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Background: The androgen receptor (AR) pathway-associated gene nuclear receptor coactivator 2 (NCOA2) has an established oncogenic role in early prostate cancer and likewise is a driver of metastatic disease and castration-resistant prostate cancer. However, its significance as a biomarker in metastatic castration-resistant prostate cancer (mCRPC), both alone and in conjunction with co-occurring AR alterations using a liquid biopsy approach has not been investigated., Methods: Ninety-one patients were included in this study, (n = 68 receiving an androgen receptor pathway inhibitor and n = 23 receiving taxane chemotherapy). Up to 30 ml of peripheral blood was collected before commencing treatment from each patient. Plasma cell-free DNA, along with a matched germline sample, underwent targeted next-generation sequencing using a validated, highly sensitive in-house prostate cancer panel. Variants in AR and NCOA2 were identified and correlated with clinical outcomes., Results: Plasma AR and NCOA2 aberrations were identified in 35% and 13% of the cohort, respectively, whilst 8% had concurrent AR and NCOA2 alterations. NCOA2 copy number gain and any NCOA2 aberration predicted for lower prostate-specific antigen (PSA) response rates. Likewise, median overall survival was shorter for NCOA2 gain (10.1 vs. 18.3 months; p = .004), remaining significant after adjusting for covariates including circulating tumor DNA fraction and tumor suppressor gene alterations. Importantly, dual AR and NCOA2 aberrations were also associated with inferior outcomes, including no PSA responses in patients treated with AR pathway inhibitors (0% vs. 64%; p = .02)., Conclusions: These data highlight the importance of identifying multiple markers of AR pathway modulation in mCRPC and represent the first instance of the assessment of plasma NCOA2 status as a prognostic biomarker for standard-of-care therapies. Further assessment is warranted to determine if NCOA2 aberrations are a marker of primary resistance to AR pathway inhibitors., (© 2021 Wiley Periodicals LLC.)

Antiseizure medications (ASMs) are frequently implicated in T cell-mediated drug hypersensitivity reactions and cause skin tropic pathologies that range in severity from mild rashes to life-threatening systemic syndromes. During the acute stages of the more severe manifestations of these reactions, drug responsive proinflammatory CD8 + T cells display classical features of Th1 cytokine production ( e.g. IFNγ) and cytolysis ( e.g. granzyme B, perforin). These T cells may be found locally at the site of pathology ( e.g. blister cells/fluid), as well as systemically ( e.g. blood, organs). What is less understood are the long-lived immunological effects of the memory T cell pool following T cell-mediated drug hypersensitivity reactions. In this study, we examine the ASM carbamazepine (CBZ) and the CBZ-reactive memory T cell pool in patients who have a history of either Stevens-Johnson syndrome (SJS) or toxic epidermal necrolysis (TEN) from 3-to-20 years following their initial adverse reaction. We show that in vitro drug restimulation of CBZ-reactive CD8 + T cells results in a proinflammatory profile and produces a mainly focused, yet private, T cell receptor (TCR) usage amongst human leukocyte antigen (HLA)-B*15:02-positive SJS or TEN patients. Additionally, we show that expression of these CBZ-reactive TCRs in a reporter cell line, lacking endogenous αβTCR, recapitulates the features of TCR activation reported for ASM-treated T cell lines/clones, providing a useful tool for further functional validations. Finally, we conduct a comprehensive evaluation of the HLA-B*15:02 immunopeptidome following ASM (or a metabolite) treatment of a HLA-B*15:02-positive B-lymphoblastoid cell line (C1R.B*15:02) and minor perturbation of the peptide repertoire. Collectively, this study shows that the CBZ-reactive T cells characterized require both the drug and HLA-B*15:02 for activation and that reactivation of memory T cells from blood results in a focused private TCR profile in patients with resolved disease., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Mifsud, Illing, Lai, Fettke, Hensen, Huang, Rossjohn, Vivian, Kwan and Purcell.)

