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.

Diverse AR-V7 cistromes in castration-resistant prostate cancer are governed by HoxB13.

The constitutively active androgen receptor (AR) splice variant 7 (AR-V7) plays an important role in the progression of castration-resistant prostate cancer (CRPC). Although biomarker studies established the role of AR-V7 in resistance to AR-targeting therapies, how AR-V7 mediates genomic functions in CRPC remains largely unknown. Using a ChIP-exo approach, we show AR-V7 binds to distinct genomic regions and recognizes a full-length androgen-responsive element in CRPC cells and patient tissues. Remarkably, we find dramatic differences in AR-V7 cistromes across diverse CRPC cells and patient tissues, regulating different target gene sets involved in CRPC progression. Surprisingly, we discover that HoxB13 is universally required for and colocalizes with AR-V7 binding to open chromatin across CRPC genomes. HoxB13 pioneers AR-V7 binding through direct physical interaction, and collaborates with AR-V7 to up-regulate target oncogenes. Transcriptional coregulation by HoxB13 and AR-V7 was further supported by their coexpression in tumors and circulating tumor cells from CRPC patients. Importantly, HoxB13 silencing significantly decreases CRPC growth through inhibition of AR-V7 oncogenic function. These results identify HoxB13 as a pivotal upstream regulator of AR-V7-driven transcriptomes that are often cell context-dependent in CRPC, suggesting that HoxB13 may serve as a therapeutic target for AR-V7-driven prostate tumors.

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.

Prostate Cancer Disseminated Tumor Cells are Rarely Detected in the Bone Marrow of Patients with Localized Disease Undergoing Radical Prostatectomy across Multiple Rare Cell Detection Platforms.

PURPOSE: Prostate circulating tumor cells escape into peripheral blood and enter bone marrow as disseminated tumor cells, representing an early step before conventionally detectable metastasis. It is unclear how frequently this occurs in localized disease and existing detection methods rely on epithelial markers with low specificity and sensitivity. We used multiple methodologies of disseminated tumor cell detection in bone marrow harvested at radical prostatectomy. MATERIALS AND METHODS: Bone marrow was harvested from 208 clinically localized cases, 16 controls and 5 metastatic cases with peripheral blood obtained from 37 metastatic cases. Samples were evaluated at 4 centers with 4 distinct platforms using antibody enrichment with the AdnaTest (Qiagen®) or VERSA (versatile exclusion based rare sample analysis), or whole sample interrogation with the RareCyte platform (Seattle, Washington) or HD-SCA (high definition single cell assay) using traditional epithelial markers and prostate specific markers. We investigated the sensitivity and specificity of these markers by evaluating expression levels in control and metastatic cases. RESULTS: EpCAM, NKX3.1 and AR were nonspecifically expressed in controls and in most samples using AdnaTest with no relation to perioperative variables. Only 1 patient with localized disease showed positive results for the prostate specific marker PSA. With the VERSA platform no localized case demonstrated disseminated tumor cells. With the RareCyte and HD-SCA platforms only a single patient had 1 disseminated tumor cell. CONCLUSIONS: Evaluation across multiple platforms revealed that epithelial markers are nonspecific in bone marrow and, thus, not suitable for disseminated tumor cell detection. Using prostate specific markers disseminated tumor cells were typically not detected in patients with localized prostate cancer.

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.).

Resistance to thyroid hormone is modulated in vivo by the nuclear receptor corepressor (NCOR1).

Mutations in the ligand-binding domain of the thyroid hormone receptor ß (TRß) lead to resistance to thyroid hormone (RTH). These TRß mutants function in a dominant-negative fashion to interfere with the transcription activity of wild-type thyroid hormone receptors (TRs), leading to dysregulation of the pituitary-thyroid axis and resistance in peripheral tissues. The molecular mechanism by which TRß mutants cause RTH has been postulated to be an inability of the mutants to properly release the nuclear corepressors (NCORs), thereby inhibiting thyroid hormone (TH)-mediated transcription activity. To test this hypothesis in vivo, we crossed Thrb(PV) mice (a model of RTH) expressing a human TRß mutant (PV) with mice expressing a mutant Ncor1 allele (Ncor1(ΔID) mice) that cannot recruit a TR or a PV mutant. Remarkably, in the presence of NCOR1ΔID, the abnormally elevated thyroid-stimulating hormone and TH levels found in Thrb(PV) mice were modestly but significantly corrected. Furthermore, thyroid hyperplasia, weight loss, and other hallmarks of RTH were also partially reverted in mice expressing NCOR1ΔID. Taken together, these data suggest that the aberrant recruitment of NCOR1 by RTH TRß mutants leads to clinical RTH in humans. The present study suggests that therapies aimed at the TR-NCOR1 interaction or its downstream actions could be tested as potential targets in treating RTH.

