Development and Optimization of a Subtraction-Normalized Immunocyte Profiling Signature for Prostate Cancer Active Surveillance Risk Stratification.

PURPOSE: Less invasive decision support tools are desperately needed to identify occult high-risk disease in men with prostate cancer (PCa) on active surveillance (AS). For a variety of reasons, many men on AS with low- or intermediate-risk disease forgo the necessary repeat surveillance biopsies needed to identify potentially higher-risk PCa. Here, we describe the development of a blood-based immunocyte transcriptomic signature to identify men harboring occult aggressive PCa. We then validate it on a biopsy-positive population with the goal of identifying men who should not be on AS and confirm those men with indolent disease who can safely remain on AS. This model uses subtraction-normalized immunocyte transcriptomic profiles to risk-stratify men with PCa who could be candidates for AS. MATERIALS AND METHODS: Men were eligible for enrollment in the study if they were determined by their physician to have a risk profile that warranted prostate biopsy. Both training (n = 1017) and validation cohort (n = 1198) populations had blood samples drawn coincident to their prostate biopsy. Purified CD2+ and CD14+ immune cells were obtained from peripheral blood mononuclear cells, and RNA was extracted and sequenced. To avoid overfitting and unnecessary complexity, a regularized regression model was built on the training cohort to predict PCa aggressiveness based on the National Comprehensive Cancer Network PCa guidelines. This model was then validated on an independent cohort of biopsy-positive men only, using National Comprehensive Cancer Network unfavorable intermediate risk and worse as an aggressiveness outcome, identifying patients who were not appropriate for AS. RESULTS: The best final model for the AS setting was obtained by combining an immunocyte transcriptomic profile based on 2 cell types with PSA density and age, reaching an AUC of 0.73 (95% CI: 0.69-0.77). The model significantly outperforms (P < .001) PSA density as a biomarker, which has an AUC of 0.69 (95% CI: 0.65-0.73). This model yields an individualized patient risk score with 90% negative predictive value and 50% positive predictive value. CONCLUSIONS: While further validation in an intended-use cohort is needed, the immunocyte transcriptomic model offers a promising tool for risk stratification of individual patients who are being considered for AS.

Prostatectomy-based validation of combined urine and plasma test for predicting high grade prostate cancer.

BACKGROUND: Distinguishing between low- and high-grade prostate cancers (PCa) is important, but biopsy may underestimate the actual grade of cancer. We have previously shown that urine/plasma-based prostate-specific biomarkers can predict high grade PCa. Our objective was to determine the accuracy of a test using cell-free RNA levels of biomarkers in predicting prostatectomy results. METHODS: This multicenter community-based prospective study was conducted using urine/blood samples collected from 306 patients. All recruited patients were treatment-naïve, without metastases, and had been biopsied, designated a Gleason Score (GS) based on biopsy, and assigned to prostatectomy prior to participation in the study. The primary outcome measure was the urine/plasma test accuracy in predicting high grade PCa on prostatectomy compared with biopsy findings. Sensitivity and specificity were calculated using standard formulas, while comparisons between groups were performed using the Wilcoxon Rank Sum, Kruskal-Wallis, Chi-Square, and Fisher's exact test. RESULTS: GS as assigned by standard 10-12 core biopsies was 3 + 3 in 90 (29.4%), 3 + 4 in 122 (39.8%), 4 + 3 in 50 (16.3%), and > 4 + 3 in 44 (14.4%) patients. The urine/plasma assay confirmed a previous validation and was highly accurate in predicting the presence of high-grade PCa (Gleason ≥3 + 4) with sensitivity between 88% and 95% as verified by prostatectomy findings. GS was upgraded after prostatectomy in 27% of patients and downgraded in 12% of patients. CONCLUSIONS: This plasma/urine biomarker test accurately predicts high grade cancer as determined by prostatectomy with a sensitivity at 92-97%, while the sensitivity of core biopsies was 78%.

The clinical and economic implications of specimen provenance complications in diagnostic prostate biopsies.

