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UTILISING PROTEOMIC PLATFORMS FOR THE IDENTIFICATION OF NOVEL SERUM MARKERS OF PROSTATE CANCER
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QUANTITATIVE MULTI-GENE EXPRESSION PROFILING OF PRIMARY PROSTATE CANCER Unversucht S.1 , Schmidt U.1 , Meye A.1 , F¨ussel S.1 , Koch R.2 , Baretton G.B.3 , Froehner M.1 , Wirth M.P.1 1 Technical University of Dresden, Department of Urology, Dresden, Germany; 2 Technical University of Dresden, Institute of Medical Informatics and Biometry, Dresden, Germany; 3 Technical University of Dresden, Institute of Pathology, Dresden, Germany
HIGH FREQUENCY OF THE TMPRSS2/ERG FUSION GENE IN PROSTATE CANCER Soller W.1 , Johansson Soller M.2 , Isaksson M.2 , Elfving P.1 , Abrahamsson P.A.1 , Lundgren R.3 , Panagopoulos I.2 1 Malm¨ o University Hospital, Department of Urology, Malm¨o, Sweden; 2 Lund University Hospital, Department of Clinical Genetics, Lund, Sweden; 3 Helsingborg Hospital, Department of Urology, Helsingborg, Sweden
INTRODUCTION & OBJECTIVES: This study describes the evaluation of the expression patterns of prostate-relatedtranscripts in 106 matched prostate tissues from prostatectomies as predictors for prostate cancer (PCa). MATERIAL & METHODS: Quantitative PCR (QPCR) assays with sitespecific hybridization probes were established for 4 housekeeping genes (GAPDH, HPRT, PBGD, TBP) and 9 prostate-relatedgenes (AibZIP, DGPCR, EZH2, PCA3, PDEF, prostein, PSA, PSCA, TRPM8). RESULTS: The relative mRNA expression levels of AibZIP, D-GPCR, EZH2, PCA3, PDEF, PSA, TRPM8 (all < 0.001) and prostein (p = 0.019) normalized to the TBP reference gene were significantly higher in malignant compared to non-malignant prostate tissues. Employing receiver-operating characteristic (ROC) analyses, PCA3 was the best single tumor marker with the highest area-under-the-curve (AUC = 0.85). A multivariate logit model for the predictability of the tumor was developed, which employed the relative expression levels of EZH2, PCA3, prostein and TRPM8 and yielded an AUC of 0.90. CONCLUSIONS: PCA3 is a powerful predictor of primary PCa but the inclusion of EZH2, prostein and TRPM8 adds even more to the diagnostic power. The finding of a significantly higher mRNA expression of 3 different genes (prostein, PSA, TRPM8) in organ-confined tumors compared to non-organ-confined tumors could be of clinical importance and should be reevaluated in prospective studies using specimens from diagnostic biopsies.
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UTILISING PROTEOMIC PLATFORMS FOR THE IDENTIFICATION OF NOVEL SERUM MARKERS OF PROSTATE CANCER
Byrne J.1 , Downes M.1 , O’Donoghue N.2 , Dunn M.J.2 , Fitzpatrick J.M.3 , Watson R.W.G.3 , Pennington S.R.2 1 Conway Institute of Biomolecular and Biomedical Research, U.C.D. and Mater Misericordiae University, Proteome Research Centre and U.C.D. School of Medicine and Medical Science, Dublin, Ireland; 2 Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Proteome Research Centre, Dublin, Ireland; 3 Mater Misericordiae University Hospital and Conway Institute of Biomolecular and Biomedical Research, UCD School of Medicine and Medical Science, Dublin, Ireland INTRODUCTION & OBJECTIVES: Current screening methods for prostate cancer rely heavily upon screening for prostate specific antigen (PSA) within peripheral blood along with digital rectal exam and biopsy. However PSA as a marker for prostate cancer lacks specificity and additional biomarkers are needed to improve the accuracy of diagnosis. In order to attempt to isolate new biomarkers, a proteomics-based approach was utilised to analyse protein expression within the serum of patients undergoing radical prostatectomy. Human serum represents a valuable potential source of biomarkers for disease. However, the range of protein concentrations within serum spans greater than 10 orders of magnitude with a small number of proteins present in high abundance, most notably albumin and immunoglobulins. In order to overcome the technical limitations associated with this fact, the serum samples were subjected to affinity depletion using the Multiple Affinity Removal System (Agilent Technologies), a system that removes the 6 most abundant proteins found in human serum – albumin, IgG, antitrypsin, IgA, transferrin and haptoglobin. Removal of these highly abundant proteins allows for the enrichment of lower abundance proteins. MATERIAL & METHODS: Twelve consented radical prostatectomy patient serum samples were divided into 2 groups based on their Gleason score (Gleason 5 and Gleason 7) and compared using Differential In-Gel Electrophoresis (DIGE), a proteomic platform which allows for highly specific differential protein expression analysis. These samples underwent depletion, were fluorescently labelled and separated by 2-D electrophoresis. The fluorescent 2-D gel images were subjected to protein expression analysis using two independent software packages: DeCyder 6.5 (GE Healthcare) and Progenesis PG240 (Nonlinear Dynamics). RESULTS: This analysis identified 63 proteins which showed differential protein expression between the two groups (p < 0.05), 13 of which were identified by both programmes. These proteins are currently undergoing identification using mass spectrometry. CONCLUSIONS: These 63 candidates, which can statistically differentiate between Gleason 5 and Gleason 7 depleted serum, could potentially hold a number of clinically relevant biomarkers which could distinguish between different grades of cancer, thus isolating patients with a higher risk of more aggressive cancer allowing for informed patient management.
