post-DRE Urine Research Articles

Increased α2,3-sialyl N-glycosylated prostate-specific membrane antigen (PSMA) in post-DRE urine is associated with high grade group prostate cancer.

Aberrant glycosylation of proteins is an important hallmark in multiple cancers. Prostate-specific membrane antigen (PSMA), a highly glycosylated protein with 10 N-linked glycosylation sites, is an Food and Drug Administration approved theranostic for prostate cancer. However, glycosylation changes in PSMA that are associated with prostate cancer disease progression have not been fully characterized. We investigated whether urinary PSMA sialylation correlate with high-grade prostate cancer. Urine samples were collected from men after digital rectal examination (DRE) before prostate biopsy. Lectin-antibody enzyme-linked immunoassay was used to quantify α2,3-sialyl PSMA in post-DRE urine samples from subjects with benign prostate tumors, Grade Group 1 prostate cancer and those with Grade Group ≥2 disease. There are significant increases in α2,3-sialylated PSMA in patients with Grade Group ≥2 disease compared to benign (p = 0.0009) and those with Grade Group 1 disease (p = 0.0063). There were no significant differences in α2,3-sialyl PSMA levels between Grade Group 1 and benign prostate tumors (p = 0.7947). Our study shows that there are significant differences in the abundance of α2,3-sialylated PSMA in post-DRE urines from disease stratified prostate cancer patients, and the increase is correlated with progression and disease severity. The detection of increased PSMA sialyation in post-DRE urines from patients with higher Grade Group ≥2 disease states provides novel untapped potential for the development of prognostic biomarkers for prostate cancer. Specifically, quantitation of α2,3-sialylated PSMA shows potential for discriminating between benign to intermediate grade disease, which is a significant clinical challenge in staging and risk stratification of prostate cancer.

Reply to Editorial Comment: Microbiomes in Post-DRE Urine Samples Are Linked to Prostate Cancer Risk

Reply to Editorial Comment: Microbiomes in Post-DRE Urine Samples Are Linked to Prostate Cancer Risk

Editorial Comment: Microbiomes in Post-DRE Urine Samples Are Linked to Prostate Cancer Risk

Editorial Comment: Microbiomes in Post-DRE Urine Samples Are Linked to Prostate Cancer Risk

Microbiomes in Post–Digital Rectal Exam Urine Samples are Linked to Prostate Cancer Risk

Abstract Purpose: Bacterial species including Cutibacterium acnes (C. acnes) have been associated with different inflammatory and neoplastic conditions in prostate cancer (PCa) tissue samples, but their clinical impact is unknown. Using next-generation sequencing (NGS)–based clinical reports, we investigated the differential abundance and incidence of microbiomes in post–digital rectal exam (DRE) urine samples from patients with PCa and a matched control group at low risk of PCa. Materials and Methods: A total of 200 post-DRE urine samples were analyzed, 100 from patients with histopathologically confirmed PCa and 100 from men at very low risk of PCa with PSA <1.5 ng/mL as controls. Bacterial and fungal communities were characterized by NGS of 16S and internal transcribed spacer (ITS) loci, respectively, with species' relative abundances provided on physicians' clinical reports. The differential abundance and incidence of species between cancer and control groups were evaluated. Results: Microbes were reported in 39% and 56% of PCa and control group samples, respectively. C. acnes had a significantly higher relative abundance in patients with PCa vs controls (P < .05), and C. acnes incidence rates were also nominally higher in patients with PCa as compared with controls (12.82% and 7.27%, respectively). By contrast, Finegoldia magna (F. magna) had a significantly higher relative abundance (P < .05) and incidence rate (P < .05) in controls as compared with patients with PCa. Conclusions: C. acnes was among the most prevalent bacterial species in PCa urine samples. F. magna identified in the low-risk group is responsible for production of equol, a soy metabolite associated with lowering risk of PCa, suggesting a role in prostate cancer chemoprevention.

MP40-01 DIFFERENTIALLY EXPRESSED GLYCOPROTEINS IN PRE- AND POST- DIGITAL RECTAL EXAMINATION URINE SAMPLES FOR DETECTING AGGRESSIVE PROSTATE CANCER

