- Email: [email protected]
130 CIRCULATING TUMOR CELLS IN PROSTATE CANCER PATIENTS: NOVEL IMMUNOMAGNETIC ENRICHMENT METHOD
e54
THE JOURNAL OF UROLOGY姞
METHODS: We analyzed micro-dissected prostate cancer and normal-adjacent to tumor (NAT) from 66 patients with prostate adenocarcinoma, and normal prostate tissue from 8 cancer-free organ donors for LINE-1 methylation levels. The methylation levels were correlated with clinicopathologic parameters. RESULTS: The mean global methylation index in prostate tumors was significantly less than methylation levels in age-matched cancer-free prostates (53.4% ⫾ 15.4% vs. 81.4% ⫾ 4.5%, respectively; p⬍0.01). Histopathologically normal prostate tissue from PCa patients had lower global methylation when compared to cancer-free prostates. Global methylation was significantly decreased in prostate tumor tissue compared to NAT (53.4% ⫾ 15.4% vs. 69.5% ⫾ 12.3%, respectively; p⬍0.05) (Figure 1). Global methylation was lowest in cancer tissue and gradually increased concomitant with distance from the tumor, indicating a marked field effect characterized by a decrease in global methylation (Figure 2). There were no significant differences in LINE-1 methylation when patients were stratified by age, Gleason grade, pathologic stage, biochemical recurrence, or lymph node status. CONCLUSIONS: Significant hypomethylation of LINE-1 retrotransposons and a marked methylation field effect were observed in prostate cancer and cancer-adjacent benign tissue, respectively.
Vol. 185, No. 4S, Supplement, Sunday, May 15, 2011
129 CYR61 IS A POTENTIAL SERUM MARKER FOR IDENTIFYING NON-ORGAN-CONFINED PROSTATE CANCER Naoki Terada*, Prakash Kulkarni, Robert Getzenberg, Baltimore, MD INTRODUCTION AND OBJECTIVES: PSA is a useful marker for detecting early prostate cancer (PCa) but not for differentiating significant from indolent PCa. Cysteine-rich angiogenic inducer 61 (Cyr61) is an extracellular matrix protein involved in the transduction of growth factor and hormone signaling, exhibiting altered expression in several types of cancers. Our tissue microarray (TMA) data demonstrated that Cyr61 expression is significantly up-regulated in PCa and that the expression levels are additive over Gleason score in being able to predict disease progression (Clin Cancer Res 2010), indicating that Cyr61 might be a clinically useful biomarker. The aim of this study was to establish ELISA methods for measuring serum Cyr61 concentrations and analyze their utility as a marker for differentiating non-organconfined PCa (NOC-PCa) from organ-confined PCa (OC-PCa). METHODS: Cyr61 protein expression levels in benign prostate and PCa cell lines (BPH1, LNCaP, DU145, PC3) were examined by immunoblotting using two different antibodies (mouse monoclonal/Cterm; Abcam and goat polyclonal/N-term; Santa Cruz). The Cyr61 concentrations in the supernatants obtained from these cell lines were measured by sandwich ELISA using these antibodies. The full-length Cyr61 recombinant protein (Abnova) was used as a standard. Thereafter, the serum Cyr61 levels were measured in blood samples obtained at the time of surgery from men that had either OC-PCa (n⫽58) or NOC-PCa (n⫽57). RESULTS: Cyr61 protein expression is low in BPH1 and LNCaP, and high in DU145 and PC3 cells. The Cyr61 concentrations in the supernatants of these cell lines were 0,0,22.6, 27.2 ng/ml, respectively. In clinical samples, there were no significant difference in serum PSA levels between OC-PCa (6.4⫾3.8ng/ml) and NOC-PCa (6.1⫾4.7/ml) (p⫽0.157). However, the serum Cyr61 levels were significantly higher in NOC-PCa (116.3⫾140.2ng/ml) than in OC-PCa (79.7⫾56.1ng/ml) (p⫽0.031). Using the cut-off values of 100ng/ml, sensitivity and specificity of Cyr61 for differentiating NOC-PCa from OC-PCa were 75.9% and 59.3%, respectively. CONCLUSIONS: Cyr61 that can be detected by sandwich ELISA in serum samples may be a potential serum marker for detecting significant PCa. Source of Funding: NIH P50 CA058236
130 CIRCULATING TUMOR CELLS IN PROSTATE CANCER PATIENTS: NOVEL IMMUNOMAGNETIC ENRICHMENT METHOD Xiaoke Huang*, Barry B. McGuire, Irene Ogden, Daniel C. O’Brien, Phillip R. Cooper, Jessica A. Banks, William J. Catalona, Raymond C. Bergan, Chicago, IL
Source of Funding: This research was supported by the Department of Defense Prostate Cancer Research Program under the award number W81XWH-41-0007 (DSOK). Views and opinions of, and endorsements by the authors do not reflect those of the U.S. Army or the Department of Defense. This research was also partly supported by NIH 5R21CA124892 (DSOK).
