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Active surveillance is a safe and effective strategy for men with lower-risk prostate cancer who want to avoid local therapy; however, many patients on active surveillance progress to active treatment (eg, prostatectomy or radiation). We hypothesized that apalutamide would decrease active surveillance attrition rates through downstaging low-grade tumors.This was an open-label, single-arm, phase II study testing 90 days of oral apalutamide 240 mg daily in men with low- to intermediate-risk prostate cancer on active surveillance. The primary objective was to determine the percentage of patients with a negative biopsy immediately following treatment. Secondary objectives were to assess long-term clinical outcomes, quality of life, safety, and biomarkers of response/resistance.Twenty-three patients enrolled and 22 completed 90 days of apalutamide with post-treatment biopsy. Fifteen (65%) had Grade Group 1 disease, and all others had Grade Group 2 disease. Seven (30%) had favorable- to intermediate-risk disease. Of 22 evaluable patients, 13 (59%) had no residual cancer on post-treatment biopsy. The median time to first positive biopsy was 364 days (95% CI: 91-742 days). The impact of apalutamide on quality of life was minimal and transient. Decipher risk classifier revealed a greater number of negative post-treatment biopsies in those with higher baseline genomic risk score (The negative repeat biopsy rate following 90 days of apalutamide was high in men with prostate cancer followed on active surveillance. Apalutamide was safe, well tolerated, and had minimal impact on quality of life. Randomized studies evaluating the effects of apalutamide in men enrolled on active surveillance are warranted.

Surrogate endpoints (SEs), such as progression-free survival (PFS) and objective response rate (ORR), are frequently used in clinical trials. The relationship between SEs and overall survival (OS) has not been well described in metastatic urothelial cancer (MUC).We evaluated trial-level data to assess the relationship between SEs and OS. We hypothesize a moderate surrogacy relationship between both PFS and ORR with OS.We systematically reviewed phase 2/3 trials in MUC with two or more treatment arms, and report PFS and/or ORR, and OS.Linear regression was performed, and the coefficient of determination (ROf 3791 search results, 59 trials and 62 comparisons met the inclusion criteria. Of the 53 trials that reported PFS, 31 (58%) reported proportional hazard regression for PFS and OS. Linear regression across trials demonstrated an RPFS provides only a moderate level of surrogacy for OS; An HRWe examined the relationship between surrogate endpoints, common outcomes in clinical trials, with survival in urothelial cancer trials. Progression-free survival is moderately correlated, while objective response rate had a poor correlation with survival and should be de-emphasized as a primary endpoint.

Immune checkpoint inhibitors are a well-established treatment option for advanced urothelial carcinoma, and biomarkers of response are needed for better patient selection. We show that metastatic disease confined to lymph nodes is associated with better outcomes, while metastases to liver, bone or both are associated with poor outcomes with immune checkpoint inhibitor therapy. Results are hypothesis-generating but relevant to practice. BACKGROUND: Sites of metastasis have prognostic significance in advanced urothelial carcinoma (aUC), but more information is needed regarding outcomes based on metastatic sites in patients treated with immune checkpoint inhibitors (ICI). We hypothesized that presence of liver/bone metastases would be associated with worse outcomes with ICI. METHODS: We identified a retrospective cohort of patients with aUC across 26 institutions, collecting demographics, clinicopathological, treatment, and outcomes information. Outcomes were compared with logistic (observed response rate; ORR) and Cox (progression-free survival; PFS, overall survival; OS) regression between patients with/without metastasis beyond lymph nodes (LN) and those with/without bone/liver/lung metastasis. Analysis was stratified by 1st or 2nd(+) line. RESULTS: We identified 917 ICI-treated patients: in the 1st line, bone/liver metastases were associated with shorter PFS (Hazard ratio; HR: 1.65 and 2.54), OS (HR: 1.60 and 2.35, respectively) and lower ORR (OR: 0.48 and 0.31). In the 2nd(+) line, bone/liver metastases were associated with shorter PFS (HR: 1.71 and 1.62), OS (HR: 1.76 and 1.56) and, for bone-only metastases, lower ORR (OR: 0.29). In the 1st line, LN-confined metastasis was associated with longer PFS (HR: 0.53), OS (HR:0.49) and higher ORR (OR: 2.97). In the 2nd(+) line, LN-confined metastasis was associated with longer PFS (HR: 0.47), OS (HR: 0.54), and higher ORR (OR: 2.79); all associations were significant. CONCLUSION: Bone and/or liver metastases were associated with worse, while LN-confined metastases were associated with better outcomes in patients with aUC receiving ICI. These findings in a large population treated outside clinical trials corroborate data from trial subset analyses.

