Association of PARP inhibitor treatment on the prevalence and progression of clonal hematopoiesis in patients with advanced prostate cancer.

BACKGROUND: Poly ADP-ribose polymerase (PARP) inhibitors are approved for the treatment of some men with advanced prostate cancer. Rare but serious side effects include myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). The impact of PARP inhibitors on clonal hematopoiesis (CH), a potential precursor lesion associated with MDS and AML, is incompletely understood in prostate cancer. We hypothesized that PARP inhibitors would increase CH prevalence and abundance. METHODS: We prospectively enrolled participants with advanced prostate cancer treated with PARP inhibitors. The presence of CH was assessed from leukocytes using an ultra-deep error-corrected dual unique molecular identifiers sequencing method targeting 49 genes most commonly mutated in CH and myeloid malignancies. Variant allele frequencies (VAF) of ≥0.5% were considered clinically significant. Blood samples were collected before and after PARP inhibitor treatment. RESULTS: Ten men were enrolled; mean age of 67 years. Six patients had Gleason 7 disease, and four had Gleason ≥8 disease at diagnosis. Nine had localized disease at diagnosis, and eight had prior treatment with radiation. The mean time between pre- and post-treatment blood samples was 11 months (range 2.6-31 months). Six patients (60%) had CH identified prior to PARP inhibitor treatment, three with multiple clones. Of 11 CH clones identified in follow-up, 5 (45%) appeared or increased after treatment. DNMT3A, TET2, and PPM1D were the most common CH alterations observed. The largest post-treatment increase involved the PPM1D gene. CONCLUSION: CH alterations are frequently found after treatment with PARP inhibitors in patients with prostate cancer and this may be one mechanism by which PARP inhibitors lead to increased risk of MDS/AML.

Characterization of structural, biochemical, pharmacokinetic, and pharmacodynamic properties of the LSD1 inhibitor bomedemstat in preclinical models.

INTRODUCTION: Lysine-specific demethylase 1 (LSD1) is emerging as a critical mediator of tumor progression in metastatic castration-resistant prostate cancer (mCRPC). Neuroendocrine prostate cancer (NEPC) is increasingly recognized as an adaptive mechanism of resistance in mCRPC patients failing androgen receptor axis-targeted therapies. Safe and effective LSD1 inhibitors are necessary to determine antitumor response in prostate cancer models. For this reason, we characterize the LSD1 inhibitor bomedemstat to assess its clinical potential in NEPC as well as other mCRPC pathological subtypes. METHODS: Bomedemstat was characterized via crystallization, flavine adenine dinucleotide spectrophotometry, and enzyme kinetics. On-target effects were assessed in relevant prostate cancer cell models by measuring proliferation and H3K4 methylation using western blot analysis. In vivo, pharmacokinetic (PK) and pharmacodynamic (PD) profiles of bomedemstat are also described. RESULTS: Structural, biochemical, and PK/PD properties of bomedemstat, an irreversible, orally-bioavailable inhibitor of LSD1 are reported. Our data demonstrate bomedemstat has >2500-fold greater specificity for LSD1 over monoamine oxidase (MAO)-A and -B. Bomedemstat also demonstrates activity against several models of advanced CRPC, including NEPC patient-derived xenografts. Significant intra-tumoral accumulation of orally-administered bomedemstat is measured with micromolar levels achieved in vivo (1.2 ± 0.45 µM at the 7.5 mg/kg dose and 3.76 ± 0.43 µM at the 15 mg/kg dose). Daily oral dosing of bomedemstat at 40 mg/kg/day is well-tolerated, with on-target thrombocytopenia observed that is rapidly reversible following treatment cessation. CONCLUSIONS: Bomedemstat provides enhanced specificity against LSD1, as revealed by structural and biochemical data. PK/PD data display an overall safety profile with manageable side effects resulting from LSD1 inhibition using bomedemstat in preclinical models. Altogether, our results support clinical testing of bomedemstat in the setting of mCRPC.

Third generation quinoline-3-carboxamide transcriptional disrupter of HDAC4, HIF-1α, and MEF-2 signaling for metastatic castration-resistant prostate cancer.