INTRODUCTION AND OBJECTIVES: Pharmacological prevention of metastases would improve quality of life for many prostate cancer (PCa) patients. To metastasize, cancer cells must invade through the basement membrane and the extracellular matrix of the organ’s stromal compartment to intravasate into the bloodstream thereby becoming circulating tumor cells (CTCs). The number of CTCs correlates with the formation of distant metastases. Rates of PCa death are ⬃10 fold lower among soy-consuming Southeast Asians than among non-soy consumers in the US. Genistein is a soy protein derivative that has been shown in vitro and in murine models to inhibit these early steps in the cascade. We implemented a Clinical Phase II Trial to determine whether genistein could decrease CTCs in men with prostate cancer (PCa). To measure this effect, we have developed a novel assay to detect and quantify CTCs in whole human blood. In addition to PSA, CTCs also express CK18. METHODS: Patients were randomized to genistein treatment (2mg/kg of genistein per day three weeks prior to radical prostatectomy) or no treatment (placebo). A whole blood sample was obtained
Vol. 185, No. 4S, Supplement, Sunday, May 15, 2011
THE JOURNAL OF UROLOGY姞
prior to surgery. After RNA extraction the expression of prostate specific antigen (PSA) and cytokeratin 18 (CK18) genes was measured by quantitative RT/PCR, and we have optimized this method by using SABiosciences qPCR Master Mix. We also employ an additional technique of immunomagnetic precipitation to extract RNA from CTCs. Specifically, we coat Dynabeads® (Invitrogen) with equal amounts of antibody to epithelial cell adhesion molecule (EpCAM) and to prostate specific membrane antigen (PSMA). RESULTS: Using a specific primer/probe set, we demonstrate that our qRT/PCR assay is linear over a wide range, and that we can detect PSA transcript in RNA from ⬍ 1 LNCaP cell. When LNCaP cells are spiked into peripheral blood mononuclear cells (PBMNCs) from men without PCa, we demonstrate that we can detect 1 human prostate cell per 10 7 PBMNCs. In our phase II clinical trial we successfully detected CTCs in 13/17 (76.5%) patients. Our technique of dual antigen pull-down can detect 1 CTC per ml of whole blood of PCa patients. PSA transcripts have not been detected in negative control blood. CONCLUSIONS: We have developed a novel, rapid and sensitive assay suitable for the detection of CTCs in peripheral blood of PCa patients. Table 1: Preliminary results in circulating tumor cell (CTC) detection in 17 patients, demonstrates successful detection in 13/17 (76.5%) patients. CTC CTC Detection Detection Patient PSA Stage Gleason PSA CK18 1 8.2 T1C 4⫹4 ⫹ NP 2
2.8
T2B/T3
5⫹4
⫹
NP
3
4.1
T2B/T3
4⫹4
⫹
NP
4
52
T1C
4⫹3
NEG
NP
5
14
T2B/T3
3⫹4
NEG
NP
6
4.9
T2A
4⫹4
⫹
NP
7
5
T2B/T3
3⫹4
NEG
NP
8
12
T1C
4⫹4
⫹
NP
9
7.16
T1C
4⫹5
⫹
NP
10
6.3
T2B
4⫹3
⫹
NP
11
16.45
T2B
4⫹5
⫹
NP
12
9.4
T1C
4⫹3
⫹
NP
13
8.4
T2A
4⫹4
⫹
NP
14
36
T3
4⫹3
NEG
⫹
15
270
T4
4⫹4
NEG
⫹
16
10
T2
4⫹4
NEG
NEG
T1C
4⫹5
⫹
⫹
17
6.2
ative steps before the devascularised prostate can be extirpated, casting doubt on the validity of this technique as a source for obtaining prostatic tissue. METHODS: We describe our biobanking process and report the RNA quality of specimens derived from robotic-assisted laparoscopic prostatectomy procedures using advanced electrophoretic techniques (RNA Integrity Numbers, RIN). We consider the impact of various clinicopathological correlates on RNA integrity using multivariate regression analysis. RESULTS: Our biobanking protocol has been used to collect ⬎1700 prostates, and retain approximately 40% of the prostate body. We generated a mean RNA yield of 6.49g per 1.5mm core, and demonstrated a mean RIN of 7.65 (standard deviation 1.56); 85% of samples had a RINⱖ7. Only prostate volume (B⫽-0.459) and Gleason score (B⫽0.597) were found to be significant predictors of RIN (p⫽0.026 & p⫽0.035, respectively). CONCLUSIONS: We demonstrate the robustness of our protocol, representing the concerted efforts of dedicated urology and pathology departments, in generating RNA of sufficient yield and quality, without compromising the histopathological evaluation and diagnosis of patients. Further studies are necessary to elucidate the impact of other clinicopathological correlates on RNA integrity, in order to direct future specimen collection and obtain the most reliable samples for research.
