Expression of Prostate-Specific Membrane Antigen in Transitional Cell Carcinoma of the Bladder: Prognostic Value?1

Vol. 6, 4049–4054, October 2000 Clinical Cancer Research 4049

Expression of Prostate-specific Membrane Antigen in Transitional Cell Carcinoma of the Bladder: Prognostic Value?1

Jean-Luc Gala,2 Sylvain Loric, Yves Guiot, PSMA mRNA PCR assay may be a useful tool to predict a Samuel Ray Denmeade, Alyssa Gady, poor outcome in TCC patients. Francis Brasseur, Michel Heusterspreute, INTRODUCTION Pascal Eschwe`ge, Philippe De Nayer, Ninety % of bladder tumors are derived from the urothe- Paul Van Cangh, and Bertrand Tombal lium, the transitional epithelium that lines the urinary bladder Applied Molecular Technology, Department of Clinical Biochemistry, and the ureter (1). The natural history of TCCs3 varies widely, Saint-Luc Clinical University, Universite´Catholique de Louvain, although it is characterized by a high rate of recurrence with an 1200 Brussels, Belgium [J-L. G., M. H., P. D. N.]; Queen Astrid Military Hospital, 1120 Brussels, Belgium [J-L. G.]; Clinical aggressive clinical course (2). Many extensive studies have been Biochemistry Laboratory, St. Antoine University Hospital, 75012 performed to determine whether particular oncogenes, tumor Paris, and Cellular Differentiation Laboratory, Pasteur Institute, suppressor genes, or chromosomal changes are involved in CNRS URA 1960, 75015 Paris, France [S. L.]; Departments of bladder tumors to identify tumors with a high risk of recurrence Pathology [Y. G.] and Urology [P. V. C., B. T.], Saint-Luc Clinical and progression. None of these markers, however, has been University, Brussels 1200, Belgium; Johns Hopkins Oncology Center, Baltimore, Maryland 21287 [S. R. D., A. G.]; Ludwig Institute for demonstrated to improve the diagnosis and treatment in indi- Cancer Research, Brussels 1200, Belgium [F. B.]; and Department of vidual patients (3). The depth of infiltration, the differentiation Urology, Hoˆpital Biceˆtre, Le Kremlin-Biceˆtre 94270, France [P. E.] grade, and the presence of concomitant carcinoma in situ are still the most common parameters to predict tumor progression and survival (3). Although ϳ80% of TCCs are initially super- ABSTRACT ficial, recurrence after localized therapy occurs in 30–90% of The expression of Prostate-specific membrane antigen the cases. Despite significant improvement in localized therapy, (PSMA) mRNA was assessed in the normal bladder urothe- intravesical chemotherapy, and immunotherapy, 15–20% of transitional cell carcinoma (TCC) specimens these recurrences evolve to invasive and/or metastatic stages ,(9 ؍ lium (n and preoperative that require radical and/or systemic therapies (2). There is ,(3 ؍ TCC-derived cell lines (n ,(52 ؍ n) Specific PSMA clearly a subgroup of patient (i.e., Ͻ50%) who will survive after .(27 ؍ blood samples from TCC patients (n mRNA was found in 100% of normal and malignant tissues radical surgery and adjuvant therapy (4). The major problem is and two cell lines. PSMA protein was detected in normal selecting those patients at risk of progression, who may benefit -Using a PSMA- from earlier aggressive treatment (i.e., radiotherapy, chemother .(4 ؍ and malignant tissues (n (3 ؍ n) specific substrate, PSMA enzymatic activity was found in apy, and immune therapy). Because no bladder urothelium- two bladder cell lines and correlated with immunostaining. specific serum marker is currently available, the diagnosis of Seven of the 27 TCC preoperative blood samples were pos- advanced TCC relies on conventional imaging technologies itive by reverse transcription-PCR. These preliminary re- (i.e., computed tomography scan, ultrasound, and magnetic res- sults, obtained on a nonrandomized cohort of patients, cor- onance imaging), the resolution sensitivities of which limit the related with tumor invasion (positive RT-PCR: 0% for pT detection to metastases Ͼ1 cm. Therefore, any biological <2 versus 41% for pT >3) and 2-year survival rate (81% in marker capable of improving the detection of early metastatic the PSMA-negative group