BACKGROUND: The quinoline-3-carboxamide, Tasquinimod (TasQ), is orally active as a maintenance therapy with an on-target mechanism-of-action via allosteric binding to HDAC4. This prevents formation of the HDAC4/NCoR1/HDAC3 complex, disrupting HIF-1α transcriptional activation and repressing MEF-2 target genes needed for adaptive survival signaling in the compromised tumor micro environment. In phase 3 clinical testing against metastatic castration-resistant prostate cancer(mCRPC), TasQ (1 mg/day) increased time-to-progression, but not overall survival. METHODS: TasQ analogs were chemically synthesized and tested for activity compared to the parental compound. These included HDAC4 enzymatic assays, qRT-PCR and western blot analyses of gene and protein expression following treatment, in vitro and in vivo efficacy against multiple prostate cancer models including PDXs, pharmacokinetic analyses,AHR binding and agonist assays, SPR analyses of binding to HDAC4 and NCoR1, RNAseq analysis of in vivo tumors, 3D endothelial sprouting assays, and a targeted kinase screen. Genetic knockout or knockdown controls were used when appropriate. RESULTS: Here, we document that, on this regimen (1 mg/day), TasQ blood levels are 10-fold lower than the optimal concentration (≥2 µM) needed for anticancer activity, suggesting higher daily doses are needed. Unfortunately, we also demonstrate that TasQ is an arylhydrocarbon receptor (AHR) agonist, which binds with an EC50 of 1 µM to produce unwanted off-target side effects. Therefore, we screened a library of TasQ analogsto maximize on-target versus off-target activity. Using this approach, we identified ESATA-20, which has ~10-fold lower AHR agonism and 5-fold greater potency against prostate cancer patient-derived xenografts. CONCLUSION: This increased therapeuticindex nominates ESATA-20 as a lead candidate forclinical development as an orally active third generation quinoline-3-carboxamide analog thatretains its on-target ability to disrupt HDAC4/HIF-1α/MEF-2-dependent adaptive survival signaling in the compromisedtumor microenvironment found in mCRPC.

Bipolar Androgen Therapy Followed by Androgen Receptor Inhibition as Sequential Therapy for Prostate Cancer.

Inhibition of androgen receptor (AR) signaling has been the mainstay of treatment of advanced prostate cancer (PCa) for the past 80 years. Combination and sequential AR-inhibiting therapies are highly effective palliative therapy, but they are not curative. All patients eventually develop resistance to primary castrating therapy [ie, castration-resistant PCa (CRPC)]. At this point, they are treated with subsequent lines of secondary AR inhibitory therapies. However, resistance to these agents also develops and patients progress to a state we have termed complete androgen inhibition-resistant PCa. This phase of the disease is associated with poor prognosis. At this point, treatment shifts to non-hormonal cytotoxic therapies (eg, chemotherapy and radiopharmaceuticals). However, the majority of PCas remain addicted to signaling through AR throughout the course of the disease. Resistant PCa cells adaptively upregulate AR activity, despite castration and AR inhibitors, via mechanisms such as AR overexpression, gene amplification, mutation, and expression of ligand-independent variants to permit sustained liganded and non-liganded AR signaling. Studies dating back nearly 30 years indicate that high expression of AR induced by prolonged castration becomes a vulnerability of CRPC cells in vitro and in mouse xenografts to supraphysiologic androgen (SPA), which induces cell death and growth arrest in this context. Based on these studies, we developed a counterintuitive treatment called bipolar androgen therapy (BAT) for patients with CRPC, in which SPA is administered intermittently to result in cycling of serum testosterone from the polar extremes of supraphysiologic to near-castrate levels. This rapid cycling is intended to disrupt the adaptive of AR regulation associated with chronic exposure to high or low levels of testosterone, while simultaneously targeting the spectrum of AR expression present in heterogeneous CRPC tumors. We have now tested BAT in >250 patients with CRPC. Here we present a review of these clinical studies, which have demonstrated collectively that BAT can be safely given to men with CRPC, improves quality of life, and produces therapeutic responses in ~30% of patients. As expected, resistance to BAT is associated with adaptive downregulation of AR expression. Intriguingly, this downregulation is associated with restoration of sensitivity to subsequent AR inhibitor therapies.

Bipolar androgen therapy (BAT): A patient's guide.

Bipolar androgen therapy (BAT) is a new treatment concept for men whose prostate cancer has become resistant to standard hormone-blocking therapy. Over the past decade, we have performed a series of clinical studies testing BAT in asymptomatic men with castration-resistant prostate cancer. The key findings from these clinical studies are that BAT (a) can be safely administered to asymptomatic patients with metastatic castrate-resistant prostate cancer; (b) does not produce symptomatic disease progression; (c) produces sustained prostate-specific antigen and objective responses in 30%-40% of patients; and (d) can resensitize and prolong response to subsequent antiandrogen therapy. The concept of BAT has generated significant interest from men with prostate cancer, their families, and their physicians. Here we provide a "Patient's Guide" that answers questions about BAT in a form that is accessible to patients, their families, and physicians. Our goal is to provide information to help patients make the most informed decisions they can regarding their prostate cancer treatment.

PSA provocation by bipolar androgen therapy may predict duration of response to first-line androgen deprivation: Updated results from the BATMAN study.

