Liquid Biopsy in Progressing Prostate Cancer Patients Starting Docetaxel with or Without Enzalutamide: A Biomarker Study of the PRESIDE Phase 3b Trial.

BACKGROUND AND OBJECTIVE: The PRESIDE (NCT02288247) randomized trial demonstrated prolonged progression-free survival (PFS) with continuing enzalutamide beyond progression in metastatic castration-resistant prostate cancer (mCRPC) patients starting docetaxel. This study aims to test the associations of PFS and circulating tumor DNA (ctDNA) prior to and after one cycle (cycle 2 day 1 [C2D1]) of docetaxel and with a liquid biopsy resistance biomarker (LBRB; plasma androgen receptor [AR] gain and/or circulating tumor cells [CTCs] expressing AR splice variant 7 [CTC-AR-V7]) prior to continuation of enzalutamide/placebo. METHODS: Patients consenting to the biomarker substudy and donating blood before starting docetaxel with enzalutamide/placebo (N = 157) were included. Sequential plasma DNA samples were characterized with a prostate-cancer bespoke next-generation-sequencing capture panel (PCF_SELECT), and CTCs were assessed for AR-V7 (Epic Sciences, San Diego, CA, USA). Cox models, Kaplan-Meier, and restricted mean survival time (RMST) at 18 mo were calculated. KEY FINDINGS AND LIMITATIONS: There was a significant association of worse PFS with pre-docetaxel ctDNA detection (N = 86 (55%), 8.1 vs 10.8 mo hazard ratio [HR] = 1.78, p = 0.004) or persistence/rise of ctDNA at C2D1 (N = 35/134, 5.5 vs 10.9 mo, HR = 1.95, 95% confidence interval [CI] = 1.15-3.30, p = 0.019). LBRB-positive patients (N = 62) had no benefit from continuing enzalutamide with docetaxel (HR = 0.78, 95% CI = 0.41-1.48, p = 0.44; RMST: 7.9 vs 7.1 mo, p = 0.50). Conversely, resistance biomarker-negative patients (N = 87) had significantly prolonged PFS (HR = 0.49, 95% CI = 0.29-0.82, p = 0.006; RMST: 11.5 vs 8.9 mo, p = 0.005). Eight patients were unevaluable. An exploratory analysis identified increased copy-number gains (CDK6/CDK4) at progression on docetaxel. Limitations included relatively low detection of CTC-AR-V7. Validation of impact on overall survival is required. CONCLUSIONS AND CLINICAL IMPLICATIONS: Liquid biopsy gives an early indication of docetaxel futility, could guide patient selection for continuing enzalutamide, and identifies cell cycle gene alterations as a potential cause of docetaxel resistance in mCRPC. PATIENT SUMMARY: In the PRESIDE biomarker study, we found that detecting circulating tumor DNA in plasma after starting treatment with docetaxel (chemotherapy) for metastatic prostate cancer resistant to androgen deprivation therapy can predict early how long patients will take to respond to treatment. Patients negative for a liquid biopsy resistance biomarker (based on the status of androgen receptor (AR) gene and AR splice variant 7 in circulating tumor cells) benefit from continuing enzalutamide in combination with docetaxel, while patients positive for the resistance biomarker did not. Additionally, we identified alterations in the cell cycle genes CDK6 and CDK4 as a potential genetic cause of resistance to docetaxel, which may support testing of specific drugs targeting these alterations.

Prognostic Implications of Blood Immune-Cell Composition in Metastatic Castration-Resistant Prostate Cancer.

The prognosis for patients with metastatic castration-resistant prostate cancer (mCRPC) varies, being influenced by blood-related factors such as transcriptional profiling and immune cell ratios. We aimed to address the contribution of distinct whole blood immune cell components to the prognosis of these patients. This study analyzed pre-treatment blood samples from 152 chemotherapy-naive mCRPC patients participating in a phase 2 clinical trial (NCT02288936) and a validation cohort. We used CIBERSORT-X to quantify 22 immune cell types and assessed their prognostic significance using Kaplan-Meier and Cox regression analyses. Reduced CD8 T-cell proportions and elevated monocyte levels were substantially connected with a worse survival. High monocyte counts correlated with a median survival of 32.2 months versus 40.3 months for lower counts (HR: 1.96, 95% CI 1.11-3.45). Low CD8 T-cell levels were associated with a median survival of 31.8 months compared to 40.3 months for higher levels (HR: 1.97, 95% CI 1.11-3.5). These findings were consistent in both the trial and validation cohorts. Multivariate analysis further confirmed the independent prognostic value of CD8 T-cell counts. This study highlights the prognostic implications of specific blood immune cells, suggesting they could serve as biomarkers in mCRPC patient management and should be further explored in clinical trials.