INTRODUCTION & OBJECTIVES: Fusion genes are involved in carcinogenesis and have often been identified through analysis of recurrent chromosomal rearrangements. In two recent studies Tomlins and co-workers (Science 2005, Cancer Res 2006) showed that a large subset of prostate cancer harbours the fusion genes TMPRSS2/ERG, TMPRSS2/ETV1 and TMPRSS2/ETV4, generated through cryptic inter- and intrachromosomal rearrangements. A gene fusion of the 5’ untranslated region of TMPRSS2 to ERG or ETV1 was identified in 23 (79%) of 29 prostate cancer samples. MATERIAL & METHODS: In the present study total RNA was extracted from 40 historical prostate adenocarcinomas, of which 18 were previously reported (Genes Chrom Cancer 2006), and RT PCR amplifications were performed. Amplified fragments were run on a 1.5% agorose gel, stained, purified and directly sequenced. The BLAST software was used for the analysis of TMPRSS2 (NM_005656) and ERG (NM_004449) sequence data. RESULTS: Nested RT-PCR with TMPRSS2forward and ERGreverse primers amplified cDNA fragments in 36 (90%) out of 40 prostate adenocarcinomas. Direct sequencing verified that the amplified fragments were chimeric TMPRSS2/ERG transcripts. In addition to the two previously reported fusion transcripts of TMPRSS2/ERG (a: (TMPRSS2 exon 1/ERG exon 4 and b: TMPRSS2 exon 1/ERG exon 2) three new junctions were detected between exons 4 and 5 of TMPRSS2 with exons 4 and 5 of ERG. Clinical data will be presented. CONCLUSIONS: Our data show the presence of the TMPRSS2/ERG chimera in 36 out of 40 prostate adenocarcinomas, confirming the findings by Tomlins et al (2005). Neither TMPRSS2/ETV1 nor TMPRSS2/ETV4 chimeric transcripts were detected in our prostate cancer series. This may be due to the small number of cases analyzed, geographic variation or a failure of the RT-PCR to detect it.