BACKGROUND: Previously, we reported results from the Phase II BATMAN study (Bipolar Androgen Therapy for Men with Androgen-ablation Naïve prostate cancer). This study (NCT01750398) was designed to evaluate the safety and efficacy of a treatment regimen consisting of a 6-month lead in-phase of androgen deprivation therapy (ADT) followed by alternating 3-month intervals of bipolar androgen therapy (BAT) and ADT alone. Here we report > 5-year follow-up related to the duration of subsequent ADT, response to first-line androgen receptor inhibitors, safety, and survival in men with castration-sensitive prostate cancer treated on the BATMAN study. METHODS: Univariate Cox regression was utilized to compare overall survival between Responders who achieved a prostate-specific antigen (PSA) level of <4 ng/ml and Non-Responders who achieved a PSA level of ≥4 ng/ml after BAT/ADT. Kaplan-Meier method and Cox regression were used to assess progression-free (PFS) and overall survival (OS) on BAT and on subsequent abiraterone or enzalutamide and on the association between PSA peak during BAT and each time to event outcome. RESULTS: Over median follow-up of 95 months, the median PFS on ADT for the entire cohort was 47.8. Median OS has not been reached (NR). Median OS for Non-Responders is 43 months versus NR (not reached) for responders (hazard ratio [HR]: 0.176, p = 0.002). Post-BAT, the PSA50 and PSA90 responses to abiraterone or enzalutamide were 94.4% and 66.7%, respectively and median PFS was 20.6 months. Patients with peak PSA level of ≥9 ng/ml after BAT had median PFS of 20.6 months versus NR for those having PSA < 9 ng/ml (HR: 0.122, p < 0.001). Median OS was 79.6 months for patients with PSA peak of ≥9 ng/ml versus NR for those having PSA peak of <9 ng/ml (HR: 0.409, p = 0.131). CONCLUSION: The use of BAT as part of first-line hormonal therapy strategy does not induce adversely affect long-term survival or induce any significant long-term adverse sequelae in patients with prostate cancer. Cycling BAT may extend the duration of ADT response and enhance response to subsequent androgen ablative therapies. The magnitude of BAT-provoked increase in PSA may predict duration of ADT response and warrants further study.

The association of sex steroid hormone concentrations with non-alcoholic fatty liver disease and liver enzymes in US men.

BACKGROUND & AIMS: This study aimed to analyse the association of sex hormone levels with liver enzyme levels and non-alcoholic fatty liver disease (NAFLD) in a nationally representative sample of men. METHODS: A total of 919 men from the US National Health and Nutrition Examination Study (NHANES) III were included in this cross-sectional analysis of data from 1988 to 1991. We used existing data on serum total and free testosterone, total and free estradiol, androstanediol glucuronide (AAG) and sex steroid-binding globulin (SHBG), and estimated their associations with aspartate aminotransferase (AST), and alanine aminotransferase (ALT) and NAFLD, as determined using ultrasound, after adjusting for possible confounders including age, race, smoking, alcohol, physical activity, waist circumference and steroid hormones. RESULTS: Lower total testosterone (TT) and higher free estradiol were associated with higher odds of NAFLD after adjusting for confounders including the other sex hormones. Lower TT was associated with higher odds of elevated AST, but not ALT. Free testosterone, total estradiol, SHBG and AAG were not associated with NAFLD or liver enzymes. CONCLUSIONS: This study supports an inverse association between TT concentration and NAFLD in men independent of other sex hormones (SHBG, AAG and estradiol) and known risk factors, such as obesity, age and lifestyle. Exploration of whether TT might be a non-invasive marker for NAFLD diagnosis is warranted.

Reversing the effects of androgen-deprivation therapy in men with metastatic castration-resistant prostate cancer.

OBJECTIVE: To investigate whether bipolar androgen therapy (BAT), involving rapid cyclic administration of high-dose testosterone, as a novel treatment for metastatic castration-resistant prostate cancer (mCRPC) promotes improvements in body composition and associated improvements in lipid profiles and quality of life. PATIENTS AND METHODS: Men from two completed trials with computed tomography imaging at baseline and after three cycles of BAT were included. Cross-sectional areas of psoas muscle, visceral and subcutaneous fat were measured at the L3 vertebral level. Functional Assessment of Chronic Illness Therapy - Fatigue questionnaire and 36-item short-form health survey were used to assess quality of life. RESULTS: The 60 included patients lost a mean (sd) of 7.8 (8.2)% of subcutaneous fat, 9.8 (18.2)% of visceral fat, and gained 12.2 (6.7)% muscle mass. Changes in subcutaneous and visceral fat were positively correlated with each other (Spearman's correlation coefficient 0.58, 95% confidence interval 0.35-0.71) independent of the effects of age, body mass index, and duration of androgen-deprivation therapy. Energy, physical function, and measures of limitations due to physical health were all significantly improved at 3 months. The improvements in body composition were not correlated with decreases in lipid levels or observed improvements in quality of life. CONCLUSIONS: In the present study, BAT was associated with significant improvements in body composition, lipid parameters, and quality of life. This has promising implications for the long-term health of men with mCRPC.

