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APTIMA® PCA3 MOLECULAR URINE TEST: DEVELOPMENT OF A METHOD TO AID IN THE DIAGNOSIS OF PROSTATE CANCER
1009
1010
DIFFERENTIATION ENHANCEMENT OF CIRCULATING IMMUNE CELLS CONTAINING INTRACELLULAR PSA: A NEW METHOD FOR DISCRIMINATION BETWEEN BENIGN AND MALIGNANT PROSTATIC DISEASE
SOLUBLE RECEPTOR OF HUMAN CYTOKINE IL-6 (SIL-6R) ON 123 PATIENTS WITH UNTREATED PROSTATE CANCER (PCA) Pina F.1, Figueiredo G.2, Lunet N.3, Tomada N.1, Silva A.1, Cruz F.1, Barros H.3 1
1
Faculdade Medicina Universidade Porto, Urology, Porto, Portugal, 2Faculdade Medicina Universidade Porto, Immunology, Porto, Portugal, 3Faculdade Medicina Universidade Porto, Epidemiology, Porto, Portugal
INTRODUCTION & OBJECTIVES: A new test measuring the percentage of PSA-positive macrophages (Mf) in peripheral blood has previously been demonstrated to be useful in discriminating between benign and malignant prostatic disease. In order to find out, if further differentiation provides better information about these cells and still enables to distinguish between benign and malignant disease, Mf of patients with prostate cancer and healthy controls were extracellularly stained for CD14 and CD16, which is a marker for immune cell activation, and intracellularly stained for detecting PSA.
INTRODUCTION & OBJECTIVES: IL-6 has important growth factor biological activity on prostate cancer (PC) through IL-6R↔gp130 receptor complex or soluble IL-6 receptor (sIL-6R). PC cells are able to constitutively produce either IL6 or sIL-6R, and pathologic conditions with high IL-6 levels (like hormone-refractory PC) may also present increased sIL-6R production. PC patients with high serum IL-6 (> 4 or > 7 ng/ml) are significantly related to progression and specific survival, and the same seems to be truth for sIL-6R. We investigate this receptor under base-line condition in a group of untreated PC patients checking for relation to several prognostic factors.
Herwig R.1, Kramer G.1, Djavan B.1, Rehder P.2, Ramoner R.2, Marberger M.1 Medical University Vienna, Urology, Vienna, Austria, 2Medical University Innsbruck, Urology, Innsbruck, Austria
MATERIAL & METHODS: Peripheral blood mononuclear cells were isolated from whole blood samples obtained from 25 patients with prostate cancer and 10 healthy controls. Cells were analysed according to extracellular signal for CD14+ and CD16+ using flow cytomety (FACS Calibur, USA). Intracellular staining was subsequently performed using a cell permeabilisation kit and monoclonal antibodies against PSA (Clone ER-PR8). Data were calculated as mean percentage with standard deviation (SD) of positive cells. RESULTS: As before, differentiation between malignant and benign disease was possible using the CD14/PSA staining. Additional staining with CD16 leads to further and better differentiation of a PSA containing CD14 / CD16 positive cell population. PSA could only be found in activated CD14+ cells. The mean percentage of PSA containing macrophages was 0.58 in healthy controls vs. 14.75% (localised prostate cancer) and 84.67% (metastatic prostate cancer) respectively. In contrast to the new method, measurement of serum total PSA levels showed such a high SD that no distinction between controls, prostate cancer and metastatic disease could be drawn. CONCLUSIONS: Measurement of the amount of intracellular PSA in peripheral blood macrophages enables clear distinction between benign and malignant disease. Moreover, localised prostate cancer significantly differed from metastatic disease with regard to the levels of circulating Mf . Thus, the highest level was found in metastatic prostate cancer and the lowest in levels in controls, suggesting that malignancy correlates with the amount of positive Mf . The population of PSA containing macrophages could be better defined and visualized as demonstrated before, leading to a better analysis of the underlying cases. Further distinction of these cells may lead to better stratification of patients and research on this field in future.