You have accessJournal of UrologyCME1 Apr 2023MP40-01 DIFFERENTIALLY EXPRESSED GLYCOPROTEINS IN PRE- AND POST- DIGITAL RECTAL EXAMINATION URINE SAMPLES FOR DETECTING AGGRESSIVE PROSTATE CANCER Indu Kohaar, Yuefan Wang, Tung-Shing Mamie Lih, Naseruddin Höti, Lori Sokoll, Gregory Chesnut, Gyorgy Petrovics, and Hui Zhang Indu KohaarIndu Kohaar More articles by this author , Yuefan WangYuefan Wang More articles by this author , Tung-Shing Mamie LihTung-Shing Mamie Lih More articles by this author , Naseruddin HötiNaseruddin Höti More articles by this author , Lori SokollLori Sokoll More articles by this author , Gregory ChesnutGregory Chesnut More articles by this author , Gyorgy PetrovicsGyorgy Petrovics More articles by this author , and Hui ZhangHui Zhang More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003278.01AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Prostate cancer (PCa) is predominantly indolent disease with a small fraction of patients undergoing metastatic progression. Therefore, it is critical to identify non-invasive diagnostic markers for the early detection of an aggressive PCa subtype. We hypothesized that non-invasive pre-digital rectal examination (pre-DRE) urine specimens harbor molecular signatures of aggressive PCa, which can help in the clinical management of the disease. Previously, we had systematically evaluated urinary glycoproteins associated with aggressive prostate cancer (AG-PCa) using post-DRE urine specimens. The goal of the study is to explore the potential of using pre-DRE urine specimens for detecting AG-CaP and validate the post-DRE findings. METHODS: This is a retrospective case cohort study based on 154 pre-DRE urine specimens collected by the Center for Prostate Disease Research under an institutional review board approved protocol from men undergoing diagnostic biopsy. We performed an optimized quantitative mass spectrometric analysis to evaluate the association of markers with aggressive PCa at diagnosis. The cohort comprised 41 patients with no cancer at biopsy, 48 patients with non-AG-PCa (Gleason score=6), and 65 patients with AG-PCa (Gleason score 7 or above). Targeted analyses of AG-PCa-associated glycoproteins previously identified in post-DRE urine samples were also applied to validate the post-DRE findings in non-DRE specimens. Data-independent acquisition mass spectrometry (DIA-MS) analysis was performed for glycosite-containing peptides. Fisher exact and Wilcoxon-Mann-Whitney tests were used for categorical and continuous variables respectively and logistic regression was evaluated using receiver operating characteristic (ROC) analysis for the urinary markers and marker panels. RESULTS: Urinary prostatic acid phosphatase (ACPP), CD97 antigen (CD97), and prostate-specific antigen (PSA) levels in pre-DRE specimens were associated with patients with AG-PCa compared to cancer-negative and non-AG-PCa. The analysis confirmed three of the previously reported post-DRE urinary glycoproteins and identified three new AG-PCa-associated glycoproteins including Fibrillin 1 (FBN1), Vitronectin (VTN) and Hemicentin 2 (HMCN2). A multi-marker panel composed of urinary PSA, VTN, HMCN2, CD97 achieved an AUC of 0.78 (p=2.69 × 10-9) for distinguishing AG-PCa from cancer-negative & Gleason 6. Analysis of differentially abundant glycopeptides in pre- and post-DRE samples showed that compared to glycopeptides from the post-DRE urine data, humoral immunity-related proteins were enriched in pre-DRE urine samples, whereas immune cell-related proteins were enriched in post-DRE urine samples. CONCLUSIONS: Comparisons between post-DRE and pre-DRE urine specimens for glycoproteins have shown that pre-DRE samples hold promise for a non-invasive and simple early option for AG-PCa detection in patients. The identified AG-PCa-associated glycoproteins may be further evaluated in large cohort of pre-DRE urine specimens for detecting clinically significant prostate cancer. Source of Funding: NCI/EDRN IAA No. ACN 17005-001-0000011 (Gregory Chesnut) © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e545 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Indu Kohaar More articles by this author Yuefan Wang More articles by this author Tung-Shing Mamie Lih More articles by this author Naseruddin Höti More articles by this author Lori Sokoll More articles by this author Gregory Chesnut More articles by this author Gyorgy Petrovics More articles by this author Hui Zhang More articles by this author Expand All Advertisement PDF downloadLoading ...

Differentially expressed glycoproteins in pre- and post-digital rectal examination urine samples for detecting aggressive prostate cancer.

Urinary glycoproteins associated with aggressive prostate cancer (AG-PCa) were previously reported using post-digital rectal examination (DRE) urine specimens. To explore the potential of using pre-DRE urine specimens for detecting AG-PCa, we compared glycoproteins between pre- and post-DRE urine specimens, verified the previously identified post-DRE AG-PCa-associated urinary glycoproteins in pre-DRE urine specimens, and explored potential new glycoproteins for AG-PCa detection in pre-DRE urine specimens. Quantitative glycoproteomic data were acquired for 154 pre-DRE urine specimens from 41 patients with no cancer at biopsy, 48 patients with non-AG-PCa (Gleason score=6), and 65 patients with AG-PCa (Gleason score 7 or above). Compared to glycopeptides from the post-DRE urine data, humoral immunity-related proteins were enriched in pre-DRE urine samples, whereas cell mediated immune response proteins were enriched in post-DRE urine samples. Analyses of AG-PCa-associated glycoproteins from pre-DRE urine revealed that the three urinary glycoproteins, prostate-specific antigen (PSA), prostatic acid phosphatase (ACPP), and CD97 antigen (CD97) that were previously identified in post-DRE urine samples, were also observed as AG-PCa associated glycoproteins in pre-DRE urine. In addition, we identified three new glycoproteins, fibrillin 1 (FBN1), vitronectin (VTN), and hemicentin 2 (HMCN2), to be potentially associated with AG-PCa in pre-DRE urine specimens. In summary, glycoprotein profiles differ between pre- and post-DRE urine specimens. The identified AG-PCa-associated glycoproteins may be further evaluated in large cohort of pre-DRE urine specimens for detecting clinically significant PCa.

Abstract 3383: Circular RNAs as non-invasive diagnostic biomarkers for prostate cancer

