biallelic mutant cases are immune ‘hot'. Here, we describe a cohort of patients with CDK12 aberration and studied the tumor infiltration of those cancers. Methods: Patients with mCRPC and available diagnostic archival and/or CRPC tumor samples were evaluated. Mutation analysis involved custom designed targeted sequencing on MiSeq sequencer. Loss of heterozygosity (LOH) was assessed for the CDK12 cases from exome sequencing using ASCAT, which include single copy lost or copy neutral LOH. Tumor infiltrating lymphocytes were assessed initially with CD3 IHC, using a deep learning-based AI analysis approach. We then subtyped the TILs using a multiplex IF approach, classifying lymphocytes using CD4, CD8 and FOXP3 positivity. Results: Overall 913 samples (between Feb/15 and Oct/19) were sequenced by targeted NGS, 42 patients presented with pathogenic alterations in CDK12 (4.6%), 27 had bi-allelic alterations, 14 mono-allelic, 1 likely biallelic. In these cases, we identified 39 frameshift alterations, 10 missense mutations mainly involving the kinase domain; with 5 having concomitant LOH. One case showed deep deletion of CDK12 and five presented with additional aberrations in other DNA repair related genes. CDK12 biallelic alterations were present in all 3 cases with both diagnostic and matched mCRPC samples available. We studied T cell infiltration in 100 (23 CDK12 biallelic alterations, 7 monoallelic, 70 controls) samples selected from within the initial targeted NGS cohort. Median intratumoral CD3+ cell density was significantly higher in CDK12 biallelic loss samples compared to matched controls in diagnostic biopsies (271.4 vs 104.683 cells/mm2, p=0.026). A similar trend was seen in mCRPC samples (142.130 vs 51.75 cells/mm2, p=0.36). Intratumoral CD4+ cell infiltration was again significantly higher in CDK12 biallelic loss samples compared to controls (98.864 vs 6.188 cells/mm2, p=0.014). Conclusions: In our analysis we show that a proportion of mCRPC patients harbor defects in CDK12, these are often alterations involving both alleles, likely present from the time of diagnosis. A majority of these CDK12 altered cancers has high CD3 infiltration compared to controls. We envision that these CDK12 aberrant ‘hot' tumors could represent a subset of mCRPC likely to respond to immune-checkpoint inhibition. Citation Format: Bora Gurel, Pasquale Rescigno, Wei Yuan, Rita Pereira, Mateus Crespo, Mattia Rediti, Ines Figueiredo, Maialen Barrero, Diletta Bianchini, Maria D. Fenor de la Maza, Khobe Chandran, Juliet Carmichael, Alec Paschalis, Adam Sharp, George Seed, Ruth Riisnaes, Claudia Bertan, Suzanne Carreira, Johann S. De Bono. CDK12-mutated lethal prostate cancers: How hot are these tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-075.

BackgroundThis case represents an exceptional response to pembrolizumab in a patient with epithelioid mesothelioma with a further response on rechallenge.Case presentationA 77-year-old woman with advanced epithelioid mesothelioma extensively pretreated with chemotherapy demonstrated a prolonged response of 45 months to 52 cycles of pembrolizumab. On rechallenge with pembrolizumab, further disease stability was achieved. Serial biopsies and analysis by immunohistochemistry and immunofluorescence demonstrated marked immune infiltration and documented the emergency of markers of immune exhaustion. Whole exome sequencing demonstrated a reduction in tumor mutational burden consistent with subclone elimination by immune checkpoint inhibitor (CPI) therapy. The relapse biopsy had missense mutation in BTN2A1.ConclusionThis case supports rechallenge of programme death receptor 1 inhibitor in cases of previous CPI sensitivity and gives molecular insights.

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.