MATERIAL & METHODS: 125 PC patients (mean age 70.2 years; mean tPSA 34.4 ng/ ml) were staged and stratified in progression risk groups of cTNM, biopsy Gleason, tPSA; 43 PC performed RP and were restaged and restratified according to pTNM, RP Gleason, tPSA and D’AMICO RP risk groups. Blood was collected for haemogram, biochemic, ALKP, tPSA, fPSA, tTestosterone, PRL, lymphocytes CD3+DR+, CD4+DR+ and CD8+DR+, human sIL-6R (DR600, m = 31,0 [14,0-46,0] pg/ml; Quantikine™/RDSystems, ELISA). Statistical Analysis: Proportions were compared with Chi2 test; Kruskal-Wallis test was used for quantitative variables. RESULTS: Among PC patients sIL-6R was detected in all cases, with a median of 31.0 pg/ml and with high levels (> 46 pg/ml) in 74%. No significant differences of sIL-6R were detected among prognostic groups of cTNM, biopsy Gleason score, tPSA, cPSA, pTNM, RP Gleason score, or D’AMICO RP groups. Median sIL-6R was significantly different between cases with f/t PSA ≤ 0.26 or > 0.26 (p=0.001). Analysis of continuous variables only showed positive significant correlation between sIL-6R and total activated (CD3+DR+) and cytotoxic activated (CD8+DR+) lymphocytes. CONCLUSIONS: IL-6sR is elevated in the great majority of PC patients. Unlike other reports we found no parallelism of increased sIL-6R values with most usual clinical or pathological prognostic risk groups. PC cases with grey zone tPSA > 0.26 and sIL-6R high values may represent a group with PC delayed diagnosis. The link of IL-6 axis to cellular cytotoxic immunity is suggested by significant association of sIL-6R and CD8+DR+ lymphocytes. Long follow-up of our patients is needed to reveal Shariat et all-2004 statement on the sIL-6R association to RP aggressive biochemical recurrence.
1011
1012
SERUM SOLUBLE UROKINASE-TYPE PLASMINOGEN ACTIVATOR RECEPTOR (SUPAR) IS A USEFUL MARKER OF PROSTATE CANCER
APTIMA® PCA3 MOLECULAR URINE TEST: DEVELOPMENT OF A METHOD TO AID IN THE DIAGNOSIS OF PROSTATE CANCER
Milanese G.1, Gasparri L.1, Dellabella M.2, Sidenius N.3, Galosi A.B.2, Minardi D.4, Blasi F.5, Fazioli F.6, Muzzonigro G.4
Fradet Y.1, Groskopf J.2, Aubin S.M. J.2, Deras I.L.2, Blase A.3, Bodrug S.2, Clark C.2, Brentano S.2, Desaulniers M.4, Rittenhouse H.2
Polytechnic University of The Marche Region, Urology, Ancona, Italy, 2A.O.U. Umberto I Lancisi - Salesi, Urology, Ancona, Italy, 3San Raffaele Scientific Institute and Ifom Foundation, Molecular Genetics Unit, Dibit, Milano, Italy, 4Polytechnic University of The Marche Region, A.O.U. Umberto I - Lancisi - Salesi, Urology, Ancona, Italy, 5San Raffaele Scientific Institute, Molecular Genetics Unit, Dibit, Milano, Italy, 6Polytechnic University of The Marche Region, Laboratory of Cellular and Molecular Biology, Ancona, Italy
1
INTRODUCTION & OBJECTIVES: Numerous studies indicate that urokinase-type plasminogen activator receptor (uPAR) is involved in cancer. We investigated the expression of uPAR in body fluids of patients with clinical suspicion of prostate cancer to evaluate its possible role as prostate cancer marker. MATERIAL & METHODS: Urine and serum from 63 consecutive patients referred to our institution for TRUS-guided sextant prostate biopsy were collected. All urine and blood samples were taken before prostate biopsy. Total PSA levels, abnormal digital rectal examination (DRE) and prostate volume were considered for each patient. Total uPAR antigen in urine and serum was measured by specific ELISA. Urinary suPAR levels were normalized for dilution by the creatinine content of the samples. Urine and serum suPAR density (urine or serum suPAR/prostate volume, D-suPAR) were also calculated. The Mann-Whitney U test and the T-test were used to compare differences between groups. Multivariate