PURPOSE: Inaccurate diagnoses of prostate cancer can result from transposition or contamination of patient biopsy specimens, which are known as specimen provenance complications. We assessed the clinical and economic burden of specimen provenance complications in prostate biopsies in the United States. MATERIALS AND METHODS: We performed a comprehensive, systematic review of the literature to approximate the effect of specimen provenance complications on direct medical costs, patient QALYs and medicolegal costs. Data were extracted from published studies on specimen provenance complications rates, prostate cancer treatment efficacy, treatment cost, litigation/settlement costs after false diagnoses of prostate biopsies and patient quality of life. Sensitivity analysis was done to identify factors that most influenced the outcomes and assess the robustness of the findings. RESULTS: Of the estimated 806,251 primary and secondary prostate biopsies performed annually in the United States 20,322 specimen provenance complications were projected to result in 4,570 clinically meaningful false diagnoses and an expected loss of 634 QALYs. The total burden of specimen provenance complications was projected to exceed $879.9 million or $3,776 per positive cancer diagnosis. This estimate was most sensitive to the indemnity cost per false-positive case and the rate of transpositions at independent reference laboratories. CONCLUSIONS: The societal burden of specimen provenance complications in patients who undergo prostate biopsy exceeds $880 million annually in the United States. This analysis framework may be useful as policy makers, health organizations and researchers seek to decrease false diagnoses of prostate cancer and the consequent effects of delayed or unnecessary treatment. Further study is warranted to quantify the economic burden among additional diseases.

The E2F1/DNMT1 axis is associated with the development of AR negative castration resistant prostate cancer.

BACKGROUND: Research on castration resistant prostate cancer (CRPC) has focused primarily on functional alterations of the androgen receptor (AR). However, little is known about the loss of AR gene expression itself and the possible contribution of AR negative cells to CRPC. METHODS: Human and murine prostate cancer tissue microarrays (TMAs) were evaluated with antibodies specific for E2F1, DNA methyltransferase 1 or AR. The human prostate cancer TMA consisted of clinical samples ranging from normal tissue to samples of metastatic disease. The murine TMA was comprised of benign, localized or metastatic prostate cancer acquired from TRAMP mice treated with castration and/or 5'-Aza-2'-deoxycytidine (5Aza). RESULTS: Immunohistochemical analysis revealed increased nuclear DNMT1 staining in localized PCa (P < 0.0001) and metastatic PCa (P < 0.0001) compared to normal tissue. Examination of specific diagnoses revealed that Gleason seven tumors exhibited greater nuclear DNMT1 staining than Gleason six tumors (P < 0.05) and that metastatic tissue exhibited greater levels of nuclear DNMT1 than Gleason seven tumors (P < 0.01). Evaluation of the murine tissue cores revealed that 8.2% and 8.1% of benign tissue cores stained positive for E2F1 and DNMT1 respectively, while 97.0% were AR positive. Conversely, 81% and 100% of tumors were positive for E2F1 and DNMT1 respectively. This was in stark contrast to only 18% of tumors positive for AR. Treatment of mice with 5Aza reduced DNMT1 staining by 30%, while AR increased by 27%. CONCLUSIONS: These findings demonstrate that the E2F1/DNMT1 inhibitory axis of AR transcription is activated during the emergence of CRPC.

Positive Impact of Implementing a Comprehensive Genetic Testing Protocol for Prostate Cancer Patients in a Multi-disciplinary Uro-oncology Practice.

INTRODUCTION: Prior to the 2017 Philadelphia Consensus Conference guidelines, genetic testing for prostate cancer was conducted based on personal and family history of malignancy pursuant to National Comprehensive Cancer Network recommendations. The updated 2019 guidelines addressed the subject of genetic testing by endorsing point-of-care genetic testing and referral to genetic counseling. However, limited literature is available regarding successful implementation of a streamlined method for genetic testing. This paper explores the benefits of implementing an on-site guideline-based genetic testing process for prostate cancer patients. METHODS: Data were retrospectively reviewed for 552 prostate cancer patients seen in a uro-oncology clinic since January 2017. Prior to September 2018 genetic testing was recommended based on National Comprehensive Cancer Network guidelines, and swabs for testing were procured off-site 1 mile from the clinic (n = 78). After September 2018 genetic testing was recommended based on the Philadelphia Consensus Conference guidelines, and swabs for testing were procured at the clinic itself (n = 474). RESULTS: A statistically significant increase in testing compliance was observed after the implementation of on-site, guideline-based testing. Genetic testing compliance increased from 33.3% to 98.7%. The time to receive the genetic test results was also reduced from 38 days to 21 days. CONCLUSIONS: The implementation of an on-site, guideline-based genetic testing model for prostate cancer patients significantly improved compliance with genetic testing to 98.7% and decreased the time to receive genetic test results by 17 days. Adopting a guideline-based model with on-site genetic testing can significantly improve the detection rate for pathogenic and actionable mutations and increase the utilization of targeted therapies.