Tumor tissue from metastatic castration-resistant prostate cancer (mCRPC) harbors frequent copy number variations (CNVs) in the PTEN-PI3K-AKT pathway. However, identifying CNVs in plasma cell-free DNA (cfDNA) has proven to be challenging. With emerging data supporting Akt inhibition in PTEN-deficient mCRPC, we profiled PTEN-PI3K-AKT pathway aberrations in patients with mCRPC using a novel cfDNA assay optimized for CNV detection., Methods: A next-generation sequencing-based cfDNA assay was used to profile 231 patients with mCRPC from two independent cohorts (Australian, n = 78; United States, n = 153). PTEN-PI3K-AKT pathway genomic aberrations were correlated with clinical outcomes, including progression-free survival and overall survival (OS)., Results: PTEN loss and PIK3CA gain were detected in 37% (85 of 231) and 17% (39 of 231) of patients, respectively. Poorer outcomes were observed in patients with PTEN-PI3K-AKT pathway aberrations, including those with dual PTEN loss and PIK3CA gain (hazard ratio 2.3, 95% CI 1.2 to 4.4). Cumulative CNV burden in the PTEN-PI3K-AKT and androgen receptor (AR) pathways was associated with significantly worse clinical outcomes (0 v 1 v ≥ 2 CNVs in Australian cohort: median OS 33.5 v 17.2 v 9.7 months, P < .001; 0 v 1 v ≥ 2 CNVs in US cohort: median OS 35.5 v 14.3 v 9.2 months, P < .001). Notably, 21% (31 of 146) of PTEN -neutral patients harbored alternative PTEN-PI3K-AKT pathway aberrations., Conclusion: PTEN-PI3K-AKT pathway CNVs were readily detected using our cfDNA assay, with the prevalence of PTEN loss comparable with tissue-based studies. Additional PTEN-PI3K-AKT pathway aberrations were found in one fifth of PTEN -neutral cases. Concurrent CNVs in the PTEN-PI3K-AKT and AR pathways portended poor survival, and identifying this high-risk patient subset for dual AR/Akt inhibition may optimize precision treatment with Akt inhibitors in mCRPC., Competing Interests: Arun A. Azad Honoraria: Janssen, Astellas Pharma, Novartis, Tolmar, Amgen, Pfizer, Bayer, Telix Pharmaceuticals, Bristol-Myers Squibb, Merck Serono, AstraZeneca, Sanofi, Ipsen, Merck Sharp & Dohme, Noxopharm Consulting or Advisory Role: Astellas Pharma, Novartis, Janssen, Sanofi, AstraZeneca, Pfizer, Bristol-Myers Squibb, Tolmar, Telix Pharmaceuticals, Merck Sharp & Dohme, Bayer, Ipsen, Merck Serono, Amgen, Noxopharm Speakers' Bureau: Astellas Pharma, Novartis, Amgen, Bayer, Janssen, Ipsen, Bristol-Myers Squibb, Merck Serono Research Funding: Astellas Pharma, Merck Serono, Novartis, Pfizer, Bristol-Myers Squibb, Sanofi, AstraZeneca, GlaxoSmithKline, Aptevo Therapeutics, MedImmune, Bionomics, Synthorx, AstraZeneca, Astellas Pharma, Ipsen, Merck Serono Travel, Accommodations, Expenses: Astellas Pharma, Sanofi, Merck Serono, Amgen, Janssen, Tolmar, Pfizer No other potential conflicts of interest were reported.Arun A. Azad Honoraria: Janssen, Astellas Pharma, Novartis, Tolmar, Amgen, Pfizer, Bayer, Telix Pharmaceuticals, Bristol-Myers Squibb, Merck Serono, AstraZeneca, Sanofi, Ipsen, Merck Sharp & Dohme, Noxopharm Consulting or Advisory Role: Astellas Pharma, Novartis, Janssen, Sanofi, AstraZeneca, Pfizer, Bristol-Myers Squibb, Tolmar, Telix Pharmaceuticals, Merck Sharp & Dohme, Bayer, Ipsen, Merck Serono, Amgen, Noxopharm Speakers' Bureau: Astellas Pharma, Novartis, Amgen, Bayer, Janssen, Ipsen, Bristol-Myers Squibb, Merck Serono Research Funding: Astellas Pharma, Merck Serono, Novartis, Pfizer, Bristol-Myers Squibb, Sanofi, AstraZeneca, GlaxoSmithKline, Aptevo Therapeutics, MedImmune, Bionomics, Synthorx, AstraZeneca, Astellas Pharma, Ipsen, Merck Serono Travel, Accommodations, Expenses: Astellas Pharma, Sanofi, Merck Serono, Amgen, Janssen, Tolmar, Pfizer No other potential conflicts of interest were reported., (© 2021 by American Society of Clinical Oncology.)