Copy number architectures define treatment-mediated selection of lethal prostate cancer clones.

Despite initial responses to hormone treatment, metastatic prostate cancer invariably evolves to a lethal state. To characterize the intra-patient evolutionary relationships of metastases that evade treatment, we perform genome-wide copy number profiling and bespoke approaches targeting the androgen receptor (AR) on 167 metastatic regions from 11 organs harvested post-mortem from 10 men who died from prostate cancer. We identify diverse and patient-unique alterations clustering around the AR in metastases from every patient with evidence of independent acquisition of related genomic changes within an individual and, in some patients, the co-existence of AR-neutral clones. Using the genomic boundaries of pan-autosome copy number changes, we confirm a common clone of origin across metastases and diagnostic biopsies, and identified in individual patients, clusters of metastases occupied by dominant clones with diverged autosomal copy number alterations. These autosome-defined clusters are characterized by cluster-specific AR gene architectures, and in two index cases are topologically more congruent than by chance (p-values 3.07 × 10-8 and 6.4 × 10-4). Integration with anatomical sites suggests patterns of spread and points of genomic divergence. Here, we show that copy number boundaries identify treatment-selected clones with putatively distinct lethal trajectories.

Clinical testing of transcriptome-wide expression profiles in high-risk localized and metastatic prostate cancer starting androgen deprivation therapy: an ancillary study of the STAMPEDE abiraterone Phase 3 trial.

Metastatic and high-risk localized prostate cancer respond to hormone therapy but outcomes vary. Following a pre-specified statistical plan, we used Cox models adjusted for clinical variables to test associations with survival of multi-gene expression-based classifiers from 781 patients randomized to androgen deprivation with or without abiraterone in the STAMPEDE trial. Decipher score was strongly prognostic (p<2×10-5) and identified clinically-relevant differences in absolute benefit, especially for localized cancers. In metastatic disease, classifiers of proliferation, PTEN or TP53 loss and treatment-persistent cells were prognostic. In localized disease, androgen receptor activity was protective whilst interferon signaling (that strongly associated with tumor lymphocyte infiltration) was detrimental. Post-Operative Radiation-Therapy Outcomes Score was prognostic in localized but not metastatic disease (interaction p=0.0001) suggesting the impact of tumor biology on clinical outcome is context-dependent on metastatic state. Transcriptome-wide testing has clinical utility for advanced prostate cancer and identified worse outcomes for localized cancers with tumor-promoting inflammation.

A correlative biomarker study and integrative prognostic model in chemotherapy-naïve metastatic castration-resistant prostate cancer treated with enzalutamide.

BACKGROUND: There is a considerable need to incorporate biomarkers of resistance to new antiandrogen agents in the management of castration-resistant prostate cancer (CRPC). METHODS: We conducted a phase II trial of enzalutamide in first-line chemo-naïve asymptomatic or minimally symptomatic mCRPC and analyzed the prognostic value of TMPRSS2-ERG and other biomarkers, including circulating tumor cells (CTCs), androgen receptor splice variant (AR-V7) in CTCs and plasma Androgen Receptor copy number gain (AR-gain). These biomarkers were correlated with treatment response and survival outcomes and developed a clinical-molecular prognostic model using penalized cox-proportional hazard model. This model was validated in an independent cohort. RESULTS: Ninety-eight patients were included. TMPRSS2-ERG fusion gene was detected in 32 patients with no differences observed in efficacy outcomes. CTC detection was associated with worse outcome and AR-V7 in CTCs was associated with increased rate of progression as best response. Plasma AR gain was strongly associated with an adverse outcome, with worse median prostate specific antigen (PSA)-PFS (4.2 vs. 14.7 m; p < 0.0001), rad-PFS (4.5 vs. 27.6 m; p < 0.0001), and OS (12.7 vs. 38.1 m; p < 0.0001). The clinical prognostic model developed in PREVAIL was validated (C-Index 0.70) and the addition of plasma AR (C-Index 0.79; p < 0.001) increased its prognostic ability. We generated a parsimonious model including alkaline phosphatase (ALP); PSA and AR gain (C-index 0.78) that was validated in an independent cohort. CONCLUSIONS: TMPRSS2-ERG detection did not correlate with differential activity of enzalutamide in first-line mCRPC. However, we observed that CTCs and plasma AR gain were the most relevant biomarkers.

