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QUANTITATIVE MULTIGENE EXPRESSION PROFILING OF PRIMARY PROSTATE CANCER
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110
COMPARISON OF GENOMIC ALTERATIONS OF DISSEMINATED TUMOUR CELLS AND THE PRIMARY TUMOUR FROM PROSTATE CARCINOMA PATIENTS
QUANTITATIVE MULTIGENE EXPRESSION PROFILING OF PRIMARY PROSTATE CANCER
Bluemke K.1, Karzek C.2, Bilkenroth U.2, Taubert H.2, Melchior A.3, Heynemann H.3, Fornara P.3 1 Martin-Luther Univ. Halle-Wittenberg, Clinic of Urology, Halle, Germany, 2Martin-Luther Univ. Hallo Wittenberg, Institute of Pathology, Halle, Germany, 3Martin-Luther Univ. Hallo Wittenberg, Clinic of Urology, Halle, Germany
INTRODUCTION & OBJECTIVES: Disseminated tumour cells (DTC) in peripheral blood are considered as the cause for the formation of metastases. Furthermore, their presence is increasingly regarded as a clinically relevant prognostic factor. Previously, we have established an immunomagnetic cell separation procedure that allows the sensitive detection of disseminated tumour cells of prostate carcinoma patients. Currently, the genomic alterations that characterize disseminated tumour cells are still unknown. The commonly used approaches to assess the status of microsatellite instability (MSI) and loss of heterozygosity (LOH) require a certain amount of DNA that is simply not available from only a limited number of tumour cells obtained from peripheral blood. MATERIAL & METHODS: In this study, we have applied the improved primer extension preamplification (I-PEP) in order to amplify the genomic DNA from DTC of prostate carcinoma patients preceding the investigation of MSI and LOH in these cells. The DNA was subsequently used to determine the status of several microsatellite markers, incl. Myc-L1, Rb, p53 Alu. The results were compared to those obtained from the primary tumour tissue, adjacent normal prostate tissue and metastases, as well as, from peripheral blood lymphocytes. RESULTS: Firstly, we could show that the I-PEP is a useful method to amplify a sufficient amount of DNA in a reproducible manner from just 3 - 5 disseminated tumour cells. We found that alterations of the marker Myc-L1 occurred in the majority of the samples; distinctive MSI and LOH of Rb occurred in far less samples. Furthermore, the disseminated tumour cells show the same alterations like the adjacent metastases. CONCLUSIONS: In summary, we show that the I-PEP in conjunction with MSI and LOH analyses is a powerful tool to amplify the genomic DNA from DTC and to determine the genetic alterations in these cells. Furthermore, we show that disseminated tumour cells from prostate carcinoma patients are characterized by specific patterns of MSI and LOH that distinguishes these cells from the originating tissue.
Meye A.1, Schmidt U.2, Fuessel S.2, Koch R.3, Baretton G.4, Froehner M.2, Wirth M.2 1
Univ. Hospital, Dept. of Urology, Dresden, Germany, 2Department of Urology, Technical University Dresden, Dresden, Germany, 3Institute of Medical Informatics and Biometry, Technical University Dresden, Dresden, Germany, 4Institute of Pathology, Technical University Dresden, Dresden, Germany INTRODUCTION & OBJECTIVES: This study describes the evaluation of the expression pattern of prostate-specific transcripts in 106 matched prostate tissues as predictors for prostate cancer (PCa). RNA was prepared from cryo-preserved paired malignant and non-malignant prostate specimens which had been removed during radical prostatectomy and examined by a trained pathologist. MATERIAL & METHODS: Quantitative PCR (QPCR) assays with site-specific hybridisation probes were established for four housekeeping genes (GAPDH, HPRT, PBGD, TBP) and nine prostate-specific genes (AibZIP, DD3/PCA3, D-GPCR, EZH2, PDEF, prostein, PSA, PSCA, TRPM8). In the analysed patient cohort, statistical differences for the commonly used housekeeping genes GAPDH (p=0.038), HPRT (p=0.036) and PBGD (p=0.00003) were observed. RESULTS: The only housekeeping gene being not differentially expressed between malignant and non-malignant prostate tissues was TBP (p=0.531). Therefore, all expression was normalized to TBP. The logarithmized relative mRNA expression of AibZIP (p<0.001), DD3/PCA3 (p<0.001), D-GPCR (p<0.001), EZH2 (p<0.001), PDEF (p<0.001), prostein (p=0.019), PSA (p<0.001) and TRPM8 (p<0.001) were significantly higher in malignant vs. non-malignant prostate tissues. Receiver operating characteristic (ROC) curves were generated, and their areas under the curve (AUC) were calculated for all single parameters. DD3/PCA3 is the marker with the highest AUC (0.85), i.e. the best single tumour marker. Furthermore, the data were analysed with regard to a multivariate model. A logit model was developed which employs the logarithmized relative expression levels of DD3/PCA3, EZH2, prostein and TRPM8 and yields an AUC of 0.90. CONCLUSIONS: It can be concluded that DD3/PCA3 is a powerful predictor of PCa but the addition of EZH2, prostein and TRPM8 adds even more to the predictive power.