Background: As potent systemic therapies transition earlier in the prostate cancer disease course, molecular biomarkers are needed to guide optimal treatment selection for metastatic hormone-sensitive prostate cancer (mHSPC). The value of whole blood RNA to detect candidate biomarkers in mHSPC remains largely undefined., Methods: In this cohort study, we used a previously optimised whole blood reverse transcription polymerase chain reaction assay to assess the prognostic utility [measured by seven-month undetectable prostate-specific antigen (PSA) and time to castration-resistance (TTCR)] of eight prostate cancer-associated gene transcripts in 43 mHSPC patients. Transcripts with statistically significant associations (P<0.05) were further investigated in a metastatic castration-resistant prostate cancer (mCRPC) cohort (n=119) receiving contemporary systemic therapy, exploring associations with PSA >50% response (PSA 50 ), progression-free survival (PFS) and overall survival (OS). Clinical outcomes were prospectively collected in a protected digital database. Kaplan-Meier estimates and multivariable Cox proportional-hazards models assessed associations between gene transcripts and clinical outcomes (mHSPC covariates: disease volume, docetaxel use and haemoglobin level; mCRPC covariates: prior exposure to chemotherapy or ARPIs, haemoglobin, performance status and presence of visceral disease). Follow-up was performed monthly during ARPI treatment, three-weekly during taxane chemotherapy, and three-monthly during androgen deprivation therapy (ADT) monotherapy. Serial PSA measurements were performed before each follow-up visit and repeat imaging was at the discretion of the investigator., Results: Detection of circulating Grainyhead-like 2 ( GRHL2 ) transcript was associated with poor outcomes in mHSPC and mCRPC patients. Detectable GRHL2 expression in mHSPC was associated with a lower rate of seven-month undetectable PSA levels (25% vs. 65%, P=0.059), and independently associated with shorter TTCR (HR 7.3, 95% CI: 1.5-36, P=0.01). In the mCRPC cohort, GRHL2 expression predicted significantly lower PSA 50 response rates (46% vs. 69%, P=0.01), and was independently associated with shorter PFS (HR 3.1, 95% CI: 1.8-5.2, P<0.001) and OS (HR 2.9, 95% CI: 1.6-5.1, P<0.001). Associations were most apparent in patients receiving ARPIs., Conclusions: Detectable circulating GRHL2 was a negative prognostic biomarker in our mHSPC and mCRPC cohorts. These data support further investigation of GRHL2 as a candidate prognostic biomarker in metastatic prostate cancer, in addition to expanding efforts to better understand a putative role in therapeutic resistance to AR targeted therapies., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/tau-20-1444). TT serves as an unpaid editorial board member of Translational Andrology and Urology from Aug 2019 to Jul 2021. EMK reports receiving honoraria from Janssen and Ipsen; travel & accommodation from Astellas Pharma, Pfizer and Ipsen; and institutional research funding from Astellas Pharma, AstraZeneca, Bristol Myers Squibb, Pfizer, and Merck Serono. AAA reports receiving compensation as a Consultant from Astellas Pharma, Janssen, and Novartis; speakers bureau for Astellas, Janssen, Novartis, Amgen, Ipsen, Bristol Myers Squibb, Merck Serono and Bayer; honoraria from Astellas, Novartis, Sanofi, AstraZeneca, Tolmar, Telix; Merck Serono; Janssen, Bristol Myers Squibb, Ipsen, Bayer, Pfizer, Amgen, Noxopharm, and Merck Sharpe Dome; research funding from Astellas (investigator), Merck Serono (investigator), Astra Zeneca (investigator), Bristol Myers Squibb (institutional), Astra Zeneca (institutional), Aptevo Therapeutics (institutional), Glaxo Smith Kline (institutional), Pfizer (institutional), MedImmune (institutional), Astellas (institutional), SYNthorx (institutional), Bionomics (institutional), Sanofi Aventis (institutional), Novartis (institutional), and Ipsen (institutional); travel and accommodation from Astellas, Merck Serono, Amgen, Novartis, Janssen, Tolmar, Pfizer;and is on the Scientific Advisory Board for Astellas, Novartis, Sanofi, AstraZeneca, Tolmar, Pfizer, Telix; Merck Serono; Janssen, Bristol Myers Squibb, Ipsen, Bayer, Merck Sharpe Dome, Amgen and Noxopharm. The other authors have no conflicts of interest to declare., (2021 Translational Andrology and Urology. All rights reserved.)