The Diagnosis of Urinary Tract Infections Using a Novel At-home Testing Protocol to Enhance Telemedicine: A Retrospective Analysis.

OBJECTIVE: To retrospectively analyze a novel courier-based home urine collection strategy for patients with symptoms of urinary tract infections (UTIs). This model was developed to provide patient care using telehealth during the coronavirus 2019 pandemic. METHODS: We analyzed data from 2206 patients with symptomatic UTIs to investigate the efficacy of a home urine collection protocol. The primary outcome was the impact of home versus office collection. RESULTS: We analyzed the results of 1112 patient samples collected in-office and 1084 patient samples collected at home. There was no difference in the rate of bacterial identification between females in the office and home collection groups. However, males in the office collection group had a higher rate of bacterial identification (p = .002). The turnaround time was significantly faster in the home collection group than the office collection group (4.08 hours shorter, p < 0.0014). Antibiotic use prior to sample collection was significantly higher in the home collection group for both males (p = .0004) and females (p = .004). Changes in antibiotics were significantly higher in the home collection group than in the office collection group for both males (p = .0009) and females (p = .0006). CONCLUSION: Our home collection protocol is a viable method to provide prompt and reliable outpatient care to urology patients suffering from UTIs. Furthermore, this approach resulted in adequate management and quicker turnaround times. Our findings demonstrate the clinical viability of a decentralized healthcare model to treat UTIs.

Evaluation of an RNAseq-Based Immunogenomic Liquid Biopsy Approach in Early-Stage Prostate Cancer.

The primary objective of this study is to detect biomarkers and develop models that enable the identification of clinically significant prostate cancer and to understand the biologic implications of the genes involved. Peripheral blood samples (1018 patients) were split chronologically into independent training (n = 713) and validation (n = 305) sets. Whole transcriptome RNA sequencing was performed on isolated phagocytic CD14+ and non-phagocytic CD2+ cells and their gene expression levels were used to develop predictive models that correlate to adverse pathologic features. The immune-transcriptomic model with the highest performance for predicting adverse pathology, based on a subtraction of the log-transformed expression signals of the two cell types, displayed an area under the curve (AUC) of the receiver operating characteristic of 0.70. The addition of biomarkers in combination with traditional clinical risk factors (age, serum prostate-specific antigen (PSA), PSA density, race, digital rectal examination (DRE), and family history) enhanced the AUC to 0.91 and 0.83 for the training and validation sets, respectively. The markers identified by this approach uncovered specific pathway associations relevant to (prostate) cancer biology. Increased phagocytic activity in conjunction with cancer-associated (mis-)regulation is also represented by these markers. Differential gene expression of circulating immune cells gives insight into the cellular immune response to early tumor development and immune surveillance.

Multiplex PCR Based Urinary Tract Infection (UTI) Analysis Compared to Traditional Urine Culture in Identifying Significant Pathogens in Symptomatic Patients.

