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Total plasma cell-free DNA (cfDNA) levels were recently shown to be prognostic in two large phase III trials of taxane chemotherapy in metastatic castration-resistant prostate cancer (mCRPC). However, whether cfDNA concentration is predictive of treatment outcomes with androgen receptor pathway inhibitors (ARPIs) is unknown. We quantified plasma cfDNA levels at baseline (n = 74) and 4 weeks on treatment (n = 56) in a prospective cohort of mCRPC patients treated with the ARPIs abiraterone acetate or enzalutamide. Elevated total cfDNA concentration (log10) at both baseline (hazard ratio [HR] 5.5, p < 0.001) and week 4 (HR 7.5, p < 0.001) was a significant negative prognostic factor for overall survival (OS), a finding maintained after adjustment for plasma circulating tumour DNA fraction. Unexpectedly, a rise in cfDNA concentration from baseline to week 4 was also associated with significantly improved OS (HR 0.14, p = 0.003). Conversely, patients with >=29.8% decrease in cfDNA from baseline (optimal cut-point) had significantly shorter median OS than the rest of the cohort (10.5 vs 25.7 mo, p = 0.03). Collectively, our findings point to the potential prognostic utility of quantifying cfDNA in mCRPC and in particular suggest that dynamic changes in total cfDNA levels may be a novel early predictive biomarker for therapeutic outcome in ARPI-treated patients. Patient Summary: We measured the levels of total cell-free DNA (cfDNA) in the plasma of patients with metastatic prostate cancer prior to and 4 weeks after starting new hormonal drugs. We found that patients with higher levels of cfDNA or a higher proportion of tumour-derived DNA at baseline had worse outcomes on hormonal therapies. Similarly, higher levels of cfDNA at 4 weeks into therapy were also associated with worse outcomes. However, a rise in total cfDNA levels at 4 weeks compared with baseline was linked with better outcomes. Measuring changes in cfDNA concentration may be a useful and technically straightf

Background: The prognostic significance of DDR alterations in mCRPC remains unclear, with conflicting data from prior reports. Whether DDR alterations are predictive of outcomes with therapeutic agents other than PARP inhibitors in mCRPC is also poorly understood. With increasing use of molecular profiling in mCRPC, understanding the full prognostic and predictive utility of plasma DDR alterations is paramount. Method(s): A next-generation sequencing Predicine liquid biopsy assay was used to profile cfDNA and germline DNA in 407 mCRPC patients (pts) from two independent international cohorts (n=162 Australia, n=245 US). DDR genes profiled were BRCA2, ATM, BRCA1, MLH1 and MSH2. Kaplan-Meier survival estimates and multivariable Cox regression analyses were used to assess associations between DDR alterations and clinical outcomes including PSA response rate (PSA RR), progression-free survival and overall survival (OS). Result(s): Median age was 74 (IQR 67-79), with median follow up 74 months and median OS 23 months. 65/407 (16%) harboured pathogenic DDR alterations, including 21 patients with 1 alteration. Frequency of genomic aberrations are shown in the table. BRCA2 alterations were further classified as heterozygous loss (66%), homozygous loss (14%), monoallelic mutation (11%) and biallelic mutation/loss of heterozygosity (9%). Aberrations in any DDR gene, ATM, MLH1 + MSH2 or BRCA2 were associated with shorter OS on univariable analysis, but only any DDR or BRCA2 aberration remained significant upon adjusting for clinical prognosticators and ctDNA fraction (Table). Pre-treatment BRCA2 aberration was associated with significantly shorter OS and lower PSA RR compared to BRCA2 wt for pts receiving an AR pathway inhibitor (ARPI) (18 vs 32 months, p=0.006; 36 vs 60%, p=0.04 respectively) but not for taxane chemotherapy (17 vs 20 months, p=0.3; 45 vs 66, p=0.1 respectively). Conclusion(s): Detection of an aberration in any DDR gene or BRCA2 was an independent poor prognos

