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DEVELOPMENT OF A MULTIPLEXED URINE ASSAY FOR PROSTATE CANCER DIAGNOSIS
Vol. 179, No. 4, Supplement, Wednesday, May 21, 2008
1991 DETECTION OF GSTP1 HYPERMETHYLATION AND HEPSIN ACTIVITY FOR PROSTATE NON-INVASIVE CANCER DIAGNOSTICS Evgenia Vasileva*, Maria Savvateeva, Ekaterina Kuznetsova, Dmitriy Fiev, Olga Abakumova, Alla Lyashenko, Andrey Vinarov, Evgeniy Severin. Moscow, Russian Federation. INTRODUCTION AND OBJECTIVE: We assess the feasibility of a urinary test for prostate cancer (PCa) detection in a high-risk patient JURXSEDVHGRQPHWK\ODWLRQVSHFL¿F3&5 PHW3&5 DQDO\VLVRIʌFODVV of glutathion S-transferases gene (GSTP1) promoter and on the analysis of amydolytic activity of hepsin, a type II transmembrane serine protease. It is known that promoter hypermethylation is a common epigenetic alteration affecting cancer-related genes. Several cDNA microarray studies have shown that hepsin is one of the highly overexpressed genes in prostate cancer tissue compared with nonmalignant and benign prostatic hyperplasia tissue. METHODS: We collected the urine specimens from the patients with suspicious on PCa immediately after DRE (digital rectal examination). Genomic DNA was isolated and methylation of GSTP1 JHQHZDVDQDO\]HGE\PHW3&5+HSVLQDFWLYLW\ZDVDQDO\]HGXVLQJ FKURPRJHQLFVXEVWUDWH6SHFWUR]\PH $PHULFDQ'LDJQRVWLFD,QF :H correlated our data with clinical information obtained from the patient record (PSA, TRUS, biopsy). RESULTS: We detected that methylation of GSTP1 occurred in cells from cancer and non-cancer patients (adenoma, prostatitis, PIN). 7KHUH ZDV QR VWDWLVWLFDOO\ VLJQL¿FDQW GLIIHUHQFH EHWZHHQ WKH JURXSV (p=0.81). It means, that it is impossible to differentiate a cancer from noncancer case by using only one marker. We did not detected methylated cytosines in GSTP1- gene in the health donors urine samples. These results suggested that methylation of GSTP1 may be used as a molecular genetic biomarkers of pathological prostate methabolism. There was no detectable hepsin activity in the normal cells, but more than 70% of urine samples from cancer patients were hepsin-positive (p=0.020). It means, that hepsin activity measurement allows distinguishing cancer from non-cancer urine samples with great probability. CONCLUSIONS: According to our investigation prognostic positive values of the markers were: 0.71 for hepsin, 0.5 for met-GSTP1 and 0.85 for combined use of these markers. Our results demonstrated that a screening test based on combined GSTP1 methylation and hepsin activity detection in the urine specimens of patients with suspected prostate malignancy may be a useful adjunct or probable substitution to serum PCA-screening tests. This molecular assay has potential application for distribution of patients into low- and high-risk groups for surveillance versus repeat biopsy. Source of Funding: None