Mipsagargin: The Beginning-Not the End-of Thapsigargin Prodrug-Based Cancer Therapeutics.

Søren Brøgger Christensen isolated and characterized the cell-penetrant sesquiterpene lactone Thapsigargin (TG) from the fruit Thapsia garganica. In the late 1980s/early 1990s, TG was supplied to multiple independent and collaborative groups. Using this TG, studies documented with a large variety of mammalian cell types that TG rapidly (i.e., within seconds to a minute) penetrates cells, resulting in an essentially irreversible binding and inhibiting (IC50~10 nM) of SERCA 2b calcium uptake pumps. If exposure to 50-100 nM TG is sustained for >24-48 h, prostate cancer cells undergo apoptotic death. TG-induced death requires changes in the cytoplasmic Ca2+, initiating a calmodulin/calcineurin/calpain-dependent signaling cascade that involves BAD-dependent opening of the mitochondrial permeability transition pore (MPTP); this releases cytochrome C into the cytoplasm, activating caspases and nucleases. Chemically unmodified TG has no therapeutic index and is poorly water soluble. A TG analog, in which the 8-acyl groups is replaced with the 12-aminododecanoyl group, afforded 12-ADT, retaining an EC50 for killing of <100 nM. Conjugation of 12-ADT to a series of 5-8 amino acid peptides was engineered so that they are efficiently hydrolyzed by only one of a series of proteases [e.g., KLK3 (also known as Prostate Specific Antigen); KLK2 (also known as hK2); Fibroblast Activation Protein Protease (FAP); or Folh1 (also known as Prostate Specific Membrane Antigen)]. The obtained conjugates have increased water solubility for systemic delivery in the blood and prevent cell penetrance and, thus, killing until the TG-prodrug is hydrolyzed by the targeting protease in the vicinity of the cancer cells. We summarize the preclinical validation of each of these TG-prodrugs with special attention to the PSMA TG-prodrug, Mipsagargin, which is in phase II clinical testing.

Extreme responses to immune checkpoint blockade following bipolar androgen therapy and enzalutamide in patients with metastatic castration resistant prostate cancer.

BACKGROUND: Immune checkpoint inhibition has been shown to have limited efficacy in patients with metastatic prostate cancer. Prostate cancers that harbor certain homologous recombination (HR) DNA repair gene mutations, inactivating CDK12 mutations or have underlying mismatch repair deficiency may be effectively treated with immunotherapy. Combination therapy may improve clinical response rates to immune checkpoint blockade. We observed profound prostate-specific antigen (PSA) and/or objective responses to immune checkpoint blockade following prior treatment with bipolar androgen therapy (BAT) and enzalutamide. METHODS: We report three cases of patients with metastatic castration resistant prostate cancer (mCRPC) undergoing therapy with anti-PD-1 inhibitors. All patients underwent both somatic molecular testing and germline genetic testing. RESULTS: Two of the three patients with mCRPC harbored an inactivating mutation in an HR DNA repair gene (BRCA2, ATM). No patient demonstrated mismatch repair deficiency, nor were CDK12 alterations present. All three patients had been treated with BAT and enzalutamide before immune checkpoint blockade, a paradoxical approach for the treatment of mCRPC developed by our group. CONCLUSIONS: These cases of mCRPC suggest that immune checkpoint blockade may have therapeutic potential in patients with prostate cancer, especially following immune activation ("priming") using BAT and enzalutamide.

A Phase I Study of Alpha-1,3-Galactosyltransferase-Expressing Allogeneic Renal Cell Carcinoma Immunotherapy in Patients with Refractory Metastatic Renal Cell Carcinoma.