Feature-region combination AUCs and benchmarking Sheet 1: Log2 fold-change, p-value, and q-value between ARPC and NEPC lines for central mean coverage in all queried (338) TFs (two tailed Mann-Whitney U test, Benjamini-Hochberg adjusted). Sheet 2: 100-fold cross-validation AUCs for all region and feature combinations subset by the ‘AR10’ overlapping features (see Methods). Sheet 3: 100-fold cross-validation AUCs for all region and feature combinations subset by the ‘Phenotype 47-defining’ overlapping features. Sheet 4: 100-fold cross-validation AUCs for all region and feature combinations (global). Sheet 5: Predictions scores, tumor fraction, depth of coverage, and subtype for benchmarking admixtures. Sheet 6: AUCs for unsupervised prediction of admixture subtypes grouped by tumor fraction and depth. Sheet 7: NEPC:ARPC ratio, tumor fraction, and ARPC and NEPC fraction predictions for mixed phenotype admixtures using Keraon (see Methods).

Supplementary Figures S1-S18 and Supplementary Table legends.

Supplementary Methods from The Androgen-Regulated Protease TMPRSS2 Activates a Proteolytic Cascade Involving Components of the Tumor Microenvironment and Promotes Prostate Cancer Metastasis

Supplementary Figure S1. TMPRSS2 expression is associated with androgen receptor (AR) levels and is regulated by androgens in vivo. Supplementary Figure S2. Characterization of the differentiation status, Tmprss2 and AR expression in Tmprss2+/+ TRAMP and Tmprss2 -/- TRAMP mice. Supplementary Figure S3. Suppression of TMPRSS2 inhibits cell proliferation and invasion. Supplementary Figure S4. TMPRSS2 influences tumor cell clearance and growth in metastatic sites. Supplementary Figure S5. Comparison of cleavage specificities for type II transmembrane serine proteases expressed in prostate epithelium. Supplementary Figure S6. Identification of putative TMPRSS2 substrates based on peptide cleavage specificities determined by PS-SCL. Supplementary Figure S7. Assessment of TMPRSS2 substrate activity. Supplementary Figure S8. Characterization of TMPRSS2 chemical inhibitors. Supplementary Figure S9. Bromhexine suppresses prostate cancer cell invasion but but does not influence proliferation in vitro.

Purpose: Abiraterone is a potent inhibitor of the steroidogenic enzyme CYP17A1 and suppresses tumor growth in patients with castration-resistant prostate cancer (CRPC). The effectiveness of abiraterone in reducing tumor androgens is not known, nor have mechanisms contributing to abiraterone resistance been established.Experimental Design: We treated human CRPC xenografts with abiraterone and measured tumor growth, tissue androgens, androgen receptor (AR) levels, and steroidogenic gene expression versus controls.Results: Abiraterone suppressed serum PSA levels and improved survival in two distinct CRPC xenografts: median survival of LuCaP35CR improved from 17 to 39 days (HR = 3.6, P = 0.0014) and LuCaP23CR from 14 to 24 days (HR = 2.5, P = 0.0048). Abiraterone strongly suppressed tumor androgens, with testosterone (T) decreasing from 0.49 ± 0.22 to 0.03 ± 0.01 pg/mg (P < 0.0001), and from 0.69 ± 0.36 to 0.03 ± 0.01 pg/mg (P = 0.002) in abiraterone-treated 23CR and 35CR, respectively, with comparable decreases in tissue DHT. Treatment was associated with increased expression of full-length AR (ARFL) and truncated AR variants (ARFL 2.3-fold, P = 0.008 and ARdel567es 2.7-fold, P = 0.036 in 23 CR; ARFL 3.4-fold, P = 0.001 and ARV7 3.1-fold, P = 0.0003 in 35CR), and increased expression of the abiraterone target CYP17A1 (∼2.1-fold, P = 0.0001 and P = 0.028 in 23CR and 35CR, respectively) and transcript changes in other enzymes modulating steroid metabolism.Conclusions: These studies indicate that abiraterone reduces CRPC growth via suppression of intratumoral androgens and that resistance to abiraterone may occur through mechanisms that include upregulation of CYP17A1, and/or induction of AR and AR splice variants that confer ligand-independent AR transactivation. Clin Cancer Res; 17(18); 5913–25. ©2011 AACR.