1992 DEVELOPMENT OF A MULTIPLEXED URINE ASSAY FOR PROSTATE CANCER DIAGNOSIS Tatiana Vener*, Carlo Derecho, Haiying Wang, Yashoda Rajpurohit, Dondapati Chowdary, Abhijit Mazumder. Warren, NJ. INTRODUCTION AND OBJECTIVE: Several studies have demonstrated the value of DNA methylation in urine-based assays for prostate cancer diagnosis. This study describes a multicenter validation with a prototype assay. METHODS: We developed a multiplexed, quantitative PHWK\ODWLRQVSHFL¿FSRO\PHUDVHFKDLQUHDFWLRQ 063 DVVD\FRQVLVWLQJ RI JOXWDWKLRQH6WUDQVIHUDVH 3 *673 UHWLQRLF DFLG UHFHSWRU ȕ 5$5ȕ DGHQRPDWRXVFROLSRO\SRVLV $3& DQGȕDFWLQLQDFORVHG tube, homogeneous assay format. We tested this format with urine samples collected post digital rectal examination (DRE) from 234 patients (PSA levels from 2.5 to 10 ng/mL) in two independent patient cohorts IURP QLQH FOLQLFDO VLWHV 7KH ¿UVW FRKRUW ZDV SDWLHQWV ELRSV\ positive and 69 biopsy-negative). Samples were shipped to Veridex at 4oC within 5 days of collection. The second cohort was 113 patients (58 biopsy-positive and 55 biopsy-negative). These samples were kept at 4oC and sedimented within 24 hours prior to shipping to Veridex. 5(68/76 ,QWKH¿UVWFRKRUWRISDWLHQWVZHGHPRQVWUDWHD FOLQLFDOVHQVLWLYLW\RIDQGVSHFL¿FLW\RIIRUGHWHFWLRQRISURVWDWH cancer with an area under the curve value of 0.691. In the second cohort
THE JOURNAL OF UROLOGY®
685
RI SDWLHQWV VHQVLWLYLW\ ZDV DQG VSHFL¿FLW\ ZDV WKXV showing comparable assay performance in both cohorts. Assay results were not used for patient management. Importantly, the GSTP1 cycle WKUHVKROG &W YDOXHEXWQRWWKRVHRI5$5ȕRU$3&GHPRQVWUDWHGD good correlation (R=0.84) with the number of cores found to contain prostate cancer or pre-malignant lesions on biopsy. Moreover, samples WKDWH[KLELWHGPHWK\ODWLRQIRUHLWKHU*673RU5$5ȕW\SLFDOO\FRQWDLQHG higher tumor volumes at prostatectomy than those samples that did not exhibit this methylation. APC methylation did not correlate to tumor YROXPH /DVWO\ RQO\ 5$5ȕ &W YDOXHV VKRZHG D PRGHVW FRUUHODWLRQ (R=0.57) with age in biopsy positive patients. CONCLUSIONS: These data demonstrate that the performance of this prototype assay has a potential to add value to the biopsy decision making process. Risk assessment of prostate cancer may be improved by using this assay as an adjunct to current screening tools. Source of Funding: None
1993 DETECTION OF TMPRSS2-ERG FUSION TRANSCRIPTS AND PCA3 IN URINARY SEDIMENTS MAY IMPROVE DIAGNOSIS OF PROSTATE CANCER Daphne Hessels*, Frank P Smit, Gerald W Verhaegh, J Alfred Witjes, Erik B Cornel, Jack A Schalken. Nijmegen, The Netherlands. INTRODUCTION AND OBJECTIVE: Early detection of prostate cancer can increase the curative success rate for prostate cancer. We studied the diagnostic usefulness of TMPRSS2-ERG fusion transcripts as well as the combination of PCA3 RNA and TMPRSS2-ERG fusion transcripts in urinary sediments after DRE. METHODS: 78 men with prostate cancer-positive biopsies and 30 men with prostate cancer-negative biopsies were included in WKLVVWXG\$IWHU'5(WKH¿UVWYRLGHGXULQHZDVFROOHFWHGDQGXULQDU\ sediments were obtained. We used semi-quantitative RT-PCR analysis IROORZHGE\6RXWKHUQEORWK\EULGL]DWLRQZLWKDUDGLRODEHOHGSUREHIRUWKH detection TMPRSS2-ERG fusion transcripts in these urinary sediments. A quantitative RT-PCR assay for PCA3 was used to determine the PCA3 score in the same sediments. RESULTS: TMPRSS2-ERG fusion transcripts were detected in the urine after DRE with a sensitivity of 37%. In this cohort of patients the PCA3-based assay had a sensitivity of 62%. When