Abstract Prostate cancer is one of the most common cancers in men. The disease is largely indolent in nature and treatment of low-risk prostate cancer is not always necessary. However, a fraction of the patients will progress to aggressive disease which accounts for most of the cancer related mortality. Detection methods accurately differentiating between high- and low-risk patients for lethal prostate cancer is a critical need and currently, prostate biopsies and subsequent histopathology and molecular profiling are the most definitive way to differentiate between these two patient populations. But biopsies are invasive procedures associated with significant morbidity. Hence, there exists a critical need to develop non-invasive and highly sensitive diagnostic biomarkers to avoid unnecessary biopsies. Here, we propose to develop a novel biomarker panel which constitutes of a class of RNA molecules called circular RNA (circRNA) to be detected in liquid biopsies. Circular RNAs are covalently closed RNA structures, which are resistant to degradation by exoribonucleases because of the absence of free termini in their nucleotide chain. Due to their increased stability compared to their linear counterparts, circRNAs are ideal candidates for biomarkers, especially in non-invasive liquid biopsies. Using MiOncoCirc, a circRNA compendium from >2,000 primary and metastatic cancer tissues representing 40 cancer types, we first identified 9 circRNAs that are specifically expressed in prostate adenocarcinoma (PRAD) compared to all other cancer types. Next, we examined if these candidates are differentially expressed between PRAD and normal prostate tissues. Based on higher levels of expression which were statistically significant in the PRAD compared to normal prostate tissues, we promoted 4 circRNAs to our biomarker panel. In addition, our panel also includes 2 circRNAs that we predicted to arise from PRAD specific lncRNAs. To study the expression of the novel 6-circRNA biomarker expression in high-risk prostate cancer patients we developed a capture-based RNA sequencing assay. For the initial round we used this assay to test our pilot post-DRE patient urine sample EDRN cohort and detected expression of the prostate cancer-specific circRNAs in a subset of these post-DRE urine samples. We are in the process of expanding our test cohort which will also contain additional clinical annotations such as Gleason score. Post cohort assembly we intend to run our 6-circRNA capture assay on these samples to determine whether the expression levels of candidate circRNAs are correlated with disease stage and other clinical parameters. Our results will facilitate the rapid development of a diagnostic test using non-invasive urine samples for the early detection of high-risk prostate cancer. Citation Format: Palak Shah, Pushpinder Bawa, Lanbo Xiao, Yelena Kleyman, Josh Vo, Saravana M. Dhanasekaran, Jason Brown, Nicolette Loeding, Alexander Koons, Javed Siddiqui, Xuhong Cao, Arul M. Chinnaiyan. Circular RNAs as non-invasive diagnostic biomarkers for prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3383.

Association of MyProstateScore (MPS) with prostate cancer grade in the radical prostatectomy specimen.

To evaluate the association between urinary MyProstateScore (MPS) and pathologic grade group (GG) at surgery in men diagnosed with GG1 prostate cancer (PCa) on biopsy. Using an institutional biospecimen protocol, we identified men with GG1 PCa on biopsy and PSA ≤10 ng/ml who underwent radical prostatectomy (RP) at the University of Michigan. MPS was retrospectively calculated using prospectively collected, post-DRE urine samples. The primary outcome was upgrading on RP pathology, defined as GG ≥ 2. The associations of MPS, PSA, and PSA density (PSAD) with upgrading were assessed on univariable logistic regression, and the predictive accuracy of each marker was estimated by the area under the receiver operating characteristic curve (AUC). There were 52 men with urinary specimens available that met study criteria, based on biopsy Gleason Grade and specimen collection. At RP, 17 men (33%) had GG1 cancer and 35 (67%) had GG ≥ 2 cancer. Preoperative MPS was significantly higher in patients with GG ≥ 2 cancer at surgery (median 37.8 [IQR, 22.2-52.4]) as compared to GG1 (19.3 [IQR, 9.2-29.4]; P = 0.001). On univariable logistic regression, increasing MPS values were significantly associated with upgrading (odds ratio 1.07 per one-unit MPS increase, 95% confidence interval 1.02-1.12, P = 0.004), while PSA and PSAD were not significantly associated with upgrading. Similarly, the discriminative ability of the MPS model (AUC 0.78) for upgrading at RP was higher compared to models based on PSA (AUC 0.52) and PSAD (AUC 0.62). In men diagnosed with GG1 PCa who underwent surgery, MPS was significantly associated with RP cancer grade. In this limited cohort of men, these findings suggest that MPS could help identify patients with undetected high-grade cancer. Additional studies are needed to better characterize this association.

