Amy Blase

PCA3: A Molecular Urine Assay for Predicting Prostate Biopsy Outcome

PURPOSE A urinary assay for PCA3, an mRNA that is highly over expressed in prostate cancer cells, has shown usefulness as a diagnostic test for this common malignancy. We further characterized PCA3 performance in different groups of men and determined whether the PCA3 score could synergize with other clinical information to predict biopsy outcome. MATERIALS AND METHODS Prospectively urine was collected following standardized digital rectal examination in 570 men immediately before prostate biopsy. Urinary PCA3 mRNA levels were quantified and then normalized to the amount of prostate derived RNA to generate a PCA3 score. RESULTS The percent of biopsy positive men identified increased directly with the PCA3 score. PCA3 assay performance was equivalent in the first vs previous negative biopsy groups with an area under the ROC curve of 0.70 and 0.68, respectively. Unlike serum prostate specific antigen the PCA3 score did not increase with prostate volume. PCA3 assay sensitivity and specificity were equivalent at serum prostate specific antigen less than 4, 4 to 10 and more than 10 ng/ml. A logistic regression algorithm using PCA3, serum prostate specific antigen, prostate volume and digital rectal examination result increased the AUC from 0.69 for PCA3 alone to 0.75 (p = 0.0002). CONCLUSIONS PCA3 is independent of prostate volume, serum prostate specific antigen level and the number of prior biopsies. The quantitative PCA3 score correlated with the probability of positive biopsy. Logistic regression results suggest that the PCA3 score could be incorporated into a nomogram for improved prediction of biopsy outcome. The results of this study provide further evidence that PCA3 is a useful adjunct to current methods for prostate cancer diagnosis.

Urine TMPRSS2:ERG Fusion Transcript Stratifies Prostate Cancer Risk in Men with Elevated Serum PSA

Urine TMPRSS2:ERG gene fusion could be used for stratification of patients at higher risk for prostate cancer. Old Gene Fusion, New Diagnostic Tricks The “PSA test” is a routine test for men over the age of 50 or for those at risk for prostate cancer. It measures the level of prostate-specific antigen (PSA) in the blood, and if that level is above a predefined cutoff, a biopsy is recommended for definitive diagnosis. This test is not perfect; benign conditions, such as an enlarged prostate, can contribute to high levels of PSA, resulting in a “false-positive” and subsequent overdiagnosis and overtreatment. Because of the high prevalence of prostate cancer (it is estimated that nearly 250,000 men will be diagnosed with the disease in 2011), it is clear that a more accurate test for prostate cancer is needed. Here, Tomlins et al. improve on the PSA test by taking a new twist on a known gene fusion, using it to stratify more than 1000 men in two multicenter cohorts based on risk for developing the disease. Recently, it was discovered that the fusion of two genes, the transmembrane protease, serine 2 (TMPRSS2) gene and the v-ets erythroblastosis virus E26 oncogene homolog (avian) (ERG) gene, known as TMPRSS2:ERG, is overexpressed in more than 50% of PSA-screened prostate cancers. The protein product of this fusion cannot be detected in serum, so the authors decided to test for the presence of TMPRSS2:ERG mRNA in urine. First, they developed a clinical-grade, transcription-mediated amplification assay for quantifying fusion mRNA—this generated a TMPRSS2:ERG “score.” Urine TMPRSS2:ERG score was linked to the presence of cancer, tumor volume, and clinically significant cancer in patients. Then, the authors combined the TMPRSS2:ERG score with the level of prostate cancer antigen 3 (PCA3) in urine. TMPRSS2:ERG+PCA3 improved the performance of the multivariate Prostate Cancer Prevention Trial risk calculator, thus demonstrating clinical utility. Who said you can’t teach an old gene fusion new tricks? By combining the cancer-specific fusion TMPRSS2:ERG score with levels of PSA (in serum) and PCA3 (in urine), Tomlins and colleagues demonstrated more accurate, individualized stratification of men at high risk for developing clinically significant prostate cancer—an important step in streamlining diagnosis and treatment. Moreover, men with extremes of TMPRSS2:ERG+PCA3 had different risks of cancer on biopsy; in combination with other clinicopathological features, urine TMPRSS2:ERG+PCA3 might also inform the urgency of biopsy after PSA screening. More than 1,000,000 men undergo prostate biopsy each year in the United States, most for “elevated” serum prostate-specific antigen (PSA). Given the lack of specificity and unclear mortality benefit of PSA testing, methods to individualize management of elevated PSA are needed. Greater than 50% of PSA-screened prostate cancers harbor fusions between the transmembrane protease, serine 2 (TMPRSS2) and v-ets erythroblastosis virus E26 oncogene homolog (avian) (ERG) genes. Here, we report a clinical-grade, transcription-mediated amplification assay to risk stratify and detect prostate cancer noninvasively in urine. The TMPRSS2:ERG fusion transcript was quantitatively measured in prospectively collected whole urine from 1312 men at multiple centers. Urine TMPRSS2:ERG was associated with indicators of clinically significant cancer at biopsy and prostatectomy, including tumor size, high Gleason score at prostatectomy, and upgrading of Gleason grade at prostatectomy. TMPRSS2:ERG, in combination with urine prostate cancer antigen 3 (PCA3), improved the performance of the multivariate Prostate Cancer Prevention Trial risk calculator in predicting cancer on biopsy. In the biopsy cohorts, men in the highest and lowest of three TMPRSS2:ERG+PCA3 score groups had markedly different rates of cancer, clinically significant cancer by Epstein criteria, and high-grade cancer on biopsy. Our results demonstrate that urine TMPRSS2:ERG, in combination with urine PCA3, enhances the utility of serum PSA for predicting prostate cancer risk and clinically relevant cancer on biopsy.