OBJECTIVE: To evaluate whether multiplex PCR-based molecular testing is noninferior to urine culture for detection of bacterial infections in symptomatic patients. METHODS: Retrospective record review of 582 consecutive elderly patients presenting with symptoms of lower urinary tract infection (UTI) was conducted. All patients had traditional urine cultures and PCR molecular testing run in parallel. RESULTS: A total of 582 patients (mean age 77; range 60-95) with symptoms of lower UTI had both urine cultures and diagnostic PCR between March and July 2018. PCR detected uropathogens in 326 patients (56%, 326/582), while urine culture detected pathogens in 217 patients (37%, 217/582). PCR and culture agreed in 74% of cases (431/582): both were positive in 34% of cases (196/582) and both were negative in 40% of cases (235/582). However, PCR and culture disagreed in 26% of cases (151/582): PCR was positive while culture was negative in 22% of cases (130/582), and culture was positive while PCR was negative in 4% of cases (21/582). Polymicrobial infections were reported in 175 patients (30%, 175/582), with PCR reporting 166 and culture reporting 39. Further, polymicrobial infections were identified in 67 patients (12%, 67/582) in which culture results were negative. Agreement between PCR and urine culture for positive cultures was 90%, exceeding the noninferiority threshold of 85% (95% conflict of interest 85.7%-93.6%). CONCLUSION: Multiplex PCR is noninferior to urine culture for detection and identification of bacteria. Further investigation may show that the accuracy and speed of PCR to diagnose UTI can significantly improve patient outcomes.

Bacterial Interactions as Detected by Pooled Antibiotic Susceptibility Testing (P-AST) in Polymicrobial Urine Specimens.

Introduction: Antimicrobial susceptibility is well characterized in monomicrobial infections, but bacterial species often coexist with other bacterial species. Antimicrobial susceptibility is often tested against single bacterial isolates; this approach ignores interactions between cohabiting bacteria that could impact susceptibility. Here, we use Pooled Antibiotic Susceptibility Testing to compare antimicrobial susceptibility patterns exhibited by polymicrobial and monomicrobial urine specimens obtained from patients with urinary tract infection symptoms. Methods: Urine samples were collected from patients who had symptoms consistent with a urinary tract infection. Multiplex polymerase chain reaction testing was performed to identify and quantify 31 bacterial species. Antibiotic susceptibility was determined using a novel Pooled Antibiotic Susceptibility Testing method. Antibiotic resistance rates in polymicrobial specimens were compared with those in monomicrobial infections. Using a logistic model, resistance rates were estimated when specific bacterial species were present. To assess interactions between pairs of bacteria, the predicted resistance rates were compared when a pair of bacterial species were present versus when just one bacterial species was present. Results: Urine specimens were collected from 3,124 patients with symptoms of urinary tract infection. Of these, multiplex polymerase chain reaction testing detected bacteria in 61.1% (1910) of specimens. Pooled Antibiotic Susceptibility Testing results were available for 70.8% (1352) of these positive specimens. Of these positive specimens, 43.9% (594) were monomicrobial, while 56.1% (758) were polymicrobial. The odds of resistance to ampicillin (p = 0.005), amoxicillin/clavulanate (p = 0.008), five different cephalosporins, vancomycin (p = <0.0001), and tetracycline (p = 0.010) increased with each additional species present in a polymicrobial specimen. In contrast, the odds of resistance to piperacillin/tazobactam decreased by 75% for each additional species present (95% CI 0.61, 0.94, p = 0.010). For one or more antibiotics tested, thirteen pairs of bacterial species exhibited statistically significant interactions compared with the expected resistance rate obtained with the Highest Single Agent Principle and Union Principle. Conclusion: Bacterial interactions in polymicrobial specimens can result in antimicrobial susceptibility patterns that are not detected when bacterial isolates are tested by themselves. Optimizing an effective treatment regimen for patients with polymicrobial infections may depend on accurate identification of the constituent species, as well as results obtained by Pooled Antibiotic Susceptibility Testing.

BK virus as a cofactor in the etiology of prostate cancer in its early stages.