PD56-09 MICROBIOMES IN POST-DIGITAL RECTAL EXAM URINE SAMPLES ARE LINKED TO PROSTATE CANCER RISK

You have accessJournal of UrologyProstate Cancer: Detection & Screening VI (PD56)1 Sep 2021PD56-09 MICROBIOMES IN POST-DIGITAL RECTAL EXAM URINE SAMPLES ARE LINKED TO PROSTATE CANCER RISK E David Crawford, Whitney N Stanton, Rick Martin, Caleb D Phillips, Adrie van Bokhoven, M Scott Lucia, Paul Arangua, Francisco G La Rosa, Zachary Grasmick, Gretchen Hoyer, Nelson N Stone, Ryan Terlecki, J Curtis Nickel, and Priya N Werahera E David CrawfordE David Crawford More articles by this author , Whitney N StantonWhitney N Stanton More articles by this author , Rick MartinRick Martin More articles by this author , Caleb D PhillipsCaleb D Phillips More articles by this author , Adrie van BokhovenAdrie van Bokhoven More articles by this author , M Scott LuciaM Scott Lucia More articles by this author , Paul AranguaPaul Arangua More articles by this author , Francisco G La RosaFrancisco G La Rosa More articles by this author , Zachary GrasmickZachary Grasmick More articles by this author , Gretchen HoyerGretchen Hoyer More articles by this author , Nelson N StoneNelson N Stone More articles by this author , Ryan TerleckiRyan Terlecki More articles by this author , J Curtis NickelJ Curtis Nickel More articles by this author , and Priya N WeraheraPriya N Werahera More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002090.09AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: We investigated the microbiome of post-digital rectal exam (DRE) urine specimens from prostate cancer (PCa) patients and a screening population with a PSA <1.5 ng/mL using PCR (polymerase chain reaction) and next generation sequencing (NGS) techniques. METHODS: 100 post-DRE urine specimens from patients with PCa were compared to 100 specimens from annual Prostate Cancer Awareness Week participants with PSA <1.5 ng/mL (low risk group). Microbial PCR and NGS was performed by MicroGenDX. RESULTS: Analysis of microbiome composition by NGS identified Cutibacterium acnes as significantly more abundant in PCa compared to low risk controls (P < 0. 05) (difference more pronounced in high grade PCa. Finegoldia magna was significantly more abundant in low risk controls (P < 0.05) compared to PCa. PCR differentially identified Klebsiella pneumoniae, Gardnerella vaginalis, Prevotella bivia, and Ureaplasma parvum, in PCa group. PCR detected Escherichia coli, Prevotella bivia, Mycoplasma hominis, Gardnerella vaginalis, and Ureaplasma parvum in the low risk control group. CONCLUSIONS: PCR and NGS analysis of post-DRE urinary microbiome showed detection and abundance differences in PCa compared to low risk controls. Cutibacterium acnes strain Type II identified in PCa in our study has been reported as the most prevalent bacterial species in PCa tissue samples. The presence of Finegoldia magna differentially identified in the group with a low risk of PCa is responsible for the production of equol, a soy metabolite associated with lowering risk of cancer. Our observations indicate the need for corroborative urine, tissue, and semen microbiome studies to identify a possible microbial contribution to prostate cancer risk. Source of Funding: Supported in parts by Schramm Foundation, Bingham Foundation, and Prostate Cancer Biorepository at the University of Colorado Anschutz Medical Campus © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e1007-e1007 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information E David Crawford More articles by this author Whitney N Stanton More articles by this author Rick Martin More articles by this author Caleb D Phillips More articles by this author Adrie van Bokhoven More articles by this author M Scott Lucia More articles by this author Paul Arangua More articles by this author Francisco G La Rosa More articles by this author Zachary Grasmick More articles by this author Gretchen Hoyer More articles by this author Nelson N Stone More articles by this author Ryan Terlecki More articles by this author J Curtis Nickel More articles by this author Priya N Werahera More articles by this author Expand All Advertisement Loading ...

Development of a Whole-urine, Multiplexed, Next-generation RNA-sequencing Assay for Early Detection of Aggressive Prostate Cancer

BackgroundDespite biomarker development advances, early detection of aggressive prostate cancer (PCa) remains challenging. We previously developed a clinical-grade urine test (Michigan Prostate Score [MiPS]) for individualized aggressive PCa risk prediction. MiPS combines serum prostate-specific antigen (PSA), the TMPRSS2:ERG (T2:ERG) gene fusion, and PCA3 lncRNA in whole urine after digital rectal examination (DRE). ObjectiveTo improve on MiPS with a novel next-generation sequencing (NGS) multibiomarker urine assay for early detection of aggressive PCa. Design, setting, and participantsPreclinical development and validation of a post-DRE urine RNA NGS assay (Urine Prostate Seq [UPSeq]) assessing 84 PCa transcriptomic biomarkers, including T2:ERG, PCA3, additional PCa fusions/isoforms, mRNAs, lncRNAs, and expressed mutations. Our UPSeq model was trained on 73 patients and validated on a held-out set of 36 patients representing the spectrum of disease (benign to grade group [GG] 5 PCa). Outcome measurements and statistical analysisThe area under the receiver operating characteristic curve (AUC) of UPSeq was compared with PSA, MiPS, and other existing models/biomarkers for predicting GG ≥3 PCa. Results and limitationsUPSeq demonstrated high analytical accuracy and concordance with MiPS, and was able to detect expressed germline HOXB13 and somatic SPOP mutations. In an extreme design cohort (n = 109; benign/GG 1 vs GG ≥3 PCa, stratified to exclude GG 2 cancer in order to capture signal difference between extreme ends of disease), UPSeq showed differential expression for T2:ERG.T1E4 (1.2 vs 78.8 median normalized reads, p < 0.00001) and PCA3 (1024 vs 2521, p = 0.02), additional T2:ERG splice isoforms, and other candidate biomarkers. Using machine learning, we developed a 15-transcript model on the training set (n = 73) that outperformed serum PSA and sequencing-derived MiPS in predicting GG ≥3 PCa in the held-out validation set (n = 36; AUC 0.82 vs 0.69 and 0.69, respectively). ConclusionsThese results support the potential utility of our novel urine-based RNA NGS assay to supplement PSA for improved early detection of aggressive PCa. Patient summaryWe have developed a new urine-based test for the detection of aggressive prostate cancer, which promises improvement upon current biomarker tests.

A novel method for detection of exfoliated prostate cancer cells in urine by RNA in situ hybridization.

In the current study, we explore the feasibility of detecting exfoliated prostate cancer cells in urine using an RNA in situ hybridization (RISH) assay. We hypothesized that robust and specific labeling of prostate cancer cells could be achieved in post-digital rectal examination (DRE) urine samples using RISH. We focused on method development, optimization, and analytical evaluation of RISH-based detection of prostate cancer in urine. We optimized a sample collection, processing, and target detection workflow for urine cytology specimens in conjunction with RNA target detection by RISH. We screened a panel of 11 prostate-specific RNA targets, and selected NKX3-1 and PRAC1 as markers for cells of prostate origin and PCA3 as a marker of prostate malignancy. Following analytical validation of a multiplexed NKX3-1/PRAC1/PCA3 assay, we evaluated whether prostate cancer cells can be detected in a pilot cohort of 19 post-DRE specimens obtained from men diagnosed with prostate cancer. Using cytology specimens prepared from spiked urine samples, we established the analytical validity of the RISH assay for detection and visualization of prostate cells in urine. Cells of prostate origin could be readily and specifically identified and separated into benign and malignant cell populations based on the multiplex test that consisted of markers specific for prostate cells (NKX3-1, PRAC1) and prostate cancer cells (PCA3). Upon evaluation of post-DRE urine from a pilot cohort of prostate cancer patients, we identified 11 samples in which prostate cells were present, 6 of which were also positive for prostate cancer cells. Multiplex RISH enables the direct visualization and molecular characterization of individual exfoliated prostate cells in urine. This proof-of-principle study provides evidence supporting the application of RISH as a potential noninvasive tool for prostate cancer detection.