analysis was performed using the binary logistic regression. RESULTS: Twenty-four patients were found to have prostate cancer and 38 had no evidence of malignancy. There was no significant difference in age or t-PSA between those men with positive and those with negative biopsies. The frequency of abnormal digital rectal examination resulted higher in patients with positive biopsies (p=0.032). The median suPAR serum levels was 2.28 ng/ml in cancer group and 1.57 ng/ml in negative group (p=0.011). The median suPAR urine levels wasn’t significantly elevated in carcinoma patients (5.68 ng/ml) compared to negative patients (3.81 ng/ml; p=0.221). Prostate cancer patients expressed significantly higher levels of serum D-suPAR (0.043 vs. 0.020; p=0.002) and of urine D-suPAR (0.095 vs. 0.039; p=0.006). In a multivariate model, t-PSA, DRE and urine D-suPAR were not predictive factors of positive biopsies, while serum D-suPAR increased the probability of positive biopsies by 13.3 times (p=0.034). CONCLUSIONS: SuPAR in serum of patients with clinical suspicion of prostate cancer might provide clinically relevant information about the state of the prostate gland; the measurement of serum D-suPAR could help to predict patients with prostate cancer.
1
Laval University, Surgery, Quebec, Canada, 2Gen-probe Incorporated, Research and Development, San Diego, United States, 3Gen-Probe Incorporated, Clinical Affairs, San Diego, United States, 4Diagnocure Incorporated, Research and Development, Quebec, Canada INTRODUCTION & OBJECTIVES: PCA3 mRNA is prostate-specific and highly overexpressed in prostate tumour cells. The APTIMA PCA3 assay currently in development uses Transcription-Mediated Amplification (TMA) to quantify PCA3 and PSA mRNAs derived from prostate cells in urine, and is being evaluated for its potential to improve the clinical evaluation of prostate disease. Sensitivity, specificity, and specimen informative rate of the prototype APTIMA PCA3 assay were assessed using male urines obtained after digital rectal examination (DRE). MATERIAL & METHODS: Urine specimens were collected following DRE from men scheduled for biopsy (n = 355) or radical prostatectomy (n = 62), and stabilized in a detergent buffer. PCA3 and PSA mRNAs were isolated, amplified and quantified. Biopsy results were correlated with the ratio of PCA3/PSA mRNA. The PSA copy level was used to normalize PCA3 signals and confirm sufficient prostate-specific mRNA in the sample. Informative rate was defined as the fraction of specimens yielding sufficient RNA for analysis. The biopsy negative population included a high percentage of prostatic intraepithelial neoplasia (PIN) and atypical small acinar proliferation (ASAP); since PCA3 mRNA expression is elevated in PIN, analysis was performed both with and without these subjects included. RESULTS: The study contained 180 biopsy-positive and 237 biopsy-negative subjects (138 with PIN or ASAP). The specimen informative rate was 95.2%. Average PCA3/PSA mRNA ratios (x1000) for Negative, PIN or ASAP, and Positive subjects were 20, 50, and 70 respectively. Receiver operating characteristics (ROC) curve analysis yielded an area under the curve of 0.680; specificity was 76% at 50% sensitivity. By comparison, serum PSA specificity was 22% for the same group. Excluding PIN or ASAP subjects, PCA3 specificity increased to 91% at 50% sensitivity, with an AUC of 0.778 (serum PSA assay specificity was unaffected). CONCLUSIONS: The high specimen informative rate and the ability to use whole urine as a specimen make the assay adaptable for use in a clinical laboratory setting. Results of this study suggest the APTIMA PCA3 assay could add increased specificity to the current algorithm for diagnosis of prostate cancer.