PCA3 Molecular Urine Assay Correlates With Prostate Cancer Tumor Volume: Implication in Selecting Candidates for Active Surveillance

PURPOSE Prostate cancer gene 3 (PCA3) has shown promise as a molecular marker in prostate cancer detection. We assessed the association of urinary PCA3 score with prostatectomy tumor volume and other clinical and pathological features. MATERIALS AND METHODS Urine specimens were collected after digital rectal examination from 59 men scheduled for prostate biopsy and 83 men scheduled for radical prostatectomy. Prostatectomy findings were evaluable for 96 men. PCA3 and prostate specific antigen mRNAs were quantified with Gen-Probe DTS 400 System. The PCA3 score was defined as the ratio of PCA3 mRNA/prostate specific antigen mRNA x10(3). RESULTS The PCA3 score in men with negative biopsies (30) and positive biopsies (29) were significantly different (median 21.1 and 31.0, respectively, p = 0.029). The PCA3 score was significantly correlated with total tumor volume in prostatectomy specimens (r = 0.269, p = 0.008), and was also associated with prostatectomy Gleason score (6 vs 7 or greater, p = 0.005) but not with other clinical and pathological features. The PCA3 score was significantly different when comparing low volume/low grade cancer (dominant tumor volume less than 0.5 cc, Gleason score 6) and significant cancer (p = 0.007). On multivariate analysis PCA3 was the best predictor of total tumor volume in prostatectomy (p = 0.001). Receiver operating characteristic curve analysis showed that the PCA3 score could discriminate low volume cancer (total tumor volume less than 0.5 cc) well with area under the curve of 0.757. CONCLUSIONS The PCA3 score appears to stratify men based on prostatectomy tumor volume and Gleason score, and may have clinical applicability in selecting men who have low volume/low grade cancer.

PCA3 Molecular Urine Test for Predicting Repeat Prostate Biopsy Outcome in Populations at Risk: Validation in the Placebo Arm of the Dutasteride REDUCE Trial

PURPOSE We determined the performance of PCA3 alone and in the presence of other covariates as an indicator of contemporaneous and future prostate biopsy results in a population with previous negative biopsy and increased serum prostate specific antigen. MATERIALS AND METHODS Urine PCA3 scores were determined before year 2 and year 4 biopsies from patients in the placebo arm of the REDUCE trial, a prostate cancer risk reduction study evaluating men with moderately increased serum prostate specific antigen results and negative biopsy at baseline. PCA3, serum prostate specific antigen and percent free prostate specific antigen results were correlated with biopsy outcome via univariate logistic regression and ROC analyses. Multivariate logistic regression was also performed including these biomarkers together with prostate volume, age and family history. RESULTS PCA3 scores were measurable from 1,072 of 1,140 subjects (94% informative rate). PCA3 scores were associated with positive biopsy rate (p <0.0001) and correlated with biopsy Gleason score (p = 0.0017). PCA3 AUC of 0.693 was greater than serum prostate specific antigen (0.612, p = 0.0077 vs PCA3). The multivariate logistic regression model yielded an AUC of 0.753 and exclusion of PCA3 from the model decreased AUC to 0.717 (p = 0.0009). PCA3 at year 2 was a significant predictor of year 4 biopsy outcome (AUC 0.634, p = 0.0002), whereas serum prostate specific antigen and free prostate specific antigen were not predictive (p = 0.3281 and 0.6782, respectively). CONCLUSIONS PCA3 clinical performance was validated in the largest repeat biopsy study to date. Increased PCA3 scores indicated increased risk of contemporaneous cancers and predicted future biopsy outcomes. Use of PCA3 in combination with serum prostate specific antigen and other risk factors significantly increased diagnostic accuracy.