Background Metastatic CC prognosis remains poor. Molecular characterisation (MC) allows better understanding of the mutations (mut) driving carcinogenesis or treatment resistance. We report the cervical tumour MC of pts in a P1 unit and its clinical implications. Methods CC pts referred to the P1 unit of The Royal Marsden Hospital from 2011-18 underwent tumour molecular profiling using OncoCartaTM (Sequenom), TruSeq® Cancer Amplicon (Illumina), GeneReadTM custom DNA damage (Qiagen) or FoundationOne® panel. Retrospective data was extracted from electronic medical records. Results Of the 37 pts (median age: 34.7 years) analysed, 45.9% had squamous histology, and 37.8% were FIGO stage 2 at diagnosis; 34 different pathogenic mut were identified with 70.3% of pts having mut. PIK3CA mut were the most common (32.4%), followed by KRAS (13.5%), APC (8.1%), TP53 (8.1%), PTEN (5.4%), KDR (5.4%), ATM (5.4%), STK11 (5.4%) and BRCA1 (5.4%). 40.5% (15/37) patients had a potentially actionable mutation. Median overall survival (mOS) was 36 months (mo) in study population, 33 mo in KRAS mut, 40 mo in PIK3CA mut, 52 mo in APC mut and 40 mo in the mut-negative group; however, this difference was not statistically significant. In the KRAS mut group, 60% were adenocarcinoma and 40% adenosquamous. All KRAS mut pts had FIGO stage 2/3 at diagnosis. 80% had visceral metastasis when referred to the P1 unit; whereas in the PIK3CA mut group, tumour histologies were 33.3% squamous, 25% adenocarcinoma, 25% adenosquamous, 8.3% mucinous, 8.3% uncommon histologies. In the PIK3CA mut group, 75% were stage 1/2 at diagnosis, and 41.6% had visceral metastasis when referred. 5.4% (2/37) of pts were allocated to a P1 trial based on the finding of a PIK3CA mut with a mOS of 40 mo compared to 36 mo for the total population (p = 0.6). One pt received an AKT inhibitor (i) but developed progressive disease (PD) after 3 cycles; the other received a PI3K i with PD after 4 cycles. Conclusions Our study corroborates existing knowledge of the association between KRAS mut and adenocarcinoma histology in CC which confers worse prognosis. The high rate of potentially actionable mut indicates the increasing scope for targeted treatment trials in CC, thus prompting early MC which may improve pt allocation to P1 trials. Legal entity responsible for the study The Institute of Cancer Research and The Royal Marsden Foundation Trust. Funding The Institute of Cancer Research and The Royal Marsden Foundation Trust. Disclosure S. Banerjee: Honoraria (self), Research grant / Funding (institution): AstraZeneca; Honoraria (self), Honoraria/reimbursement: Tesaro; Honoraria (self), Honoraria/reimbursement: Clovis; Honoraria (self), Honoraria/reimbursement: Merck Serono; Honoraria (self), Honoraria/reimbursement: Nucana; Honoraria (self), Honoraria/reimbursement: Immunogen; Honoraria (self), Honoraria/reimbursement: Seattle Genetics; Honoraria (self), Honoraria/reimbursement: Roche; Honoraria (self), Honoraria/reimbursement: Gamamabs. J.S. de Bono: Honoraria (self), Non-remunerated activity/ies: Astellas Pharma; Honoraria (self), Research grant / Funding (institution), Non-remunerated activity/ies: AstraZeneca; Honoraria (self): Genentech/Roche; Honoraria (self): Pfizer; Honoraria (self), Non-remunerated activity/ies: Sanofi; Honoraria (self): Bayer; Honoraria (self): Boehringer Ingelheim; Honoraria (self): Merck Serono; Honoraria (self): Merck Sharp & Dohme; Non-remunerated activity/ies: Genmab; Non-remunerated activity/ies: GlaxoSmithKline; Non-remunerated activity/ies: Orion Pharma GmbH; Non-remunerated activity/ies: Qiagen; Non-remunerated activity/ies: Taiho Pharmaceutical; Non-remunerated activity/ies: Vertex; Licensing / Royalties, Royalties paid to Institution, no personal income: Abiraterone Rewards to Inventors; Licensing / Royalties, Royalties paid to Institution, no personal income: PARP inhibitors and DNA repair defects. U. Banerji: Honoraria (self), Precision Medicine - Advanced Prostate Cancer Meeting, London UK -30/11/2018: Astellas; Honoraria (self), Advisory / Consultancy, Translational Clinical Oncology Advisory Board, London, 24/07/2018; FIH Precision Oncology SAB, Frankfurt, 10-11/11/2015: Novartis; Advisory / Consultancy, Clinical Advisory Board, London, 19/03/2018 and 12/02/2015: Karus Therapeutics; Advisory / Consultancy, GI Advisory Board, London, 2/12/2017: Phoenix ACT; Honoraria (self), European Digestive Oncology Research Forum, London, 27-28/11/2017: Eli Lilly; Honoraria (self), KOL ERK Workshop, New Orleans, 17/04/2016; HSP90 Workshop, Washington DC,8/03/2014: Astex; Honoraria (self), AUY922 meeting, New Jersey, USA,12/02/2015: Vernalis; Research grant / Funding (institution), ONX-0801:Funding for investigator-initiated Phase I trial: Onyx Pharmaceuticals; Research grant / Funding (institution), ONX-0801:top-up funding for investigator-initiated phase I trial: BTG International; Research grant / Funding (institution), RAF/MEK: funding for investigator-initiated phase I trial: Chugai; Research grant / Funding (institution), TAX-TORC: funding for investigator-initiated phase I trial: AstraZeneca; Research grant / Funding (institution), FRAME: funding for investigator-initiated phase I trial: Verastem. A. Minchom: Travel / Accommodation / Expenses: Loxo; Advisory / Consultancy: Janssen; Honoraria (self): Faron; Speaker Bureau / Expert testimony: Bayer Media Event. J. Lopez: Advisory / Consultancy: Genmab; Advisory / Consultancy: Novartis; Research grant / Funding (institution), Travel / Accommodation / Expenses: Roche/Genentech; Research grant / Funding (institution), Travel / Accommodation / Expenses: Basilea. All other authors have declared no conflicts of interest.