versus 29% in the PSMA-positive disease would be of great clinical benefit. group). Although the clinical usefulness of this assay re- PSMA is a Mr 100,000 type II membrane glycoprotein quires confirmation in larger prospective randomized trials, identified by Horoszewicz et al. (5) from crude membrane current preliminary results suggest that a blood-borne extract of the androgen-dependent prostate cancer cell line, LNCaP. The expression of PSMA is low in the normal prostatic epithelium, increases markedly in prostate cancer, and is main- tained in poorly differentiated tumors and prostate cancer metastasis (6). In contrast to secreted prostate-specific antigen, Received 6/21/99; revised 7/13/00; accepted 7/24/00. PSMA is a membrane-bound antigen, the expression of which The costs of publication of this article were defrayed in part by the increases after androgen ablation protein (reviewed in Ref. 7). payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to Several successful strategies have been developed to detect indicate this fact. PSMA protein or mRNA to identify and target prostatic cells (8, 1 Supported by Grant 3.4574.98 from the Fonds National de la Recher- che Scientifique, a grant from l’Oeuvre Belge du Cancer, and Grant 1367 from the Association de Recherche Contre le Cancer. 2 To whom requests for reprints should be addressed, at Applied Mo- lecular Technology, Department of Clinical Biochemistry, Clos 3 The abbreviations used are: TCC, transitional cell carcinoma; PSMA, Chapelle-aux-Champs, 30-UCL/30.46, B-1200 Brussels, Belgium. prostate-specific membrane antigen; RT-PCR, reverse transcription- Phone: 00/32-2-764-31-65 or 00/32-2-764-39-26; Fax: 00/32-2-764-31- PCR; NAALADase, N-acetylated-␣-linked acidic dipeptidase; NAAG, 66; E-mail: [email protected]. N-acetyl-aspartyl-glutamate.

9). Initially believed to be highly prostate specific, PSMA has 10% FCS (Life Technologies, Inc., Gent, Belgium) in a 90%

been shown recently, however, to be expressed in nonprostatic air/5% CO2 incubator at 37°C. tissues such as breast carcinoma, duodenum, and in normal and PSMA RT-PCR Assay. Total RNA from bladder tissue malignant renal tissues (10–12). was obtained by guanidinium/thiocyanate/phenol/chloroform In the present study, the expression of PSMA mRNA, extraction technique (14). Total RNA from cell lines and buffy protein, and enzymatic activity is demonstrated in the normal coats obtained after centrifugation on Ficoll Hypaque of blood bladder, in TCC specimens, and in primary cell lines established samples was extracted using Trizol reagent (Life Technologies, in vitro from TCC specimens. In addition, preliminary clinical Inc., Grand Island, NY) according to the manufacturer’s instruc- data suggest that circulating PSMA-expressing cells can be tion. Details regarding reverse transcription, amplification, and detected in TCC and that detection of PSMA transcript seems to control measurements for preventing carryover have been de- correlate with stage and survival. scribed previously (15). To simplify the assay procedure, the previously described nested RT-PCR, with two rounds of 19 cycles each, was replaced by a single round of 32 cycles. MATERIALS AND METHODS Amplification was performed in a DNA thermal cycler 480 Human Bladder Specimens and Blood Samples. Blad- (Perkin-Elmer, Foster City, CA) by using the previously de- der and blood samples were obtained after informed patient scribed internal PSMA primers (15). The limit of detection for consent. Surgical specimens were collected from 9 normal blad- PSMA with the single-round, 32-cycle PCR assay was assessed ders and 52 TCCs (25 Յ pT1, 27 Ն pT3). Specimens were on serial dilution of LNCaP in blood, LNCaP in the PSMA- snap-frozen in liquid nitrogen for RNA analysis and formalin- negative cell line K562, and finally with serial dilution of 1.2-kb fixed/paraffin-embedded for pathological analysis and immuno- PSMA cDNA plasmid pJRD184, as detailed previously. The histochemical detection of PSMA. Blood samples were obtained limit of detection was defined as the last dilution