LESSONS LEARNED: HyperAcute Renal immunotherapy was well tolerated and demonstrated antitumor activity in patients requiring salvage-line treatment for metastatic renal cell carcinoma (mRCC). HyperAcute Renal immunotherapy was safely administered with concomitant salvage-line treatments for mRCC, and it may be a candidate for inclusion in novel combinations for salvage treatment of mRCC because of its unique mechanism of action. BACKGROUND: HyperAcute Renal (HAR) immunotherapy exploits a naturally occurring barrier to xenotransplantation and zoonotic infections in humans to immunize patients against metastatic renal cell carcinoma (mRCC) cells. HAR consists of two allogeneic renal cancer cell lines genetically modified to express α(1,3)Gal, to which humans have an inherent pre-existing immunity. METHODS: Patients with refractory mRCC were eligible for this phase I dose-escalation trial. Concomitant treatment was permitted after the initial 2 months of HAR monotherapy. HAR was injected intradermally weekly for 4 weeks then biweekly for 20 weeks, totaling 14 immunizations. The primary endpoint was safety and determination of a maximum tolerated dose (MTD). RESULTS: Among 18 patients enrolled, two grade 3 adverse events (AEs) were attributed to HAR, lymphopenia and injection site reaction, and no grade 4/5 AEs occurred. The recommended phase II dose (RP2D) was 300 million cells. One patient had a partial response and eight patients had stable disease, for a disease control rate of 50% (9/18). Median overall survival with low-dose HAR was 14.2 months and was 25.3 months with high-dose HAR. CONCLUSION: In pretreated mRCC, HAR immunotherapy was well tolerated and demonstrated antitumor activity. HAR immunotherapy may be a candidate for inclusion in novel combinations for salvage treatment of mRCC.

An IL-2 proaerolysin fusion toxin that selectively eliminates regulatory t cells to enhance antitumor immune response.

BACKGROUND: Recent success with immune-checkpoint inhibitors in some tumor types has highlighted the power of the immune system to control and eradicate human cancer cells. However, these therapies have demonstrated a limited activity in prostate cancer, which has a more immunosuppressive microenvironment that can be because of the presence of a variety of inhibitory cell types, such as myeloid-derived suppressor cells, mesenchymal stem cells, and regulatory T cells (Tregs). One strategy to improve the efficacy of immune-based therapies for prostate cancer is to selectively eliminate these immunosuppressive cells within the tumor microenvironment. METHODS: We developed and characterized a chimeric protein consisting of the cytokine IL-2 fused to binding mutant of the highly toxic bacterial toxin proaerolysin (ie IL2-R336A). RESULTS: The IL2-R336A fusion protein selectively kills immunosuppressive Tregs that express the IL-2 receptor while having little to no effect on cells negative for this target. IL2-R336A depleted Tregs in both tumor bearing and nontumor bearing mice. Tumor bearing mice vaccinated with a GMCSF-expressing CT-26 GVAX vaccine had reduced tumor growth when given IL2-R336A before vaccination. IL2-R336A also enhanced immune response to a model hemagglutinin antigen (HA) in HA-tolerized mice. CONCLUSION: These results suggest that this IL2-R336A toxin may be a useful in improving the therapeutic efficacy of antitumor vaccines by enhancing the immune response against target tumor antigens.

Risk of development of visceral metastases subsequent to abiraterone vs placebo: An analysis of mode of radiographic progression in COU-AA-302.

BACKGROUND: Abiraterone increases survival in prostate cancer, but tumors resistant to abiraterone can exhibit a hormonally resistant, aggressive phenotype. We hypothesized that the therapeutic pressure of abiraterone is resulting in more clinically aggressive disease at progression, characterized by increased visceral metastases. Our objective was to determine whether abiraterone increased the risk of development of visceral metastases at the time of progression compared with placebo in a randomized phase III trial. METHODS: We performed a post hoc analysis of the COU-AA-302 trial of abiraterone plus prednisone vs placebo plus prednisone in patients with metastatic castration-resistant prostate cancer. The primary outcome was the development of visceral metastases. The cumulative incidences of visceral metastases were calculated by the Kaplan-Meier method and compared using log-rank testing. Multivariable Cox regression analysis assessed for the independent association of abiraterone with the development of visceral metastases. RESULTS: Eighty-four of 1088 patients developed visceral metastases during study. Log-rank testing and Cox regression showed no difference in time to visceral metastases between groups (HR 1.01 [95% confidence interval (CI), 0.65-1.56]; P = .97). Abiraterone treatment was not associated with the development of visceral metastases in multivariable analysis (HR 0.89 [95% CI, 0.57-1.40]; P = .62). The study was limited by censoring of radiographic outcomes at the time of completion of primary study therapy; longer term risks were not assessed. CONCLUSIONS: Abiraterone was not associated with increased risk of visceral metastatic disease at the time of progression compared with placebo.

A pilot study of prostate-specific membrane antigen (PSMA) dynamics in men undergoing treatment for advanced prostate cancer.