5002 Background: Prostate-specific membrane antigen (PSMA) is a promising target for theranostics in metastatic castration resistant prostate cancer (mCRPC). Methods: Membranous PSMA (mPSMA) expression was immunohistochemically evaluated in castration sensitive (CSPC) (n = 38) and mCRPC (n = 60) tissue biopsies, and associations with molecular aberrations (next-generation sequencing; NGS) and clinical outcome were determined. Results: mPSMA expression was significantly higher (p = 0.005) in mCRPC biopsies (median H-score [interquartile range]; 55.0 [2.8-117.5]) compared to CSPC biopsies (17.5 [0.0-60.0]). Furthermore, patients with higher mPSMA expression ( > median H-score) at diagnosis had higher Gleason Grade (p = 0.04) and shorter OS (p = 0.006). Critically, 42% (16/38) of CSPC biopsies and 27% (16/60) of mCRPC biopsies were completely negative for mPSMA expression. In addition, CSPC and mCRPC biopsies expressing mPSMA demonstrated marked intra-tumor heterogeneity in expression levels, commonly exhibiting areas without detectable PSMA (CSPC – 100%; mCRPC – 84%), while heterogeneous mPSMA expression between metastases from the same patient was also observed. Subsequent genomic analysis showed that mCRPC patients with deleterious DNA damage repair (DDR) aberrations have higher (p = 0.016) mPSMA expression (87.5 [25.0-247.5]) than those without these (20 [0.3-98.8]). Furthermore, 9 of the 11 patients (82%) responding to PARP inhibition had a mPSMA H-Score above the median. The association between mPSMA expression and DDR aberrations was validated in an independent cohort with known DDR aberrations. Tumors with DDR aberrations had significantly higher mPSMA (ATM 212.5 [136.3-300] p = 0.005; BRCA2 300 [165-300] p = < 0.001) than unselected mCRPC biopsies (55.0 [2.75-117.5]). Finally, analyses of 122 mCRPC biopsy transcriptomes confirmed a negative correlation between PSMA and BRCA2 mRNA expression (p = 1.5x10-5). Conclusions: mPSMA expression in CSPC and mCRPC exhibits marked intra- and inter-patient heterogeneity, limiting the clinical utility of PSMA-targeted theranostics. We show for the first time that DDR gene aberrations associate with high mPSMA expression and may serve as predictive biomarkers for PSMA-targeted therapies.