Predicting Prostate Cancer Risk Through Incorporation of Prostate Cancer Gene 3

PURPOSE The online Prostate Cancer Prevention Trial risk calculator combines prostate specific antigen, digital rectal examination, family and biopsy history, age and race to determine the risk of prostate cancer. In this report we incorporate the biomarker prostate cancer gene 3 into the Prostate Cancer Prevention Trial risk calculator. MATERIALS AND METHODS Methodology was developed to incorporate new markers for prostate cancer into the Prostate Cancer Prevention Trial risk calculator based on likelihood ratios calculated from separate case control or cohort studies. The methodology was applied to incorporate the marker prostate cancer gene 3 into the risk calculator based on a cohort of 521 men who underwent prostate biopsy with measurements of urinary prostate cancer gene 3, serum prostate specific antigen, digital rectal examination and biopsy history. External validation of the updated risk calculator was performed on a cohort of 443 European patients, and compared to Prostate Cancer Prevention Trial risks, prostate specific antigen and prostate cancer gene 3 by area underneath the receiver operating characteristic curve, sensitivity and specificity. RESULTS The AUC of posterior risks (AUC 0.696, 95% CI 0.641-0.750) was higher than that of prostate specific antigen (AUC 0.607, 95% CI 0.546-0.668, p = 0.001) and Prostate Cancer Prevention Trial risks (AUC 0.653, 95% CI 0.593-0.714, p <0.05). Although it was higher it was not statistically significantly different from that of prostate cancer gene 3 (AUC 0.665, 95% CI 0.610-0.721, p >0.05). Sensitivities of posterior risks were higher than those of prostate cancer gene 3, prostate specific antigen and Prostate Cancer Prevention Trial risks. CONCLUSIONS New markers for prostate cancer can be incorporated into the Prostate Cancer Prevention Trial risk calculator by a novel approach. Incorporation of prostate cancer gene 3 improved the diagnostic accuracy of the Prostate Cancer Prevention Trial risk calculator.

Evaluation of the ETS-Related Gene mRNA in Urine for the Detection of Prostate Cancer

Purpose: Prevalent gene fusions in prostate cancer involve androgen-regulated promoters (primarily TMPRSS2) and ETS transcription factors (predominantly ETS-regulated gene (ERG)], which result in tumor selective overexpression of ERG in two thirds of patients. Because diverse genomic fusion events lead to ERG overexpression in prostate cancer, we reasoned that it may be more practical to capture such alterations using an assay targeting ERG sequences retained in such gene fusions. This study evaluates the potential of an assay quantitating ERG mRNA in post–digital rectal exam (DRE) urine for improving prostate cancer detection. Experimental Design: Patients scheduled to undergo transrectal ultrasound-guided needle biopsy of the prostate were prospectively enrolled. On the day of biopsy, patients provided a urine sample immediately following a DRE. Urine ERG mRNA was measured and normalized to urine prostate-specific antigen (PSA) mRNA using the DTS 400 system. Demographic traits, clinical characteristics and biopsy results were analyzed for association with urine ERG score. Results: The study was conducted on 237 patients. Prostate cancer was shown on biopsy in 40.9% of study subjects. A higher urine ERG score associated significantly with malignancy on biopsy (P = 0.0145), but not with clinical stage or Gleason score. Urine ERG score performed best in Caucasians and in men with a PSA of ≤4 ng/mL (area under the curve = 0.8). Conclusions: A higher urine ERG score in post-DRE urine is associated with the diagnosis of prostate cancer on biopsy. Urine ERG score performed particularly well in men with a PSA of ≤4.0 ng/mL, a segment of the screening population in which further diagnostic markers are needed to determine in whom biopsy should be done. Clin Cancer Res; 16(5); 1572–6

Prostate cancer gene 3 score predicts prostate biopsy outcome in men receiving dutasteride for prevention of prostate cancer: results from the REDUCE trial.