giving a preoperatively from an additional series of 27 consecutive pa- positive PCR result with an inter-assay coefficient variation tients scheduled to undergo radical cystoprostatectomy for in- Ͻ20% (15). Blood specimens used as negative controls were vasive or recurrent TCC. All patients were males, ages 49–82 obtained from 200 healthy blood donors (100 females and 100 (mean, 67) years. Preoperative stratification and follow-up of males). TCC disease were performed by chest, abdominal, and pelvic Northern Blotting. The pSPORT plasmid coding for the computed tomography scan, abdominal ultrasounds, and liver 2.6-kb PSMA cDNA probe is a generous gift from W. D. W. tests. None of the patients had detectable metastases preopera- Heston (Memorial Sloan-Kettering Cancer Center, New York, tively. Screening for concomitant prostate cancer was based on NY). Northern blot and hybridization were performed on nylon digital rectal examination and serum prostate-specific antigen membranes with UV-fixed poly(Aϩ) RNA (2 ␮g/lane) ex- value. All patients diagnosed with ՆpT3 have received a stand- tracted from normal adult tissues (Clontech, Palo Alto, CA) ard adjuvant MVAC regimen (methotrexate, vinblastine, Adria- according to the manufacturer’s instruction. Gels were autora- mycin, cisplatin). Prostatic and bladder specimens were ana- diographed, and results were normalized for actin expression. lyzed separately, and tumor invasion was stratified according to Scanning densitometry was performed to semiquantitatively as- the Tumor-Node-Metastasis classification. Local and invasive sess the level of expression. node metastatic disease was investigated both by performing DNA Sequencing and Analysis. The PSMA cDNA pathological analysis on tissues obtained at surgery as well as by product from TCC lines (n ϭ 3), TCC surgical specimens (n ϭ using radiological and bone scan investigations. Venous blood 10), and normal bladder urothelia (n ϭ 5) were sequenced in (2 ϫ 10 ml; Sarstedt, Nu¨mbrecht, Germany) was collected in both orientations. The sequencing reactions were carried out on EDTA-treated tubes. All tubes were processed within1hof an automated ABI 373 A apparatus by using the Taq Dye Deoxy collection. Terminator Cycle Sequencing kit (Applied Biosystems) accord- Cell Lines and Reagents. LB831, LB796, and LB905 ing to the manufacturer’s instruction. The specificity of the bladder carcinoma cell lines are primary cultures established remaining PSMA cDNA PCR products was assessed by South- from invasive TCC tumors and were generously provided by the ern transfer, probe radiolabeling, hybridization, and autoradiog- Ludwig Institute for Cancer Research (Brussels, Belgium). raphy as described (15). LB831 has been characterized recently (13). Cells were grown Immunohistochemical Detection of PSMA. Paraffin- in Iscove’s modified Dulbecco’s medium containing 10% FCS, embedded sections, 5-␮m thick, from 3 normal and 4 malignant 116 ␮g/ml L-arginine, 36 ␮g/ml L-asparagine, 219 ␮g/ml L- urothelial tissues, and 3 lymph node metastases from prostatic glutamine, and antibiotics (200 units/ml penicillin and 100 adenocarcinoma were dewaxed, rehydrated, and processed as ␮ g/ml streptomycin) in a 95% air/5% CO2 incubator at 37°C. described previously (16). Cells growing in culture were col- Androgen-dependent human prostate cancer cell lines LNCaP, lected, cytospun onto glass slides, fixed in 4% paraformalde- androgen-independent human prostate cancer cell line PC-3, and hyde, and permeabilized. Cells were incubated overnight at 4°C myeloblastic leukemia cell line K562 were obtained from Amer- with an anti-PSMA monoclonal antibody, CYT-351 (dilution ican Type Tissue Collection (Manassas, VA). An androgen- 1:300), generously provided by Cytogen Corp. (Princetown, independent human prostate cancer cell line, TSU, was gener- NJ), and revealed with an anti-mouse EnVision-Peroxidase sys- ously provided by Dr. John Isaacs (Johns Hopkins Oncology tem (Dako, Golstrup, Denmark). A prostate cancer lymph node Center, Baltimore, MD). Cell lines were grown in RPMI 1640 metastasis and LNCaP cell line were used as positive controls. supplemented with L-glutamine, nonessential amino acids, and Myeloid K562 and prostate PC-3 cell lines were used as nega-