both markers were combined the sensitivity increased to 73%. In a cohort of men with persistently elevated serum PSA levels and history of negative biopsies the positive predictive value of TMPRSS2-ERG fusion transcripts was 94%, suggesting that detection of TMPRSS2-ERG fusion transcripts could give a better indication which patients require repeat biopsies. &21&/86,216 ,QWKLVUHSRUWZHXVHGIRUWKH¿UVWWLPHWKH FRPELQDWLRQRIWKHSURVWDWHFDQFHUVSHFL¿FELRPDUNHUV703566(5* DQG3&$GHPRQVWUDWLQJDVLJQL¿FDQWLPSURYHPHQWLQWKHVHQVLWLYLW\ for prostate cancer diagnosis. Source of Funding: None
1994 QUANTITATION OF TMPRSS2:ERG FUSION TRANSCRIPTS IN FROZEN AND PARAFFIN EMBEDDED PROSTATE CANCERS (PC) Dean A Troyer*, Hongyin Bu, Rosario Mendez-Meza, Roble G Bedolla, Aamir Ehsan, Hongxin Fan. San Antonio, TX. INTRODUCTION AND OBJECTIVE: Using quantitative real time RT-PCR (QR-PCR), rearrangements between TMPRSS2 gene (21q22.3) and the ETS transcription factor family members ERG (21q22.2), ETV1 (7p21.2), ETV4 (17q21) are detected in PC..We studied TMPRSS2:ERG fusion, the most common of these. METHODS: Sixty-one PC samples from 22 subjects were DQDO\]HG3DLUHGIUR]HQDQGIRUPDOLQ¿[HGSDUDI¿QHPEHGGHGWLVVXH (FFPE) were evaluated. Total RNA was extracted using the BioRobot® (=ZRUNVWDWLRQDQG(=51$8QLYHUVDO7LVVXH.LW 4LDJHQ IRUIUR]HQ tissues, or RNeasy FFPE Kit (Qiagen) for FFPE. TMRPSS2:ERG fusion transcripts were detected by either TaqMan (targeting T1/E4 transcript, PRGL¿HGIURP/X[PDQ%HWDO RU6<%(*UHHQ WDUJHWLQJ7( Tomlins et al, 2005) QR-PCR. ERG levels were detected by SYBE Green QR-PCR (targeting E5-6/E6, Tomlins et al, 2005) using an ABI PRISM 7900HT (Applied Biosystems). Fusion and house keeping (HK) gene
1991 DETECTION OF GSTP1 HYPERMETHYLATION AND HEPSIN ACTIVITY FOR PROSTATE NON-INVASIVE CANCER DIAGNOSTICS Evgenia Vasileva*, Maria Savvateeva, Ekaterina Kuznetsova, Dmitriy Fiev, Olga Abakumova, Alla Lyashenko, Andrey Vinarov, Evgeniy Severin. Moscow, Russian Federation. INTRODUCTION AND OBJECTIVE: We assess the feasibility of a urinary test for prostate cancer (PCa) detection in a high-risk patient JURXSEDVHGRQPHWK\ODWLRQVSHFL¿F3&5 PHW3&5 DQDO\VLVRIʌFODVV of glutathion S-transferases gene (GSTP1) promoter and on the analysis of amydolytic activity of hepsin, a type II transmembrane serine protease. It is known that promoter hypermethylation is a common epigenetic alteration affecting cancer-related genes. Several cDNA microarray studies have shown that hepsin is one of the highly overexpressed genes in prostate cancer tissue compared with nonmalignant and benign prostatic hyperplasia tissue. METHODS: We collected the urine specimens from the patients with suspicious on PCa immediately after DRE (digital rectal examination). Genomic DNA was isolated and methylation of GSTP1 JHQHZDVDQDO\]HGE\PHW3&5+HSVLQDFWLYLW\ZDVDQDO\]HGXVLQJ FKURPRJHQLFVXEVWUDWH6SHFWUR]\PH $PHULFDQ'LDJQRVWLFD,QF :H correlated our data with clinical information obtained from the patient record (PSA, TRUS, biopsy). RESULTS: We detected that methylation of GSTP1 occurred in cells from cancer and non-cancer patients (adenoma, prostatitis, PIN). 