Accumulation of copy number alterations and clinical progression across advanced prostate cancer.

BACKGROUND: Genomic copy number alterations commonly occur in prostate cancer and are one measure of genomic instability. The clinical implication of copy number change in advanced prostate cancer, which defines a wide spectrum of disease from high-risk localised to metastatic, is unknown. METHODS: We performed copy number profiling on 688 tumour regions from 300 patients, who presented with advanced prostate cancer prior to the start of long-term androgen deprivation therapy (ADT), in the control arm of the prospective randomised STAMPEDE trial. Patients were categorised into metastatic states as follows; high-risk non-metastatic with or without local lymph node involvement, or metastatic low/high volume. We followed up patients for a median of 7 years. Univariable and multivariable Cox survival models were fitted to estimate the association between the burden of copy number alteration as a continuous variable and the hazard of death or disease progression. RESULTS: The burden of copy number alterations positively associated with radiologically evident distant metastases at diagnosis (P=0.00006) and showed a non-linear relationship with clinical outcome on univariable and multivariable analysis, characterised by a sharp increase in the relative risk of progression (P=0.003) and death (P=0.045) for each unit increase, stabilising into more modest increases with higher copy number burdens. This association between copy number burden and outcome was similar in each metastatic state. Copy number loss occurred significantly more frequently than gain at the lowest copy number burden quartile (q=4.1 × 10-6). Loss of segments in chromosome 5q21-22 and gains at 8q21-24, respectively including CHD1 and cMYC occurred more frequently in cases with higher copy number alteration (for either region: Kolmogorov-Smirnov distance, 0.5; adjusted P<0.0001). Copy number alterations showed variability across tumour regions in the same prostate. This variance associated with increased risk of distant metastases (Kruskal-Wallis test P=0.037). CONCLUSIONS: Copy number alteration in advanced prostate cancer associates with increased risk of metastases at diagnosis. Accumulation of a limited number of copy number alterations associates with most of the increased risk of disease progression and death. The increased likelihood of involvement of specific segments in high copy number alteration burden cancers may suggest an order underlying the accumulation of copy number changes. TRIAL REGISTRATION: ClinicalTrials.gov NCT00268476 , registered on December 22, 2005. EudraCT  2004-000193-31 , registered on October 4, 2004.

Allele-informed copy number evaluation of plasma DNA samples from metastatic prostate cancer patients: the PCF_SELECT consortium assay.

Sequencing of cell-free DNA (cfDNA) in cancer patients' plasma offers a minimally-invasive solution to detect tumor cell genomic alterations to aid real-time clinical decision-making. The reliability of copy number detection decreases at lower cfDNA tumor fractions, limiting utility at earlier stages of the disease. To test a novel strategy for detection of allelic imbalance, we developed a prostate cancer bespoke assay, PCF_SELECT, that includes an innovative sequencing panel covering ∼25 000 high minor allele frequency SNPs and tailored analytical solutions to enable allele-informed evaluation. First, we assessed it on plasma samples from 50 advanced prostate cancer patients. We then confirmed improved detection of genomic alterations in samples with <10% tumor fractions when compared against an independent assay. Finally, we applied PCF_SELECT to serial plasma samples intensively collected from three patients previously characterized as harboring alterations involving DNA repair genes and consequently offered PARP inhibition. We identified more extensive pan-genome allelic imbalance than previously recognized in prostate cancer. We confirmed high sensitivity detection of BRCA2 allelic imbalance with decreasing tumor fractions resultant from treatment and identified complex ATM genomic states that may be incongruent with protein losses. Overall, we present a framework for sensitive detection of allele-specific copy number changes in cfDNA.

Blood-based liquid biopsies for prostate cancer: clinical opportunities and challenges.

Liquid biopsy has been established as a powerful, minimally invasive, tool to detect clinically actionable aberrations across numerous cancer types in real-time. With the development of new therapeutic agents in prostate cancer (PC) including DNA repair targeted therapies, this is especially attractive. However, there is unclarity on how best to screen for PC, improve risk stratification and ultimately how to treat advanced disease. Therefore, there is an urgent need to develop better biomarkers to help guide oncologists' decisions in these settings. Circulating tumour cells (CTCs), exosomes and cell-free DNA/RNA (cfDNA/cfRNA) analysis, including epigenetic features such as methylation, have all shown potential in prognostication, treatment response assessment and detection of emerging mechanisms of resistance. However, there are still challenges to overcome prior to implementing liquid biopsies in routine clinical practice such as preanalytical considerations including blood collection and storage, the cost of CTC isolation and enrichment, low-circulating tumour content as a limitation for genomic analysis and how to better interpret the sequencing data generated. In this review, we describe an overview of the up-to-date clinical opportunities in the management of PC through blood-based liquid biopsies and the next steps for its implementation in personalised treatment guidance.