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EXPRESSION OF RECEPTOR AND NON-RECEPTOR TYROSINE KINASES IN PROSTATE CANCER
COMPARATIVE GENOMIC HYBRIDISATION (CGH) AND SPECTRAL KARYOTYPING (SKY) ANALYSIS OF LNCAP SUBLINES
Bastian P.J.1, Kummer S.2, Heukamp L.C.2, Müller S.C.1, Alexander V.R.2
Thalmann G.N.1, Philips J.2, Afonso A.3, Chung L.W.K.4
1
1
Rheinische Friedrich - Wilhelms Universität, Klinik Und Poliklinik für Urologie, Bonn, Germany, 2Rheinische Friedrich-Wilhelms Universität, Institut für Pathologie, Bonn, Germany INTRODUCTION & OBJECTIVES: Tyrosine kinases represent a group of enzymes that are crucial in carcinogenesis. They act at important relay points, interfering with a complex network of communicating signalling molecules with various effects on proliferation, differentiation, cell motility, cell death and apoptosis. Consequently, they are object to numerous examinations investigating the molecular basis of carcinogenesis and progression. Until now, no systematic expression profile of all tyrosine kinases in prostate cancer has been published. We present our initial expression data from 12 patients who underwent radical retropubic prostatectomy. MATERIAL & METHODS: We examined the expression of all tyrosine kinases (n=89) in prostatic tissue using real-time PCR based TaqMan Low Density Array technology (Applied Biosystems) after highly standardised tissue sampling and examination, RNA-isolation and cDNA synthesis. Patients have been classified into patients with benign prostatic disease (n=3) and patients with prostate cancer (n=9), the latter ranked according to their Gleason score. Expression data were processed using a straightforward combination of different software solutions for normalization and data processing. RESULTS: In this pilot experiment we could identify 12 tyrosine kinases that were clearly differentially expressed in benign and malignant prostatic tissue with a minimal 5-fold difference according to our expression data (AATK, ABL2, EGFR, FGFR1, FGFR2, VEGFR2, NTRK1, NTRK3, PDGFR1, PDGFR2, ROR2, TYRO3). CONCLUSIONS: Our prelimineray results suggest a number of new promising candidate genes that have not been examined in prostatic tissue up to now. Further analyses based on a larger cohort as well as immunohistochemical validation will further prove the role of these genes in prostate carcinogenesis and progression. Eur Urol Suppl 2006;5(2):50
University Hospital of Berne, Department of Urology, Berne, Switzerland, 2Mt. Sinai Hospital, Department of Medicine, New York, United States, 3Beth Israel Medical Center, Department of Urology, New York, United States, 4Emory University, Department of Urology, Atlanta, United States INTRODUCTION & OBJECTIVES: The aim of this study was to correlate copy number changes using comparative genomic hybridisation (CGH) and structural aberrations using spectral karyotyping (SKY) in androgen-independent (AI) cell lines forming osteoblastic bone metastases derived from androgen-dependent (AD) LNCaP cells. MATERIAL & METHODS: Biotin-labelled tumour DNA and digoxigenin-labelled normal donor DNA (sex-matched) was co-hybridized to sex-matched normal human lymphocyte metaphase chromosomes for CGH. Changes were detected, and images acquired and analysed with Q-CGH software. Metaphases were obtained for SKY and SKY kits were hybridized to metaphase chromosomes. Images were acquired with SkyView software using a spectral cube and a CCD camera connected to a DMRXA microscope with a custom designed SKY-3 optical filter. DNA was prepared using high salt extraction methods. RESULTS: CGH: LNCaP contained loss of Y, 1p, 2, 4q, 6p, 13q, 20p and gain of 3q. C4, compared to LNCaP, has no discernible changes. C4-2, compared to C4, has loss of 18. Compared to C4-2, the sublines C4-2B2 - 5 have loss of 6q