Abstract B02: Genomic and transcriptomic profiling of urine in prostate cancer

Abstract Prostate cancer (CaP) affects one in 8 men in the US and Canada, but many of these men could be spared aggressive treatment, often associated with significant morbidity, if diagnostic tools could more accurately assess the risk that the cancer will metastasize. Low pathologic/Gleason grade (Gleason Grade 3+3=6, equivalent to WHO Grade Group 1) is the main defining feature of nonlethal low-risk CaP, and active surveillance (AS) is the standard of care for such men. However, current diagnostic methods cannot accurately separate low and higher risk CaP based on core biopsies, presenting a major dilemma as repeated biopsies during AS carry significant risks. At present no diagnostic tools, either imaging-based or biomarker-based, can safely supplant repeated invasive biopsies. Genomic profiling of liquid biospecimens (“liquid biopsies”) is quickly becoming translated as part of contemporary clinical trials and is being translated for use in the diagnostic setting. Less invasive than traditional solid tissue biopsy approaches, liquid biopsy specimens have the potential to be used not only for early detection but also to monitor therapeutic response and progression. Leveraging the advantage of the anatomic relationship between the prostate and urinary tract, we obtained 11 post-digital rectal exam (DRE) urine samples from patients who presented at the Kingston Health Sciences Centre (Kingston, Ontario) to be evaluated for prostate cancer. Through a pan-Canadian research consortium called PRONTO, we developed a tissue-based classifier (PRONTO-T) differentiating between low- and high-risk cancer with an independently validated AUC of 0.85. This proof-of-concept study tests post-digital rectal examination (DRE) urine samples for genomic features associated with aggressive CaP, including features included in the PRONTO-T risk classifier. DNA and RNA were extracted from 11 post-DRE urine samples. Profiling with a customized NGS-based targeted-methylation panel by Thermo Fisher demonstrated robust signals from urine-derived DNA. Similarly, urine samples profiled with the pan-cancer Oncomine Comprehensive Assay (v3) detected potentially actionable mutations in MET and PIK3CA. RNA obtained from post-DRE urines met quality control standards for transcriptional profiling using the NanoString nCounter System and revealed patterns of gene expression changes consistent with prostate cancer biology. These encouraging preliminary results demonstrate that the profiling of urine can provide sufficient diagnostic and potentially prognostic information to significantly impact the clinical management of prostate cancer. Citation Format: Jane Bayani, Palak G. Patel, Dan Dion, Vanessa Bratti, Ania Ahmadian-Namin, Tamara Jamaspishvili, Jason Izard, D. Robert Siemens, David M. Berman, John M. S. Bartlett. Genomic and transcriptomic profiling of urine in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B02.

Urinary prostate-specific antigen and microseminoprotein-beta levels in men with and without prostate cancer: A prospective cohort study.

Introduction:The role of urinary proteomics in the diagnosis of prostate cancer (PCa) is undefined. Levels of urinary biomarkers such as prostate-specific antigen (PSA) and microseminoprotein-beta (MSMB) may differ between men with and without PCa. We tested this hypothesis using urine samples before and after digital rectal examination (DRE) in men with an indication for prostate biopsy.Materials and Methods:In an institutional ethics committee approved prospective cohort study, men with elevated PSA or a nodule on DRE underwent a pre- and post-DRE urine sample examination for urinary PSA and MSMB levels. Levels were compared between men who had PCa diagnosed on biopsy (Group A) and those with a negative biopsy (Group B).Results:Seventy-seven patients were recruited of whom 32 had PCa (Group A) and 45 had no cancer (Group B) on biopsy. The median (interquartile range) serum PSA was 49.6 (0.2–254) ng/ml. The median urine PSA (29.5 vs. 26.4 mg/dl) and MSMB (1.7 vs. 2.4 mg/dl) were similar in both groups at baseline. However, post-DRE, both these metabolites rose in Group B but not in Group A, resulting in significantly higher post-to-pre values in Group B versus Group A. The post-DRE urine PSA/MSMB ratio was also significantly different between the groups.Conclusions:Urinary PSA and MSMB rose significantly after DRE only in men without PCa. Post-DRE urine PSA, MSMB, and PSA/MSMB ratio can differentiate PCa from benign pathology in men with an indication for prostate biopsy.