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CORRELATION OF THE VASCULARIZATION AND THE UPTAKE OF THE HYPOXIA TRACER [18F] EF5 IN PROSTATE CANCER TUMOURS OVEREXPRESSINNG VEGF AND FGF8B Tuomela J.1 , Gr¨onroos T.2 , Sepp¨anen J.1 , Forsback S.2 , Minn H.3 , Solin O.2 , H¨ark¨onen P.4 1 University of Turku, Anatomy, Turku, Finland; 2 University of Turku, Turku PET Centre, Turku, Finland; 3 Turku University Central Hospital, Department of Oncology and Radiotherapy, Turku, Finland; 4 Lund University, Department of Laboratory Medicine, Malm¨o, Sweden INTRODUCTION & OBJECTIVES: We have studied the role of the angiogenic growth factors, vascular endothelial growth factor (VEGF) and fibroblast growth factor 8b (FGF8b) in growth and spread of experimental PC-3 prostate tumours in nude mice. Immunohistochemical staining with the endothelial marker CD31 showed that the density of the capillary network was increased in tumours formed by PC-3 cells transfected with VEGF (PC-3/VEGF) or FGF8b (PC-3/FGF8b) compared to those formed by control vector transfected PC-3 (PC-3/mock) cells. We have evaluated the uptake of the hypoxia tracer [18F]EF5 into these tumours and correlated the uptake with the vascularization of tumours using the perfusion marker (Hoechst 33342) and antibodies against markers for hypoxia and non-labelled EF5. MATERIAL & METHODS: PC-3/VEGF, PC-3/FGF8b or PC-3/mock cells were inoculated subcutaneously into nude mice. Tumours were grown for 4 weeks. [18F]EF5 was injected intravenously (5.9±0.9 MBq) into non-anesthetized tumour bearing mice and the marker was allowed to distribute for 120 minutes. EF5 and the perfusion marker Hoechst were injected intravenously into mice 120 minutes and 2 minutes before sacrifice, respectively. After sacrifice blood, muscle and tumour tissues were removed, counted for 18F-radioactivity and weighed. The spatial distribution of 18F-radioactivity in frozen tumour sections was determined with digital autoradiography. Adjacent sections were immunohistochemically stained for EF5 and CD31. The results were compared to glucose metabolism in these tumours using [18F]FDG RESULTS: The number of CD31 positive vessels was significantly higher in PC-3/VEGF and PC-3/FGF8b tumours compared with PC-3/mock control tumours (p < 0.001). The distribution of Hoechst showed a different pattern in the various tumour groups as well. Furthermore, a negative correlation between the EF5 and the Hoechst distribution was seen. A variation in the mean tumourto-muscle 18F-radioactivity uptake ratios was seen between the different tumour groups. Our results demonstrate that the [18F]EF5 uptake was significantly (p < 0.05) lower in the PC-3/VEGF tumours as compared to the uptake seen in PC-3/FGF8b or PC-3/mock tumours. The results will be correlated to staining with antibodies against hypoxia markers. CONCLUSIONS: Our results suggest that the oxygenation status can vary in tumours which show a highly vascular morphology. The uptake of both [18F]EF5 and EF5 can reveal differences in the distribution of vascular network and function of vessels in tumours. We conclude that [18F]EF5 is an appropriate hypoxia tracer to be used with PET.
Eur Urol Suppl 2006;5:789
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QUANTITATIVE MULTI-GENE EXPRESSION PROFILING OF PRIMARY PROSTATE CANCER Unversucht S.1 , Schmidt U.1 , Meye A.1 , F¨ussel S.1 , Koch R.2 , Baretton G.B.3 , Froehner M.1 , Wirth M.P.1 1 Technical University of Dresden, Department of Urology, Dresden, Germany; 2 Technical University of Dresden, Institute of Medical Informatics and Biometry, Dresden, Germany; 3 Technical University of Dresden, Institute of Pathology, Dresden, Germany
HIGH FREQUENCY OF THE TMPRSS2/ERG FUSION GENE IN PROSTATE CANCER Soller W.1 , Johansson Soller M.2 , Isaksson M.2 , Elfving P.1 , Abrahamsson P.A.1 , Lundgren R.3 , Panagopoulos I.2 1 Malm¨ o University Hospital, Department of Urology, Malm¨o, Sweden; 2 Lund University Hospital, Department of Clinical Genetics, Lund, Sweden; 3 Helsingborg Hospital, Department of Urology, Helsingborg, Sweden
INTRODUCTION & OBJECTIVES: This study describes the evaluation of the expression patterns of prostate-relatedtranscripts in 106 matched prostate tissues from prostatectomies as predictors for prostate cancer (PCa). MATERIAL & METHODS: Quantitative PCR (QPCR) assays with sitespecific hybridization probes were established for 4 housekeeping genes (GAPDH, HPRT, PBGD, TBP) and 9 prostate-relatedgenes (AibZIP, DGPCR, EZH2, PCA3, PDEF, prostein, PSA, PSCA, TRPM8). RESULTS: The relative mRNA expression levels of AibZIP, D-GPCR, EZH2, PCA3, PDEF, PSA, TRPM8 (all < 0.001) and prostein (p = 0.019) normalized to the TBP reference gene were significantly higher in malignant compared to non-malignant prostate tissues. Employing receiver-operating characteristic (ROC) analyses, PCA3 was the best single tumor marker with the highest area-under-the-curve (AUC = 0.85). A multivariate logit model for the predictability of the tumor was developed, which employed the relative expression levels of EZH2, PCA3, prostein and TRPM8 and yielded an AUC of 0.90. CONCLUSIONS: PCA3 is a powerful predictor of primary PCa but the inclusion of EZH2, prostein and TRPM8 adds even more to the diagnostic power. The finding of a significantly higher mRNA expression of 3 different genes (prostein, PSA, TRPM8) in organ-confined tumors compared to non-organ-confined tumors could be of clinical importance and should be reevaluated in prospective studies using specimens from diagnostic biopsies.