Background: With the evolving use of targeted therapies exploiting genomic vulnerabilities in mCRPC, screening patients for sensitizing alterations is of increasing relevance. However, the influence of age on the detection of relevant genomic alterations is incompletely understood. In this study, we compared the cell-free DNA (cfDNA) profiles of younger (age <70) versus older (age >=70) men with mCRPC and assessed the relative prognostic impact of common genomic alterations. Method(s): A next-generation sequencing-based Predicine cfDNA assay was used to profile 276 mCRPC patients from two independent international cohorts. The frequency of genomic alterations across age categories was compared using the chi-squared test, while circulating tumour DNA fraction (ctDNA%) was compared using the Mann-Whitney U test. Cox proportional hazards analysis with interaction testing was used to assess the association between age, genomic alteration and overall survival. Result(s): The median age of the combined cohort was 72 years (IQR 66-78), with 170 (62%) patients >=70 years old. Despite differences in baseline characteristics (poorer performance status in older patients, higher Gleason score and de novo metastatic disease in younger patients), there was no significant difference in ctDNA% (median 22% vs 30%, p=0.6). We observed similar frequencies of genomic alterations across the 10 most commonly aberrant genes, including AR (<70 vs >=70: 46% vs 49%, p=0.6), TP53 (40% vs 36%, p=0.6), BRCA2 (29% vs 21%, p=0.13) and PTEN (39% vs 35%, p=0.5). This similarity persisted when analysing mutations and copy number alterations in isolation. While driver alterations in both age categories were strongly associated with unfavourable outcomes (Table), an exploratory analysis revealed that copy number gain in PIK3CA may have a worse prognostic impact in younger men (p for interaction = 0.03), with a lesser effect observed in AR gain (p = 0.1) and MYC gain (p = 0.1). Conclusion(s): The genom

Background: Tumour tissue from metastatic castration-resistant prostate cancer (mCRPC) harbors frequent copy number variations (CNVs) in the phosphatidylinositol-3-kinase (PI3K)/Aktsignaling pathway. However, identifying CNVs in plasma cell-free DNA (cfDNA) has proven challenging. With emerging data supporting Akt inhibition in PTEN-deficient mCRPC, cfDNA assays for robustly identifying PI3K/Akt pathway aberrations including CNVs are urgently required. Method(s): In this multi-institutional prospective biomarker study, we used the Predicine cfDNA assay, optimised for CNV detection, to perform targeted sequencing in 231 mCRPC patients in two independent cohorts (Australian, n = 78; US, n = 153). Kaplan-Meier survival estimates and multivariable Cox regression analysis were used to assess associations between genomic aberrations and progression-free survival (PFS) and overall survival (OS). Result(s): PTEN loss and PIK3CAgain were detected in 37% (85/231) and 17% (39/231) of patients, respectively. Poorer outcomes were observed in patients with PI3K/Akt pathway aberrations, including those with dual PTEN loss and PIK3CA gain (HR 2.3, 95% CI 1.2-4.4). Similarly, cumulative CNV burden in the PI3K/Akt and AR pathways (0 vs 1 vs >=2 CNVs in Australian cohort: median OS 33.5 vs 17.2 vs 9.7 months, p< 0.001; 0 vs 1 vs >=2 CNVs in US cohort: median OS 35.5 vs 14.3 vs 9.2 months, p< 0.001) was associated with significantly worse clinical outcomes. Notably, 21% (31/146) of PTEN-neutral patients harbored other alterations in the PI3K/Akt pathway. Conclusion(s): Our cfDNA assay readily detected PI3K/Akt pathway CNVs, with the prevalence of PTEN loss comparable to prior tissue sequencing studies. CNVs in the PI3K/Akt pathway were associated with deleterious clinical outcomes, especially when concurrent with AR gain. Additional PI3K/Akt pathway aberrations were found in approximately one-fifth of PTEN-neutral mCRPC. Collectively, our data demonstrate the potential utility of profi

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. Method(s): 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 (PSA50), 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. Result(s): 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 a