Abstract 3008: PSMA glycosylation and aggressive prostate cancer progression

Abstract Introduction: Prostate cancer has a heterogeneous spectrum of clinical outcomes with phenotypes ranging from indolent asymptomatic cases to very aggressive metastatic and lethal forms. Our long term objective is to identify molecules and characterize mechanisms for PCa progression to aggressive lethal disease. PSMA, a membrane bound glycoprotein is significantly over-expressed in more aggressive and metastatic prostate carcinomas with a negative correlation with patient prognosis. The role of its glycans in disease progression and in the development of biomarkers for the disease and potential therapeutics are currently unknown. Aberrant glycosylation of glycoproteins has been observed in many malignancies, and these changes influence disease progression. Our rationale is that differential glycosylation of PSMA correlates with PCa aggressive disease progression. In this study, we characterize N-linked glycosylation profiles in disease stratified PCa cells and prostate proximal fluids show differential expression of specific PSMA conjugated glycan structures that correlates with aggressive phenotypes. Methods/Results: PSMA was isolation from PCa cell lysates and post-DRE urines by column based affinity chromatography followed by SDS-PAGE and Western Blot and Coomassie staining. The fractionated PSMA protein band was excised and processed for proteomic and glycomic analysis. Isolated PSMA was trypsin digested and treated with PNGaseF and other glycosidases to cleave of N-linked glycans from the peptides. Isolated glycans were subjected to permethylation before MALDI-TOF-MS and high resolution, high mass accuracy and high sensitivity LC/MS/MS on an Orbitrap Fusion Lumos Tribrid MS instrument with multiple fragmentation capabilities (CID, HCD, ETD and EThcD) for glycan identification and quantitation. Validation of differential glycan expression was further performed by targeted glycan based MS and lectin based biochemical technologies. Conclusions: Our preliminary studies show that there is differential expression of PSMA N-linked glycans with disease severity in prostate cancer cells and post DRE urine samples. Further analyses are currently underway using an extensive cohort of post DRE urine samples and additional studies are focusing on uncovering the mechanisms that are modulated by PSMA glycosylation that are responsible for PCa disease progression. These could assist in the development of novel diagnostic and therapeutic strategies for the disease. Citation Format: Tanya C. Burch, Stephen S. Mackay, Ian O. Oduor, Joseph J. Otto, Raymond S. Lance, Dean A. Troyer, Oliver John Semmes, Julius O. Nyalwidhe. PSMA glycosylation and aggressive prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3008.

Abstract 914: Development of a whole-urine, next-generation sequencing-based assay for early detection of aggressive prostate cancer

Abstract Despite advances in biomarker development, early detection of aggressive prostate cancer (PCa) remains challenging. Existing biomarkers show modest improvement over models based on serum prostate specific antigen (PSA). We have previously developed a clinical-grade laboratory-developed test, named MiProstate Score (MiPS), for individualized risk prediction of aggressive prostate cancer. It uses transcription-mediated amplification to quantify the gene-fusion TMPRSS2:ERG (T2:ERG) (T1E4 splice isoform) and the lncRNA PCA3 from whole-urine obtained after a digital rectal exam (DRE), combined with serum PSA. To improve MiPS, we describe here the pre-clinical development and validation of a targeted next generation sequencing assay (NGS-MiPS) using post-DRE urine RNA to asses ~90 PCa transcriptomic biomarkers. These include those in MiPS as well as many isoforms of common PCa gene fusions, mRNA, and lncRNA candidate biomarkers nominated by our large-scale PCa tissue RNAseq and other sources. We have obtained a 98% informative sample rate from 2.5 mL of urine and high technical reproducibility (Pearson r=0.99). Risk scores for having PCa [or high-grade PCa (Gleason Score &amp;gt;6)] on biopsy, as determined by clinical MiPS vs. the clinical MiPS model using NGS data, were highly concordant, Pearson’s r=0.74 (and r=0.81). Urine from patients with benign or Gleason 6 vs. Gleason ≥ 4+3=7 cancer on biopsy (extreme design) showed expected differences in the levels of T2:ERG T1E4 (p=0.00003) and PCA3 (p=0.07), with additional T2:ERG splice isoforms and other biomarkers also being significantly different between low vs. high grade cancer. Feature selection and logistic regression trained in an extreme design cohort (n=73) yielded a 29-transcript model that outperformed MiPS and serum PSA in two validation cohorts: 1. A held-out set from the extreme design cohort n=36, AUC 0.81 vs. 0.76 and 0.63 respectively; 2. A separate active surveillance cohort n=45, AUCs 0.66 vs. 0.56 and 0.53 respectively. These results support the potential utility of our urine based targeted NGS assay to supplement serum PSA for the early detection of aggressive prostate cancer. Citation Format: Andi K. Cani, Kevin Hu, Javed Siddiqui, Sumin Han, Daniel H. Hovelson, Chia-Jen Liu, Simpa S. Salami, Ganesh S. Palapattu, Todd M. Morgan, John T. Wei, Arul M. Chinnaiyan, Scott A. Tomlins. Development of a whole-urine, next-generation sequencing-based assay for early detection of aggressive prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 914.

SelectMDx versus Prostate Health Index in the identification of high-grade prostate cancer.

30 Background: A major clinical challenge is to identify patients at increased risk of high-grade prostate cancer (PCa) (Gleason scores ≥ 7) before biopsy. Thus, we evaluated the clinical utility of SelectMDx and Prostate Health Index (phi) tests for diagnosis of high-grade PCa as compared with transperineal mapping biopsy (TMB) results, where an average of 80 prostate needle biopsies are taken every 5 mm for a diagnostic accuracy of 98%. Methods: 70 patients were selected who had TMB. They were evaluated with both SelectMDx and phi tests from before or after TMB; all had serum and post-digital rectal examination urine samples collected prior to treatment, stored in our biorepository. phi was evaluated using proPSA, free PSA and total PSA in serum. SelectMDx test measured mRNA levels of the HOXC6 and DLX1 in post-DRE urine. Published data shows that phi &lt;27 and SelectMDx score = 0% correlate with patients free of HG PCa. Test results were compared against TMB histopathology data. Multivariate logistic regression (MLS) analyses and receiver operating characteristic (ROC) curves were used to determine diagnostic accuracy. DeLong test was used to determine statistical significance of ROC curves. All analyses were done for diagnosis of any PCa and high-grade PCa. Results: TMB histopathology showed 17/70 patients with no PCa and 22/53 with high-grade PCa. The sensitivity, specificity, positive (PPV) and negative (NPV) predictive values of each test are shown in the table below. Pairwise ROC comparison showed no statistically significant difference in the area under the curve of diagnosing PCa (0.75 vs 0.65) and high-grade PCa (0.71 vs 0.81) by phi and SelectMDx tests respectively. MLS analyses showed phi was significantly better than SelectMDx for diagnosing PCa (β = 0.054; p=0.005) and SelectMDx was significantly better than phi for diagnosing high-grade PCa (β = 8.45; p=0.0002). Conclusions: With high sensitivity and NPV, SelectMDx test is more useful than phi for screening patients at risk of high-grade PCa prior to biopsy. [Table: see text]