10
UTILISING PROTEOMIC PLATFORMS FOR THE IDENTIFICATION OF NOVEL SERUM MARKERS OF PROSTATE CANCER
Byrne J.1 , Downes M.1 , O’Donoghue N.2 , Dunn M.J.2 , Fitzpatrick J.M.3 , Watson R.W.G.3 , Pennington S.R.2 1 Conway Institute of Biomolecular and Biomedical Research, U.C.D. and Mater Misericordiae University, Proteome Research Centre and U.C.D. School of Medicine and Medical Science, Dublin, Ireland; 2 Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Proteome Research Centre, Dublin, Ireland; 3 Mater Misericordiae University Hospital and Conway Institute of Biomolecular and Biomedical Research, UCD School of Medicine and Medical Science, Dublin, Ireland INTRODUCTION & OBJECTIVES: Current screening methods for prostate cancer rely heavily upon screening for prostate specific antigen (PSA) within peripheral blood along with digital rectal exam and biopsy. However PSA as a marker for prostate cancer lacks specificity and additional biomarkers are needed to improve the accuracy of diagnosis. In order to attempt to isolate new biomarkers, a proteomics-based approach was utilised to analyse protein expression within the serum of patients undergoing radical prostatectomy. Human serum represents a valuable potential source of biomarkers for disease. However, the range of protein concentrations within serum spans greater than 10 orders of magnitude with a small number of proteins present in high abundance, most notably albumin and immunoglobulins. In order to overcome the technical limitations associated with this fact, the serum samples were subjected to affinity depletion using the Multiple Affinity Removal System (Agilent Technologies), a system that removes the 6 most abundant proteins found in human serum – albumin, IgG, antitrypsin, IgA, transferrin and haptoglobin. Removal of these highly abundant proteins allows for the enrichment of lower abundance proteins. MATERIAL & METHODS: Twelve consented radical prostatectomy patient serum samples were divided into 2 groups based on their Gleason score (Gleason 5 and Gleason 7) and compared using Differential In-Gel Electrophoresis (DIGE), a proteomic platform which allows for highly specific differential protein expression analysis. These samples underwent depletion, were fluorescently labelled and separated by 2-D electrophoresis. The fluorescent 2-D gel images were subjected to protein expression analysis using two independent software packages: DeCyder 6.5 (GE Healthcare) and Progenesis PG240 (Nonlinear Dynamics). RESULTS: This analysis identified 63 proteins which showed differential protein expression between the two groups (p < 0.05), 13 of which were identified by both programmes. These proteins are currently undergoing identification using mass spectrometry. CONCLUSIONS: These 63 candidates, which can statistically differentiate between Gleason 5 and Gleason 7 depleted serum, could potentially hold a number of clinically relevant biomarkers which could distinguish between different grades of cancer, thus isolating patients with a higher risk of more aggressive cancer allowing for informed patient management.