Plasma tumor DNA is associated with increased risk of venous thromboembolism in metastatic castration-resistant cancer patients.

Cancer is a risk factor for venous thromboembolism (VTE). Plasma tumor DNA (ptDNA) is an independent predictor of outcome in metastatic castration-resistant prostate cancer (mCRPC). We aimed to investigate the association between ptDNA and VTE in mCRPC. This prospective biomarker study included 180 mCRPC patients treated with abiraterone and enzalutamide from April 2013 to December 2018. We excluded patients with a previous VTE history and/or ongoing anticoagulation therapy. Targeted next-generation sequencing was performed to determine ptDNA fraction from pretreatment plasma samples. VTE risk based on survival analysis was performed using cumulative incidence function and estimating sub-distributional hazard ratio (SHR). At a median follow-up of 58 months (range 0.5-111.0), we observed 21 patients who experienced VTE with a cumulative incidence at 12 months of 17.1% (95% confidence interval [CI] 10.3-23.9). Elevated ptDNA, visceral metastasis, prior chemotherapy and lactate dehydrogenase (LDH) were significantly associated with higher VTE incidence compared to patients with no thrombosis (12-month estimate, 18.6% vs 3.5%, P = .0003; 44.4% vs 14.8%, P = .015; 24.7% vs 4.5%, P = .006; and 30.0% vs 13.5%, P = .05, respectively). In the multivariate analysis including ptDNA level, visceral metastases, number of lesions and serum LDH, high ptDNA fraction was the only independent factor associated with the risk of thrombosis (HR 5.78, 95% CI 1.63-20.44, P = .006). These results first suggest that baseline ptDNA fraction in mCRPC patients treated with abiraterone or enzalutamide may be associated with increased VTE risk. These patients may be followed-up more closely for the VTE risk, and the need for a primary thromboprophylaxis should be taken into account in mCRPC with elevated ptDNA.

Plasma androgen receptor and response to adapted and standard docetaxel regimen in castration-resistant prostate cancer: A multicenter biomarker study.

BACKGROUND: Plasma AR status has been identified as a potential biomarker of response in metastatic castration-resistant prostate cancer (mCRPC) patients receiving docetaxel or AR-targeted therapies. However, the relevance of plasma AR in the overall management of CRPC patients receiving different docetaxel doses is unknown. PATIENTS AND METHODS: This was a multi-institution study of associations between baseline plasma AR copy number status, assessed by droplet digital PCR, and outcome in 325 mCRPC patients receiving docetaxel at standard or adapted regimen at the discretion of the treating physician. Upon analysis, patients were assigned randomly to either a training (n = 217) or validation (n = 108) cohort. RESULTS: In the training cohort, AR-gained patients treated with adapted docetaxel regimen had a significantly worse median progression-free survival (PFS) (3.8 vs 6.3 months, hazard ratio [HR] 2.58, 95% confidence interval [CI] 1.34-4.95, p < 0.0001), median overall survival (10.8 vs 20.6 months, HR 1.98, 95% CI 1.09-3.62, p = 0.0064) and PSA response (PSA > -50%: odds ratio 4.88 95%CI 1.55-14.32, p = 0.013) as compared to plasma AR normal patients. These findings were all confirmed in the validation cohort. However, in patients treated with standard docetaxel regimen, these differences were not seen. The interaction between AR CN status and dose reduction of docetaxel was considered as independent factor for PFS in both the training and validation cohort (HR 2.84, 95% CI 1.41-5.73, p = 0.003, and HR 4.79, 95% CI 1.79-12.82, p = 0.002). CONCLUSION: Despite the retrospective non-randomised design of this study, our hypothesis-generating findings could suggest plasma AR as a potential biomarker for optimal docetaxel timing and dose in mCRPC patients. Prospective trials are warranted.

Circulating Androgen Receptor for Prognosis and Treatment Selection in Prostate Cancer.