5005 Background: We previously reported the antitumor activity of olaparib (400mg BID) against molecularly unselected mCRPC (TOPARP-A; Mateo et al NEJM 2015). We now report TOPARP-B, a phase II trial for patients with mCRPC preselected for putatively pathogenic DDR alterations. Methods: Patients with mCRPC progressing after ≥ 1 taxane chemotherapy underwent targeted sequencing of tumor biopsies and were deemed eligible when alterations (germline or somatic; mono- or bi-allelic) in any DDR gene were detected. Patients were randomized 1:1 under a “pick-the-winner” design to 400mg or 300mg of olaparib BID, aiming to exclude ≤30% response rate (RR) in either arm. The primary endpoint RR was defined as radiological response (RECIST 1.1) and/or PSA50% fall and/or CTC count conversion (Cellsearch; ≥5 to < 5), confirmed after 4-weeks. Analyses of RR per gene alteration subgroup was pre-planned. Secondary endpoints included progression-free survival (PFS), tolerability. Results: Overall, 98 patients (median age 67.6y) were randomized, with 92 patients treated and evaluable for the primary endpoint (70 RECIST-evaluable; 89 PSA50%-evaluable; 55 CTC-evaluable). All had progressed on ADT; 99% were post-docetaxel, 90% post-abiraterone/enzalutamide, 38% post-cabazitaxel. The overall RR was 54% (95%CI 39-69%, meeting threshold for primary endpoint) in the 400mg cohort and 37% (95%CI 23-53%) in the 300mg cohort. With a median follow-up of 17.6 months (mo), the overall median PFS (mPFS) was 5.4 mo. Subgroup analyses per altered gene identified indicated response rates for: BRCA1/2 of 80% (24/30; mPFS 8.1mo); PALB2 57% (4/7; mPFS 5.3mo); ATM 37% (7/19; mPFS 6.1mo); CDK12 25% (5/20; mPFS 2.9mo); others [ATRX, CHEK1, CHEK2, FANCA, FANCF, FANCG, FANCI, FANCM, RAD50, WRN] 20% (4/20; mPFS 2.8mo). The highest PSA50% response rates were observed in the BRCA1/2 (22/30; 73%) and PALB2 (4/6; 67%) subgroups. Conclusions: Olaparib has antitumor activity against heavily pre-treated mCRPC with DDR gene defects, with BRCA1/2 aberrant tumors being most sensitive but with confirmed responses in patients with other DDR alterations. Clinical trial information: NCT01682772.

Few data are available outlining outcomes of panitumumab in advanced colorectal cancer patients benefiting from prior cetuximab-based regimens.Thirty patients with KRAS wild type metastatic colorectal cancer with clinical benefit from prior cetuximab-based regimens between May 2004 and October 2011 were reviewed at nine Italian Institutions. Inclusion key criteria included interruption of cetuximab for reasons other than progressive disease. Patients were classified according to prior regimens (0 or ≥1), prior response or stabilization, surgery of metastases, and Köhne prognostic score. At the time of subsequent progression, patients were treated with single agent panitumumab until progressive disease, unacceptable toxicity, or consent withdrawal.Panitumumab obtained 67% disease control rate and 30% objective response rate, with median PFS of 4.2 and median OS of 9.6 mo. Patients with BRAF/NRAS/PI3KCA and KRAS (by mutant enriched technique) wild-type tumors had the best chance of response to panitumumab.Single agent panitumumab provided significant clinical benefit in heavily pretreated patients without acquired resistance to prior cetuximab-based regimens.