Eur Urol Suppl 2006;5(2):275
1010
DIFFERENTIATION ENHANCEMENT OF CIRCULATING IMMUNE CELLS CONTAINING INTRACELLULAR PSA: A NEW METHOD FOR DISCRIMINATION BETWEEN BENIGN AND MALIGNANT PROSTATIC DISEASE
SOLUBLE RECEPTOR OF HUMAN CYTOKINE IL-6 (SIL-6R) ON 123 PATIENTS WITH UNTREATED PROSTATE CANCER (PCA) Pina F.1, Figueiredo G.2, Lunet N.3, Tomada N.1, Silva A.1, Cruz F.1, Barros H.3 1
1
Faculdade Medicina Universidade Porto, Urology, Porto, Portugal, 2Faculdade Medicina Universidade Porto, Immunology, Porto, Portugal, 3Faculdade Medicina Universidade Porto, Epidemiology, Porto, Portugal
INTRODUCTION & OBJECTIVES: A new test measuring the percentage of PSA-positive macrophages (Mf) in peripheral blood has previously been demonstrated to be useful in discriminating between benign and malignant prostatic disease. In order to find out, if further differentiation provides better information about these cells and still enables to distinguish between benign and malignant disease, Mf of patients with prostate cancer and healthy controls were extracellularly stained for CD14 and CD16, which is a marker for immune cell activation, and intracellularly stained for detecting PSA.
INTRODUCTION & OBJECTIVES: IL-6 has important growth factor biological activity on prostate cancer (PC) through IL-6R↔gp130 receptor complex or soluble IL-6 receptor (sIL-6R). PC cells are able to constitutively produce either IL6 or sIL-6R, and pathologic conditions with high IL-6 levels (like hormone-refractory PC) may also present increased sIL-6R production. PC patients with high serum IL-6 (> 4 or > 7 ng/ml) are significantly related to progression and specific survival, and the same seems to be truth for sIL-6R. We investigate this receptor under base-line condition in a group of untreated PC patients checking for relation to several prognostic factors.
Herwig R.1, Kramer G.1, Djavan B.1, Rehder P.2, Ramoner R.2, Marberger M.1 Medical University Vienna, Urology, Vienna, Austria, 2Medical University Innsbruck, Urology, Innsbruck, Austria
MATERIAL & METHODS: Peripheral blood mononuclear cells were isolated from whole blood samples obtained from 25 patients with prostate cancer and 10 healthy controls. Cells were analysed according to extracellular signal for CD14+ and CD16+ using flow cytomety (FACS Calibur, USA). Intracellular staining was subsequently performed using a cell permeabilisation kit and monoclonal antibodies against PSA (Clone ER-PR8). Data were calculated as mean percentage with standard deviation (SD) of positive cells. RESULTS: As before, differentiation between malignant and benign disease was possible using the CD14/PSA staining. Additional staining with CD16 leads to further and better differentiation of a PSA containing CD14 / CD16 positive cell population. PSA could only be found in activated CD14+ cells. The mean percentage of PSA containing macrophages was 0.58 in healthy controls vs. 14.75% (localised prostate cancer) and 84.67% (metastatic prostate cancer) respectively. In contrast to the new method, measurement of serum total PSA levels showed such a high SD that no distinction between controls, prostate cancer and metastatic disease could be drawn. CONCLUSIONS: Measurement of the amount of intracellular PSA in peripheral blood macrophages enables clear distinction between benign and malignant disease. Moreover, localised prostate cancer significantly differed from metastatic disease with regard to the levels of circulating Mf . Thus, the highest level was found in metastatic prostate cancer and the lowest in levels in controls, suggesting that malignancy correlates with the amount of positive Mf . The population of PSA containing macrophages could be better defined and visualized as demonstrated before, leading to a better analysis of the underlying cases. Further distinction of these cells may lead to better stratification of patients and research on this field in future.
