783 DEVELOPMENT OF A MIRNA ASSAY FOR PROSTATE CANCER DETECTION

781

Comparison of miRNA profiles in high and intermediate risk prostate carcinoma – role of mir-16 in development and progression of prostate carcinoma



782

The antiproliferative B cell translocation gene 2 ( BTG2) is under post-transcriptional control of miR-21 in prostate cancer: Implications for tumorigenesis

Kneitz B.1, Kneitz S.2, Stenger N.1, Ströbel P.3, Riedmiller H.1, Spahn M.1

Bonci D.1, Coppola V.1, Musumeci M.1, Addario A.1, D’Urso L.2, Collura D.2, De Maria R.1, Muto G.3

University Medical School, University of Würzburg, Dept. of Urology and Pediatric Urology, Würzburg, Germany, 2University of Würzburg, Micro-Array Core Unite, Izkf (Interdisciplinary Center for Clinical Research), Würzburg, Germany, 3University Medical Center Mannheim, University of Heidelberg, Institute of Pathology, Würzburg, Germany

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Introduction & Objectives: To identify prostate carcinoma specific and risk / progression associated miRNAs we analysed the expression of microRNAs in intermediate risk (IRPCa) and high risk prostate carcinoma (HR-PCa). Material & Methods: Expression of 665 miRNAs was initially analysed in 8 IR-PCa, 6 HR-PCa and 6 benign prostate hyperplasias (BPH) using microarrays. MiRNA expression were confirmed by qRT-PCR. Expression of mir-16 was analysed in a large study group (n=94) including a statistic relevant number of high risk patients (Gleason >9, clinical recurrence <5 years). BCL-2 protein expression was analysed by immunhistochemistry in mir-16 dysregulated carcinomas and DU-145 cells were transiently transfected with precursor miRNAs to analyse proliferation and bcl-2 expression. Results: Widespread downregulation of miRNAs (52 down-, 1 upregulated in PCA vs. BPH) was detected in prostate carcinoma by micro array experiments characterizing a list of prostate carcinoma specific miRNAs. Hierarchical clustering identified PCA specific signatures differentiating PCA from BPH tissue and IR-PCa from HR-PCa. Mir-16 was frequently detected to be downregulated in IR-PCa cases. Mir-16 downregulation in carcinomas was also associated to Gleason score and overexpression of Bcl-2 in primary carcinoma. Using cox regression analysis we performed statistical analysis of mir-16 expression as marker for prostate carcinoma development. We further could show that transient overexpression of miR-16 in prostate carcinoma cell lines resulted in a proliferation inhibition combined with a significant reduction of Bcl-2 transcription. Conclusions: HR-PCA can be separated by a specific miRNA signature indicating a role of miRNAs in progression of PCA. Downregulation of mir-16 occurs in a subpopulation of IR-PCa indicating a potential role of mir-16 as prognostic marker. Our results further indicate that both the detection of miRNA signatures analyzed by microarrays and the RT-PCR based identification of aberrant expression of specific miRNAs e.g. mir-16 might be useful tools for diagnosis and prognosis in prostate cancer.



783

Istituto Superiore Sanità, Dept. of Haematology, Oncology and Molecular Medicine, Rome, Italy, 2 Ospedale San Giovanni Bosco, Dept. of Urology, Turin, Italy, 3St. Giovanni Bosco Hospital, Dept. of Urology, Turin, Italy Introduction & Objectives: The understanding of the molecular mechanisms involved in the pathogenesis of prostate cancer is necessary to develop effective therapeutic strategies and new techniques for diagnosis. One of the early events found in the prostate carcinogenesis is the functional inactivation of BTG2, an antiproliferative protein that acts both as effector of p53 by preventing the growth of cells that have suffered genetic damage as well as negative modulator of cell cycle through ‘Action on pRB. The loss of BTG2 has been described as a post-transcriptional event already present at level of HGPIN. We have assumed that such deregulation could be brought up-regulation of one or more microRNAs (miRNAs), small non-coding RNA involved in the modulation of gene expression by inhibiting the translation of specific target genes. Of growing importance is in fact revealing the role played by microRNA in tumorigenic process, through posttranscriptional deregulation of oncogenes or tumor suppressor. Material & Methods: The bioinformatics research looking for potential miR able to adjust BTG2 was done. On this basis our attention focused on microRNA 21. Then we tested the over expression of miR 21 in a large number of tumor samples from patients undergoing radical prostatectomy for prostate cancer. Levels of miR were compared to those of healthy prostate tissue in control harm. Were then evaluated the effects of miR 21 overexpression in a model of normal epithelial prostate cells, RWPE-1. To assess the effects of target-specific suppression modulated by miR21, lentiviral carrier BTG2 TW 3’UTR was used, by cloning the BTG2 region downstreaming the EGFP reporter gene 3’UTR. Prostate cells have been infected with the RWPE-2, yielding miR 21 levels twice the RWPE-1. Results: Here we report that 70% of the 20 tumors analyzed showed miR-21 up-regulation. miR21 is deregulated also in the early stages of prostate cancer including low Gleason grade cases. We found that BTG2 is a new target for miR-21 and its expression is inversely correlated with the miR-level in prostate cancer cells. miR-21 overexpression, in a prostate normal cell line, accelerates cell growth and promotes docetaxel resistance. miR-21 knock-down rescues the expression of BTG-2, blocking in vitro proliferation and in vivo tumor formation of transformed prostate cells in immunodeficient NOD/SCID mice. Conclusions: The BTG2 expression profile candidates as a tumor suppressor in prostate cells particularly in basal compartment. miR-21 controls at post-transcriptional level BTG2 also in the early tumor lesions, indicating it has a role in basal compartment expansion and tumor formation. These findings might have significant therapeutic implications and may be exploited in the future for novel treatments of prostate cancer, particularly for patients with low risk tumors