BACKGROUND: Prostate-specific membrane antigen (PSMA) is a rational target for noninvasive detection of recurrent prostate cancer (PCa) and for therapy of metastatic castration-resistant prostate cancer (mCRPC) with PSMA-targeted agents. Here we conducted serial measurements of PSMA expression on circulating tumor cells (CTCs) to evaluate patterns of longitudinal PSMA dynamics over the course of multiple sequential therapies. METHODS: A retrospective investigation of men with mCRPC undergoing evaluation at medical oncology clinics at our institution assessed the dynamics of PSMA expression in the context of different systemic treatments administered sequentially. Eligibility included patients who began systemic therapies with androgen receptor (AR)-directed agents or taxane agents for whom peripheral blood samples were tested for CTC mRNA of AR splice variant-7 (AR-V7), prostate-specific antigen (PSA), and PSMA (with >2 CTC + results) in a CLIA-accredited laboratory. RESULTS: From August 2015 to November 2017, we identified 96 eligible men. Fifteen had greater than or equal to 2 sequential therapies and evaluable CTC samples, greater than or equal to 1 expressing PSMA (PSMA+). Among the 15 patients included in this analysis, a total of 54 PSMA status evaluations were performed in the context of 48 therapies during a median follow-up of 18 months. At baseline, PSMA signal was detected ("positive") in 11 of 15 (73.3%) patients, while for 4 of 15 (26.7%) patients PSMA signal was undetectable ("negative"). In all but two patients, the baseline collection corresponded with a change in treatment. On the second assessment, PSMA increases were detected in all 4/4 (100%) PSMA-negative patients and 8 of 11 (72.7%) PSMA-positive patients. PSMA significantly decreased in a patient treated with 177 Lu-PSMA-617. Serum PSA declines were seen in 7 of 8 (88%) of the treatment periods where PSMA decreased. CONCLUSIONS: PSMA expression in CTCs is a dynamic marker. PSMA transcript declines appear to be associated with concurrent decreases in serum PSA. Sequential CTC sampling could provide a noninvasive response assessment to systemic treatment for mCRPC.

Bipolar androgen therapy in men with metastatic castration-resistant prostate cancer after progression on enzalutamide: an open-label, phase 2, multicohort study.

BACKGROUND: Prostate cancer that progresses after enzalutamide treatment is poorly responsive to further antiandrogen therapy, and paradoxically, rapid cycling between high and low serum testosterone concentrations (bipolar androgen therapy [BAT]) in this setting might induce tumour responses. We aimed to evaluate BAT in patients with metastatic castration-resistant prostate cancer that progressed after enzalutamide. METHODS: We did this single-centre, open-label, phase 2, multicohort study in the USA. We included patients aged 18 years or older who had histologically confirmed and radiographically documented metastatic castration-resistant prostate cancer, with no more than two previous second-line hormonal therapies, and a castrate concentration of testosterone. Patients were asymptomatic, with Eastern Cooperative Oncology Group performance status of 0-2, and did not have high-risk lesions for tumour flare (eg, >5 sites of visceral disease or bone lesions with impending fracture). For the cohort reported here, we required patients to have had progression on enzalutamide with a continued prostate-specific antigen (PSA) rise after enzalutamide treatment discontinuation. Patients received BAT, which consisted of intramuscular testosterone cipionate 400 mg every 28 days until progression and continued luteinising hormone-releasing hormone agonist therapy. Upon progression after BAT, men were rechallenged with oral enzalutamide 160 mg daily. The co-primary endpoints were investigator-assessed 50% decline in PSA concentration from baseline (PSA50) for BAT (for all patients who received at least one dose) and for enzalutamide rechallenge (based on intention-to-treat analysis). These data represent the final analysis for the post-enzalutamide cohort, while two additional cohorts (post-abiraterone and newly castration-resistant prostate cancer) are ongoing. The trial is registered with ClinicalTrials.gov, number NCT02090114. FINDINGS: Between Aug 28, 2014, and May 18, 2016, we accrued 30 eligible patients and treated them with BAT. Nine (30%; 95% CI 15-49; p<0·0001) of 30 patients achieved a PSA50 to BAT. 29 patients completed BAT and 21 proceeded to enzalutamide rechallenge, of whom 15 (52%; 95% CI 33-71; p<0·0001) achieved a PSA50 response. During BAT, the only grade 3-4 adverse event occurring in more than one patient was hypertension (three [10%] patients). Other grade 3 or worse adverse events occurring during BAT in one [3%] patient each were pulmonary embolism, myocardial infarction, urinary obstruction, gallstone, and sepsis. During enzalutamide retreatment, no grade 3-4 toxicities occurred in more than one patient. No treatment-related deaths were reported during either BAT or enzalutamide retreatment. INTERPRETATION: BAT is a safe therapy that resulted in responses in asymptomatic men with metastatic castration-resistant prostate cancer and also resensitisation to enzalutamide in most patients undergoing rechallenge. Further studies with BAT are needed to define the potential clinical role for BAT in the management of metastatic castration-resistant prostate cancer and the optimal strategy for sequencing between androgen and antiandrogen therapies in metastatic castration-resistant prostate cancer to maximise therapeutic benefit to patients. FUNDING: National Institutes of Health and National Cancer Institute.