Background: The treatment paradigm for metastatic castration-resistant prostate cancer (mCRPC) has evolved significantly in recent years. Identifying predictive and/or prognostic biomarkers in the context of this rapidly expanding therapeutic armamentarium remains a pressing and unmet clinical need., Objective: To develop a prognostic whole-blood gene signature for mCRPC patients., Design, Setting, and Participants: As part of an ongoing prospective, multicentre biomarker research study (Australian Prostate Biomarker Alliance), we enrolled 115 mCRPC patients commencing chemotherapy (n = 34) or androgen receptor (AR) pathway inhibitors therapy (n = 81) and obtained pretreatment whole-blood samples in PAXgene RNA tubes. Gene expression was assessed using reverse transcription-polymerase chain reaction., Outcome Measurements and Statistical Analysis: Gene transcripts correlating with overall survival (OS) at p < 0.10 in univariate Cox regression models were incorporated into a multigene signature. Kaplan-Meier survival estimates and multivariate analyses were used to assess association with clinical outcomes. Prognostic strength of the signature was estimated using a concordance probability estimate (CPE)., Results and Limitations: Based on univariate analysis for OS, the following genes were incorporated into a multigene signature: AR splice variant 7 (AR-V7), and three androgen-regulated genes: GRHL2, HOXB13, and FOXA1. The number of positive transcripts clearly stratified survival outcomes (median OS: not reached vs 24.8 mo vs 16.2 mo for 0, 1, and ≥2 transcripts, respectively; p = 0.0052). Notably, this multigene signature retained prognostic significance on multivariable analysis (hazard ratio, 2.1; 95% confidence interval, 1.1-4.0; p = 0.019). Moreover, CPE for this model was 0.78, indicating strong discriminative capacity. Limitations include short follow-up time., Conclusions: Our data demonstrate the prognostic utility of a novel whole-blood AR-based signature in mCRPC patients commencing contemporary systemic therapies. Our pragmatic assay requires minimal processing, can be performed in most hospital laboratories, and could represent a key prognostic tool for risk stratification in mCRPC., Patient Summary: We found that expression of certain genes associated with the androgen receptor could help determine how long men with advanced prostate cancer survive after starting modern drug therapies., (Copyright © 2019 European Association of Urology. Published by Elsevier B.V. All rights reserved.)

Background: The androgen receptor (AR) remains a critical driver in metastatic castration-resistant prostate cancer (mCRPC). Profiling AR aberrations in both circulating DNA and RNA may identify key predictive and/or prognostic biomarkers in the context of contemporary systemic therapy., Objective: To profile AR aberrations in circulating nucleic acids and correlate with clinical outcomes., Design, Setting, and Participants: We prospectively enrolled 67 mCRPC patients commencing AR pathway inhibitors (ARPIs; n = 41) or taxane chemotherapy (n = 26). Using a first-in-class next-generation sequencing-based assay, we performed integrated cell-free DNA (cfDNA) and cell-free RNA (cfRNA) profiling from a single 10 ml blood tube., Outcome Measurements and Statistical Analysis: Kaplan-Meier survival estimates and multivariable Cox regression analyses were used to assess associations between clinical outcomes and the following AR aberrations: copy number variation, splice variants (AR-V7 and AR-V9) and somatic mutations., Results and Limitations: Cell-free DNA and cfRNA were successfully sequenced in 67 (100%) and 59 (88%) patients, respectively. Thirty-six (54%) patients had one or more AR aberrations. AR gain and cumulative number of AR aberrations were independently associated with clinical/radiographic progression-free survival (PFS; hazard ratio [HR] 3.2, p = 0.01 and HR 3.0 for 0 vs ≥2, p = 0.04) and overall survival (HR 2.8, p = 0.04 and HR 2.9 for 0 vs ≥2, p = 0.03). Notably, concurrent AR gain and AR splice variant expression (AR gain/AR-V+) was associated with shorter prostate-specific antigen PFS on both ARPIs (HR 6.7, p = 0.009) and chemotherapy (HR 3.9, p = 0.04). Importantly, key findings were validated in an independent cohort of mCRPC patients (n = 40), including shorter OS in AR gain/AR-V+ disease (HR 3.3, p = 0.02). Limitations include sample size and follow-up period., Conclusions: We demonstrate the utility of a novel, multianalyte liquid biopsy assay capable of simultaneously detecting AR alterations in cfDNA and cfRNA. Concurrent profiling of cfDNA and cfRNA may provide vital insights into disease biology and resistance mechanisms in mCRPC., Patient Summary: In this study of men with advanced prostate cancer, DNA and RNA abnormalities in the androgen receptor detected in blood were associated with poor outcomes on available drug treatments. This information could be used to better guide treatment of advanced prostate cancer., (Copyright © 2020 European Association of Urology. Published by Elsevier B.V. All rights reserved.)