The Impact of Circulating Tumor Cell HOXB13 RNA Detection in Men with Metastatic Castration-Resistant Prostate Cancer (mCRPC) Treated with Abiraterone or Enzalutamide.

PURPOSE: HOXB13 is an androgen receptor (AR) coregulator specifically expressed in cells of prostatic lineage. We sought to associate circulating tumor cell (CTC) HOXB13 expression with outcomes in men with mCRPC treated with abiraterone or enzalutamide. EXPERIMENTAL DESIGN: We conducted a retrospective analysis of the multicenter prospective PROPHECY trial of mCRPC men (NCT02269982, n = 118) treated with abiraterone/enzalutamide. CTC detection and HOXB13 complementary DNA (cDNA) expression was measured using a modified Adnatest, grouping patients into 3 categories: CTC 0 (undetectable); CTC+ HOXB13 CTC low (<4 copies); or CTC+ HOXB13 CTC high. The HOXB13 threshold was determined by maximally selected rank statistics for prognostic associations with overall survival (OS) and progression-free survival (PFS). RESULTS: We included 102 men with sufficient CTC HOXB13 cDNA, identifying 25%, 31%, and 44% of patients who were CTC 0, CTC+ HOXB13 low, and CTC+ HOXB13 high, respectively. Median OS were 25.7, 27.8, and 12.1 months whereas the median PFS were 9.0, 7.7, and 3.8 months, respectively. In subgroup analysis among men with CellSearch CTCs ≥5 copies/mL and adjusting for prior abi/enza treatment and Halabi clinical risk score, the multivariate HR for HOXB13 CTC detection was 2.39 (95% CI, 1.06-5.40) for OS and 2.78 (95% CI, 1.38-5.59) for PFS, respectively. Low HOXB13 CTC detection was associated with lower CTC PSA, PSMA, AR-FL, and AR-V7 detection, and more liver/lung metastases (41% vs. 25%). CONCLUSIONS: Higher CTC HOXB13 expression is associated with AR-dependent biomarkers in CTCs and is adversely prognostic in the context of potent AR inhibition in men with mCRPC.

Uncommon and Challenging Phenotypes of High-Risk Human Papillomavirus-Associated Head and Neck Carcinomas Revealed by High-Throughput Studies.

BACKGROUND: HPV- associated squamous cell carcinoma (SCC) is uncommon in non-oropharynx sites and not well characterized. This study aims to investigate uncommon phenotypes of HPV-associated head and neck carcinoma, the prevalence and morphologic spectrum of HPV-associated SCC in the oral cavity, larynx and hypopharynx. METHOD: P16 immunostaining and HPV E6/7 in situ hybridization (ISH) were performed on tissue microarrays comprised of SCCs from different anatomic sites: oropharynx (n = 270), hypopharynx (n = 52), oral cavity (n = 95) and larynx (n = 123). Tumors were classified as HPV-associated based on a positive E6/7 ISH testing. RNA sequencing was performed on several selected cases. RESULT: 66% oropharynx SCCs (OPSCCs) were HPV-associated; all were p16/HPV testing concordant except one which was p16 negative. The p16-/HPV + OPSCC resembled similar gene expression signature with p16+/HPV + OPSCCs by transcriptome analysis. 6/95 (6%) oral cavity SCCs were HPV-associated, all from male patients and 5/6 (83%) arose from the floor of mouth. Morphologically, 3/6 (50%) showed keratinizing SCC and 5/6 (83%) demonstrated HPV-associated squamous dysplasia in adjacent mucosa. 1/123 (less than 1%) larynx SCCs and 0/52 hypopharynx SCCs were HPV-associated. CONCLUSION: Although uncommon, p16 negative HPV-associated OPSCC can occur, emphasizing the importance of judicious HPV testing. The morphology of HPV-associated oral cavity SCCs may deviate from prototypic nonkeratinizing SCC, making them difficult to recognize. Presence of HPV-associated squamous dysplasia could serve as a morphologic clue.