NP ⫽ Not performed, Neg ⫽ Negative.
Source of Funding: Supported in part by the Urological Research Foundation, Prostate SPORE Grant (P50 CA9038605S2) and the Robert H. Lurie Comprehensive Cancer Center Grant (P30 CA60553)
131 BIOBANKING OF TISSUE OBTAINED FROM ROBOTIC-ASSISTED RADICAL PROSTATECTOMY CAN BE SUCCESSFULLY IMPLEMENTED WITHOUT COMPROMISING RNA INTEGRITY Harveer Dev*, Prasanna Sooriakumaran, Naoki Kitabayashi, Robert Kim, Mark Rubin, Ashutosh Tewari, New York, NY INTRODUCTION AND OBJECTIVES: Translational research in prostate cancer demands high specimen quality in order to map the genetic signatures of malignant tissues. RNA quality is believed to decrease with ischaemia time, and therefore, traditional open radical prostatectomy has benefited from allowing the retrieval of the prostate immediately after its devascularisation. In contrast, prostatectomies which utilise robotic platforms require the completion of several oper-
e55
Source of Funding: None.
THE JOURNAL OF UROLOGY姞
METHODS: We analyzed micro-dissected prostate cancer and normal-adjacent to tumor (NAT) from 66 patients with prostate adenocarcinoma, and normal prostate tissue from 8 cancer-free organ donors for LINE-1 methylation levels. The methylation levels were correlated with clinicopathologic parameters. RESULTS: The mean global methylation index in prostate tumors was significantly less than methylation levels in age-matched cancer-free prostates (53.4% ⫾ 15.4% vs. 81.4% ⫾ 4.5%, respectively; p⬍0.01). Histopathologically normal prostate tissue from PCa patients had lower global methylation when compared to cancer-free prostates. Global methylation was significantly decreased in prostate tumor tissue compared to NAT (53.4% ⫾ 15.4% vs. 69.5% ⫾ 12.3%, respectively; p⬍0.05) (Figure 1). Global methylation was lowest in cancer tissue and gradually increased concomitant with distance from the tumor, indicating a marked field effect characterized by a decrease in global methylation (Figure 2). There were no significant differences in LINE-1 methylation when patients were stratified by age, Gleason grade, pathologic stage, biochemical recurrence, or lymph node status. CONCLUSIONS: Significant hypomethylation of LINE-1 retrotransposons and a marked methylation field effect were observed in prostate cancer and cancer-adjacent benign tissue, respectively.