Prostate cancer has been projected to cause almost 10% of all male cancer deaths in the United States in 2007. The incidence of mutations in the tumor suppressor genes Rb1 and p53, especially in the early stages of the disease, is low compared to those for other cancers. This has led to the hypothesis that a human virus such as BK virus (BKV), which establishes a persistent subclinical infection in the urinary tract and encodes oncoproteins that interfere with these tumor suppressor pathways, is involved. Previously, we detected BKV DNA in the epithelial cells of benign and proliferative inflammatory atrophy ducts of cancerous prostate specimens. In the present report, we demonstrate that BKV is present at a much lower frequency in noncancerous prostates. Additionally, in normal prostates, T-antigen (TAg) expression is observed only in specimens harboring proliferative inflammatory atrophy and prostatic intraepithelial neoplasia. We further demonstrate that the p53 gene from atrophic cells expressing TAg is wild type, whereas tumor cells expressing detectable nuclear p53 contain a mix of wild-type and mutant p53 genes, suggesting that TAg may inactivate p53 in the atrophic cells. Our results point toward a role for BKV in early prostate cancer progression.

Targeting CCL2 with systemic delivery of neutralizing antibodies induces prostate cancer tumor regression in vivo.

The identification of novel tumor-interactive chemokines and the associated insights into the molecular and cellular basis of tumor-microenvironment interactions have continued to stimulate the development of targeted cancer therapeutics. Recently, we have identified monocyte chemoattractant protein 1 (MCP-1; CCL2) as a prominent regulator of prostate cancer growth and metastasis. Using neutralizing antibodies to human CCL2 (CNTO888) and the mouse homologue CCL2/JE (C1142), we show that treatment with anti-CCL2/JE antibody (2 mg/kg, twice weekly i.p.) attenuated PC-3Luc-mediated overall tumor burden in our in vivo model of prostate cancer metastasis by 96% at 5 weeks postintracardiac injection. Anti-CCL2 inhibition was not as effective as docetaxel (40 mg/kg, every week for 3 weeks) as a single agent, but inhibition of CCL2 in combination with docetaxel significantly reduced overall tumor burden compared with docetaxel alone, and induced tumor regression relative to initial tumor burden. These data suggest an interaction between tumor-derived chemokines and host-derived chemokines acting in cooperation to promote tumor cell survival, proliferation, and metastasis.

Urinary Molecular Biomarker Test Impacts Prostate Biopsy Decision Making in Clinical Practice.

INTRODUCTION: There is an unmet need for noninvasive methods to better identify patients at increased risk for clinically significant prostate cancer. SelectMDx® is a molecular urine test validated for the detection of Gleason score 7 and higher cancers (ISUP [International Society of Urological Pathology] Grade Group 2-5). In this multicenter trial we evaluated the test's impact on prostate biopsy decision making in clinical practice. METHODS: The study involved 5 U.S. community urology practices which sequentially enrolled 418 patients who received a SelectMDx test between May 2016 and April 2017 while undergoing evaluation for initial prostate biopsy. All tests were ordered by the urologist for patient management. We determined biopsy and prostate cancer detection rates in patients with SelectMDx positive versus SelectMDx negative results. RESULTS: Of the 418 subjects evaluated with SelectMDx 253 (61%) had negative results and 165 (39%) had positive results. Subsequent biopsy rates for SelectMDx positive and negative cases were 60% (99) and 12% (32), respectively (p <0.001). Time from SelectMDx result to biopsy was shorter for those with positive vs negative results (median 2 vs 5 months, p=0.001). Of patients who underwent biopsy within 3 months of testing 71 (43%) with positive results underwent biopsy and 27 had cancers identified, including 10 greater than Grade Group 2. Of 9 patients with SelectMDx negative results (3.6%) who underwent biopsy 4 were diagnosed with cancer, all Grade Group 2 or less. CONCLUSIONS: SelectMDx had a significant impact on initial prostate biopsy decision making. Biopsy rates in SelectMDx positive cases were fivefold higher than in SelectMDx negative cases. These results describe the clinical utility of SelectMDx in real-world community urology practice.

Clinical outcomes in men with prostate cancer who selected active surveillance using a clinical cell cycle risk score.