Validation of a two-gene mRNA urine test for detection of high-grade prostate cancer in German men.

96 Background: There is an unmet need for non-invasive methods that can accurately identify patients at increased risk for clinically significant prostate cancer (PCa). SelectMDx is a urine-based molecular test that has been clinically validated for the detection of high-grade PCa. We evaluated SelectMDx clinical performance in a cohort of German men undergoing initial prostate biopsy. Methods: The study population consisted of 443 men sequentially enrolled men who underwent initial prostate biopsy between July 2009 and December 2014 due to suspected PCa. Post-DRE urine was collected from all subjects prior to biopsy, and samples stored at -70C. Urinary HOXC6 and DLX-1 mRNAs were quantified by PCR in May 2018, and RNA results combined with clinical risk factors to determine the likelihood that biopsy would identify ISUP grade group (GG) ≥ 2 (Gleason Score ≥ 7) PCa. We assessed SelectMDx performance for detection of GG ≥ 2 PCa, compared to the PCPT Risk Calculator Version 2.0 (PCPTRC, http://myprostatecancerrisk.com , accessed Oct 7, 2018). Results: For the 443 subjects enrolled, average age was 66 years (median 66, interquartile range 61 to 71), and average serum PSA level 8.8 ng/mL (6.4, 4.8 to 9.7). Cancer was detected in 243/443 (55%) men biopsied (43% GG1, 36% GG2, 9% GG3 and 12% GG4-5). The prevalence of GG2-5 PCa in this population was 31.4% (139/443). For detection of GG2 or higher PCa versus GG1 or no PCa at biopsy, SelectMDx AUC was 0.82 (95% C.I. 0.78-0.86) and the PCPTRC yielded AUC 0.75 (0.70-0.80), P &lt; 0.001. SelectMDx sensitivity was 94% (89-98%), specificity 46% (40-52%), positive predictive value 45% (42-47%) and negative predictive value (NPV) 95% (90-97%). If the initial biopsy had been performed based on SelectMDx results alone, 46% of potentially unnecessary biopsies and 34% of all biopsies would have been avoided, while 5.8% of men with biopsy-detectable high-grade PCa (seven GG2, one GG3) may have had their diagnosis delayed. Conclusions: In this first validation study of SelectMDx in German men, the test’s clinical performance was comparable to the published EU validation study, showing a high NPV for detection of GG2 or higher PCa. These results provide further evidence for the clinical validity of SelectMDx.

Abstract 651: Development of an RNA in situ hybridization assay for the detection of prostate cancer cells in urine

Abstract Urine-based detection of prostate cancer represents a promising resource for non-invasive diagnostics. The value of urine as a biospecimen in the management of prostate cancer has already been established, but the utility of urine has yet to be fully explored. The objective of our study was to develop a RNA in situ hybridization (RISH) method for detecting prostate cells in urine and to evaluate the feasibility of using this assay for cancer detection in clinical specimens. We hypothesized that robust and specific labeling of prostate cancer cells could be achieved using this approach, providing benefit over existing urine detection methods, namely the FDA-approved PCA3 test, by allowing for the direct visualization and molecular characterization of individual cells. We optimized the following collection, processing, and preparation procedure for detection of prostate cells in fresh urine specimens collected from patients following digital rectal exam (DRE): the entire urine volume was centrifuged to collect the sediment, which was then formalin fixed, cytocentrifuged onto two slides, and slides were stored at -20°C until proceeding to staining and cytological analysis. Cellularity and sample adequacy was assessed by Papanicolaou staining. For identification of prostate cells in post-DRE urine via RISH, single-probe chromogenic labeling for NKX3.1 or HOXB13 was performed. Pretreatment conditions and staining parameters for RISH were optimized using urine samples spiked with 22Rv1 cells prior to staining patient specimens. We were able to readily identify cells of prostate origin in both spiked urines and patient samples. To further discriminate normal prostate cells from cancer cells, fluorescent multiplex RISH was pursued. We screened a candidate panel of prostate-specific RNA targets that included NKX3.1, HOXB13, KLK3, PRAC1, PRAC2, MALAT1, HOXC6, AMACR, and PCA3. Based on detection specificity, NKX3.1 and PRAC1 were selected as markers to identify cells of prostate origin and PCA3 as a marker of malignancy. Multiplex RISH with these three markers was carried out on 19 patient specimens. Prostate cells, classified as those positive for PRAC1 and/or NKX3.1, were identified in 11 samples, 6 of which were also positive for PCA3. These observations demonstrate that multiplex RISH can be used for the specific detection and visualization of prostate cancer cells in post-DRE urine samples. Although further refinement and validation are necessary, this study provides the first evidence supporting the application of a urinary RISH assay as a potential non-invasive method for prostate cancer detection. Ongoing and future studies will include characterizing performance of additional biomarker panels and validating clinical utility of this technique in a diagnostic setting. Citation Format: Jillian N. Eskra, Daniel Rabizadeh, Christian P. Pavlovich, Leslie Mangold, Elizabeth Fabian, William B. Isaacs, Jun Luo. Development of an RNA in situ hybridization assay for the detection of prostate cancer cells in urine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 651.