MATERIAL & METHODS: 125 PC patients (mean age 70.2 years; mean tPSA 34.4 ng/ ml) were staged and stratified in progression risk groups of cTNM, biopsy Gleason, tPSA; 43 PC performed RP and were restaged and restratified according to pTNM, RP Gleason, tPSA and D’AMICO RP risk groups. Blood was collected for haemogram, biochemic, ALKP, tPSA, fPSA, tTestosterone, PRL, lymphocytes CD3+DR+, CD4+DR+ and CD8+DR+, human sIL-6R (DR600, m = 31,0 [14,0-46,0] pg/ml; Quantikine™/RDSystems, ELISA). Statistical Analysis: Proportions were compared with Chi2 test; Kruskal-Wallis test was used for quantitative variables. RESULTS: Among PC patients sIL-6R was detected in all cases, with a median of 31.0 pg/ml and with high levels (> 46 pg/ml) in 74%. No significant differences of sIL-6R were detected among prognostic groups of cTNM, biopsy Gleason score, tPSA, cPSA, pTNM, RP Gleason score, or D’AMICO RP groups. Median sIL-6R was significantly different between cases with f/t PSA ≤ 0.26 or > 0.26 (p=0.001). Analysis of continuous variables only showed positive significant correlation between sIL-6R and total activated (CD3+DR+) and cytotoxic activated (CD8+DR+) lymphocytes. CONCLUSIONS: IL-6sR is elevated in the great majority of PC patients. Unlike other reports we found no parallelism of increased sIL-6R values with most usual clinical or pathological prognostic risk groups. PC cases with grey zone tPSA > 0.26 and sIL-6R high values may represent a group with PC delayed diagnosis. The link of IL-6 axis to cellular cytotoxic immunity is suggested by significant association of sIL-6R and CD8+DR+ lymphocytes. Long follow-up of our patients is needed to reveal Shariat et all-2004 statement on the sIL-6R association to RP aggressive biochemical recurrence.
1011
1012
SERUM SOLUBLE UROKINASE-TYPE PLASMINOGEN ACTIVATOR RECEPTOR (SUPAR) IS A USEFUL MARKER OF PROSTATE CANCER
APTIMA® PCA3 MOLECULAR URINE TEST: DEVELOPMENT OF A METHOD TO AID IN THE DIAGNOSIS OF PROSTATE CANCER
Milanese G.1, Gasparri L.1, Dellabella M.2, Sidenius N.3, Galosi A.B.2, Minardi D.4, Blasi F.5, Fazioli F.6, Muzzonigro G.4
Fradet Y.1, Groskopf J.2, Aubin S.M. J.2, Deras I.L.2, Blase A.3, Bodrug S.2, Clark C.2, Brentano S.2, Desaulniers M.4, Rittenhouse H.2
Polytechnic University of The Marche Region, Urology, Ancona, Italy, 2A.O.U. Umberto I Lancisi - Salesi, Urology, Ancona, Italy, 3San Raffaele Scientific Institute and Ifom Foundation, Molecular Genetics Unit, Dibit, Milano, Italy, 4Polytechnic University of The Marche Region, A.O.U. Umberto I - Lancisi - Salesi, Urology, Ancona, Italy, 5San Raffaele Scientific Institute, Molecular Genetics Unit, Dibit, Milano, Italy, 6Polytechnic University of The Marche Region, Laboratory of Cellular and Molecular Biology, Ancona, Italy
1
INTRODUCTION & OBJECTIVES: Numerous studies indicate that urokinase-type plasminogen activator receptor (uPAR) is involved in cancer. We investigated the expression of uPAR in body fluids of patients with clinical suspicion of prostate cancer to evaluate its possible role as prostate cancer marker. MATERIAL & METHODS: Urine and serum from 63 consecutive patients referred to our institution for TRUS-guided sextant prostate biopsy were collected. All urine and blood samples were taken before prostate biopsy. Total PSA levels, abnormal digital rectal examination (DRE) and prostate volume were considered for each patient. Total uPAR antigen in urine and serum was measured by specific ELISA. Urinary suPAR levels were normalized for dilution by the creatinine content of the samples. Urine and serum suPAR density (urine or serum suPAR/prostate volume, D-suPAR) were also calculated. The Mann-Whitney U test and the T-test were used to compare differences between groups. Multivariate analysis was