Aim: To evaluate active surveillance (AS) selection, safety and durability among men with low-risk prostate cancer assessed using the clinical cell cycle risk (CCR) score, a combined clinical and molecular score. Patients & methods: Initial treatment selection (AS vs treatment) and duration of AS were evaluated for men with low-risk prostate cancer according to the CCR score and National Comprehensive Cancer Network guidelines. Adverse events included biochemical recurrence and metastasis. Results: 82.4% (547/664) of men initially selected AS (median follow-up: 2.2 years), 0.4% (2/547) of whom experienced an adverse event. Two-thirds of patients remained on AS for more than 3 years; patient choice was the most common reason for leaving AS. Conclusion: The CCR score may aid in the identification of men who can safely defer prostate cancer treatment.

Papillary renal cell carcinoma with oncocytic cells and nonoverlapping low grade nuclei: expanding the morphologic spectrum with emphasis on clinicopathologic, immunohistochemical and molecular features.

Papillary renal cell carcinoma (PRCC), a morphologically and genetically distinct subtype of RCC, is morphologically separated into 2 subtypes, type 1 and 2, for prognostic purposes. Type 1 PRCC (single layer of small cells, scant pale cytoplasm) is more common and has a favorable prognosis compared with type 2 (pseudostratified high-grade nuclei, abundant eosinophilic/oncocytic cytoplasm). We report the clinicopathologic, immunohistochemical, and molecular data of 7 adult papillary tumors with morphological features distinct from type 1 or 2 PRCC. All tumors demonstrated predominant papillary architecture, lined by cells with oncocytic cytoplasm, and nonoverlapping low Fuhrman grade nuclei (1 or 2). Foamy macrophages were noted in 2 of 7 tumors. No case demonstrated necrosis or psammoma bodies. Most tumors (6/7) were small (mean size, 2.0 cm; range, 0.8-5.7 cm) and limited to the kidney. No tumor recurrence or metastasis was identified (median follow-up, 22 months). All tumors demonstrated trisomy for 7 and 17 by fluorescence in situ hybridization analysis and uniform CK 7, CD10, and alpha-methylacyl-coenzyme A racemase expression, characteristic of PRCC. These results suggest that these tumors are distinct from type 1 (owing to oncocytic cells) and type 2 (owing to low-grade nonstratified nuclei, low stage, and good outcome). Awareness of this favorable spectrum of PRCC is important to avoid its potential misinterpretation as an aggressive type 2 PRCC (owing to oncocytic cells) or rarely as an oncocytoma (owing to oncocytic cells and low-grade nuclei). Morphologic spectrum of these PRCCs emphasizes that the future prognostic model of PRCC may need to be based primarily on the nuclear characteristics, irrespective of the cytoplasmic features.

5-aza-2'-deoxycytidine delays androgen-independent disease and improves survival in the transgenic adenocarcinoma of the mouse prostate mouse model of prostate cancer.

PURPOSE: We have previously shown that 5-aza-2'-deoxycytidine (5-aza) is an effective chemopreventive agent capable of preventing early disease progression in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model. The purpose of this study was to determine the effect of 5-aza on preexisting TRAMP prostate cancers and prevention of androgen-independent prostate cancer. EXPERIMENTAL DESIGN: TRAMP mice with established prostate cancers were treated with 5-aza, castration, castration + 5-aza, or vehicle control (PBS). One cohort of 22 mice per treatment was euthanized after 10 weeks of treatment, whereas a second cohort of 14 mice per group was followed until death to determine survival. Histologic sections of prostate, pelvic lymph nodes, lung, and liver were blinded and analyzed by a certified genitourinary pathologist (K.J.W.). RESULTS: Combined treatment (castration + 5-aza) provided significant survival benefits over either single treatment (combined versus castration P = 0.029, combined versus 5-aza P = 0.036). At 24 weeks of age, 86% of mice within the PBS cohort exhibited histologic evidence of prostate cancer, whereas only 47% of the combined cohort exhibited malignant disease (P < 0.0001). Additionally, whereas 43% of the PBS treatment group exhibited lymph node metastases, these were only observed in 21% of the combined treatment mice. CONCLUSIONS: This is the first study to examine the effect of 5-aza and combined castration + 5-aza on preexisting prostate cancer in an animal model. Based on these preclinical findings, we suggest that 5-aza treatment may prolong the time to an androgen-independent status and thus survival in a hormone-deprived setting in prostate cancer.