Supplemental Methods, Figure Legends, Table Legends, References

Figure S1. Targeted siRNA knockdown of DPP4 decreases DPP4 antibody positivity in western blots and IHC; Figure S2. Immunoscoring of AR, AR-V7, P-AR(S81), and PSA protein expression in serially biopsied VCaP xenografts; Figure S3. Ingenuity Pathway, Hierarchical Clustering, and Principal Component analysis of Pre-Cx, CRPC, and AER, serial biopsies; Figure S4. DPP4 is an AR-stimulated gene; Figure S5. DPP4 expression is decreased in Post-ADT therapy samples in several clinical and one preclinical data set compared to Pre-Cx; Figure S6. DPP4 expression is decreased in clinical CRPC sections, despite normal levels of PSA; Figure S7. CpG islands in the DPP4 promoter and exon 1 are unmethylated; Figure S8. lncRNA-OIS1 is expressed at similar levels in Pre-Cx and AER VCaP tumors; Figure S9. Androgen treatment of VCaP CRPC xenografts restores DPP4 expression; Figure S10. The volume ratio (End Volume/Starting Volume) of VCaP tumors shows a trend toward positive correlation with tumor DPP4 protein levels.

Purpose:Enzalutamide is a second-generation androgen receptor (AR) inhibitor that has improved overall survival (OS) in metastatic castration-resistant prostate cancer (CRPC). However, nearly all patients develop resistance. We designed a phase II multicenter study of enzalutamide in metastatic CRPC incorporating tissue and blood biomarkers to dissect mechanisms driving resistance.Patients and Methods:Eligible patients with metastatic CRPC underwent a baseline metastasis biopsy and then initiated enzalutamide 160 mg daily. A repeat metastasis biopsy was obtained at radiographic progression from the same site when possible. Blood for circulating tumor cell (CTC) analysis was collected at baseline and progression. The primary objective was to analyze mechanisms of resistance in serial biopsies. Whole-exome sequencing was performed on tissue biopsies. CTC samples underwent RNA sequencing.Results:A total of 65 patients initiated treatment, of whom 22 (33.8%) had received prior abiraterone. Baseline biopsies were enriched for alterations in AR (mutations, amplifications) and tumor suppression genes (PTEN, RB1, and TP53), which were observed in 73.1% and 92.3% of baseline biopsies, respectively. Progression biopsies revealed increased AR amplifications (64.7% at progression vs. 53.9% at baseline) and BRCA2 alterations (64.7% at progression vs. 38.5% at baseline). Genomic analysis of baseline and progression CTC samples demonstrated increased AR splice variants, AR-regulated genes, and neuroendocrine markers at progression.Conclusions:Our results demonstrate that a large proportion of enzalutamide-treated patients have baseline and progression alterations in the AR pathway and tumor suppressor genes. We demonstrate an increased number of BRCA2 alterations post-enzalutamide, highlighting the importance of serial tumor sampling in CRPC.

Figure S1: Assessment for neuroendocrine features by histology and IHC; Figure S2: Scatter plots depicting relationships between the 267-gene AR activity ssGSEA score and the 131-gene proliferation ssGSEA score for multiple genesets; Figure S3: Plots depicting correlations with AR activity ssGSEA score, and assessing GR expression; Figure S4: Segmented read depth array CGH-style plots for chromosomes showing alterations to cancer-related genes; Figure S5: Euler diagrams depicting shared and distinct somatic genomic events for cases 003, 007, 501, 503, 506, 507, 509, 512, and 520.

Characterization of ENZ-resistant cell lines

Color figure 3 for supplement.