Vol. 185, No. 4S, Supplement, Sunday, May 15, 2011
129 CYR61 IS A POTENTIAL SERUM MARKER FOR IDENTIFYING NON-ORGAN-CONFINED PROSTATE CANCER Naoki Terada*, Prakash Kulkarni, Robert Getzenberg, Baltimore, MD INTRODUCTION AND OBJECTIVES: PSA is a useful marker for detecting early prostate cancer (PCa) but not for differentiating significant from indolent PCa. Cysteine-rich angiogenic inducer 61 (Cyr61) is an extracellular matrix protein involved in the transduction of growth factor and hormone signaling, exhibiting altered expression in several types of cancers. Our tissue microarray (TMA) data demonstrated that Cyr61 expression is significantly up-regulated in PCa and that the expression levels are additive over Gleason score in being able to predict disease progression (Clin Cancer Res 2010), indicating that Cyr61 might be a clinically useful biomarker. The aim of this study was to establish ELISA methods for measuring serum Cyr61 concentrations and analyze their utility as a marker for differentiating non-organconfined PCa (NOC-PCa) from organ-confined PCa (OC-PCa). METHODS: Cyr61 protein expression levels in benign prostate and PCa cell lines (BPH1, LNCaP, DU145, PC3) were examined by immunoblotting using two different antibodies (mouse monoclonal/Cterm; Abcam and goat polyclonal/N-term; Santa Cruz). The Cyr61 concentrations in the supernatants obtained from these cell lines were measured by sandwich ELISA using these antibodies. The full-length Cyr61 recombinant protein (Abnova) was used as a standard. Thereafter, the serum Cyr61 levels were measured in blood samples obtained at the time of surgery from men that had either OC-PCa (n⫽58) or NOC-PCa (n⫽57). RESULTS: Cyr61 protein expression is low in BPH1 and LNCaP, and high in DU145 and PC3 cells. The Cyr61 concentrations in the supernatants of these cell lines were 0,0,22.6, 27.2 ng/ml, respectively. In clinical samples, there were no significant difference in serum PSA levels between OC-PCa (6.4⫾3.8ng/ml) and NOC-PCa (6.1⫾4.7/ml) (p⫽0.157). However, the serum Cyr61 levels were significantly higher in NOC-PCa (116.3⫾140.2ng/ml) than in OC-PCa (79.7⫾56.1ng/ml) (p⫽0.031). Using the cut-off values of 100ng/ml, sensitivity and specificity of Cyr61 for differentiating NOC-PCa from OC-PCa were 75.9% and 59.3%, respectively. CONCLUSIONS: Cyr61 that can be detected by sandwich ELISA in serum samples may be a potential serum marker for detecting significant PCa. Source of Funding: NIH P50 CA058236
130 CIRCULATING TUMOR CELLS IN PROSTATE CANCER PATIENTS: NOVEL IMMUNOMAGNETIC ENRICHMENT METHOD Xiaoke Huang*, Barry B. McGuire, Irene Ogden, Daniel C. O’Brien, Phillip R. Cooper, Jessica A. Banks, William J. Catalona, Raymond C. Bergan, Chicago, IL
Source of Funding: This research was supported by the Department of Defense Prostate Cancer Research Program under the award number W81XWH-41-0007 (DSOK). Views and opinions of, and endorsements by the authors do not reflect those of the U.S. Army or the Department of Defense. This research was also partly supported by NIH 5R21CA124892 (DSOK).
INTRODUCTION AND OBJECTIVES: Pharmacological prevention of metastases would improve quality of life for many prostate cancer (PCa) patients. To metastasize, cancer cells must invade through the basement membrane and the extracellular matrix of the organ’s stromal compartment to intravasate into the bloodstream thereby becoming circulating tumor cells (CTCs). The number of CTCs correlates with the formation of distant metastases. Rates of PCa death are ⬃10 fold lower among soy-consuming Southeast Asians than among non-soy consumers in the US. Genistein is a soy protein derivative that has been shown in vitro and in murine models to inhibit these early steps in the cascade. We implemented a Clinical Phase II Trial to determine whether genistein could decrease CTCs in men with prostate cancer (PCa). To measure this effect, we have developed a novel assay to detect and quantify CTCs in whole human blood. In addition to PSA, CTCs also express CK18. METHODS: Patients were randomized to genistein treatment (2mg/kg of genistein per day three weeks prior to radical prostatectomy) or no treatment (placebo). A whole blood sample was obtained