Inhibition of DNA methyltransferase activity prevents tumorigenesis in a mouse model of prostate cancer.

Transcriptional silencing of tumor suppressor genes by DNA methylation plays an important role in tumorigenesis. These aberrant epigenetic modifications may be mediated in part by elevated DNA methyltransferase levels. DNA methyltransferase 1 (DNMT1), in particular, is overexpressed in many tumor types. Recently, we showed that Dnmt1 is transcriptionally regulated by E2F transcription factors and that retinoblastoma protein (pRb) inactivation induces Dnmt1. Based on these observations, we investigated regulation of Dnmt1 by polyomavirus oncogenes, which potently inhibit the pRb pocket protein family. Infection of primary human prostate epithelial cells with BK polyomavirus dramatically induced Dnmt1 transcription following large T antigen (TAg) translation and E2F activation. For in vivo study of Dnmt1 regulation, we used the transgenic adenocarcinoma of the mouse prostate (TRAMP) model, which expresses the SV40 polyomavirus early region, including TAg, under control of a prostate-specific promoter. Analysis of TRAMP prostate lesions revealed greatly elevated Dnmt1 mRNA and protein levels beginning in prostatic intraepithelial neoplasia and continuing through advanced prostate cancer and metastasis. Interestingly, when TRAMP mice were treated in a chemopreventive manner with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza), 0 of 14 mice developed prostate cancer at 24 weeks of age, whereas 7 of 13 (54%) control-treated mice developed poorly differentiated prostate cancer. Treatment with 5-aza also prevented the development of lymph node metastases and dramatically extended survival compared with control-treated mice. Taken together, these data suggest that Dnmt1 is rapidly activated by pRb pathway inactivation, and that DNA methyltransferase activity is required for malignant transformation and tumorigenesis.

Elevated E2F1 inhibits transcription of the androgen receptor in metastatic hormone-resistant prostate cancer.

Activation of E2F transcription factors, through disruption of the retinoblastoma (Rb) tumor-suppressor gene, is a key event in the development of many human cancers. Previously, we showed that homozygous deletion of Rb in a prostate tissue recombination model exhibits increased E2F activity, activation of E2F-target genes, and increased susceptibility to hormonal carcinogenesis. In this study, we examined the expression of E2F1 in 667 prostate tissue cores and compared it with the expression of the androgen receptor (AR), a marker of prostate epithelial differentiation, using tissue microarray analysis. We show that E2F1 expression is low in benign and localized prostate cancer, modestly elevated in metastatic lymph nodes from hormone-naïve patients, and significantly elevated in metastatic tissues from hormone-resistant prostate cancer patients (P = 0.0006). In contrast, strong AR expression was detected in benign prostate (83%), localized prostate cancer (100%), and lymph node metastasis (80%), but decreased to 40% in metastatic hormone-resistant prostate cancer (P = 0.004). Semiquantitative reverse transcription-PCR analysis showed elevated E2F1 mRNA levels and increased levels of the E2F-target genes dihyrofolate reductase and proliferating cell nuclear antigen in metastatic hormone-independent prostate cancer cases compared with benign tissues. To identify a role of E2F1 in hormone-independent prostate cancer, we examined whether E2F1 can regulate AR expression. We show that exogenous expression of E2F1 significantly inhibited AR mRNA and AR protein levels in prostate epithelial cells. E2F1 also inhibited an AR promoter-luciferase construct that was dependent on the transactivation domain of E2F1. Furthermore, using chromatin immunoprecipitation assays, we show that E2F1 and the pocket protein family members p107 and p130 bind to the AR promoter in vivo. Taken together, these results show that elevated E2F1, through its ability to repress AR transcription, may contribute to the progression of hormone-independent prostate cancer.

Specimen Provenance Testing Identifies Contamination That Affects Molecular Prognostic Assay Results in Prostate Cancer Biopsy Specimens.