Liquid biopsy components from blood, such as cell free DNA (cfDNA) and circulating tumor cells (CTCs), are prognostic for overall survival in advanced prostate cancer patients and allow the study of clonal evolution. cfDNA is easily obtained and has been widely used for molecular characterization and reflects pooled genomic profiles in a patient, but has limitations regarding gene copy number calls. CTC single-cell genomic studies generate precise gene copy number calls and elucidate intrapatient intercellular genomic heterogeneity. The main limitation of CTC analyses has been the low CTC count found in many cancer patients. We elected to study whether liquid biopsy by apheresis in advanced prostate cancer patients increases the yield of CTC to study tumor genomics, intrapatient heterogeneity, and ex vivo organotypic 3D models. Advanced metastatic prostate cancer patients being considered for clinical trials were invited to consent to apheresis. Apheresis CTC counts using CellSearchTM (Menarini) were acquired from 16 patients. The contents of the CellSearch cartridges were sorted into pure single cells by fluorescence-activated cell sorting and subsequently assessed by array comparative genomic hybridization (aCGH, Agilent Technology) for copy number aberrations (CNA). Exome and aCGH from tissue biopsies were compared to the single cell aCGH results. We generated patient-derived organoid (PDOs) cultures from apheresis products by preenrichment using density gradient (Lymphoprep) and subsequent CTC enrichment by EpCAM positive selection (EasySep StemCell Technologies). PDOs were characterized by immunofluorescence (IF) as DAPI+/CK+/EpCAM+ and CD45- cells and subsequently by aCGH for CNA. All sixteen patients (median age of 70 years; range 60-77 years) tolerated apheresis without any adverse effects. CTC counts from peripheral blood (PB) prior to apheresis ranged from 13 to 711 (median = 96), and did not significantly change post apheresis. The estimated CTC yield per apheresis ranged from 660-35473 per apheresis product (median = 3351). This constitutes an increase of 102-fold when compared to median CTC capture from 7.5mL of PB. A total of 170 single CTCs from 15 apheresis patients were genomically profiled and the copy number aberration profiles confirmed prostate cancer with multiple genomic hallmarks including CNAs such as AR amplification, chromosome 8q gain (MYC locus), and PTEN, RB1, BRCA2, TP53, CHD1 loss. CNA profiles of PDOs showed similar genomic aberrations to same patient CTCs and also reflected intrapatient heterogeneity detected by single CTC analysis. In conclusion, apheresis from advanced prostate cancer patients is a well-tolerated procedure and in our study increased the CTC yield by 102-fold when compared to PB. CTC and PDOs from apheresis products shared similar CNA profile compared with tissues biopsies and furthermore gave us an insight of the tumor heterogeneity and clonal evolution. Citation Format: Maryou B.K. Lambros, Veronica Gil, Mateus Crespo, Mariane S. Fontes, Alan Mackay, Gemma Folwer, Gemma Folwer, Berni Ebbs, Rui Neves, Penny Flohr, Susana Miranda, Semini Sumanasuriya, Daniel N. Rodrigues, Rita Pereira, Geroge Seed, Wei Yuan, Joanne Hunt, Deirdre Moloney, Dionne Ayanda, Niven Mehra, Jane Goodall, Claudia Bertan, Suzanne Carreira, Nikolas H. Stoecklein, Leon W.M.M. Terstappen, Gunther Boysen, Joahnn S. De Bono. Liquid biopsy by apheresis: Molecular characterization of circulating tumor cells and their organoid culture reflects intrapatient heterogeneity and clonal evolution [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A051.

5013Background: mCRPC is enriched for genomic aberrations in TP53, RB1, AR, PTEN and DNA damage repair (DDR) compared to localized prostate cancer. Here we pursued NGS of 470 primary prostate tumor...

5070Background: Cyclin-dependent kinase 12 (CDK12) mutations have been described in lethal prostate cancer (PCa) and are reported to impact DNA repair including homologous recombination repair, and...