and a possible gain of 18. In addition, C4 -2B4 shows gains of 3p, 5, and 8. SKY: All cells were near tetraploid with a modal chromosome number of 71-105. Recurrent marker chromosomes in C4, C4-2, and C4-2B3 cell lines were a non-reciprocal translocation involving chromosomes 4, 6, and 10 [der(4)t(4;6;10) and der(6)t(4;6)] and a reciprocal translocation involving chromosomes 1 and 15 [t(1;15) and der(15)t(1;15)]. A recurrent marker involving chromosomes 6 and 16 was found in C4 and C4-2 [(der(16)t(6;16)] but not in C4-2B3. A translocation involving 10 and 16 (der(10)t(10;16)) was only seen in C4-2. A der(16)t(6;16;10) and a der(14)t(14;20) was seen in C4-2B3 and not in C4 or C4-2. CONCLUSIONS: During tumour progression tumour aneuploidy remains remarkably stable. Changes in DNA copy number in LNCaP sublines are due largely to non-reciprocal translocations involving chromosomes 4q, 6q, and 16q in the background of a near-tetraploid genome. Chromosomal breakpoints that result may prove to be important for the progression of hormone resistant prostate cancer, such as the deletion of ATBF1, a potential tumour suppressor located at 16q22.
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COMPARISON OF GENOMIC ALTERATIONS OF DISSEMINATED TUMOUR CELLS AND THE PRIMARY TUMOUR FROM PROSTATE CARCINOMA PATIENTS
QUANTITATIVE MULTIGENE EXPRESSION PROFILING OF PRIMARY PROSTATE CANCER
Bluemke K.1, Karzek C.2, Bilkenroth U.2, Taubert H.2, Melchior A.3, Heynemann H.3, Fornara P.3 1 Martin-Luther Univ. Halle-Wittenberg, Clinic of Urology, Halle, Germany, 2Martin-Luther Univ. Hallo Wittenberg, Institute of Pathology, Halle, Germany, 3Martin-Luther Univ. Hallo Wittenberg, Clinic of Urology, Halle, Germany
INTRODUCTION & OBJECTIVES: Disseminated tumour cells (DTC) in peripheral blood are considered as the cause for the formation of metastases. Furthermore, their presence is increasingly regarded as a clinically relevant prognostic factor. Previously, we have established an immunomagnetic cell separation procedure that allows the sensitive detection of disseminated tumour cells of prostate carcinoma patients. Currently, the genomic alterations that characterize disseminated tumour cells are still unknown. The commonly used approaches to assess the status of microsatellite instability (MSI) and loss of heterozygosity (LOH) require a certain amount of DNA that is simply not available from only a limited number of tumour cells obtained from peripheral blood. MATERIAL & METHODS: In this study, we have applied the improved primer extension preamplification (I-PEP) in order to amplify the genomic DNA from DTC of prostate carcinoma patients preceding the investigation of MSI and LOH in these cells. The DNA was subsequently used to determine the status of several microsatellite markers, incl. Myc-L1, Rb, p53 Alu. The results were compared to those obtained from the primary tumour tissue, adjacent normal prostate tissue and metastases, as well as, from peripheral blood lymphocytes. RESULTS: Firstly, we could show that the I-PEP is a useful method to amplify a sufficient amount of DNA in a reproducible manner from just 3 - 5 disseminated tumour cells. We found that alterations of the marker Myc-L1 occurred in the majority of the samples; distinctive MSI and LOH of Rb occurred in far less samples. Furthermore, the disseminated tumour cells show the same alterations like the adjacent metastases. CONCLUSIONS: In summary, we show that the I-PEP in conjunction with MSI and LOH analyses is a powerful tool to amplify the genomic DNA from DTC and to determine the genetic alterations in these cells. Furthermore, we show that disseminated tumour cells from prostate carcinoma patients are characterized by specific patterns of MSI and LOH that distinguishes these cells from the originating tissue.