Clinical and Functional Analyses of an African-ancestry Gain-of-function HOXB13 Variant Implicated in Aggressive Prostate Cancer.

BACKGROUND: Recent reports have uncovered a HOXB13 variant (X285K) predisposing to prostate cancer in men of West African ancestry. The clinical relevance and protein function associated with this inherited variant are unknown. OBJECTIVE: To determine the clinical relevance of HOXB13 (X285K) in comparison with HOXB13 (G84E) and BRCA2 pathogenic/likely pathogenic (P/LP) variants, and to elucidate the oncogenic mechanisms of the X285K protein. DESIGN, SETTING, AND PARTICIPANTS: Real-world data were collected from 21,393 men with prostate cancer undergoing genetic testing from 2019 to 2022, and in vitro cell-line models were established for the evaluation of oncogenic functions associated with the X285K protein. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Genetic testing results were compared among patient groups according to self-reported race/ethnicity, Gleason scores, and American Joint Committee on Cancer stages using the exact test. Oncogenic functions of X285K were evaluated by RNA sequencing, chromatin immunoprecipitation sequencing, and Western blot analyses. RESULTS AND LIMITATIONS: HOXB13 (X285K) was significantly enriched in self-reported Black (1.01%) versus White (0.01%) patients. We observed a trend of more aggressive disease in the HOXB13 (X285K) and BRCA2 P/LP carriers than in the HOXB13 (G84E) carriers. Replacement of the wild-type HOXB13 protein with the X285K protein resulted in a gain of an E2F/MYC signature, validated by the elevated expression of cyclin B1 and c-Myc, without affecting the androgen response signature. Elevated expression of cyclin B1 and c-Myc was explained by enhanced binding of the X285K protein to the promoters and enhancers of these genes. The limitations of the study are the lack of complete clinical outcome data for all patients studied and the use of a single cell line in the functional analysis. CONCLUSIONS: HOXB13 (X285K) is significantly enriched in self-reported Black patients, and X285K carriers detected in the real-world clinical setting have aggressive prostate cancer features similar to the BRCA2 carriers. Functional studies revealed a unique gain-of-function oncogenic mechanism of X285K protein in regulating E2F/MYC signatures. PATIENT SUMMARY: The HOXB13 (X285K) variant is clinically and functionally linked to aggressive prostate cancer, supporting genetic testing for X285K in Black men and early disease screening of carriers of this variant.

RNA profiling of circulating tumor cells systemically captured from diagnostic leukapheresis products in prostate cancer patients.

The application of circulating tumor cells (CTCs) in both clinical practice and research has been continuously limited by the rare number of targets that can be found in a tube of peripheral blood. Diagnostic leukapheresis (DLA) was used to increase the sampling volume. AdnaTest was used to process the whole leukopak, and the RNAs of captured CTCs was then profiled by NanoString nCounter platform. Spike-in experiments and leukopaks from patients with metastatic prostate cancer were used to validate this new strategy. The whole leukopak was further concentrated five times to reduce the total volume from 150 â€‹mL to 30 â€‹mL, which enabled it to be processed by 3 separate AdnaTest kits. The spike-in experiment demonstrated a reliable capture when there were more than 100 cancer cells/10 â€‹mL of concentrated leukopak. In 1 out of 5 real patient samples, CTCs were only detected in the leukopak, but not in peripheral blood. The RNA profiling of DLA CTCs indicated a more aggressive phenotype of CTCs occurred when the patient was experiencing a disease relapse, even when the serum prostate specific antigen (PSA) level was still relatively low and CTCs in peripheral blood were not detectable. We established a new protocol, integrating DLA, AdnaTest and NanoString nCounter technology, to profile RNAs from CTCs captured from a large blood screening volume. The new protocol can process the whole leukopak with sensitive CTC capture. The RNA profiling of CTCs can provide valuable information for disease monitoring.

AR Splicing Variants and Resistance to AR Targeting Agents.