PSA-alpha-2-macroglobulin complex is enzymatically active in the serum of patients with advanced prostate cancer and can degrade circulating peptide hormones.

BACKGROUND: Prostate cancer cells produce high levels of the serine protease Prostate-Specific Antigen (PSA). PSA is enzymatically active in the tumor microenvironment but is presumed to be enzymatically inactive in the blood due to complex formation with serum protease inhibitors α-1-antichymotrypsin and α-2-macroglobulin (A2M). PSA-A2M complexes cannot be measured by standard ELISA assays and are also rapidly cleared from the circulation. Thus the exact magnitude of PSA production by prostate cancer cells is not easily measured. The PSA complexed to A2M is unable to cleave proteins but maintains the ability to cleave small peptide substrates. Thus, in advanced prostate cancer, sufficient PSA-A2M may be in circulation to effect total A2M levels, levels of cytokines bound to A2M and hydrolyze small circulating peptide hormones. METHODS: Total A2M levels in men with advanced prostate cancer and PSA levels above 1000 ng/mL were measured by ELISA and compared to controls. Additional ELISA assays were used to measure levels of IL-6 and TGF-beta which can bind to A2M. The ability of PSA-A2M complexes to hydrolyze protein and peptide substrates was analyzed ± PSA inhibitor. Enzymatic activity of PSA-A2M in serum of men with high PSA levels was also assayed. RESULTS: Serum A2M levels are inversely correlated with PSA levels in men with advanced prostate cancer. Il-6 Levels are significantly elevated in men with PSA >1000 ng/mL compared to controls with PSA <0.1 ng/mL. PSA-A2M complex in serum of men with PSA levels >1000 ng/mL can hydrolyze small fluorescently labeled peptide substrates but not large proteins that are PSA substrates. PSA can hydrolyze small peptide hormones like PTHrP and osteocalcin. PSA complexed to A2M retains the ability to degrade PTHrP. CONCLUSIONS: In advanced prostate cancer with PSA levels >1000 ng/mL, sufficient PSA-A2M is present in circulation to produce enzymatic activity against circulating small peptide hormones. Sufficient PSA is produced in advanced prostate cancer to alter total A2M levels, which can potentially alter levels of a variety of growth factors such as IL-6, TGF-beta, basic FGF, and PDGF. Alterations in levels of these cytokines and proteolytic degradation of small peptide hormones may have profound effect on host-cancer interaction.

Androgen Receptor Splice Variants Are Not Substrates of Nonsense-Mediated Decay.

BACKGROUND: Androgen receptor (AR) splice variants have been clinically associated with progressive cancer, castration-resistance, and resistance to AR antagonists and androgen synthesis inhibitors. AR variants can be generated by genomic alterations and alternative splicing, and their expression is androgen-regulated. There has been a suggestion that AR variants bearing premature termination codons and coding for truncated proteins should be regulated by the nonsense-mediated decay (NMD) mRNA surveillance pathway, suggesting that either the NMD pathway is dysfunctional in variant-expressing cell lines or that variants are somehow able to evade degradation by NMD. METHODS: We first used siRNA knockdown of the NMD regulator, UPF1, in an NMD reporter assay to determine if this surveillance pathway is functioning normally in AR variant-expressing cell lines. We then used UPF1 knockdown to determine if expression of the AR variants ARV3 and ARV7 is affected by inhibition of NMD. Next, we analyzed androgen regulation of UPF1 and used transcript expression analysis to determine if there is any association between UPF1 expression, resistance, and ARV3 or ARV7 expression. RESULTS: We found that the NMD pathway functions normally in the AR variant-expressing cell line 22Rv1 and that inhibition of NMD does not increase expression of ARV3 or ARV7. Furthermore, we found that expression of UPF1 is not androgen-regulated. We also found that UFP1 expression levels do not differentiate castration-sensitive from resistant cell line and that UPF1 expression does not correlate with expression of ARV3 or ARV7 in cells in which these variants are highly expressed. CONCLUSION: This study eliminates a possible mechanism of regulation of certain AR variants. Future research into the regulation of AR variants should focus on other mechanisms to better understand the origin of these variants and to possibly inhibit their expression for the resensitization of resistant cancers. Prostate 77:829-837, 2017. © 2017 Wiley Periodicals, Inc.

AR-V7 and resistance to enzalutamide and abiraterone in prostate cancer.