Clinical characteristics of prostate cancer patients with matched diagnostic (archival) castration-sensitive and metastatic castration-resistant tissue biopsies

The standard treatment for metastatic prostate cancer, androgen deprivation therapy (ADT), is designed to suppress androgen receptor (AR) activity. However, men invariably progress to castration-resistant prostate cancer (CRPC), and AR reactivation contributes to progression in most cases. To identify mechanisms that may drive CRPC, we examined a VCaP prostate cancer xenograft model as tumors progressed from initial androgen sensitivity prior to castration to castration resistance and then on to relapse after combined therapy with further AR-targeted drugs (abiraterone plus enzalutamide). AR activity persisted in castration-resistant and abiraterone/enzalutamide–resistant xenografts and was associated with increased expression of the AR gene and the AR-V7 splice variant. We then assessed expression of individual AR-regulated genes to identify those that persisted, thereby contributing to tumor growth, versus those that decreased and may therefore exhibit tumor suppressor activities. The most significantly decreased AR target gene was dipeptidyl peptidase 4 (DPP4), which encodes a membrane-anchored protein that cleaves dipeptides from multiple growth factors, resulting in their increased degradation. DPP4 mRNA and protein were also decreased in clinical CRPC cases, and inhibition of DPP4 with sitagliptin enhanced the growth of prostate cancer xenografts following castration. Significantly, DPP4 inhibitors are frequently used to treat type 2 diabetes as they increase insulin secretion. Together, these results implicate DPP4 as an AR-regulated tumor suppressor gene whose loss enhances growth factor activity and suggest that treatment with DPP4 inhibitors may accelerate emergence of resistance to ADT.Significance: These findings identify DPP4 as an AR-stimulated tumor suppressor gene that is downregulated during progression to castration-resistant prostate cancer, warning that treatment with DPP4 inhibitors, commonly used to treat type 2 diabetes, may accelerate prostate cancer progression following androgen deprivation therapy. Cancer Res; 78(22); 6354–62. ©2018 AACR.

Purpose: Prostate cancer is dependent on androgen receptor (AR) activation. Optimal AR antagonism may effectively cytoreduce local disease and suppress or eliminate micrometastases. We evaluated neoadjuvant therapy prior to prostatectomy with the potent AR antagonist enzalutamide (enza) either alone or in combination with dutasteride (dut) and leuprolide (enza/dut/luteinizing hormone-releasing hormone analogues [LHRHa]).Experimental Design: Forty-eight of 52 men with intermediate or high-risk localized prostate cancer proceeded to prostatectomy after neoadjuvant enzalutamide or enza/dut/LHRHa for 6 months. We assessed pathologic complete response (pCR), minimal residual disease (MRD; ≤3 mm maximum diameter of residual disease), residual cancer burden (RCB), and expression of PSA and serum and tissue androgen concentrations. We compared the proportion of patients with pCR in each treatment arm with a historical control rate of 5%, based on previous reports of flutamide with LHRHa.Results: In the enzalutamide arm, none of the 25 patients achieved pCR or MRD. In the enza/dut/LHRHa arm, one of 23 patients (4.3%) achieved pCR and 3 of 23 (13.0%) achieved MRD. Median RCB was higher in the enzalutamide arm than in the enza/dut/LHRHa arm (0.41 cm3 vs. 0.06 cm3, respectively). Tissue testosterone and dihydrotestosterone levels correlated with RCB. No adverse events leading to study drug discontinuation were reported.Conclusions: Combination therapy with enza/dut/LHRHa resulted in pCR and MRD rates comparable with historical controls. Evidence of continued AR activity in residual tumor suggests that AR signaling may contribute to survival. Strategies to more effectively ablate AR activity are warranted to determine whether more substantial antitumor effects are observed. Clin Cancer Res; 23(9); 2169–76. ©2016 AACR.