MP35-10 DETECTION OF PROSTATE CANCER-ASSOCIATED TRANSCRIPTS IN URINARY EXTRACELLULAR VESICLES

You have accessJournal of UrologyProstate Cancer: Markers I1 Apr 2018MP35-10 DETECTION OF PROSTATE CANCER-ASSOCIATED TRANSCRIPTS IN URINARY EXTRACELLULAR VESICLES Kathryn Pellegrini, Dattatraya Patil, Kristen Douglas, Ella Anastasiades, Kristin Larsen, Frances Kim, Almira Catic, Mersiha Torlak, Eugene Huang, Carlos Moreno, and Martin Sanda Kathryn PellegriniKathryn Pellegrini More articles by this author , Dattatraya PatilDattatraya Patil More articles by this author , Kristen DouglasKristen Douglas More articles by this author , Ella AnastasiadesElla Anastasiades More articles by this author , Kristin LarsenKristin Larsen More articles by this author , Frances KimFrances Kim More articles by this author , Almira CaticAlmira Catic More articles by this author , Mersiha TorlakMersiha Torlak More articles by this author , Eugene HuangEugene Huang More articles by this author , Carlos MorenoCarlos Moreno More articles by this author , and Martin SandaMartin Sanda More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.1123AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The relatively high rate of negative prostate biopsy demonstrates the need for additional non-invasive biomarkers to improve the diagnosis and classification of prostate cancers prior to biopsy. We have previously established that post-DRE extracellular vesicles (EVs) are enriched for prostate-specific RNAs, making them an ideal target for prostate cancer biomarker discovery. In our current study, we aimed to identify genes with increased expression in the post-DRE urine EVs of patients with aggressive prostate cancer. METHODS We developed and refined a method for the targeted analysis of over 260 different RNA transcripts in post-DRE urine EVs. Genes were selected for inclusion in our assay based on their prior inclusion on tissue biomarker panels of aggressive prostate cancer, or from analysis of TCGA datasets to identify genes with both significant over-expression in aggressive prostate cancer and minimal expression in normal genitourinary tissues. RESULTS We prepared RNA from the post-DRE urine EVs of 29 men and used our targeted RNA sequencing assay to measure expression of over 260 different transcripts. We detected 92 transcripts (34%) in all 29 specimens, with only 19 transcripts (7%) being undetectable in any specimen. Comparison of patients with no evidence of disease (n = 15) and patients with Gleason Score ≥7 prostate cancer (n = 14) identified 25 genes with significantly higher expression in patients with aggressive prostate cancer, including known prostate cancer-associated genes such as PCA3, AMACR, and SPINK1, while KLK3 showed no significant difference in expression (Figure 1). CONCLUSIONS Post-DRE urine EVs contain transcripts that are indicative of prostate cancer status and represent a novel source for biomarker discovery and development. Further investigation of patient specimens with this method offers the potential to determine combinations of transcripts that could be used to non-invasively detect and monitor prostate cancer status. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e450-e451 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Kathryn Pellegrini More articles by this author Dattatraya Patil More articles by this author Kristen Douglas More articles by this author Ella Anastasiades More articles by this author Kristin Larsen More articles by this author Frances Kim More articles by this author Almira Catic More articles by this author Mersiha Torlak More articles by this author Eugene Huang More articles by this author Carlos Moreno More articles by this author Martin Sanda More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Evaluation of prostate cancer risk in men with PSA &lt; 1.5.

64 Background: Prostate Specific Antigen (PSA) screening remains controversial primarily because of over detection and treatment. There is an unmet clinical need to identify patients at increased risk for high-grade (HG – Gleason Score ≥7) prostate cancer (PCa) since PSA has low sensitivity. Combining PSA with well-validated prostate cancer biomarkers (PCM) can improve risk assessment. We investigated the performance of three PCMs (phi – prostate health index, 4KScore, and SelectMDx) on patients with PSA levels &lt; 1.5 ng/mL that represent a “safe zone” where risk of any PCa is rare Methods: 652 men were screened for PCa during the annual Prostate Cancer Awareness Week at the University of Colorado Hospital. This study was supported by Prostate Condition Education Council and the Schramm Foundation. phi is evaluated using p2PSA, total PSA, and free PSA in serum. Phi &lt; 52.7 suggests absence of HG PCa. 4KScore incorporates four kallikrein protein biomarkers: total PSA, free PSA, intact PSA, human kallikrein protein, and clinical information. A 4KScore &lt; 20% suggests absence of HG PCa. The SelectMDx post-DRE urine test measures mRNA levels of the homeobox C6 and distal-less homeobox 1 biomarkers. SelectMDx score of 0% indicates absence of HG PCa. Results: No patients with a PSA &lt; 1.5 had SelectMDx &gt; 0% and/or phi &gt; 52.7. One patient had a 4KScore of 27%, indicating a risk for HG PCa. For patients with PSA between 1.5-3.99, 2.9% (4/135), 7.4% (4/54), and 2.3% (2/85) had positive phi, 4KScore, and SelectMDx results, respectively. Conclusions: Men with PSA &lt;1.5 ng/mL are at very low risk for HG PCa. Men with PSA between 1.5-3.99 with positive PCM results may be referred for further evaluation. [Table: see text]