Over the past decade, advances in prostate cancer research have led to discovery and development of novel biomarkers and effective treatments. As treatment options diversify, it is critical to further develop and use optimal biomarkers for the purpose of maximizing treatment benefit and minimizing unwanted adverse effects. Because most treatments for prostate cancer target androgen receptor (AR) signaling, aberrations affecting this drug target are likely to emerge following the development of castration-resistant prostate cancer (CRPC), and it is conceivable that such aberrations may play a role in drug resistance. Among the many AR aberrations, we and others have been studying androgen receptor splice variants (AR-Vs), especially AR-V7, and have conducted preclinical and clinical studies to develop and validate the clinical utility of AR-V7 as a prognostic and potential predictive biomarker. In this review, we first describe mechanisms of AR-V generation, regulation and their functions from a molecular perspective. We then discuss AR-Vs from a clinical perspective, focusing on the significance of AR-Vs detected in different types of human specimens and AR-Vs as potential therapeutic targets.

Inhibition of mTORC1 signaling reduces tumor growth but does not prevent cancer progression in a mouse model of thyroid cancer.

Selective drugs targeting dysregulated oncogenic pathways are promising cancer therapies. Because the mammalian target of rapamycin complex 1 (mTORC1) pathway is hyperactivated in human follicular thyroid cancer (FTC), we hypothesized that its inhibition could block cancer development and progression. We, therefore, analyzed the effect of a treatment with a specific mTORC1 inhibitor (RAD001) in a faithful mouse model of FTC with constitutive mTORC1 activation (TRbeta(PV/PV)Pten(+/-) mice). The treatment did not prevent capsular and vascular invasion of the thyroid and the occurrence of lung metastasis. However, it substantially decelerated thyroid tumor growth, thereby prolonging TRbeta(PV/PV)Pten(+/-) mouse life span. RAD001 efficiently inhibited mTORC1 activity, as shown by the reduced phosphorylation of its downstream targets involved in the activity of the translation machinery, such as ribosomal S6 kinase (p70(S6K)), eukaryotic translation initiation factor 4E binding protein (4E-BP1) and the eukaryotic translation initiation factors eIF-4B and eIF-4G. Whereas mTORC1 signaling inhibition did not alter cell apoptosis, it induced a significant decrease in cell proliferation that was associated with the reduced abundance and altered activity of key regulators of cell cycle progression. Altogether, our data indicate that mTORC1 signaling plays a major role in the integration of the mitogenic signal in FTC. Therefore, our preclinical study with a relevant mouse model of FTC demonstrates for the first time that RAD001 efficaciously stabilizes cancer growth although it does not prevent its fatal outcome. In conclusion, our work underscores that in the treatment of FTC patients, RAD001 can only be used in combination with drugs and therapies inducing tumor shrinkage and blocking metastasis.

Nuclear receptor corepressor is a novel regulator of phosphatidylinositol 3-kinase signaling.

The nuclear receptor corepressor (NCoR) regulates the activities of DNA-binding transcription factors. Recent observations of its distribution in the extranuclear compartment raised the possibility that it could have other cellular functions in addition to transcription repression. We previously showed that phosphatidylinositol 3-kinase (PI3K) signaling is aberrantly activated by a mutant thyroid hormone beta receptor (TRbetaPV, hereafter referred to as PV) via physical interaction with p85alpha, thus contributing to thyroid carcinogenesis in a mouse model of follicular thyroid carcinoma (TRbetaPV/PV mouse). Since NCoR is known to modulate the actions of TRbeta mutants in vivo and in vitro, we asked whether NCoR regulates PV-activated PI3K signaling. Remarkably, we found that NCoR physically interacted with and competed with PV for binding to the C-terminal SH2 (Src homology 2) domain of p85alpha, the regulatory subunit of PI3K. Confocal fluorescence microscopy showed that both NCoR and p85alpha were localized in the nuclear as well as in the cytoplasmic compartments. Overexpression of NCoR in thyroid tumor cells of TRbetaPV/PV mouse reduced PI3K signaling, as indicated by the decrease in the phosphorylation of its immediate downstream effector, p-AKT. Conversely, lowering cellular NCoR by siRNA knockdown in tumor cells led to overactivated p-AKT and increased cell proliferation and motility. Furthermore, NCoR protein levels were significantly lower in thyroid tumor cells than in wild-type thyrocytes, allowing more effective binding of PV to p85alpha to activate PI3K signaling and thus contributing to tumor progression. Taken together, these results indicate that NCoR, via protein-protein interaction, is a novel regulator of PI3K signaling and could serve to modulate thyroid tumor progression.

Androgen receptor variant-driven prostate cancer II: advances in laboratory investigations.

