1. Updated Abstract T2:ERG Correlation with PCa Aggressiveness Conclusions T2:ERG Assay Clinical Performance The T2:ERG assay described has not been approved or cleared by FDA. Purpose: There are several unmet needs in diagnosing and stratifying the aggressiveness of prostate cancer (PCa). Although serum prostate-specific antigen (sPSA) has high sensitivity for predicting prostate biopsy outcome, it has poor specificity leading to >60% negative biopsies. Once diagnosed, better prognostic tools to stratify the PCa aggressiveness are needed to guide treatment options. In this multi-site study, we evaluated the performance of a quantitative TMPRSS2:ERG (T2:ERG) gene fusion urine test for predicting prostate biopsy outcome. We also correlated T2:ERG mRNA levels in urine with indicators of PCa aggressiveness. Experimental Design: Post-DRE first void urine specimens were prospectively collected from 556 consenting patients scheduled for prostate biopsy from the University of Michigan, San Diego VA Medical Center and Université Laval. T2:ERG mRNA copies in samples were quantified using transcription-mediated amplification assays and results normalized to PSA mRNA copies to generate a T2:ERG Score. T2:ERG mRNA levels were correlated with biopsy outcome, biopsy Gleason Score, % positive cores, % involvement and PSA density. Results: PCa was detected in 226/556 men (41%). The T2:ERG urine test performed equivalently at three different sites (sens/spec=~42%/~85%). T2:ERG copy levels were significantly correlated with biopsy Gleason Score, % positive cores, % involvement and indolent vs. significant PCa as defined by the Epstein criteria, but not with PSA density. Conclusions: In a prospective multi-center study, the T2:ERG urine test demonstrated high specificity for predicting biopsy outcome; sensitivity was similar to the prevalence of T2:ERG gene fusions in prostate tumors. Importantly, T2:ERG copy levels significantly correlated with prognostic indicators of PCa aggressiveness. The T2:ERG urine test may therefore have utility for discriminating between patients requiring aggressive treatment and those who could be managed conservatively through active surveillance. Figure 1) T2:ERG assay clinical performance to predict prostate biopsy. The quantitative T2:ERG assay performs similarly in three different geographic cohorts. At a T2:ERG Score cutoff of 65, the sensitivity for the combined cohort was 42% and specificity 84%. TMPRSS2:ERG Gene Fusion Molecular Urine Test Correlates with Indicators of Prostate Cancer Aggressiveness Sheila M. J. Aubin 1, Sarah Williamsen 1, Petrea Hodge 1, Jessica Meinke 1, Javed Siddiqui 2, Laurie Sefton-Miller 2, Jonathan Silberstein 3, Kimberly Christensen 3, Amy Blase 1, Carol Kashefi 3, Yves Fradet 4, Harry Rittenhouse 1, Kyoko Sakamoto 3, Arul Chinnaiyan 2, Jack Groskopf 1 1 Gen-Probe Inc., San Diego, CA, 2 University of Michigan, Ann Arbor, MI, 3 San Diego VA Medical Center, San Diego, CA, 4 Université Laval, Quebec, Canada Figure 2) Correlation of T2:ERG copy levels with aggressive cancer, n = 92: T2:ERG copy levels were significantly correlated to the Epstein criteria for classifying aggressiveness of prostate cancer. The Epstein criteria defines insignificant cancer as biopsy Gleason score <6, % PCa involvement <50, % positive biopsy cores <33% and PSA density <0.15. San Diego A prototype quantitative T2:ERG urine test has been developed. Clinical performance vs. biopsy outcome: – High specificity (82-85%) for detection of cancer – Sensitivity (40-45%) similar to T2:ERG prevalence in prostate tumors – Equivalent performance from three clinical sites T2:ERG levels in urine correlate with criteria for indolent vs. significant cancer. Methods Assay Steps Pipet samples manually Add target capture reagent Incubate at 62°C, RT Wash magnetic particles with wash buffer using target capture system (TCS) Add oil, amplification and enzyme reagents Incubate at 42°C for TMA Add probe, selection and detection reagents for HPA Read using a luminometer Target Capture System Luminometer Assay Overview Messenger RNA targets are purified by magnetic separation using oligonucleotides and magnetic microparticles to isolate target nucleic acid for amplification and to eliminate potentially interfering substances. Gen-Probe's Transcription-Mediated Amplification (TMA) reliably targets RNA sequences and generates a billion fold copies of target nucleic acid in 15-30 minutes. The Hybridization Protection Assay (HPA) uses a specific DNA probe that hybridizes with the nucleic acid target to emit a chemiluminescent signal for detection and differentiation. Subject Demographics Demographics n115213228 No. biopsy positive (%)4943%9243%8537% Previously negative biopsy (%)28%39%13% Median/ave age (range)65/67(47 - 86)64/64(45 - 91)64/64(42 - 85) No. Ethnicity (%): White7869%213100%19387% Black1110%146% Hispanic1110% Asian98%115% Native American/Alaskan22% Unknown22%4 Median/ave ng/ml PSA (range)4.2/5.3(0.2 - 59.6)5.7/8.2(0.68 - 118)5.1/8.3(0.4 - 257.6) Median/ave cc TRUS prostate vol (range)45.0/55.6(19 - 232)38.0/42.0(12 - 145)47.1/58.7(9.4 - 253.0) Université Laval San Diego VA Medical Center Universitiy of Michigan ** Cancer significance based on Epstein criteria (JAMA 271:368-74, Cancer 101:2001-5) *Wilcoxonranksum test- Gleason Score %PCainvolvement % pos cores PSA density Cancer Significance** nMedian T2:ERG Scorep value* 6 52 19 0.033 >6 4063 50% 69 21 0.034 >50%2382 33% 6117 0.004 >33%3179 0.15 36 30 0.608 >0.15 5637 "Indolent" 19 6 0.024 "Significant" 73 47 DRE First-catch urine sample (25 - 30 mL) Transfer urine to transport tube Specimen Collection and Processing Male urine samples were collected after a digital rectal exam (DRE). The DRE was performed by applying firm pressure from the base to apex and from the lateral to the median line for each lobe (exactly 3 strokes per lobe). Urine was kept on ice and processed within 4 hours. Whole urine samples were processed by adding an equal volume of urine transport medium (UTM, buffered detergent). Combined5560.717 65 150 42% 30% 84% 90% nAUCCutoffSensitivitySpecificity Laval/SDVA3280.7036540%85% U of Michigan2280.7446545%82%