tive controls (8, 15). Staining intensity was semiquantitatively expressed as described (17). PSMA Enzymatic Assay. In previous studies, PSMA has been demonstrated to express a folate hydrolase enzymatic activity (18) and an NAALADase activity (19). Non- PSMA ␥-glutamyl hydrolases are ubiquitously found in the lysosomes of cells and are also secreted by many tumor cell types (20). To specifically determine PSMA enzymatic activity, the NAALADase activity of PSMA was assayed using [3H]NAAG, adapted as described previously by Tiffany et al. (21). The NAALADase activity of LNCaP human prostate cancer cells has been characterized previously, and membranes from these cells were used as positive controls. The Fig. 1 Northern blotting of normal human tissues. Upper lanes, hybridization with PSMA probe; lower lanes, hybridization with control prostate cancer cell lines TSU and PC-3, which do not ␤-actin probe. Lane 1, colon; Lane 2, small intestine; Lane 3, bladder; express PSMA by immunohistochemical or enzymatic anal- Lane 4, heart; Lane 5, stomach; Lane 6, prostate. ysis, were used as negative controls (21). Membranes were prepared and amount of [3H]NAAG hydrolysis determined for each cell type assessed. Briefly, LNCaP, TSU, PC-3, LB905, LB831, and LB796 cell lines were grown to RESULTS AND DISCUSSION 80–90% confluence in serum-containing media as described Limit of Detection of the RT-PCR Assay. The single- above. Ten to 50 million cells were scraped in cold HBSS and round assay consistently detected 10 LNCaP cells in 1 ml of ϫ pelleted at 1000 g at 4°C. The cell pellet was resuspended in venous blood or 1.9 LNCaP cells in 106 peripheral blood mono- cold 50 mM Tris (pH 7.4), sonicated three times for 5 min each, nuclear cells and 1 LNCaP cell in 104 K562. One thousand ϫ and then centrifuged at 100,000 g for 10 min at 4°C. The copies of the cDNA coding PSMA plasmid, pJRD184, were pelleted membranes were resuspended in 50 mM Tris and as- consistently detected, whereas dilutions of 100 and 10 copies sayed for protein content, and additional Tris buffer was added produced, respectively, a weaker consistent band or an incon- to bring the concentration of protein to 0.5 mg/ml. Samples were sistent barely detectable band (data not shown). In comparison, stored at Ϫ80°C before use. the limit of detection of the TCC cell line LB905 diluted in the Regarding the determination of NAALADase activity, PSMA-negative K562 was 1:100. No positive signal was re- 100 ␮g of protein (i.e., 200 ␮l) from each cell line were corded in the 200 blood specimens from normal male/female ␮ ␮ added to PSMA assay buffer (100 lof10mM CoCl2, 250 l blood donors. These results confirm our previous data demon- of 200 mM Tris, pH 7.4), and the volume was brought to strating the absence of illegitimate transcript amplification at 950 ␮l. To determine PSMA-specific activity, the highly such a limit of detection (15). potent PSMA inhibitor 2-(phosphonomethyl)-pentanedioic Expression of PSMA mRNA in a Panel of Normal acid(Alexis, San Diego, CA; Ref. 22) was added to a final Human Tissues. Northern blotting from normal human tis- concentration of 1 ␮M to a second identical sample. After a sues confirmed the expression of PSMA mRNA in several 10-min incubation at room temperature, 50 ␮lof1␮m tissues. Besides a very strong prostate expression, PSMA tran- [3H]NAAG (DuPont NEN, Boston, MA) were added, and scripts were also clearly detected in RNAs extracted from the reactions incubated for 28 h. For assays using LNCaP mem- small intestine, the bladder (Fig. 1), and in extracts from several branes, 5 ␮g of total protein were used per assay, and other tissues including kidney, brain, and liver (data not shown). [3H]NAAG hydrolysis was assayed after only 3 h. At the end Scanning densitometry of the detected products demonstrates of the incubation period, 200 ␮l from each assay were loaded that PSMA expression was ϳ500 times lower in the normal on a column constructed by placing a 3-mm solid glass bead bladder than in the normal prostatic tissue. into a 9-inch Pasteur pipette to which 5 cm of ion exchange Expression of PSMA mRNA in Normal Urothelium,