OBJECTIVES To examine the ability of the urinary prostate cancer gene 3 (PCA3) assay to predict biopsy-detected cancers in men receiving dutasteride in the Reduction by Dutasteride of Prostate Cancer Events (REDUCE) study cohort. METHODS Urine and serum samples from 930 men in the active arm were acquired at years 2 and 4 of the biopsy visits. In addition to univariate logistic regression and receiver operating characteristic analysis, multivariate analysis for association with biopsy outcome was performed for PCA3 score in the presence of serum prostate-specific antigen (PSA), age, prostate volume, and family history of prostate cancer. RESULTS At year 2, the univariate PCA3 score area under the receiver operating characteristic curve (AUC) was 0.668 versus 0.603 for PSA. At year 4, the PCA3 assay significantly predicted the biopsy outcome (AUC 0.628, 95% confidence interval 0.556-0.700), and the PSA level was not predictive (AUC 0.556, 95% confidence interval 0.469-0.642). The year 2 multivariate model yielded an AUC of 0.712. Removing the PCA3 score decreased the AUC to 0.660 (P = .0166 vs the full model). The median PCA3 scores in the dutasteride arm were not different from those in the 1072 men in the placebo arm (16.2 and 17.2 at year 2, P = .1755; and 18.8 and 18.1 at year 4, P = .2340, respectively). However, the PSA values were reduced >50% in the dutasteride arm at both visits (both P < .0001 vs placebo). At a PCA3 score cutoff of 35, the sensitivity and specificity were equivalent between the 2 arms. CONCLUSIONS In the present study, the PCA3 assay outperformed PSA for cancer detection in men undergoing dutasteride treatment and improved the diagnostic accuracy when combined with the PSA level and other clinical variables. In addition, no adjustment in PCA3 score was needed to yield equivalent clinical performance between the dutasteride and placebo arms. These findings are particularly important in light of the potential role of dutasteride for prostate cancer chemoprevention.

The long and winding road to FDA approval of a novel prostate cancer test: our story.

Only a very small fraction of the many tumor biomarkers discovered are successfully translated from the academic research laboratory into clinical practice. The pathway has not always been linear and has involved people with expertise in a wide range of specialties, including basic research, assay development, clinical affairs, regulatory affairs, marketing, business, oncology, and executive-management leadership. The experience with a tumor biomarker, the PCA3 [prostate cancer gene 3 (non-protein coding)] gene, illustrates the long and winding road that must be navigated. Here we reflect, from a historical point of view, on how the interface between academic science, industry, urologists, and clinical laboratories has been essential to advance biomarkers from solid basic science to a validated clinical laboratory test. PCA3 was first described as DD3 in 1999 by Bussemakers and colleagues (1). Researchers in the Isaacs laboratory at Johns Hopkins University used differential-display analysis to compare patterns of mRNA production in benign and malignant prostate tissue, with the goal of identifying unknown genes involved in prostate tumorigenesis. PCA3 expression was further characterized in the Schalken laboratory at Radboud University, Nijmegen (2), and this research confirmed 2 important properties of PCA3 as a prostate cancer (PCa)6 marker: PCA3 expression is prostate tissue specific, and PCA3 is highly overexpressed in PCa compared with benign tissue. This PCa-specific overexpression led the Nijmegen researchers to assess the feasibility of a PCA3 -based urine test for PCa detection. In 2001, a license for PCA3 was obtained by DiagnoCure, a company in Quebec City, Canada, to develop the assay and provide a clinical application. Transfer of a cancer biomarker to industry requires both the appropriate technology and a proper cancer strategy. The Nijmegen methodology was converted from PCR to nucleic acid sequence–based amplification (NASBA), a …

Abstract 2815: Urine TMPRSS2:ERG for prostate cancer risk stratification in men with elevated serum PSA

Background: Over 1,000,000 men undergo prostate biopsy each year in the U.S., most for “elevated” serum PSA. Given the lack of sensitivity and specificity, and unclear mortality benefit of PSA testing, methods to individualize management of elevated PSA are needed. We evaluated urine expression of TMPRSS2:ERG, a gene fusion occurring in 50% of prostate cancers, for risk-stratifying men presenting for biopsy. Methods: TMPRSS2:ERG was measured by a clinical grade, transcription-mediated-amplification assay in prospectively collected whole-urine from 1,094 men undergoing biopsy at 10 academic and community clinics. Findings: Urine TMPRSS2:ERG was associated with indicators of clinically significant cancer at biopsy and prostatectomy, including tumor size, high prostatectomy Gleason score and upgrading at prostatectomy. TMPRSS2:ERG in combination with urine PCA3, improved the multivariate PCPT risk calculator performance for predicting cancer on biopsy (AUC in test set, 0.79 vs. 0.64, p Interpretation: Urine TMPRSS2:ERG, in combination with urine PCA3, enhances the utility of serum PSA for predicting prostate cancer and clinically relevant cancer on biopsy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2815. doi:10.1158/1538-7445.AM2011-2815