Analysis of androgen receptor (AR) status, particularly AR copy number, in plasma DNA is a minimally invasive method with the potential to identify treatment resistance in patients with castration-resistant prostate cancer (CRPC) starting enzalutamide or abiraterone. Patients with elevated plasma AR do not have worse outcomes than patients with normal plasma AR when treated with taxanes. Consequently, circulating AR may improve clinical decision-making between AR-directed therapies versus taxanes and probably also between adapted versus standard taxane regimens. The evidence indicates that circulating AR could have a role in overall CRPC management. Promising clinical implications of plasma AR testing are measurement in earlier stages of prostate cancer, disease monitoring, and within the context of a multiplex biomarker strategy to improve treatment selection for CRPC patients. PATIENT SUMMARY: Measurement of the copy number of androgen receptor genes in plasma is a promising tool for guiding personalised treatment in patients with castration-resistant prostate cancer. However, prospective trials to validate these findings are needed.

Plasma tumour DNA as an early indicator of treatment response in metastatic castration-resistant prostate cancer.

BACKGROUND: Plasma tumour DNA (ptDNA) levels on treatment are associated with response in a variety of cancers. However, the role of ptDNA in prostate cancer monitoring remains largely unexplored. Here we characterised on-treatment ptDNA dynamics and evaluated its potential for early assessment of therapy efficacy for metastatic castration-resistant prostate cancer (mCRPC). METHODS: Between 2011 and 2016, 114 sequential plasma samples from 43 mCRPC abiraterone-treated patients were collected. Targeted next-generation sequencing was performed to determine ptDNA fraction. ptDNA progressive disease was defined as a rise in the fraction compared to the pre-treatment. RESULTS: A ptDNA rise in the first on-treatment sample (interquartile range (IQR) 2.6-3.7 months) was significantly associated with increased risk of early radiographic or any prostate-specific antigen (PSA) rise (odds ratio (OR) = 15.8, 95% confidence interval (CI) 3.5-60.2, p = 0.0002 and OR = 6.0, 95% CI 1.6-20.0, p = 0.01, respectively). We also identified exemplar cases that had a rise in PSA or pseudoprogression secondary to bone flare but no rise in ptDNA. In an exploratory analysis, initial ptDNA change was found to associate with the duration of response to prior androgen deprivation therapy (p < 0.0001) but not to prior taxanes (p = 0.32). CONCLUSIONS: We found that ptDNA assessment for therapy monitoring in mCRPC is feasible and provides data relevant to the clinical setting. Prospective evaluation of these findings is now merited.

Genome-wide plasma DNA methylation features of metastatic prostate cancer.

Tumor DNA circulates in the plasma of cancer patients admixed with DNA from noncancerous cells. The genomic landscape of plasma DNA has been characterized in metastatic castration-resistant prostate cancer (mCRPC) but the plasma methylome has not been extensively explored. Here, we performed next-generation sequencing (NGS) on plasma DNA with and without bisulfite treatment from mCRPC patients receiving either abiraterone or enzalutamide in the pre- or post-chemotherapy setting. Principal component analysis on the mCRPC plasma methylome indicated that the main contributor to methylation variance (principal component one, or PC1) was strongly correlated with genomically determined tumor fraction (r = -0.96; P < 10-8) and characterized by hypermethylation of targets of the polycomb repressor complex 2 components. Further deconvolution of the PC1 top-correlated segments revealed that these segments are comprised of methylation patterns specific to either prostate cancer or prostate normal epithelium. To extract information specific to an individual's cancer, we then focused on an orthogonal methylation signature, which revealed enrichment for androgen receptor binding sequences and hypomethylation of these segments associated with AR copy number gain. Individuals harboring this methylation pattern had a more aggressive clinical course. Plasma methylome analysis can accurately quantitate tumor fraction and identify distinct biologically relevant mCRPC phenotypes.

Identification of single nucleotide variants using position-specific error estimation in deep sequencing data.

BACKGROUND: Targeted deep sequencing is a highly effective technology to identify known and novel single nucleotide variants (SNVs) with many applications in translational medicine, disease monitoring and cancer profiling. However, identification of SNVs using deep sequencing data is a challenging computational problem as different sequencing artifacts limit the analytical sensitivity of SNV detection, especially at low variant allele frequencies (VAFs). METHODS: To address the problem of relatively high noise levels in amplicon-based deep sequencing data (e.g. with the Ion AmpliSeq technology) in the context of SNV calling, we have developed a new bioinformatics tool called AmpliSolve. AmpliSolve uses a set of normal samples to model position-specific, strand-specific and nucleotide-specific background artifacts (noise), and deploys a Poisson model-based statistical framework for SNV detection. RESULTS: Our tests on both synthetic and real data indicate that AmpliSolve achieves a good trade-off between precision and sensitivity, even at VAF below 5% and as low as 1%. We further validate AmpliSolve by applying it to the detection of SNVs in 96 circulating tumor DNA samples at three clinically relevant genomic positions and compare the results to digital droplet PCR experiments. CONCLUSIONS: AmpliSolve is a new tool for in-silico estimation of background noise and for detection of low frequency SNVs in targeted deep sequencing data. Although AmpliSolve has been specifically designed for and tested on amplicon-based libraries sequenced with the Ion Torrent platform it can, in principle, be applied to other sequencing platforms as well. AmpliSolve is freely available at https://github.com/dkleftogi/AmpliSolve .