INTRODUCTION & OBJECTIVES: Fusion genes are involved in carcinogenesis and have often been identified through analysis of recurrent chromosomal rearrangements. In two recent studies Tomlins and co-workers (Science 2005, Cancer Res 2006) showed that a large subset of prostate cancer harbours the fusion genes TMPRSS2/ERG, TMPRSS2/ETV1 and TMPRSS2/ETV4, generated through cryptic inter- and intrachromosomal rearrangements. A gene fusion of the 5’ untranslated region of TMPRSS2 to ERG or ETV1 was identified in 23 (79%) of 29 prostate cancer samples. MATERIAL & METHODS: In the present study total RNA was extracted from 40 historical prostate adenocarcinomas, of which 18 were previously reported (Genes Chrom Cancer 2006), and RT PCR amplifications were performed. Amplified fragments were run on a 1.5% agorose gel, stained, purified and directly sequenced. The BLAST software was used for the analysis of TMPRSS2 (NM_005656) and ERG (NM_004449) sequence data. RESULTS: Nested RT-PCR with TMPRSS2forward and ERGreverse primers amplified cDNA fragments in 36 (90%) out of 40 prostate adenocarcinomas. Direct sequencing verified that the amplified fragments were chimeric TMPRSS2/ERG transcripts. In addition to the two previously reported fusion transcripts of TMPRSS2/ERG (a: (TMPRSS2 exon 1/ERG exon 4 and b: TMPRSS2 exon 1/ERG exon 2) three new junctions were detected between exons 4 and 5 of TMPRSS2 with exons 4 and 5 of ERG. Clinical data will be presented. CONCLUSIONS: Our data show the presence of the TMPRSS2/ERG chimera in 36 out of 40 prostate adenocarcinomas, confirming the findings by Tomlins et al (2005). Neither TMPRSS2/ETV1 nor TMPRSS2/ETV4 chimeric transcripts were detected in our prostate cancer series. This may be due to the small number of cases analyzed, geographic variation or a failure of the RT-PCR to detect it.
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CORRELATION OF THE VASCULARIZATION AND THE UPTAKE OF THE HYPOXIA TRACER [18F] EF5 IN PROSTATE CANCER TUMOURS OVEREXPRESSINNG VEGF AND FGF8B Tuomela J.1 , Gr¨onroos T.2 , Sepp¨anen J.1 , Forsback S.2 , Minn H.3 , Solin O.2 , H¨ark¨onen P.4 1 University of Turku, Anatomy, Turku, Finland; 2 University of Turku, Turku PET Centre, Turku, Finland; 3 Turku University Central Hospital, Department of Oncology and Radiotherapy, Turku, Finland; 4 Lund University, Department of Laboratory Medicine, Malm¨o, Sweden INTRODUCTION & OBJECTIVES: We have studied the role of the angiogenic growth factors, vascular endothelial growth factor (VEGF) and fibroblast growth factor 8b (FGF8b) in growth and spread of experimental PC-3 prostate tumours in nude mice. Immunohistochemical staining with the endothelial marker CD31 showed that the density of the capillary network was increased in tumours formed by PC-3 cells transfected with VEGF (PC-3/VEGF) or FGF8b (PC-3/FGF8b) compared to those formed by control vector transfected PC-3 (PC-3/mock) cells. We have evaluated the uptake of the hypoxia tracer [18F]EF5 into these tumours and correlated the uptake with the vascularization of tumours using the perfusion marker (Hoechst 33342) and antibodies against markers for hypoxia and non-labelled EF5. MATERIAL & METHODS: PC-3/VEGF, PC-3/FGF8b or PC-3/mock cells were inoculated subcutaneously into nude mice. Tumours were grown for 4 weeks. [18F]EF5 was injected intravenously (5.9±0.9 MBq) into non-anesthetized tumour bearing mice and the marker was allowed to distribute for 120 minutes. EF5 and the perfusion marker Hoechst were injected intravenously into mice 120 minutes and 2 minutes before sacrifice, respectively. After sacrifice blood, muscle and tumour tissues were removed, counted for 18F-radioactivity and weighed. The spatial distribution of 18F-radioactivity in frozen tumour sections was determined with digital autoradiography. Adjacent sections were immunohistochemically stained for EF5 and CD31. The results were compared to glucose metabolism in these tumours using [18F]FDG RESULTS: The number of CD31 positive vessels was significantly higher in PC-3/VEGF and PC-3/FGF8b tumours compared with PC-3/mock control tumours (p < 0.001). The distribution of Hoechst showed a different pattern in the various tumour groups as well. Furthermore, a negative correlation between the EF5 and the Hoechst distribution was seen. A variation in the mean tumourto-muscle 18F-radioactivity uptake ratios was seen between the different tumour groups. Our results demonstrate that the [18F]EF5 uptake was significantly (p < 0.05) lower in the PC-3/VEGF tumours as compared to the uptake seen in PC-3/FGF8b or PC-3/mock tumours. The results will be correlated to staining with antibodies against hypoxia markers. CONCLUSIONS: Our results suggest that the oxygenation status can vary in tumours which show a highly vascular morphology. The uptake of both [18F]EF5 and EF5 can reveal differences in the distribution of vascular network and function of vessels in tumours. We conclude that [18F]EF5 is an appropriate hypoxia tracer to be used with PET.
Eur Urol Suppl 2006;5:789