performed using the binary logistic regression. RESULTS: Twenty-four patients were found to have prostate cancer and 38 had no evidence of malignancy. There was no significant difference in age or t-PSA between those men with positive and those with negative biopsies. The frequency of abnormal digital rectal examination resulted higher in patients with positive biopsies (p=0.032). The median suPAR serum levels was 2.28 ng/ml in cancer group and 1.57 ng/ml in negative group (p=0.011). The median suPAR urine levels wasn’t significantly elevated in carcinoma patients (5.68 ng/ml) compared to negative patients (3.81 ng/ml; p=0.221). Prostate cancer patients expressed significantly higher levels of serum D-suPAR (0.043 vs. 0.020; p=0.002) and of urine D-suPAR (0.095 vs. 0.039; p=0.006). In a multivariate model, t-PSA, DRE and urine D-suPAR were not predictive factors of positive biopsies, while serum D-suPAR increased the probability of positive biopsies by 13.3 times (p=0.034). CONCLUSIONS: SuPAR in serum of patients with clinical suspicion of prostate cancer might provide clinically relevant information about the state of the prostate gland; the measurement of serum D-suPAR could help to predict patients with prostate cancer.
1
Laval University, Surgery, Quebec, Canada, 2Gen-probe Incorporated, Research and Development, San Diego, United States, 3Gen-Probe Incorporated, Clinical Affairs, San Diego, United States, 4Diagnocure Incorporated, Research and Development, Quebec, Canada INTRODUCTION & OBJECTIVES: PCA3 mRNA is prostate-specific and highly overexpressed in prostate tumour cells. The APTIMA PCA3 assay currently in development uses Transcription-Mediated Amplification (TMA) to quantify PCA3 and PSA mRNAs derived from prostate cells in urine, and is being evaluated for its potential to improve the clinical evaluation of prostate disease. Sensitivity, specificity, and specimen informative rate of the prototype APTIMA PCA3 assay were assessed using male urines obtained after digital rectal examination (DRE). MATERIAL & METHODS: Urine specimens were collected following DRE from men scheduled for biopsy (n = 355) or radical prostatectomy (n = 62), and stabilized in a detergent buffer. PCA3 and PSA mRNAs were isolated, amplified and quantified. Biopsy results were correlated with the ratio of PCA3/PSA mRNA. The PSA copy level was used to normalize PCA3 signals and confirm sufficient prostate-specific mRNA in the sample. Informative rate was defined as the fraction of specimens yielding sufficient RNA for analysis. The biopsy negative population included a high percentage of prostatic intraepithelial neoplasia (PIN) and atypical small acinar proliferation (ASAP); since PCA3 mRNA expression is elevated in PIN, analysis was performed both with and without these subjects included. RESULTS: The study contained 180 biopsy-positive and 237 biopsy-negative subjects (138 with PIN or ASAP). The specimen informative rate was 95.2%. Average PCA3/PSA mRNA ratios (x1000) for Negative, PIN or ASAP, and Positive subjects were 20, 50, and 70 respectively. Receiver operating characteristics (ROC) curve analysis yielded an area under the curve of 0.680; specificity was 76% at 50% sensitivity. By comparison, serum PSA specificity was 22% for the same group. Excluding PIN or ASAP subjects, PCA3 specificity increased to 91% at 50% sensitivity, with an AUC of 0.778 (serum PSA assay specificity was unaffected). CONCLUSIONS: The high specimen informative rate and the ability to use whole urine as a specimen make the assay adaptable for use in a clinical laboratory setting. Results of this study suggest the APTIMA PCA3 assay could add increased specificity to the current algorithm for diagnosis of prostate cancer.
Eur Urol Suppl 2006;5(2):275