Background: Metastatic castration-resistant prostate cancer (mCRPC) disproportionately affects older individuals. While the molecular landscape of mCRPC is well established, less is understood about how genomic alterations differ with age. We compared cell-free DNA (cfDNA) profiles of younger (age<70) and older (age>=70) men with mCRPC and assessed the relative prognostic impact of genomic alterations between age categories. Method(s): Plasma cfDNA from 276 patients across two independent prospective cohorts (Australian, n=123; US, n=153) was sequenced using the PredicineTM cfDNA assay. Genomic alteration frequency (copy number variants and mutations) and circulating tumour DNA fraction (ctDNA%) were compared between age categories using the chi-squared and Wilcoxon rank-sum tests, respectively. Kaplan-Meier survival estimates, and Cox regression analysis were used to assess associations between age, genomic alterations, and overall survival. Result(s): Median age was 72 years (IQR 66-78), with 170 (62%) patients >=70. There was no significant difference in ctDNA% between older and younger patients (median 22% vs 30%, p=0.6). Similarly, no significant difference in alteration frequency was observed in prostate cancer driver genes, including AR (<70 vs >=70: 46% vs 49%, p=0.6), PTEN (39% vs 31%, p=0.2), BRCA2 (29% vs 21%, p=0.13) and ATM (17% vs 14%, p=0.4). The impact of established poor prognostic alterations was evident in both age categories. In an exploratory analysis, certain alterations may have a more pronounced effect in younger patients, including PIK3CA gain (HR 4.2 for <70, HR 1.9 for >=70 years; HR for interaction 2.2, p = 0.03) and MYC gain (HR 3.6 for <70, HR 2.1 for >=70 years; HR for interaction 1.8, p = 0.1). Conclusion(s): The frequency of clinically relevant genomic alterations was comparable between older and younger mCRPC patients. Screening for alterations that confer sensitivity to precision medicine therapies should be considered regardless o

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. Method(s): 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. Result(s): 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). Conclusion(s): 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 r

PURPOSE Tumor tissue frommetastatic 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-deficientmCRPC, we profiled PTENPI3K- 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.Copyright © 2021 American Society of Clinical Oncology. All rights reserved.

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 (PSA50), 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 assoc

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 resis

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(s): 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. Conclusion(s): We demonstrate the utility of a novel, multianalyte liquid biopsy assay ca

Background: Multiple tumour tissue studies have demonstrated the prognostic utility of CNVs in mCRPC. However, accurate assessment of CNVs in plasma cfDNA remains challenging, and prognostic significance has not been well characterized. Using a large customized panel, we correlated plasma CNVs with clinical outcomes in a contemporary cohort of mCRPC patients. Method(s): Deep targeted sequencing was performed using a 180-gene cfDNA panel (Predicine) in 56 patients commencing AR pathway inhibitors (enzalutamide or abiraterone; n = 34) or taxane chemotherapy (n = 22) at two Australian institutions. Kaplan-Meier estimates and Cox proportional-hazards models were used to correlate CNVs with progression-free survival (PFS) and overall survival (OS). Significant results were validated in an independent cohort (Mayo Clinic, n = 144). Result(s): Median follow-up was 19.4 months (mo; IQR 11.3-31.9). The most common CNVs in the Australian cohort are shown (Table). OS was significantly decreased in patients with PI3KCA gain (median 21.7 mo vs 6.6 mo, p < 0.0001 ), PTEN loss (24.8 mo vs 11.7 mo, p = 0.0019) and AR gain (21.7 mo vs 12.0 mo, p = 0.0083). Furthermore, all three alterations independently predicted for poor survival in multivariable analyses (MVA; Table). Findings in the independent cohort showed similar OS results in MVA: PIK3CA gain (HR 2.0, p = 0.07), PTEN loss (HR 1.7, p = 0.08) and AR gain (HR 1.7, p = 0.03). Conclusion(s): Sequencing of plasma cfDNA revealed that PTEN loss, and PIK3CA and AR gain are associated with inferior clinical outcomes in patients commencing contemporary systemic treatment. These data support therapeutic strategies co-targeting the PI3K and AR pathways in mCRPC. Covariates in MVA: circulating tumour DNA fraction > 2%, prior treatment, visceral disease, baseline pain and ECOG >2.

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).Copyright © 2020 Arun Azad.