Plasma AR status and cabazitaxel in heavily treated metastatic castration-resistant prostate cancer.

BACKGROUND: Plasma androgen receptor (AR) copy number status has been identified as a potential biomarker of response in patients with metastatic castration-resistant prostate cancer (mCRPC) receiving docetaxel or the AR-targeted therapies abiraterone or enzalutamide. However, the relevance of plasma AR status in the context of cabazitaxel therapy is unknown. PATIENTS AND METHODS: Between September 2011 and January 2018, pretherapy plasma samples were collected from 155 patients treated with second- or third-line cabazitaxel at standard or reduced dose in different biomarker protocols. Droplet digital polymerase chain reaction was used to identify plasma AR gain and normal samples. The primary objective was to evaluate associations of plasma AR status with treatment outcome. In an exploratory analysis, a comparison between plasma AR and treatment type was investigated by incorporating updated data from our prior study of 85 post-docetaxel patients receiving abiraterone or enzalutamide. RESULTS: We observed a shorter median overall survival (OS) and progression-free survival (PFS) in AR-gained compared to AR-normal patients (OS 10.5 versus 14.1 months, hazard ratio (HR) = 1.44, 95% confidence interval [CI] 0.98-2.13, P = 0.064 and PFS 4.0 versus 5.0 months, HR = 1.47, 95% CI 1.05-2.07, P = 0.024). In patients with mCRPC receiving second-line therapies, a significant treatment interaction was observed between plasma AR and cabazitaxel versus AR-directed therapies for OS (P = 0.041) but not PFS (P = 0.244). In an exploratory analysis, AR-gained patients treated with initial reduced dose of cabazitaxel had a significantly shorter median OS (7.3 versus 11.5 months, HR = 1.95, 95% CI 1.13-3.38, P = 0.016) and PFS (2.7 versus 5.0 months, HR = 2.27, 95% CI 1.39-3.71, P = 0.001). CONCLUSION: Plasma AR status has a potential clinical utility in patients being considered for cabazitaxel. Validation of these findings in prospective trials is warranted.

Plasma Androgen Receptor and Docetaxel for Metastatic Castration-resistant Prostate Cancer.

Plasma androgen receptor (AR) gain identifies metastatic castration-resistant prostate cancer (mCRPC) patients with worse outcome on abiraterone/enzalutamide, but its relevance in the context of taxane chemotherapy is unknown. We aimed to evaluate whether docetaxel is active regardless of plasma AR and to perform an exploratory analysis to compare docetaxel with abiraterone/enzalutamide. This multi-institutional study was a pooled analysis of AR status, determined by droplet digital polymerase chain reaction, on pretreatment plasma samples. We evaluated associations between plasma AR and overall/progression-free survival (OS/PFS) and prostate-specific antigen (PSA) response rate in 163 docetaxel-treated patients. OS was significantly shorter in case of AR gain (hazard ratio [HR]=1.61, 95% confidence interval [CI]=1.08-2.39, p=0.018), but not PFS (HR=1.04, 95% CI 0.74-1.46, p=0.8) or PSA response (odds ratio=1.14, 95% CI=0.65-1.99, p=0.7). We investigated the interaction between plasma AR and treatment type after incorporating updated data from our prior study of 73 chemotherapy-naïve, abiraterone/enzalutamide-treated patients, with data from 115 first-line docetaxel patients. In an exploratory analysis of mCRPC patients receiving first-line therapies, a significant interaction was observed between plasma AR and docetaxel versus abiraterone/enzalutamide for OS (HR=0.16, 95% CI=0.06-0.46, p<0.001) and PFS (HR=0.31, 95% CI=0.12-0.80, p=0.02). Specifically, we reported a significant difference for OS favoring abiraterone/enzalutamide for AR-normal patients (HR=1.93, 95% CI=1.19-3.12, p=0.008) and a suggestion favoring docetaxel for AR-gained patients (HR=0.53, 95% CI=0.24-1.16, p=0.11). These data suggest that AR-normal patients should receive abiraterone/enzalutamide and AR-gained could benefit from docetaxel. This treatment selection merits prospective evaluation in a randomized trial. PATIENT SUMMARY: We investigated whether plasma androgen receptor (AR) predicted outcome in metastatic castration-resistant prostate cancer (mCRPC) patients treated with docetaxel, and we performed an exploratory analysis in patients treated with docetaxel or AR-directed drugs as first-line mCRPC therapy. We showed that plasma AR normal favored hormonal treatment, whilst plasma AR-gained patients may have had a longer response to docetaxel, suggesting that plasma AR status could be a useful treatment selection biomarker.