Plasma circulating tumor DNA (ctDNA) analysis has emerged as a minimally invasive means to perform molecular tumor typing. Here we developed a custom ultra-sensitive ctDNA next-generation sequencing assay using molecular barcoding technology and off-the-shelf reagents combined with bioinformatics tools for enhanced ctDNA analysis. Assay performance was assessed via a spike-in experiment and the technique was applied to analyze 41 plasma samples from men with advanced prostate cancer. Orthogonal validation was performed using a commercial assay. Sensitivity and specificity of 93 and 99.5% were recorded for ultra-rare somatic variants (<1%), with high concordance observed between the in-house and commercial assays. The optimized protocol dramatically improved the efficiency of the assay and enabled the detection of low-frequency somatic variants from plasma cell-free DNA (cfDNA).

Background: Metastatic prostate cancer is a clonally heterogeneous disease state characterized by progressive somatic perturbations. The aim of this study was to identify cell free DNA- (cfDNA-) based alterations and their associations with outcomes in progressive metastatic prostate cancer., Methods: In this longitudinal prospective cohort study plasma cfDNA/circulating tumor DNA (ctDNA) was analyzed before, during, and after androgen deprivation therapy (ADT) in 4 independent patient groups ranging from untreated metastatic hormone sensitive prostate cancer (mHSPC) to metastatic castrate resistant prostate cancer (mCRPC). Next generation sequencing was performed on ctDNA and germline DNA to characterize alterations and associations with clinical outcomes were determined for each group., Findings: cfDNA yields were different in progressive mHSPC and mCRPC states (P < .001). In mHSPC, a higher than median ctDNA fraction was predictive of shorter time to ADT failure (HR, 2.29 [95% CI, 1.13-4.65]; Log-Rank P  =  .02). cfDNA, ctDNA taken with volume of metastatic disease in mHSPC and with alkaline phosphatase levels prognosticated survival better than clinical factors alone in mHSPC and mCRPC states (Log Rank P = 0.03). ctDNA-based AR, APC mutations were increased in mCRPC compared to mHSPC (P < ·05).TP53 mutations, RB1 loss, and AR gene amplifications correlated with poorer survival in mCRPC. Mutations in multiple DNA repair genes (ATM, BRCA1, BRCA2, CHEK2) were associated with time to ADT treatment failure and survival in mHSPC., Interpretation: ctDNA fraction can further refine clinical prognostic factors in metastatic prostate cancer. Somatic ctDNA alterations have potential prognostic, predictive, and therapeutic implications in metastatic prostate cancer management., Funding: Several funding sources have supported this study. A full list is provided in the Acknowledgments. No funding was received from Predicine, Inc. during the conduct of the study., Competing Interests: Declaration of Competing Interest Manish Kohli received travel/accommodation from Celgene; Tiantian Zheng, Amy Wang, Carlos Montesinos, Calven Wong, Pan Du, Shidong Jia, and Jianjun Yu are stockholders in Predicine, Inc.; Lisa Horvath received research funding from Astellas Pharma, travel/accommodation from Astellas Pharma and Pfizer, and is on the Scientific Advisory Board for Imagion; Kate Mahon received travel/accommodation from Astellas Pharma; Edmond M Kwan received honoraria from Janssen, research funding from Astellas Pharma and AstraZeneca, and travel /accommodations from Astellas Pharma, Pfizer, and Ipsen; Arun A Azad is a consultant for Astellas Pharma, Janssen, and Novartis, is on the speakers bureau for Astellas Pharma, Janssen, Novartis and Amgen received honoraria from Astellas Pharma, Janssen, Novartis, Tolmar, Amgen, Pfizer, and Telix, is on the Scientific Advisory Board for Astellas Pharma, Novartis, Sanofi, AstraZeneca, Tolmar, Pfizer, and Telix, and received research funding from Astellas Pharma, and Merck Serono., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: The field of liquid biopsies in oncology is rapidly expanding, with the application of cell-free circulating tumour DNA (ctDNA) showing promise in this era of precision medicine. Compared with traditional clinical and radiographic tumour monitoring methods, the analysis of ctDNA provides a minimally-invasive and technically feasible approach to assess temporal and spatial molecular evolutions of the tumour landscape. The constantly advancing technological platforms available for ctDNA extraction and analysis allow greater analytical sensitivities than ever before. The potential translational impact of ctDNA as a blood-based biomarker for the identification, characterization and monitoring of cancer has been demonstrated in numerous proof-of-concept studies, with ctDNA analysis beginning to be applied clinically across multiple facets of oncology., Conclusions: In this review we discuss the biology, recent advancements, technical considerations and clinical implications of ctDNA in the context of cancer, and highlight important challenges and future directions for the integration of ctDNA into standardised patient care.