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: The expression of androgen receptor splice variants (AR-V) in mCRPC patients has widely been regarded as a predictive biomarker for non-response to AR-targeted therapies. However, recent data from our group and others has questioned this association. We report new data from our whole blood assay, correlating baseline and end-of-treatment (EOT) AR-V7 and AR-V9 expression with clinical outcomes in mCRPC patients treated with abiraterone or enzalutamide. Method(s): We have previously developed a whole-blood, quantitative polymerase chain reaction assay for detecting circulating AR-V7 and AR-V9. The assay was applied to samples prospectively collected from 48 mCRPC patients immediately prior to commencing abiraterone or enzalutamide, and where available, at treatment cessation. Patients positive for either AR-V7 or AR-V9 were defined as AR-V-positive, and AR-Vnegative if neither variant was detected. All AR-V-positive samples underwent confirmatory DNA sequencing to confirm variant expression. AR-V expression was correlated with PSA response rate (chi-square test) and PSA progression-free survival (PSA-PFS) (logrank test). Result(s): The median follow-up was 10.6 mo. Twelve out of 48 patients (25%) were AR-V-positive, with no samples positive for both variants at baseline. Similar response rates were observed in AR-V-positive (7/12) and AR-V-negative (23/36) patients (50% vs. 64%, p = 0.7). PSA-PFS did not differ significantly between groups (8.1 mo vs. not reached, p = 0.5). EOT samples were available in nine patients, with four exhibiting AR-V-positivity. Of these, two (50%) patients were AR-V-positive at baseline, and two (50%) patients converted from AR-V-negative to AR-V-positive at treatment cessation (PSA-PFS 3.4 and 2.6 mo respectively). One of the conversion patients transitioned from V7-/V9- to V7+/V9+. Neither AR-V7 nor AR-V9 were detected in any of 13 normal male controls. Conclusion(s): With extended follow-up, whole blood expression of AR-V7 an

Background: Identifying predictive biomarkers for mCRPC patients receiving androgen receptor signalling inhibitors (ARSI) or chemotherapy remains an unmet clinical need. FOLH1 encodes for ProstateSpecific Membrane Antigen (PSMA), a type II glycoprotein highly expressed on prostate cancer cells. We designed a whole blood assay to detect FOLH1 mRNA, and correlated expression with clinical outcomes in patients commencing ARSI (abiraterone or enzalutamide) or chemotherapy (docetaxel or cabazitaxel). Method(s): mCRPC patients commencing ARSI or chemotherapy were prospectively recruited at three Australian centres from June 2016 to July 2018. A quantitative reverse transcription polymerase chain reaction assay was used to detect FOLH1 transcript from whole blood samples collected in PAXgene RNA tubes. Pre-treatment FOLH1 expression was correlated with PSA response rate (Fisher's exact test) and PSA progression-free survival (PSA-PFS) (log-rank test). Result(s): Median follow-up was 13.6 months (IQR 9.7-19.3). In total, 88 pretreatment samples were analysed, of which 75 (85%) were FOLH1-positive. In patients receiving ARSI, outcomes favoured FOLH1-positive patients compared to FOLH1negative patients, with higher PSA response rates (39/60, 65% vs. 2/7, 29%; p = 0.1) and longer PSA-PFS (median 9.0 months [95% CI, 7.210.8] vs. 2.8 months [95% CI, 2.33.3] ; p = 0.03). Conversely, in chemotherapy-treated patients, inferior outcomes were observed in FOLH1-positive patients compared to FOLH1negative patients, with lower PSA response rates (4/15, 27% vs. 5/6, 83%, p = 0.05) and shorter PSA-PFS (median 2.9 months [95% CI, 2.83.0] vs. 4.1 months [95% CI, 3.74.5] ; p = 0.32). Conclusion(s): Pre-treatment FOLH1 expression may differentiate between outcomes on ARSI vs. chemotherapy in mCRPC patients. The utility of FOLH1 as a predictive biomarker in mCRPC warrants further evaluation in larger, independent cohorts. (Table Presented) .

Objective: Identifying predictive and/or prognostic biomarkers in the context of an ever-expanding therapeutic armamentarium for metastatic castrate-resistant prostate cancer (mCRPC) patients remains an unmet clinical need. Our objective was to develop a prognostic whole blood gene signature for mCRPC patients commencing systemic therapy. Method(s): We obtained pre-treatment whole blood samples in PAXgene RNA tubes from 115 mCRPC patients commencing androgen receptor (AR) pathway inhibitors (n = 81) or chemotherapy (n = 34) across threeAustralian centres from June 2016 to July 2018. The presence of 10 prostate cancer-associated transcripts was assessed using reverse transcription polymerase chain reaction (RT-PCR). Gene transcripts correlating with overall survival (OS) at P < .10 in univariate Cox regression models were incorporated into a multigene signature, before assessing for association with clinical outcomes. The prognostic strength of the signature was further evaluated using a concordance probability estimate (CPE). Result(s): A total of four genes were incorporated into a multigene signature: AR splice variant 7 (AR-V7), and three androgen-regulated genes: GRHL2, HOXB13 and FOXA1. Increasing number of positive transcripts stratified for survival outcomes (median OS: not reached vs 24.8 months vs 16.2 months for 0, 1 and >=2 transcripts, respectively; P = .0052). Strikingly, thismultigene signature retained prognostic significance on multivariable analysis (HR 2.1, 95% CI 1.1-4.0, P = .019). The CPE for this model was 0.78, indicating that the signature was a strong discriminator of patient prognosis. Conclusion(s): In this prospective, multicentre study, a novel whole blood AR-based multigene signature demonstrated strong prognostic utility in a cohort of mCRPC patients commencing contemporaneous systemic therapies. This signature may play a valuable role in upfront risk stratification of mCRPC patients, which in turn may inform on design of future clinic