Multimodal Approach to Outcome Prediction in Metastatic Castration-Resistant Prostate Cancer by Integrating Functional Imaging and Plasma DNA Analysis.

PURPOSE: Biomarkers for treatment personalization in metastatic castration-resistant prostate cancer (mCRPC) could help improve patient outcomes. Multiple tests on blood have reported associations with poorer outcome, including serum lactate dehydrogenase (LDH), chromogranin A (CGA), neutrophil:lymphocyte ratio (NLR), and, recently, copy number (CN) of androgen receptor (AR) in plasma DNA. Biologic data suggest an association between choline uptake and AR signaling. We aimed to integrate 18F-fluorocholine (FCH) uptake on positron emission tomography/computed tomography (PET/CT) scanning with plasma AR CN and other routinely obtained circulating biomarkers to evaluate their association with outcome. MATERIALS AND METHODS: We determined plasma AR CN by digital droplet polymerase chain reaction from 105 mCRPC samples collected before abiraterone (n = 65) or enzalutamide (n = 40) therapy in the before (n = 26) and after (n = 79) chemotherapy settings. Pretreatment serum LDH, CGA, and NLR were also measured. FCH-PET/CT scan was performed at baseline, and maximum standardized uptake value (SUVmax), total lesion activity (TLA), and metabolic tumor volume (MTV) were calculated. Main end points were the correlation of FCH-PET/CT parameters with circulating biomarkers and their impact on outcome. RESULTS: Plasma AR CN gain was observed in 27 patients (25.7%), and it correlated significantly with higher median SUVmax, TLA, and MTV values (P < .001). Kaplan-Meier curves showed significantly worse progression-free survival and overall survival in patients with plasma AR gain and higher SUVmax, TLA, and MTV values (P < .001 in each prognostic group). Conversely, no association was reported for prostate-specific antigen response. On multivariable analysis of overall survival, we showed as independent factors AR gain (hazard ratio [HR], 1.92; 95% CI, 1.07 to 3.47; P = .029), presence of visceral metastasis (HR, 3.04; 95% CI, 1.66 to 5.58; P = < .001), LDH (HR, 2.95; 95% CI, 1.72 to 5.05; P < .001), NLR (HR, 3.51; 95% CI, 2.14 to 5.74; P < .001), serum CGA (HR, 3.36; 95% CI, 1.99 to 5.67; P < .001), and MTV (HR, 2.09; 95% CI, 1.25 to 3.50; P = .005). CONCLUSION: Our results indicate the potential usefulness of integrating functional imaging with plasma DNA analysis and other noninvasive biomarkers as a tool to improve treatment selection for CRPC. A larger prospective evaluation is warranted.

Plasma Androgen Receptor Copy Number Status at Emergence of Metastatic Castration-Resistant Prostate Cancer: A Pooled Multicohort Analysis.