Meye A.1, Schmidt U.2, Fuessel S.2, Koch R.3, Baretton G.4, Froehner M.2, Wirth M.2 1
Univ. Hospital, Dept. of Urology, Dresden, Germany, 2Department of Urology, Technical University Dresden, Dresden, Germany, 3Institute of Medical Informatics and Biometry, Technical University Dresden, Dresden, Germany, 4Institute of Pathology, Technical University Dresden, Dresden, Germany INTRODUCTION & OBJECTIVES: This study describes the evaluation of the expression pattern of prostate-specific transcripts in 106 matched prostate tissues as predictors for prostate cancer (PCa). RNA was prepared from cryo-preserved paired malignant and non-malignant prostate specimens which had been removed during radical prostatectomy and examined by a trained pathologist. MATERIAL & METHODS: Quantitative PCR (QPCR) assays with site-specific hybridisation probes were established for four housekeeping genes (GAPDH, HPRT, PBGD, TBP) and nine prostate-specific genes (AibZIP, DD3/PCA3, D-GPCR, EZH2, PDEF, prostein, PSA, PSCA, TRPM8). In the analysed patient cohort, statistical differences for the commonly used housekeeping genes GAPDH (p=0.038), HPRT (p=0.036) and PBGD (p=0.00003) were observed. RESULTS: The only housekeeping gene being not differentially expressed between malignant and non-malignant prostate tissues was TBP (p=0.531). Therefore, all expression was normalized to TBP. The logarithmized relative mRNA expression of AibZIP (p<0.001), DD3/PCA3 (p<0.001), D-GPCR (p<0.001), EZH2 (p<0.001), PDEF (p<0.001), prostein (p=0.019), PSA (p<0.001) and TRPM8 (p<0.001) were significantly higher in malignant vs. non-malignant prostate tissues. Receiver operating characteristic (ROC) curves were generated, and their areas under the curve (AUC) were calculated for all single parameters. DD3/PCA3 is the marker with the highest AUC (0.85), i.e. the best single tumour marker. Furthermore, the data were analysed with regard to a multivariate model. A logit model was developed which employs the logarithmized relative expression levels of DD3/PCA3, EZH2, prostein and TRPM8 and yields an AUC of 0.90. CONCLUSIONS: It can be concluded that DD3/PCA3 is a powerful predictor of PCa but the addition of EZH2, prostein and TRPM8 adds even more to the predictive power.