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(s): 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. Conclusion(s): Our data demonstrate the prognostic utility of a novel whole-blood AR-based signatur, We demonstrated that a whole-blood androgen receptor-based multigene signature was an important prognostic tool in a cohort of metastatic castration-resistant prostate cancer (mCRPC)patients receiving contemporaneous systemic treatment. This signature may have a valuable role in upfront risk stratification and prognostication of mCRPC patients. 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(s): 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 retain

Background: Androgen receptor splice variant (AR-V) expression has previously been regarded as a negative predictive biomarker for response to abiraterone and enzalutamide in mCRPC patients. However, recent data questions this association. We designed a whole blood assay to detect AR-V7 and AR-V9, the two most abundantly expressed AR-Vs, and correlated expression with clinical outcomes in patients commencing abiraterone or enzalutamide. Method(s): We developed a quantitative real-time polymerase chain reaction assay to detect AR-V7 and AR-V9 from whole blood collected in PAXgene tubes. The assay was applied to samples prospectively collected from 37 mCRPC patients prior to commencing abiraterone or enzalutamide, and at treatment cessation. Patients positive for either AR-V7 or AR-V9 were defined as AR-V-positive, and AR-V-negative if neither variant was detected. AR-V expression was correlated with PSA response rate (chi-square test) and PSA progression-free survival (PSA-PFS) (log-rank test). Assay sensitivity was determined by serially diluting RNA from VCaP prostate cancer cells (known to express AR-V7) to establish a lower limit of detection. Result(s): The median follow-up was 7.29 months (IQR 4.21-10.55); 9 of 37 patients (24%) were AR-V-positive. We observed similar response rates in AR-V-positive (6/9) and AR-V-negative (18/28) patients (66% vs. 64%, p = 0.896). PSA-PFS did not differ significantly between groups (9.2 months vs. not reached, p = 0.355). Two patients converted from AR-V-negative to AR-V-positive (PSA-PFS 3.35 and 0.60 months respectively), and one patient remained AR-V-positive at baseline and end-of-treatment sampling. The lower limit of detection for AR-V7 was 0.1%, and AR-V7/V9 was not detected in any of the 13 normal male controls. Conclusion(s): We developed a sensitive and specific whole blood assay for AR-V7 and AR-V9 detection in patients with mCRPC. Neither PSA response rates nor PSA-PFS differed significantly between AR-V-positive a

In 2014, a landmark study was published demonstrating that androgen receptor splice variant (AR-V) AR-V7 expression was a negative predictive biomarker for response to abiraterone acetate and enzalutamide in metastatic castration-resistant prostate cancer (mCRPC) patients. However, these results were not supported by the recently reported ARMOR3-SV phase III clinical trial, which employed an identical circulating tumor cell assay to assess AR-V7 expression. Therefore, the predictive utility of AR-V7 expression in mCRPC remains uncertain, as does any potential association between other AR splice variants and treatment response. To further investigate, we designed a highly sensitive and specific whole blood assay for detecting AR-V7 and AR-V9. We then examined for a correlation between baseline AR-V7/V9 status and treatment outcome in 37 mCRPC patients commencing abiraterone or enzalutamide. Blood was taken in a PAXgene RNA tube at baseline prior to the commencement of therapy and at end of treatment (if applicable). 24% (9/37) of patients were AR-V-positive. Notably, PSA response rates did not significantly differ between AR-V-positive (6/9) and AR-V-negative (18/28) patients (66% vs. 64%, p=0.896). Likewise, median PSA progression-free survival was not significantly different between AR-V-positive and AR-V-negative patients (9.2 months vs. not reached; p=0.894). Additionally, we identified 2 patients who were AR-V7 negative at baseline but had converted to AR-V7 positive in their end-of-treatment sample. This was associated with much shorter PSA-PFS than those that remained negative (2.58 months vs. no reached), suggesting gain of AR-V expression may be a marker of acquired resistance. These data, which support the findings of the pivotal ARMOR3-SV clinical trial, suggest that baseline AR-V expression does not predict outcomes in mCRPC patients receiving abiraterone or enzalutamide.