7KHUH ZDV QR VWDWLVWLFDOO\ VLJQL¿FDQW GLIIHUHQFH EHWZHHQ WKH JURXSV (p=0.81). It means, that it is impossible to differentiate a cancer from noncancer case by using only one marker. We did not detected methylated cytosines in GSTP1- gene in the health donors urine samples. These results suggested that methylation of GSTP1 may be used as a molecular genetic biomarkers of pathological prostate methabolism. There was no detectable hepsin activity in the normal cells, but more than 70% of urine samples from cancer patients were hepsin-positive (p=0.020). It means, that hepsin activity measurement allows distinguishing cancer from non-cancer urine samples with great probability. CONCLUSIONS: According to our investigation prognostic positive values of the markers were: 0.71 for hepsin, 0.5 for met-GSTP1 and 0.85 for combined use of these markers. Our results demonstrated that a screening test based on combined GSTP1 methylation and hepsin activity detection in the urine specimens of patients with suspected prostate malignancy may be a useful adjunct or probable substitution to serum PCA-screening tests. This molecular assay has potential application for distribution of patients into low- and high-risk groups for surveillance versus repeat biopsy. Source of Funding: None
1992 DEVELOPMENT OF A MULTIPLEXED URINE ASSAY FOR PROSTATE CANCER DIAGNOSIS Tatiana Vener*, Carlo Derecho, Haiying Wang, Yashoda Rajpurohit, Dondapati Chowdary, Abhijit Mazumder. Warren, NJ. INTRODUCTION AND OBJECTIVE: Several studies have demonstrated the value of DNA methylation in urine-based assays for prostate cancer diagnosis. This study describes a multicenter validation with a prototype assay. METHODS: We developed a multiplexed, quantitative PHWK\ODWLRQVSHFL¿FSRO\PHUDVHFKDLQUHDFWLRQ 063 DVVD\FRQVLVWLQJ RI JOXWDWKLRQH6WUDQVIHUDVH 3 *673 UHWLQRLF DFLG UHFHSWRU ȕ 5$5ȕ DGHQRPDWRXVFROLSRO\SRVLV $3& DQGȕDFWLQLQDFORVHG tube, homogeneous assay format. We tested this format with urine samples collected post digital rectal examination (DRE) from 234 patients (PSA levels from 2.5 to 10 ng/mL) in two independent patient cohorts IURP QLQH FOLQLFDO VLWHV 7KH ¿UVW FRKRUW ZDV SDWLHQWV ELRSV\ positive and 69 biopsy-negative). Samples were shipped to Veridex at 4oC within 5 days of collection. The second cohort was 113 patients (58 biopsy-positive and 55 biopsy-negative). These samples were kept at 4oC and sedimented within 24 hours prior to shipping to Veridex. 5(68/76 ,QWKH¿UVWFRKRUWRISDWLHQWVZHGHPRQVWUDWHD FOLQLFDOVHQVLWLYLW\RIDQGVSHFL¿FLW\RIIRUGHWHFWLRQRISURVWDWH cancer with an area under the curve value of 0.691. In the second cohort
THE JOURNAL OF UROLOGY®
685
RI SDWLHQWV VHQVLWLYLW\ ZDV DQG VSHFL¿FLW\ ZDV WKXV showing comparable assay performance in both cohorts. Assay results were not used for patient management. Importantly, the GSTP1 cycle WKUHVKROG &W YDOXHEXWQRWWKRVHRI5$5ȕRU$3&GHPRQVWUDWHGD good correlation (R=0.84) with the number of cores found to contain prostate cancer or pre-malignant lesions on biopsy. Moreover, samples WKDWH[KLELWHGPHWK\ODWLRQIRUHLWKHU*673RU5$5ȕW\SLFDOO\FRQWDLQHG higher tumor volumes at prostatectomy than those samples that did not exhibit this methylation. APC methylation did not correlate to tumor YROXPH /DVWO\ RQO\ 5$5ȕ &W YDOXHV VKRZHG D PRGHVW FRUUHODWLRQ (R=0.57) with age in biopsy positive patients. CONCLUSIONS: These data demonstrate that the performance of this prototype assay has a potential to add value to the biopsy decision making process. Risk assessment of prostate cancer may be improved by using this assay as an adjunct to current screening tools. Source of Funding: None