BACKGROUND: The androgen-receptor isoform encoded by splice variant 7 lacks the ligand-binding domain, which is the target of enzalutamide and abiraterone, but remains constitutively active as a transcription factor. We hypothesized that detection of androgen-receptor splice variant 7 messenger RNA (AR-V7) in circulating tumor cells from men with advanced prostate cancer would be associated with resistance to enzalutamide and abiraterone. METHODS: We used a quantitative reverse-transcriptase-polymerase-chain-reaction assay to evaluate AR-V7 in circulating tumor cells from prospectively enrolled patients with metastatic castration-resistant prostate cancer who were initiating treatment with either enzalutamide or abiraterone. We examined associations between AR-V7 status (positive vs. negative) and prostate-specific antigen (PSA) response rates (the primary end point), freedom from PSA progression (PSA progression-free survival), clinical or radiographic progression-free survival, and overall survival. RESULTS: A total of 31 enzalutamide-treated patients and 31 abiraterone-treated patients were enrolled, of whom 39% and 19%, respectively, had detectable AR-V7 in circulating tumor cells. Among men receiving enzalutamide, AR-V7-positive patients had lower PSA response rates than AR-V7-negative patients (0% vs. 53%, P=0.004) and shorter PSA progression-free survival (median, 1.4 months vs. 6.0 months; P<0.001), clinical or radiographic progression-free survival (median, 2.1 months vs. 6.1 months; P<0.001), and overall survival (median, 5.5 months vs. not reached; P=0.002). Similarly, among men receiving abiraterone, AR-V7-positive patients had lower PSA response rates than AR-V7-negative patients (0% vs. 68%, P=0.004) and shorter PSA progression-free survival (median, 1.3 months vs. not reached; P<0.001), clinical or radiographic progression-free survival (median, 2.3 months vs. not reached; P<0.001), and overall survival (median, 10.6 months vs. not reached, P=0.006). The association between AR-V7 detection and therapeutic resistance was maintained after adjustment for expression of full-length androgen receptor messenger RNA. CONCLUSIONS: Detection of AR-V7 in circulating tumor cells from patients with castration-resistant prostate cancer may be associated with resistance to enzalutamide and abiraterone. These findings require large-scale prospective validation. (Funded by the Prostate Cancer Foundation and others.).

A phase II randomized trial of Observation versus stereotactic ablative RadiatIon for OLigometastatic prostate CancEr (ORIOLE).

BACKGROUND: We describe a randomized, non-blinded Phase II interventional study to assess the safety and efficacy of stereotactic ablative radiotherapy (SABR) for hormone-sensitive oligometastatic prostate adenocarcinoma, and to describe the biology of the oligometastatic state using immunologic, cellular, molecular, and functional imaging correlates. 54 men with oligometastatic prostate adenocarcinoma will be accrued. The primary clinical endpoint will be progression at 6 months from randomization with the hypothesis that SABR to all metastases will forestall progression by disrupting the metastatic process. Secondary clinical endpoints will include local control at 6 months post-SABR, toxicity and quality of life, and androgen deprivation therapy (ADT)-free survival (ADT-FS). Further fundamental analysis of the oligometastatic state with be achieved through correlation with investigational 18F-DCFPyL PET/CT imaging and measurement of circulating tumor cells, circulating tumor DNA, and circulating T-cell receptor repertoires, facilitating an unprecedented opportunity to characterize, in isolation, the effects of SABR on the dynamics of and immunologic response to oligometastatic disease. METHODS/DESIGN: Patients will be randomized 2:1 to SABR or observation with minimization to balance assignment by primary intervention, prior hormonal therapy, and PSA doubling time. Progression after 6 months will be compared using Fisher's exact test. Hazard ratios and Kaplan-Meier estimates of progression free survival (PFS), ADT free survival (ADT-FS), time to locoregional progression (TTLP) and time to distant progression (TTDP) will be calculated based on an intention-to-treat. Local control will be assessed using Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 criteria. Withdrawal from the study prior to 6 months will be counted as progression. Adverse events will be summarized by type and grade. Quality of life pre- and post- SABR will be measured by Brief Pain Inventory. DISCUSSION: The ORIOLE trial is the first randomized, non-blinded Phase II interventional study in the North America evaluating the safety and efficacy of SABR in oligometastatic hormone-sensitive prostate cancer. Leading-edge laboratory and imaging correlates will provide unique insight into the effects of SABR on the oligometastatic state. TRIAL REGISTRATIONS: ClinicalTrials.gov Identifier: NCT02680587. URL of Registry: https://clinicaltrials.gov/show/NCT02680587 Date of Registration: 02/08/2016. Date of First Participant Enrollment: 05/23/2016.