Objective: Recent data have demonstrated the utility of baseline plasma cell-free DNA (cfDNA) concentration as an independent prognostic indicator for metastatic castrate-resistant prostate cancer (mCRPC) patients receiving systemic chemotherapy.1 However, the clinical utility of this biomarker is still unknown in the androgen receptor signalling inhibitor (ARSI)-treated population. Our objective was to correlate baseline cfDNA concentration and clinical outcomes in patients commencing ARSI for mCRPC. Method(s): Blood for cfDNA analysis was prospectively collected from 42 mCRPC patients prior to commencing abiraterone or enzalutamide. Evaluable patients were required to have a minimum of 12 weeks follow-up. cfDNA was extracted from plasma using the QIAamp circulating nucleic acid kit as per the manufacturer's protocol, then quantified using fluorescence spectrometry (Qubit). Univariable analysis was conducted using logistic regression to determine if there was an association between baseline cfDNA concentration (expressed as log10 concentration) and prostate serum antigen (PSA) response. In addition, Cox proportional hazard models tested for associations between cfDNA concentration (plus other baseline prognostic variables) and the composite endpoint of clinical/radiological progression-free survival (PFS). All P-values < 0.05 were considered significant. Result(s): The median baseline cfDNA concentration in this cohort was 8.2 ng/mL (IQR 4.7-14.9 ng/mL). Median follow-up was 9.2 mo. Baseline log10 cfDNA concentration was not associated with a confirmed PSA response (odds ratio [OR] = 0.4; P = 0.2). Median clinical/rPFS was not reached. In univariate analysis, baseline log10 cfDNA concentration significantly correlated with shorter clinical/rPFS (hazard ratio [HR] = 3.1; 95% CI: 1.2-7.7; P = 0.02). Other baseline prognostic factors correlating with shorter clinical/rPFS included log10 LDH concentration (P = 0.002) and log10 neutrophil-lymphocyte ratio (P = 0.008). C

In 2014, a landmark study was published demonstrating that the expression of androgen receptor splice variant (AR-V) 7 was a negative predictive biomarker for response to abiraterone acetate and enzalutamide in metastatic castration-resistant prostate cancer (mCRPC) patients. However, these results were not supported by the recently reported ARMOR3-SV phase III clinical trial, which employed an identical circulating tumour cell assay to assess AR-V7 expression. Therefore, the predictive utility of AR-V7 expression in mCRPC remains uncertain, as does any potential association between other AR-Vs and treatment response. To further investigate, we designed a highly sensitive and specific whole blood assay for detecting AR-V7 and AR-V9. We then examined for a correlation between baseline AR-V7/V9 status and treatment outcome in 37 mCRPC patients commencing abiraterone or enzalutamide. Of the patients, 24% (9/37) were AR-V-positive. Notably, prostate-specific antigen (PSA) response rates did not significantly differ between AR-V-positive (6/9) and AR-V-negative (18/28) patients (66% vs 64%, p = 0.9). Likewise, median PSA progression-free survival was not significantly different between AR-V-positive and AR-V-negative patients (9.2 mo vs not reached; p = 0.9). These data, which support the findings of the pivotal ARMOR3-SV clinical trial, suggest that baseline AR-V expression does not predict outcomes in mCRPC patients receiving abiraterone or enzalutamide. Patient Summary: Detection of androgen receptor splice variants (AR-Vs) in circulating tumour cells of advanced prostate cancer patients has been linked to resistance to abiraterone and enzalutamide. We designed a blood test to detect AR-Vs that can be performed more routinely than tests involving circulating tumour cells and found that patients with AR-Vs still benefit from these effective treatments. We designed a whole blood assay for the detection of androgen receptor splice variant (AR-V) 7 and AR-V9. The presence