1993 DETECTION OF TMPRSS2-ERG FUSION TRANSCRIPTS AND PCA3 IN URINARY SEDIMENTS MAY IMPROVE DIAGNOSIS OF PROSTATE CANCER Daphne Hessels*, Frank P Smit, Gerald W Verhaegh, J Alfred Witjes, Erik B Cornel, Jack A Schalken. Nijmegen, The Netherlands. INTRODUCTION AND OBJECTIVE: Early detection of prostate cancer can increase the curative success rate for prostate cancer. We studied the diagnostic usefulness of TMPRSS2-ERG fusion transcripts as well as the combination of PCA3 RNA and TMPRSS2-ERG fusion transcripts in urinary sediments after DRE. METHODS: 78 men with prostate cancer-positive biopsies and 30 men with prostate cancer-negative biopsies were included in WKLVVWXG\$IWHU'5(WKH¿UVWYRLGHGXULQHZDVFROOHFWHGDQGXULQDU\ sediments were obtained. We used semi-quantitative RT-PCR analysis IROORZHGE\6RXWKHUQEORWK\EULGL]DWLRQZLWKDUDGLRODEHOHGSUREHIRUWKH detection TMPRSS2-ERG fusion transcripts in these urinary sediments. A quantitative RT-PCR assay for PCA3 was used to determine the PCA3 score in the same sediments. RESULTS: TMPRSS2-ERG fusion transcripts were detected in the urine after DRE with a sensitivity of 37%. In this cohort of patients the PCA3-based assay had a sensitivity of 62%. When both markers were combined the sensitivity increased to 73%. In a cohort of men with persistently elevated serum PSA levels and history of negative biopsies the positive predictive value of TMPRSS2-ERG fusion transcripts was 94%, suggesting that detection of TMPRSS2-ERG fusion transcripts could give a better indication which patients require repeat biopsies. &21&/86,216 ,QWKLVUHSRUWZHXVHGIRUWKH¿UVWWLPHWKH FRPELQDWLRQRIWKHSURVWDWHFDQFHUVSHFL¿FELRPDUNHUV703566(5* DQG3&$GHPRQVWUDWLQJDVLJQL¿FDQWLPSURYHPHQWLQWKHVHQVLWLYLW\ for prostate cancer diagnosis. Source of Funding: None
1994 QUANTITATION OF TMPRSS2:ERG FUSION TRANSCRIPTS IN FROZEN AND PARAFFIN EMBEDDED PROSTATE CANCERS (PC) Dean A Troyer*, Hongyin Bu, Rosario Mendez-Meza, Roble G Bedolla, Aamir Ehsan, Hongxin Fan. San Antonio, TX. INTRODUCTION AND OBJECTIVE: Using quantitative real time RT-PCR (QR-PCR), rearrangements between TMPRSS2 gene (21q22.3) and the ETS transcription factor family members ERG (21q22.2), ETV1 (7p21.2), ETV4 (17q21) are detected in PC..We studied TMPRSS2:ERG fusion, the most common of these. METHODS: Sixty-one PC samples from 22 subjects were DQDO\]HG3DLUHGIUR]HQDQGIRUPDOLQ¿[HGSDUDI¿QHPEHGGHGWLVVXH (FFPE) were evaluated. Total RNA was extracted using the BioRobot® (=ZRUNVWDWLRQDQG(=51$8QLYHUVDO7LVVXH.LW 4LDJHQ IRUIUR]HQ tissues, or RNeasy FFPE Kit (Qiagen) for FFPE. TMRPSS2:ERG fusion transcripts were detected by either TaqMan (targeting T1/E4 transcript, PRGL¿HGIURP/X[PDQ%HWDO RU6<%(*UHHQ WDUJHWLQJ7( Tomlins et al, 2005) QR-PCR. ERG levels were detected by SYBE Green QR-PCR (targeting E5-6/E6, Tomlins et al, 2005) using an ABI PRISM 7900HT (Applied Biosystems). Fusion and house keeping (HK) gene