BACKGROUND: The androgen receptor (AR) is a key prostate cancer drug target. Suppression of AR signaling mediated by the full-length AR (AR-FL) is the therapeutic goal of all existing AR-directed therapies. AR-targeting agents impart therapeutic benefit, but lead to AR aberrations that underlie disease progression and therapeutic resistance. Among the AR aberrations specific to castration-resistant prostate cancer (CRPC), AR variants (AR-Vs) have emerged as important indicators of disease progression and therapeutic resistance. METHODS: We conducted a systemic review of the literature focusing on recent laboratory studies on AR-Vs following our last review article published in 2016. Topics ranged from measurement and detection, molecular origin, regulation, genomic function, and preclinical therapeutic targeting of AR-Vs. We provide expert opinions and perspectives on these topics. RESULTS: Transcript sequences for 22 AR-Vs have been reported in the literature. Different AR-Vs may arise through different mechanisms, and can be regulated by splicing factors and dictated by genomic rearrangements, but a low-androgen environment is a prerequisite for generation of AR-Vs. The unique transcript structures allowed development of in situ and in-solution measurement and detection methods, including mRNA and protein detection, in both tissue and blood specimens. AR-V7 remains the main measurement target and the most extensively characterized AR-V. Although AR-V7 coexists with AR-FL, genomic functions mediated by AR-V7 do not require the presence of AR-FL. The distinct cistromes and transcriptional programs directed by AR-V7 and their coregulators are consistent with genomic features of progressive disease in a low-androgen environment. Preclinical development of AR-V-directed agents currently focuses on suppression of mRNA expression and protein degradation as well as targeting of the amino-terminal domain. CONCLUSIONS: Current literature continues to support AR-Vs as biomarkers and therapeutic targets in prostate cancer. Laboratory investigations reveal both challenges and opportunities in targeting AR-Vs to overcome resistance to current AR-directed therapies.

Androgen receptor variant-driven prostate cancer II: advances in clinical investigation.

BACKGROUND: Approximately 10-30% of men with mCRPC will test positive for AR-V7 using one of two analytically and clinically validated circulating tumor cell (CTC)-based assays. These men have poor outcomes with approved AR-targeting therapies but may retain sensitivity to chemotherapy. Here, we discuss the clinical implications of testing and strategies that may benefit AR splice variant (AR-V)-positive men and discuss whether such variants are passengers or drivers of aggressive clinical behavior. METHODS: We conducted a systemic review of the literature, covering updates since our 2016 review on androgen receptor variants in mCRPC, outcomes, and existing and novel approaches to therapy. We provide an expert opinion about management strategies for AR-V7-positive men and key unanswered research questions. RESULTS: AR-V7-positive men, defined by Epic nuclear protein detection or the modified AdnaTest mRNA detection in CTCs, identify a subset of men with mCRPC that have a low probability of response to AR-targeting therapy with short progression-free and overall survival in multivariable analyses. AR-variants do not exist in isolation, but rather in the context of a complex, heterogeneous, and evolving mCRPC genome and phenotype as well as patient-specific clinical heterogeneity, and multiple mechanisms of resistance likely exist in patients regardless of AR-V7 detection. Efforts to develop broader resistance assays are needed, and effective treatment strategies beyond taxanes are needed to address the causal driver role of AR-variants and to benefit patients with AR-V-expressing prostate cancer. CONCLUSIONS: CTC AR-V7 detection using the AdnaTest mRNA or Epic nuclear protein assays represents the first analytically and prospective clinically validated liquid biopsy assays that may inform treatment decisions in men with mCRPC, particularly after failure of first-line AR-therapy. The importance of AR-variants is likely to increase with the earlier use of AR-targeting strategies in other settings, and novel interventions for these men are needed.

The MAO inhibitors phenelzine and clorgyline revert enzalutamide resistance in castration resistant prostate cancer.

The antiandrogen enzalutamide (Enz) has improved survival in castration resistant prostate cancer (CRPC) patients. However, most patients eventually develop Enz resistance that may involve inducing the androgen receptor (AR) splicing variant 7 (ARv7). Here we report that high expression of monoamine oxidase-A (MAO-A) is associated with positive ARv7 detection in CRPC patients following Enz treatment. Targeting MAO-A with phenelzine or clorgyline, the FDA-approved drugs for antidepression, resensitize the Enz resistant (EnzR) cells to Enz treatment and further suppress EnzR cell growth in vitro and in vivo. Our findings suggest that Enz-increased ARv7 expression can transcriptionally enhance MAO-A expression resulting in Enz resistance via altering the hypoxia HIF-1α signals. Together, our results show that targeting the Enz/ARv7/MAO-A signaling with the antidepressants phenelzine or clorgyline can restore Enz sensitivity to suppress EnzR cell growth, which may indicate that these antidepression drugs can overcome the Enz resistance to further suppress the EnzR CRPC.