784

Development of a miRNA assay for prostate cancer detection

TMPRSS2-ERG gene fusion defines a metastatic phenotype of prostate cancer

Larne O.S.1, Edsjö A.2, Bjartell A.3, Ceder A.Y.1

Perner S.1, Svensson M.2, Hossain R.2, Day J.3, Groskopf J.3, Hofer M.D.4, Kuefer R.5, Rickman D.S.2, Rubin M.A.2

Lund University, Clinical Chemistry, Dept. of Laboratory Medicine, Malmö, Sweden, Lund University, Clinical Pathology and Cytology, Malmö, Sweden, 3Lund University, Dept. of Clinical Medicine, Malmö, Sweden 1

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Introduction & Objectives: Prostate cancer is the most frequent malignancy in Swedish men. Today, elevated level of prostate specific antigen, PSA, in serum, is used as a biomarker for prostate cancer. However, elevated PSA level cannot discriminate between aggressive forms of prostate cancer and benign diseases. Subsequently, high rate of overdetection arise and most cases will never experience cancer symptoms during their lifetime. MicroRNAs, miRNAs, are small non-coding RNAs which control the translation of mRNA. In prostate cancer altered levels of miRNA give raise to a changed protein expression profile which is believed to contribute to the progression of the cancer. We want to investigate wherever the different miRNA levels in prostate cancer tissues can be detected and hence used as a clinical marker of the disease. The aim of this study was to set up a robust assay to measure miRNA levels. Material & Methods: RNA from 14 prostate samples embedded in paraffin were extracted; both originating from patients with prostate cancer or benign prostate hyperplasia. Two RNA extraction kits were compared: mirVanaTM miRNA Isolation Kit (Ambion) and RecoverAll TM Total Nucleic Acid Isolation (Ambion). To measure the levels of miRNA we used a two step miRNA specific Taqman based qRT-PCR (Applied Biosystems). To find good controls we compared 10 endogenous small RNA controls; RNU6b, RNU19, RNU24, RNU38b, RNU44, RNU48, RNU49, RNU66, U47and Z30. Analyses in geNorm reveal the best endogenous controls. Results: Our results show that a slightly modified version of mirVanaTM miRNA Isolation Kit (Ambion) is better suited for extraction of small RNAs. RNU48, RNU66 and U47 have the least variation in gene expression in benign and cancerous prostate tissue according to our qRT-PCR results. We investigated how sensitive the assay was and found that it could detect 100 miRNA copies or 2.3x10-15 pg miRNA. We also investigated the stability of the assay and found that the biological replicates deviated more than technical replicates. Conclusions: Our analyses indicate that RNU48, RNU66 and U47 are best suited as endogen controls for miRNA detection in prostatic disease. We also found that the stability and sensitivity of the assay are very high.

Eur Urol Suppl 2009;8(4):316

Weill Cornell Medical Center, Dept. of Pathology and Laboratory Medicine, Tübingen, Germany, 2Weill Cornell Medical Center, Dept. of Pathology and Laboratory Medicine, New York, United States of America, 3Gen-Probe, Inc., San Diego, United States of America, 4 Brigham and Women’s Hospital, Dept. of Pathology, Boston, United States of America, 5 University Hospital of Ulm, Dept. of Urology, Ulm, Germany

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Introduction & Objectives: Histologic and molecular heterogeneity is a hallmark of prostate cancer (PCa), suggesting that distinct cancer foci may arise independently. Previously work demonstrated that clonal intrafocal homogeneity and interfocal heterogeneity of TMPRSS2-ERG fusion status exists in multifocal PCa. This poses a significant limitation to the development of prognostic and predictive biomarkers as diagnostic needle biopsies may inaccurately represent the driving tumor(s). PCa progression is closely tied to the development of metastatic disease and previous work suggests that TMPRSS2-ERG fused PCa have a more aggressive natural history. The aim of this study was to interrogate multifocal PCa to determine if the TMPRSS2-ERG tumor foci were more apt to metastasize than fusion negative foci . Material & Methods: We studied samples from 33 PCa patients who underwent radical prostatectomy and lymphadenectomy and that had at least two distinct cancer foci in the prostate and at least one lymph node (LN) metastasis. Each focus (PCa and LN) was assigned a primary and secondary Gleason grade and assessed for fusion status by fluorescence in-situ hybridization (FISH). Fusion transcript was assessed by quantitative RT-PCR in a subset of cases. Results: 15 out of the 26 cases (58%) exhibited interfocal homogeneity with regard to fusion status (9 cases being fusion negative in all foci and 6 cases being fusion positive in all foci). Two out of the 26 cases revealed interfocal heterogeneity with regard to fusion mechanism. 9 out of the 26 cases (42%) revealed interfocal heterogeneity with regard to fusion status. In all PCa cases that were fusion positive regardless of diameter we found that the corresponding LN metastasis were also fusion positive. The fusion status in the LN metastasis did not necessarily correspond to the fusion status of the largest PCa focus or to the fusion status of the focus with the highest Gleason score. Conclusions: In this unique cohort, we found that tumor size and Gleason score was not the best predictor of pelvic LN metastasis. In this cohort, TMPRSS2-ERG fusion status was the strongest determinant of metastatic dissemination. These findings may have important clinical implications for the screening and diagnosis of PCa.