PURPOSE: Increases in androgen receptor (AR) copy number (CN) can be detected in plasma DNA when patients develop metastatic castration-resistant prostate cancer. We aim to evaluate the association between AR CN as a continuous variable and clinical outcome. PATIENTS AND METHODS: PCR2023 was an international, multi-institution, open-label, phase II study of abiraterone acetate plus prednisolone (AAP) or abiraterone acetate plus dexamethasone that included plasma AR assessment as a predefined exploratory secondary end point. Plasma AR CN data (ClinicalTrials.gov identifier: NCT01867710) from this study (n = 133) were pooled with data from the following three other cohorts: cohort A, which was treated with either AAP or enzalutamide (n = 73); the PREMIERE trial (ClinicalTrials.gov identifier: NCT02288936) of biomarkers for enzalutamide (n = 94); and a phase II trial from British Columbia (ClinicalTrials.gov identifier: NCT02125357) that randomly assigned men to either AAP or enzalutamide (n = 201). The primary outcome measures for the biomarker analysis were overall survival and progression-free survival. RESULTS: Using multivariable fractional polynomials analysis using Cox regression models, a nonlinear relationship between plasma AR CN and outcome was identified for overall survival, where initially for small incremental gains in CN there was a large added hazard ratio that plateaued at higher CN. The CN cut point associated with the highest local hazard ratio was 1.92. A similar nonlinear association was observed with progression-free survival. In an exploratory analysis of PCR2023, the time from start of long-term androgen-deprivation therapy to start of AAP or abiraterone acetate plus dexamethasone was significantly shorter in patients with plasma AR CN of 1.92 or greater than patients with plasma AR CN of less than 1.92 (43 v 130 weeks, respectively; P = .005). This was confirmed in cohort A (P = .003), the PREMIERE cohort (P = .03), and the British Colombia cohort (P = .003). CONCLUSION: Patients with metastatic castration-resistant prostate cancer can be dichotomized by a plasma AR CN cut point of 1.92. Plasma AR CN value of 1.92 or greater identifies aggressive disease that is poorly responsive to AR targeting and is associated with a prior short response to primary androgen-deprivation therapy.

Circulating tumor DNA in advanced prostate cancer: transitioning from discovery to a clinically implemented test.

The genomic landscape of metastatic castration-resistant prostate cancer (mCRPC) differs from that of the primary tumor and is dynamic during tumor progression. The real-time and repeated characterization of this process via conventional solid tumor biopsies is challenging. Alternatively, circulating cell-free DNA (cfDNA) containing circulating tumor DNA (ctDNA) can be obtained from patient plasma using minimally disruptive blood draws and is amenable to sequential analysis. ctDNA has high overlap with the genomic sequences of biopsies from metastases and has the advantage of being representative of multiple metastases. The availability of techniques with high sensitivity and specificity, such as next-generation sequencing (NGS) and digital PCR, has greatly contributed to the development of the cfDNA field and enabled the detection of genomic alterations at low ctDNA fractions. In mCRPC, a number of clinically relevant genomic alterations have been tracked in ctDNA, including androgen receptor (AR) aberrations, which have been shown to be associated with an adverse outcome to novel antiandrogen therapies, and alterations in homologous recombination repair (HRR) genes, which have been associated with a response to PARP inhibitors. Several clinical applications have been proposed for cfDNA analysis, including its use as a prognostic tool, as a predictive biomarker, to monitor tumor response and to identify novel mechanisms of resistance. To date, the cfDNA analysis has provided interesting results, but there is an urgent need for these findings to be confirmed in prospective clinical trials.

Plasma androgen receptor and serum chromogranin A in advanced prostate cancer.

Recently, mixed forms between adenocarcinoma and neuroendocrine prostate cancer (NEPC) have emerged that are characterized by persistent androgen receptor (AR)-signalling and elevated chromogranin A (CgA) levels. The main aim of this study was to analyze castration-resistant prostate cancer (CRPC) patients treated with abiraterone or enzalutamide, assessing progression-free/overall survival (PFS/OS) in association with circulating AR and CgA. AR aberrations were analyzed by droplet digital PCR in pre-treatment plasma samples collected from two biomarker protocols [197 patients from a retrospective study (REC 2192/2013) and 59 from a prospective trial (REC 6798/2015)]. We subdivided patients into three groups according to CgA by receiver-operating characteristic (ROC) curves. In the primary cohort, plasma AR gain and mutations (p.L702H/p.T878A) were detected in 78 (39.6%) and 16 (8.1%) patients, respectively. We observed a significantly worse PFS/OS in patients with higher-CgA than in patients with normal-CgA, especially those with no AR-aberrations. Multivariable analysis showed AR gain, higher-CgA and LDH levels as independent predictors of PFS [hazard ratio (HR) = 2.16, 95% confidence interval (95% CI) 1.50-3.12, p < 0.0001, HR = 1.73, 95% CI 1.06-2.84, p = 0.026, and HR = 2.13, 95% CI 1.45-3.13, p = 0.0001, respectively) and OS (HR = 1.72, 95% CI 1.15-2.57, p = 0.008, HR = 3.63, 95% CI 2.13-6.20, p < 0.0001, and HR = 2.31, 95% CI 1.54-3.48, p < 0.0001, respectively). These data were confirmed in the secondary cohort. Pre-treatment CgA detection could be useful to identify these mixed tumors and would seem to have a prognostic role, especially in AR-normal patients. This association needs further evaluation in larger prospective cohorts.