218 Background: CRPC is enriched for germline mutations in DNA damage repair genes (gDDRm). BRCA2 mutations (g BRCA2m) associate with poor prognosis from localized PC, but prognostic and predictive value for standard therapy in CRPC is unclear. We reviewed clinical outcome of 390 patients previously tested for gDDRm. Methods: Patient records were reviewed for 372 patients from 3 institutions (Royal Marsden UK, Weill-Cornell NY, University of Washington, WA) with gDDRm status previously published (Pritchard et al, NEJM 2016) and 18 g BRCA1/2m carriers from KConFab consortium (Australia). Baseline characteristics and survival were annotated. Response (PSA50%/RECIST) and PFS (RECIST/PSA progression or start of a new therapy due to clinical progression) were collected for Abiraterone, Enzalutamide and Docetaxel. To account for potential differences between cohorts, a mixed effect model (Weibull distribution) with random intercept per cohort was pursued. Results: dDDRm status was available for n = 390 (60 gDDRm+, including 37 g BRCA2m, and 330 gDDRm-). Overall, 74% and 69% received Docetaxel and Abiraterone/Enzalutamide respectively; 47% gDDRm+ and 34% gDDRm- received PARPi and/or platinum. Median overall survival from CRPC was 3.0 vs 3.2 years in gDDRm+ vs gDDRm- (p = 0.73; g BRCA2m = 3.0 years, p = 0.72). Age and Gleason score at diagnosis were associated with survival from castration-resistance in multivariate analysis. Median PFS on Docetaxel for gDDRm+ (6.8 months; 6.3 for g BRCA2m) and gDDRm- (5.1 months) were not significantly different (p = 0.2). Similarly, RR to Docetaxel was similar for the two groups (61% vs 54% in gDDRm+ vs gDDRm-; 63% g BRCA2m). Median PFS and RR on first Abiraterone/Enzalutamide were similar across groups (PFS: 8.3 months gDDRm+, 8.3 months for gDDRm-; p = 0.9; RR 46% vs 56% respectively) The Interaction of PARPi/platinum therapy among gDDRm+ patients resulted in an aHR for OS from CRPC of 0.59 (95%CI 0.28-1.25; p = 0.17). Conclusions: In this retrospective analysis, CRPC patients with gDDRm still benefited from standard therapies similarly to non-mutation carriers; interpretation of survival data should consider the high proportion treated with PARPi/platinum.

1 Unit of Medical Statistics, Biometry and Bioinformatics, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 2 Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 3 Molecular Genetics of Cancer, Fondazione Istituto FIRC di Oncologia Molecolare, Milan, Italy 4 Department of Pathology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 5 Scientific Directorate, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy

INTRODUCTION: A subset of prostate cancers (PCs) has a hypermutated phenotype. We hypothesized that lethal PCs with a high mutational load (ML) have DNA repair defects including mismatch-repair (MMR) aberrations and that such aberrations may impact outcome. METHODS: Diagnostic and metastatic castration resistant prostate cancer (mCRPC) tissues were tested by A) immunohistochemistry (IHC) for MSH2, MSH6, MLH1, and PMS2; B) microsatellite instability (MSI) (Promega v1.2 assay); C) targeted next generation sequencing (NGS) of the coding regions of 113 genes, a panel enriched for DNA-repair genes that includes MMR genes. Utilizing NGS data we also evaluated ML and MSI by NGS (MSINGS; Pritchard et al, 2014). IHC was used as reference for sensitivity (SE) and specificity (SP) of methods B) and C).Youden’s index was used to determine the optimal cut off for the ROC for IHC in relation to MSINGS and ML. Demographics and clinical data were retrospectively collected from electronic records. Relationship between ML in diagnostic and mCRPC samples was evaluated by paired t-test. The relationship between ML and MMR deficient tumors on IHC (MMRd) and/or MSI tumors by PCR (MMRd/MSI) was analyzed by negative binomial regression model. The relationship between MMRd/MSI tumors and overall survival (OS) was analyzed using univariate and multivariate Cox regression, adjusting for radical treatment (prostatectomy or radiotherapy), Gleason score, age, PSA and stage and presence of metastatic disease at diagnosis. RESULTS: We analyzed 306 PC biopsies (180 hormone-sensitive [HS] and 126 castration resistant [CR]) from 208 patients. Overall 16 patients (7.7%) had either MMRd or MSI. Matched, same-patient, HSPC and mCRPC tumor biopsies were available for 82 patients. Only one case with MMRd status in HS was not confirmed in matched mCRPC. This patient was considered MMR proficient for the survival analysis. MSI had a SE of 80% and a SP of 97%. An MSI by NGS cut-off of 0.096 had SE of 60% and SP of 99%, with an area under ROC curve of 0.83. A ML cut-off of 11 mutations per panel had SE of 60% and SP of 99%, with an area under ROC curve of 0.79. Although ML in diagnostic samples correlates with ML in mCRPC, ML was significantly higher in mCRPC MMRd/MSI cases (p CONCLUSION: We provide evidence that MMRd/MSI status represents a negative prognostic factor for survival for metastatic CRPC. MMRd/MSI status in diagnostic biopsies accurately identifies MMRd/MSI in mCRPC. MSINGS and ML can detect MMRd/MSI tumors. Citation Format: Pasquale Rescigno, Daniel Nava Rodrigues, Wei Yuan, Suzanne Carreira, Maryou Lambros, George Seed, Ruth Riisnaes, Susana Miranda, David Dolling, Matthew Clarke, Mateus Crespo, Claudia Bertan, Gunther Boysen, Joaquin Mateo, Ana Ferreira, Adam Sharp, Ines Figueiredo, Semini Sumanasuriya, Mariane Sousa Fontes, Diletta Bianchini, Zafeiris Zafeiriou, Johann Sebastian de Bono. Mismatch repair defects in lethal prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4679. doi:10.1158/1538-7445.AM2017-4679