Vol. 185, No. 4S, Supplement, Sunday, May 15, 2011
THE JOURNAL OF UROLOGY姞
prior to surgery. After RNA extraction the expression of prostate specific antigen (PSA) and cytokeratin 18 (CK18) genes was measured by quantitative RT/PCR, and we have optimized this method by using SABiosciences qPCR Master Mix. We also employ an additional technique of immunomagnetic precipitation to extract RNA from CTCs. Specifically, we coat Dynabeads® (Invitrogen) with equal amounts of antibody to epithelial cell adhesion molecule (EpCAM) and to prostate specific membrane antigen (PSMA). RESULTS: Using a specific primer/probe set, we demonstrate that our qRT/PCR assay is linear over a wide range, and that we can detect PSA transcript in RNA from ⬍ 1 LNCaP cell. When LNCaP cells are spiked into peripheral blood mononuclear cells (PBMNCs) from men without PCa, we demonstrate that we can detect 1 human prostate cell per 10 7 PBMNCs. In our phase II clinical trial we successfully detected CTCs in 13/17 (76.5%) patients. Our technique of dual antigen pull-down can detect 1 CTC per ml of whole blood of PCa patients. PSA transcripts have not been detected in negative control blood. CONCLUSIONS: We have developed a novel, rapid and sensitive assay suitable for the detection of CTCs in peripheral blood of PCa patients. Table 1: Preliminary results in circulating tumor cell (CTC) detection in 17 patients, demonstrates successful detection in 13/17 (76.5%) patients. CTC CTC Detection Detection Patient PSA Stage Gleason PSA CK18 1 8.2 T1C 4⫹4 ⫹ NP 2
2.8
T2B/T3
5⫹4
⫹
NP
3
4.1
T2B/T3
4⫹4
⫹
NP
4
52
T1C
4⫹3
NEG
NP
5
14
T2B/T3
3⫹4
NEG
NP
6
4.9
T2A
4⫹4
⫹
NP
7
5
T2B/T3
3⫹4
NEG
NP
8
12
T1C
4⫹4
⫹
NP
9
7.16
T1C
4⫹5
⫹
NP
10
6.3
T2B
4⫹3
⫹
NP
11
16.45
T2B
4⫹5
⫹
NP
12
9.4
T1C
4⫹3
⫹
NP
13
8.4
T2A
4⫹4
⫹
NP
14
36
T3
4⫹3
NEG
⫹
15
270
T4
4⫹4
NEG
⫹
16
10
T2
4⫹4
NEG
NEG
T1C
4⫹5
⫹
⫹
17
6.2
ative steps before the devascularised prostate can be extirpated, casting doubt on the validity of this technique as a source for obtaining prostatic tissue. METHODS: We describe our biobanking process and report the RNA quality of specimens derived from robotic-assisted laparoscopic prostatectomy procedures using advanced electrophoretic techniques (RNA Integrity Numbers, RIN). We consider the impact of various clinicopathological correlates on RNA integrity using multivariate regression analysis. RESULTS: Our biobanking protocol has been used to collect ⬎1700 prostates, and retain approximately 40% of the prostate body. We generated a mean RNA yield of 6.49g per 1.5mm core, and demonstrated a mean RIN of 7.65 (standard deviation 1.56); 85% of samples had a RINⱖ7. Only prostate volume (B⫽-0.459) and Gleason score (B⫽0.597) were found to be significant predictors of RIN (p⫽0.026 & p⫽0.035, respectively). CONCLUSIONS: We demonstrate the robustness of our protocol, representing the concerted efforts of dedicated urology and pathology departments, in generating RNA of sufficient yield and quality, without compromising the histopathological evaluation and diagnosis of patients. Further studies are necessary to elucidate the impact of other clinicopathological correlates on RNA integrity, in order to direct future specimen collection and obtain the most reliable samples for research.
NP ⫽ Not performed, Neg ⫽ Negative.
Source of Funding: Supported in part by the Urological Research Foundation, Prostate SPORE Grant (P50 CA9038605S2) and the Robert H. Lurie Comprehensive Cancer Center Grant (P30 CA60553)
131 BIOBANKING OF TISSUE OBTAINED FROM ROBOTIC-ASSISTED RADICAL PROSTATECTOMY CAN BE SUCCESSFULLY IMPLEMENTED WITHOUT COMPROMISING RNA INTEGRITY Harveer Dev*, Prasanna Sooriakumaran, Naoki Kitabayashi, Robert Kim, Mark Rubin, Ashutosh Tewari, New York, NY INTRODUCTION AND OBJECTIVES: Translational research in prostate cancer demands high specimen quality in order to map the genetic signatures of malignant tissues. RNA quality is believed to decrease with ischaemia time, and therefore, traditional open radical prostatectomy has benefited from allowing the retrieval of the prostate immediately after its devascularisation. In contrast, prostatectomies which utilise robotic platforms require the completion of several oper-
e55
Source of Funding: None.