OBJECTIVE: To determine if tissue contamination in histologic specimens can significantly affect the results of prognostic molecular markers that are routinely used as confirmatory tests to safely assign appropriate candidates to prostate cancer active surveillance protocols. MATERIALS AND METHODS: This study evaluates 2134 cases from a single, large urology practice that were successfully tested for DNA specimen provenance verification using short tandem repeat analysis for the presence of a significant level of contaminating DNA. After removal of the contamination, 5 of the samples were retested, and the results of the molecular diagnostic test were compared. RESULTS: Forty-nine of the 2134 cases (2.3%) sent for DNA provenance analysis were found to possess significant levels of contamination. Of these 49 cases, 7 were resent for a repeat molecular diagnostic test after being decontaminated. Five of these prostate cancer specimens had sufficient tissue and RNA to give a more accurate cell cycle progression (CCP) score. The average absolute change in these patients' CCP scores was 0.48, with a minimum of 0.1-unit and a maximum of 1.0-unit difference. These changes in CCP scores are significant enough to cause meaningful alterations in a patient's calculated 10-year mortality rate, as defined by their combined risk score. CONCLUSION: DNA contamination in unstained tissue sections sent for prognostic prostate cancer molecular diagnostic testing occurs in 2.3% of the cases, and can be of a magnitude that affects the results and subsequent clinical decision of appropriateness for active surveillance.

Clinical Utility of Gene Expression Classifiers in Men With Newly Diagnosed Prostate Cancer.

PURPOSE: Tissue-based gene expression classifiers (GECs) may assist with management decisions in patients with newly diagnosed prostate cancer. We sought to assess the current use of GEC tests and determine how the test results are associated with primary disease management. METHODS: In this observational study, patients diagnosed with localized prostate cancer were tracked through the Michigan Urological Surgery Improvement Collaborative registry. The utilization and results of three GECs (Decipher Prostate Biopsy, Oncotype DX Prostate, and Prolaris) were prospectively collected. Practice patterns, predictors of GEC use, and effect of GEC results on disease management were investigated. RESULTS: Of 3,966 newly diagnosed patients, 747 (18.8%) underwent GEC testing. The rate of GEC use in individual practices ranged from 0% to 93%, and patients undergoing GEC testing were more likely to have a lower prostate-specific antigen level, lower Gleason score, lower clinical T stage, and fewer positive cores (all P < .05). Among patients with clinical favorable risk of cancer, the rate of active surveillance (AS) differed significantly among patients with a GEC result above the threshold (46.2%), those with a GEC result below the threshold (75.9%), and those who did not undergo GEC (57.9%; P < .001 for comparison of the three groups). This results in an estimate that, for every nine men with favorable risk of cancer who undergo GEC testing, one additional patient may have their disease initially managed with AS. On multivariable analysis, patients with favorable-risk prostate cancer who were classified as GEC low risk were more likely to be managed on AS than those without testing (odds ratio, 1.84; P = .006). CONCLUSION: There is large variability in practice-level use and GEC tests ordered in patients with newly diagnosed, localized prostate cancer. In patients with clinical favorable risk of cancer, GEC testing significantly increased the use of AS. Additional follow-up will help determine whether incorporation of GEC testing into initial patient care favorably affects clinical outcomes.

Expression of the cytoskeleton linker protein ezrin in human cancers.

Expression of the metastasis-associated protein, ezrin, in over 5,000 human cancers and normal tissues was analyzed using tissue microarray immunohistochemistry. Ezrin staining was compared between cancers and their corresponding normal tissues, between cancers of epithelial and mesenchymal origin, in the context of the putative inhibitor protein, merlin, and against clinicopathological data available for breast, lung, prostate cancers and sarcomas. Ezrin was found in most cancers and normal tissues at varying levels of intensity. In general ezrin was expressed at higher levels in sarcomas than in carcinomas. By normalizing the expression of ezrin in each cancer using ezrin expression found in the corresponding normal tissue, significant associations between ezrin were found in advancing histological grade in sarcomas (P = 0.02) and poor outcome in breast cancer (P = 0.025). Clinicopathologic associations were not changed by simultaneous assessment of ezrin and merlin in each patient sample for the cancer types examined. These data support a role for ezrin in the biology of human cancers and the need for additional studies in breast cancer and sarcoma patients that may validate ezrin as a marker of cancer progression and as a potential target for cancer therapy.