There is conflicting evidence on the predictive role of KRAS status when cetuximab is added to oxaliplatin-based regimens. This study investigated the impact of KRAS, NRAS, BRAF, PI3KCA and TP53 status on outcome of elderly metastatic colorectal cancer patients enrolled in TEGAFOX-E (cetuximab, oxaliplatin and oral uracil/ftorafur—UFT) phase II study. Twenty-eight patients were enrolled and all were evaluable for safety and activity. Twenty-three specimens were analysed for KRAS, BRAF, NRAS, PI3KCA and TP53 mutational status by means of polymerase chain reaction and correlated with objective response, progression-free survival and overall survival. An evident increase of response rate was noted in KRAS/NRAS wild-type cases (70 versus 33 %, P = 0.198). KRAS/NRAS wild-type status showed an independent association with a longer progression-free survival (44 versus 9 weeks, P = 0.009). Considering the combined assessment of BRAF, KRAS/NRAS and TP53, a trend towards an increase of response rate was noted in patients without mutations (83 versus 33 %, P = 0.063). Moreover, patients with all wild-type genes had significantly longer progression-free survival than patients with any mutation (48 versus 10 weeks, P = 0.007). As a single biomarker, only KRAS/NRAS proteins maintained an independent value for outcome prediction. Patients with KRAS/NRAS, BRAF and TP53 wild-type tumours could derive the maximal benefits from treatment with cetuximab, oxaliplatin and UFT.

Metformin, a widely used and well-tolerated antidiabetic drug, may reduce cancer risk and improve prognosis of certain malignancies. In vitro and in vivo studies on several cancer models have evaluated the potential antitumorigenic effects of metformin and many clinical trials are looking to its use for suppressing tumor growth. However, the mechanism for its anti-cancer effect remains uncertain and needs to be clarified. Here we investigated the anti-cancer activity of metformin on colorectal cancer growth by treating three colorectal cancer cell lines, HT29, HCT116, HCT116p53-/-, with the drug. We found that, at a dose of 5mM, metformin reduced cell proliferation in all the colorectal cancer cells analyzed, as shown by BrdU incorporation assays. Metformin induced cell cycle arrest in G0/G1 phase that was accompanied by a strong decrease of cyclin D1, c-Myc expression, and inhibition of pRB phosphorylation. The antiproliferative activity of the drug resulted mediated by suppression of mTOR and IGF-1/AKT pathways. In particular, we observed inactivation of mTOR and of its downstream targets S6 and 4EBP1, both in an AMPK dependent and independent way, and also downregulation of the activity of IGF1R. We analysed the biological mechanisms by which metformin inhibited cell proliferation by evaluating its ability to induce apoptosis, autophagy or senescence, as observed in other cancer cell lines. Unexpectedly, we found that metformin did not stimulate any of these mechanisms in colorectal cancer cells. In support of these results, a colony formation assay showed that metformin did not arrest growth of cells, but only slowed down cell ability of forming colonies. Our findings highlight that metformin inhibits the proliferation of colorectal cancer cell lines as a consequence of promoting cell cycle arrest in the G0/G1 phase, but does not induce cell death. Further investigations are needed to better elucidate the mechanisms altered by the drug in colorectal cancer and caution should be used when treating cancer patients with metformin. Citation Format: Angela Mogavero, Maria Valeria Maiorana, Claudia Bertan, Fabio Bozzi, Marco A. Pierotti, Manuela Gariboldi. Metformin has an inhibitory effect on cell proliferation but does not induce death in colorectal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1161. doi:10.1158/1538-7445.AM2015-1161