111
112
EXPRESSION OF RECEPTOR AND NON-RECEPTOR TYROSINE KINASES IN PROSTATE CANCER
COMPARATIVE GENOMIC HYBRIDISATION (CGH) AND SPECTRAL KARYOTYPING (SKY) ANALYSIS OF LNCAP SUBLINES
Bastian P.J.1, Kummer S.2, Heukamp L.C.2, Müller S.C.1, Alexander V.R.2
Thalmann G.N.1, Philips J.2, Afonso A.3, Chung L.W.K.4
1
1
Rheinische Friedrich - Wilhelms Universität, Klinik Und Poliklinik für Urologie, Bonn, Germany, 2Rheinische Friedrich-Wilhelms Universität, Institut für Pathologie, Bonn, Germany INTRODUCTION & OBJECTIVES: Tyrosine kinases represent a group of enzymes that are crucial in carcinogenesis. They act at important relay points, interfering with a complex network of communicating signalling molecules with various effects on proliferation, differentiation, cell motility, cell death and apoptosis. Consequently, they are object to numerous examinations investigating the molecular basis of carcinogenesis and progression. Until now, no systematic expression profile of all tyrosine kinases in prostate cancer has been published. We present our initial expression data from 12 patients who underwent radical retropubic prostatectomy. MATERIAL & METHODS: We examined the expression of all tyrosine kinases (n=89) in prostatic tissue using real-time PCR based TaqMan Low Density Array technology (Applied Biosystems) after highly standardised tissue sampling and examination, RNA-isolation and cDNA synthesis. Patients have been classified into patients with benign prostatic disease (n=3) and patients with prostate cancer (n=9), the latter ranked according to their Gleason score. Expression data were processed using a straightforward combination of different software solutions for normalization and data processing. RESULTS: In this pilot experiment we could identify 12 tyrosine kinases that were clearly differentially expressed in benign and malignant prostatic tissue with a minimal 5-fold difference according to our expression data (AATK, ABL2, EGFR, FGFR1, FGFR2, VEGFR2, NTRK1, NTRK3, PDGFR1, PDGFR2, ROR2, TYRO3). CONCLUSIONS: Our prelimineray results suggest a number of new promising candidate genes that have not been examined in prostatic tissue up to now. Further analyses based on a larger cohort as well as immunohistochemical validation will further prove the role of these genes in prostate carcinogenesis and progression. Eur Urol Suppl 2006;5(2):50
University Hospital of Berne, Department of Urology, Berne, Switzerland, 2Mt. Sinai Hospital, Department of Medicine, New York, United States, 3Beth Israel Medical Center, Department of Urology, New York, United States, 4Emory University, Department of Urology, Atlanta, United States INTRODUCTION & OBJECTIVES: The aim of this study was to correlate copy number changes using comparative genomic hybridisation (CGH) and structural aberrations using spectral karyotyping (SKY) in androgen-independent (AI) cell lines forming osteoblastic bone metastases derived from androgen-dependent (AD) LNCaP cells. MATERIAL & METHODS: Biotin-labelled tumour DNA and digoxigenin-labelled normal donor DNA (sex-matched) was co-hybridized to sex-matched normal human lymphocyte metaphase chromosomes for CGH. Changes were detected, and images acquired and analysed with Q-CGH software. Metaphases were obtained for SKY and SKY kits were hybridized to metaphase chromosomes. Images were acquired with SkyView software using a spectral cube and a CCD camera connected to a DMRXA microscope with a custom designed SKY-3 optical filter. DNA was prepared using high salt extraction methods. RESULTS: CGH: LNCaP contained loss of Y, 1p, 2, 4q, 6p, 13q, 20p and gain of 3q. C4, compared to LNCaP, has no discernible changes. C4-2, compared to C4, has loss of 18. Compared to C4-2, the sublines C4-2B2 - 5 have loss of 6q and a possible gain of 18. In addition, C4 -2B4 shows gains of 3p, 5, and 8. SKY: All cells were near tetraploid with a modal chromosome number of 71-105. Recurrent marker chromosomes in C4, C4-2, and C4-2B3 cell lines were a non-reciprocal translocation involving chromosomes 4, 6, and 10 [der(4)t(4;6;10) and der(6)t(4;6)] and a reciprocal translocation involving chromosomes 1 and 15 [t(1;15) and der(15)t(1;15)]. A recurrent marker involving chromosomes 6 and 16 was found in C4 and C4-2 [(der(16)t(6;16)] but not in C4-2B3. A translocation involving 10 and 16 (der(10)t(10;16)) was only seen in C4-2. A der(16)t(6;16;10) and a der(14)t(14;20) was seen in C4-2B3 and not in C4 or C4-2. CONCLUSIONS: During tumour progression tumour aneuploidy remains remarkably stable. Changes in DNA copy number in LNCaP sublines are due largely to non-reciprocal translocations involving chromosomes 4q, 6q, and 16q in the background of a near-tetraploid genome. Chromosomal breakpoints that result may prove to be important for the progression of hormone resistant prostate cancer, such as the deletion of ATBF1, a potential tumour suppressor located at 16q22.