Purpose: Despite the efficacy of abiraterone, a CYP17A1 inhibitor, in metastatic castration-resistant prostate cancer (CRPC), nearly all patients develop resistance. The purpose of this phase II study was to evaluate mechanisms of resistance to more complete androgen synthesis inhibition with abiraterone and dutasteride.Experimental Design: Eligible patients with metastatic CRPC underwent a baseline metastasis biopsy. Patients received abiraterone and prednisone for two 4-week cycles. After this time, high-dose dutasteride (3.5 mg daily) was added. Patients continued therapy until study withdrawal or radiographic progression. Repeat metastasis biopsy was obtained at progression. The primary endpoint was to assess mechanisms of resistance. Serum hormone and abiraterone levels were assessed. Tissue was assessed for androgen receptor (AR) and AR splice variant-7 (ARV7) expression.Results: Forty patients were enrolled. Sixty percent (n = 24) achieved a ≥50% reduction in prostate-specific antigen (PSA). The median time to radiographic progression was 11 months. Nearly all baseline (n = 29 of 31) and posttreatment (n = 16 of 16) tumors tested for AR nuclear expression were positive. Of those tested, ARV7 expression was present in 48% (n = 10 of 21) of baseline and 42% (n = 5 of 12) of treatment discontinuation specimens. Compared with patients with higher serum abiraterone levels at treatment discontinuation, patients with lower levels had higher circulating androgens.Conclusions: Despite increased androgen synthesis inhibition, we demonstrate that tumor AR axis remains important in disease progression. We highlight that abiraterone metabolism and pharmacokinetics may play a role in resistance. The noncomparative design limits conclusions on the efficacy of dual therapy with abiraterone and dutasteride, but the results support development of further multifaceted approaches toward AR inhibition. Clin Cancer Res; 23(4); 935–45. ©2016 AACR.

Purpose: Germline variation in solute carrier organic anion (SLCO) genes influences cellular steroid uptake and is associated with prostate cancer outcomes. We hypothesized that, due to its steroidal structure, the CYP17A inhibitor abiraterone may undergo transport by SLCO-encoded transporters and that SLCO gene variation may influence intracellular abiraterone levels and outcomes.Experimental Design: Steroid and abiraterone levels were measured in serum and tissue from 58 men with localized prostate cancer in a clinical trial of LHRH agonist plus abiraterone acetate plus prednisone for 24 weeks prior to prostatectomy. Germline DNA was genotyped for 13 SNPs in six SLCO genes.Results: Abiraterone levels spanned a broad range (serum median 28 ng/mL, 108 nmol/L; tissue median 77 ng/mL, 271 nmol/L) and were correlated (r = 0.355, P = 0.001). Levels correlated positively with steroids upstream of CYP17A (pregnenolone, progesterone), and inversely with steroids downstream of CYP17A (DHEA, AED, testosterone). Serum PSA and tumor volumes were higher in men with undetectable versus detectable tissue abiraterone at prostatectomy (median 0.10 vs. 0.03 ng/dL, P = 0.02; 1.28 vs. 0.44 cc, P = 0.09, respectively). SNPs in SLCO2B1 associated with significant differences in tissue abiraterone (rs1789693, P = 0.0008; rs12422149, P = 0.03) and higher rates of minimal residual disease (tumor volume < 0.5 cc; rs1789693, 67% vs. 27%, P = 0.009; rs1077858, 46% vs. 0%, P = 0.03). LNCaP cells expressing SLCO2B1 showed two- to fourfold higher abiraterone levels compared with vector controls (P < 0.05).Conclusions: Intraprostatic abiraterone levels and genetic variation in SLCO genes are associated with pathologic responses in high-risk localized prostate cancer. Variation in SLCO genes may serve as predictors of response to abiraterone treatment. Clin Cancer Res; 23(16); 4592–601. ©2017 AACR.

A cluster analysis of adenocarcinoma and neuronal CRPC metastases (n=71). The CHGA+/SYP+ sites cluster together (highlighted in orange (AR+) and yellow (AR-)). Genes between CHGA- vs. CHGA+/SYP+ were compared and considered differentially expressed when SAM score was >3 or

Mutations in 3 cases with only 1 focus dissected

AgilentTM oligo array expression analysis of thirty-seven genes with >2.5-fold differential expression common to 5 CHGA+/SYP+/AR- and 5 CHGA+/SYP+/AR+ CRPC metastases when compared to 61 CHGA- or CHGA+/SYP- CRPC metastases. Mean-centered ratios of genes are colored according to scale (Supplemental Figure 2).

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