202 Background: Codon 12 KRAS and BRAF mutations may have a role in the mucinous differentiation pathway in colorectal cancer (Bazan V et al, Ann Oncol 2002; Tran et al, Cancer 2011). Elucidating the molecular mechanisms driving this disease is a priority for PMP, with the goal of identifying new markers and therapeutic targets needed to improve disease management. Due to the fact that neoplastic cells may be deceptively bland and sparse histologically within an abundant mucinous background, the adoption of high-sensitivity gene sequencing techniques may be extremely helpful. Methods: Mucinous tumors from 10 PMP cases of appendiceal origin treated in a multidisciplinary team at the National Cancer Institute of Milan were histologically classified according to Bradley classification. Following microdissection and DNA extraction, mutational analysis of KRAS exon 2-4 and BRAF exon 15 were performed by means of PCR, while KRAS exon 2 status was further confirmed through a specific mutant enriched PCR, shown to be a more sensitive approach in colorectal cancer (Molinari et al, Clin Cancer Res 2011). Results: Histological classification was low-grade in 6 and high-grade in 4 tumors. Codon 12 KRAS mutations were detected in 5/10 cases (50%). In the remaining 5 cases: sequencing of exon 4 lead to identification of a first-described A146V mutation in 1 case; with mutant enriched technique, we identified 3 additional codon 12 mutated cases. Due to use of high sensibility techniques and gene sequencing outside hotspot regions, we were able to identify KRAS mutations in 9/10 (90%) specimens. No BRAF exon 15 mutation was detected. Conclusions: The prognostic role of KRAS mutation in PMP patients is under investigation on a larger data-set of National Cancer Institute of Milan and will be presented at the Meeting. However, these preliminary data suggest a major role of KRAS in the carcinogenesis of mucinous tumors of appendiceal origin with PMP.

529 Background: Pathologic response of HCRM to neoadjuvant tx is associated to better outcome. In retrospective analyses Bev-based tx improved tumor regression grade (TRG), while cetuximab (Cmab) increased RECIST response and R0 resections in prospective studies. The aim of this analysis was to assess the effect of Bev vs Cmab-based tx in terms of HCRM pathological response and outcome. Methods: Fifty-eight pts with HCRM were resected at National cancer Institute of Milan from 2007 to 2012 following Bev (n=30) or Cmab (n=28) based neoadjuvant tx. Pathological response was classified from TRG 1 for pathological complete response (pCR), to TRG5 for no response; TRG 1-3, for histological response; TRG 4-5 for scarce/no response (Rubbia-Brandt L, Ann Oncol 2007). Results: KRAS missing in 9 pts (3 Bev/6 Cmab), mutated in 18 pts (60%) in Bev vs 4 (14%) in Cmab group.Median number of resected metastases: 4.6 vs 3.9; mean TRG: 2.9 (2.2-4) in Bev vs 3.8 (3-5) in Cmab group (p=0.04 at Kruskal-Wallis test). Pathological response rate was higher for Bev vs Cmab (70% vs 39%; p=0.037 at Chi-square test). Bev also increased pCRs (20% vs 0%, p=0.024 at Fisher’s test). With generalized estimating equation for all resected nodules, mean TRG was lower in Bev vs Cmab group: 2.6 (2-4) vs 3.7 (3-4), p