Prospective Multicenter Study of Circulating Tumor Cell AR-V7 and Taxane Versus Hormonal Treatment Outcomes in Metastatic Castration-Resistant Prostate Cancer.

PURPOSE: Androgen receptor splice variant 7 (AR-V7) detection in circulating tumor cells (CTCs) is associated with a low probability of response and short progression-free (PFS) and overall survival (OS) in men with metastatic castration-resistant prostate cancer (mCRPC) treated with enzalutamide or abiraterone. However, it is unclear whether such men benefit from taxane chemotherapy. PATIENTS AND METHODS: PROPHECY is a multicenter prospective blinded study of patients with poor-risk mCRPC starting abiraterone or enzalutamide and observed through subsequent progression and taxane chemotherapy. We assessed AR-V7 status using the Johns Hopkins modified AdnaTest CTC AR-V7 messenger RNA assay and the Epic Sciences CTC nuclear-localized AR-V7 protein assay before treatment. The primary objective was to validate the independent prognostic value of CTC AR-V7 status based on radiographic/clinical PFS. OS, confirmed prostate-specific antigen (PSA), and objective radiologic responses were secondary end points. RESULTS: We enrolled 118 men with mCRPC treated with abiraterone or enzalutamide, 51 of whom received subsequent docetaxel or cabazitaxel. Pretreatment CTC AR-V7 status by the Johns Hopkins and Epic Sciences assays was independently associated with worse PFS (hazard ratio [HR], 1.7; 95% CI, 1.0 to 2.9 and HR, 2.1; 95% CI, 1.0 to 4.4, respectively) and OS (HR, 3.3; 95% CI, 1.7 to 6.3 and HR, 3.0; 95% CI, 1.4 to 6.3, respectively) and a low probability of confirmed PSA responses, ranging from 0% to 11%, during treatment with abiraterone or enzalutamide. At progression, subsequent CTC AR-V7 detection was not associated with an inferior PSA or radiographic response or worse PFS or OS with subsequent taxane chemotherapy after adjusting for CellSearch CTC enumeration and clinical prognostic factors. CONCLUSION: Detection of AR-V7 in CTCs by two different blood-based assays is independently associated with shorter PFS and OS with abiraterone or enzalutamide, but such men with AR-V7-positive disease still experience clinical benefits from taxane chemotherapy.

Nongenomic activation of phosphatidylinositol 3-kinase signaling by thyroid hormone receptors.

Thyroid hormone (T3) is critical in growth, development, differentiation, and maintenance of metabolic homeostasis. Recent studies suggest that thyroid hormone receptors (TRs) not only mediate the biological activities of T3 via nucleus-initiated transcription, but also could act via nongenomic pathways. The striking phenotype of thyroid cancer exhibited by a knockin mutant mouse that harbors a dominant negative TRbeta mutant (TRbeta(PV/PV) mouse) allows the elucidation of novel oncogenic activity of a TRbeta mutant (PV) via extra-nuclear actions. PV physically interacts with the regulatory p85alpha subunit of phosphatidylinositol 3-kinase (PI3K) to activate the downstream AKT-mammalian target of rapamycin (mTOR) and p70(S6K) and PI3K-integrin-linked kinase-matrix metalloproteinase-2 signaling pathways. The PV-mediated PI3K activation results in increased cell proliferation, motility, migration, and metastasis. Remarkably, a nuclear receptor corepressor (NCoR) was found to regulate the PV-activated PI3K signaling by competing with PV for binding to the C-terminal SH2 domain of p85alpha. Over-expression of NCoR in thyroid tumor cells of TRbeta(PV/PV) mice reduces AKT-mTOR-p70(S6K) signaling. Conversely, lowering cellular NCoR by siRNA knockdown in tumor cells leads to over-activated PI3K-AKT signaling to increase cell proliferation and motility. Furthermore, NCoR protein levels are significantly lower in thyroid tumor cells than in wild type thyrocytes, allowing more effective binding of PV to p85alpha to activate PI3K signaling, thereby contributing to tumor progression. Thus, PV, an apo-TRbeta, could act via direct protein-protein interaction to mediate critical oncogenic actions. These studies also uncovered a novel extra-nuclear role of NCoR in modulating the nongenomic actions of a mutated TRbeta in controlling thyroid carcinogenesis.

Expression pattern of androgen receptor and AR-V7 in androgen-deprivation therapy-naïve salivary duct carcinomas.

Androgen-deprivation therapy has been used to treat salivary duct carcinoma (SDC). The androgen receptor splice variant-7 (AR-V7) has been detected in castration-resistant prostate cancer and implicated in resistance to androgen receptor (AR)-targeted therapies. Given the potential role of AR/AR-V7 in SDC treatment, this study focuses on AR/AR-V7 expression in SDC specimens collected before androgen-deprivation therapy. RNA in situ hybridization (ISH) and immunohistochemistry (IHC) to detect total AR and AR-V7 were performed on formalin-fixed, paraffin-embedded SDC specimens from 23 patients. Full-length AR and AR-V7 transcripts were quantified in a subset of tumors by reverse-transcription polymerase chain reaction. Twenty SDCs were positive for total AR by ISH and IHC. Among AR-positive SDCs, 70% (14/20) were positive for AR-V7 messenger RNA by ISH, whereas 15% (3/20) were positive for AR-V7 protein by IHC. The 3 SDCs that expressed the highest levels of AR-V7 were all from female patients; one of them expressed a significant amount of AR-V7 and barely detectable full-length AR transcripts by reverse-transcription polymerase chain reaction. IHC expression of Forkhead box protein A1, prostate-specific antigen, prostatic acid phosphatase, and NKX3.1 was observed in some SDCs regardless of patient sex. Five SDCs demonstrated strong human epidermal growth factor receptor 2 expression. We conclude that treatment-naïve SDCs may express AR-V7 at levels comparable to or even exceeding the levels detected in castration-resistant prostate cancer. Our data support the feasibility to incorporate AR-V7 assessment via ISH and/or IHC in the ongoing clinical trials evaluating the therapeutic benefit of AR-targeted therapies in SDC patients.

Prospective Multicenter Validation of Androgen Receptor Splice Variant 7 and Hormone Therapy Resistance in High-Risk Castration-Resistant Prostate Cancer: The PROPHECY Study.

PURPOSE: Androgen receptor splice variant 7 (AR-V7) results in a truncated receptor, which leads to ligand-independent constitutive activation that is not inhibited by anti-androgen therapies, including abiraterone or enzalutamide. Given that previous reports suggested that circulating tumor cell (CTC) AR-V7 detection is a poor prognostic indicator for the clinical efficacy of secondary hormone therapies, we conducted a prospective multicenter validation study. PATIENTS AND METHODS: PROPHECY ( ClinicalTrials.gov identifier: NCT02269982) is a multicenter, prospective-blinded study of men with high-risk mCRPC starting abiraterone acetate or enzalutamide treatment. The primary objective was to validate the prognostic significance of baseline CTC AR-V7 on the basis of radiographic or clinical progression free-survival (PFS) by using the Johns Hopkins University modified-AdnaTest CTC AR-V7 mRNA assay and the Epic Sciences CTC nuclear-specific AR-V7 protein assay. Overall survival (OS) and prostate-specific antigen responses were secondary end points. RESULTS: We enrolled 118 men with mCRPC who were starting abiraterone or enzalutamide treatment. AR-V7 detection by both the Johns Hopkins and Epic AR-V7 assays was independently associated with shorter PFS (hazard ratio, 1.9 [95% CI, 1.1 to 3.3; P = .032] and 2.4 [95% CI, 1.1 to 5.1; P = .020], respectively) and OS (hazard ratio, 4.2 [95% CI, 2.1 to 8.5] and 3.5 [95% CI, 1.6 to 8.1], respectively) after adjusting for CTC number and clinical prognostic factors. Men with AR-V7-positive mCRPC had fewer confirmed prostate-specific antigen responses (0% to 11%) or soft tissue responses (0% to 6%). The observed percentage agreement between the two AR-V7 assays was 82%. CONCLUSION: Detection of AR-V7 in CTCs by two blood-based assays is independently associated with shorter PFS and OS with abiraterone or enzalutamide, and such men with mCRPC should be offered alternative treatments.