resin preconditioned with distilled H2O. The column was TCC Surgical Specimens, and TCC-derived Cell Lines. washed with 2 ml of 1 M formic acid into 10 ml of scintil- Specific PSMA mRNA transcripts were detected in each of the lation mixture, and total counts were determined using a 9 normal urothelia, the 52 TCC surgical specimens, and in 2 of Beckman LS 8100 scintillation counter. Specific PSMA ac- the 3 TCC-derived cell lines (Fig. 2). The sequence analysis of tivity was determined by subtracting activity in samples the PCR products from 5 normal urothelia, 10 TCC specimens, containing membranes plus inhibitor from samples contain- and the 2 positive TCC-derived cell lines confirmed that these ing membranes only. A standard curve was plotted using PSMA mRNA transcripts were 100% homologous in length and increasing concentrations of [3H]NAAG to convert measured cDNA sequence to the PSMA mRNA transcripts extracted from counts to pmol [3H]glutamate released. Results were ex- the prostate (8). Southern blot on the remaining samples con- pressed in pmol [3H]glutamate released/(min ϫ mg) of total firmed the specificity of PSMA PCR products (data not shown). protein and are the mean of three separate assays Ϯ SE. Each These results demonstrate that PSMA mRNA is significantly experiment was done in duplicate. Activity was considered expressed in the normal urothelium and TCC tissues or derived not detectable if Ͻ0.001 pmol/(min ϫ mg). cell lines.

Table 1 CYT-351 immunostaining of normal and malignant urothelial cells Intensity and pattern of CYT-351 immunostaining are given for bladder tissues, including bladder-derived cell lines (LB905, LB831, and LB796) as well as normal urothelium and malignant TCC. Results are compared with negative (myeloid K562 and prostate PC-3 cell lines) and positive (prostate LNCaP cell line and surgical specimen of adeno- Fig. 2 Amplification of PSMA transcripts from bladder tissues (dupli- carcinoma) controls. cate samples) with a single-round, 32-cycle PCR assay. Photograph of Cell lines and tissular samples Staining intensity Staining pattern an ethidium bromide-stained gel showing the 193-bp PCR bands: Lanes 1–4, normal urothelium; Lanes 5–7, TCC samples; Lanes 8 and 9, Negative control bladder cell lines LB 905 and LB 831, respectively; Lane 10, negative K562 0 control cell line K562; Lane 11, positive control cell line LNCaP (cDNA PC-3 0 diluted 1:10). A 123-bp molecular weight marker is disclosed at the left Bladder and right borders of the figure. LB905 1–3ϩ Highly heterogeneous LB831 1–2ϩ Heterogeneous LB796 0 Normal urothelium 2–3ϩ Homogeneous Malignant TCC 2–3ϩ Highly heterogeneous PSMA Protein Expression in the Normal Urothelium, Prostate ϩ TCC Surgical Specimens, and in TCC Cell Lines. A posi- LNCaP 5 Homogeneous Adenocarcinoma 5ϩ Homogeneous tive homogeneous staining intensity and pattern of expression were consistently detected in normal and malignant urothelia specimens (Table 1; Fig. 3, A and B). Heterogeneous staining was detected in two of the three TCC-derived cell lines, LB905 and LB831 (Table 1; Fig. 3C). In comparison, PSMA expression prostatic carcinoma (i.e., pT2a) and invasive bladder cancer in LNCaP and prostatic adenocarcinoma specimens was more (i.e., pT3b). Nine of 10 patients with both TCC and localized intense than any of the urothelial-derived tissues (Table 1; prostate cancer had no detectable PSMA mRNA in the blood Fig. 3D). (i.e., 5 TCC Յ pT2 and 4 TCC Ն pT3). These results confirm Measurement of PSMA Enzymatic Activity in Bladder the previously published low rate of RT-PCR detection of Cancer-derived Cell Lines. Using the described assay, a PSMA transcripts in the blood of patients with localized prostate small amount of [3H]NAAG hydrolysis was consistently de- cancer (i.e., 15–25%; reviewed in Ref. 23). TCC specimens tected in LB905 [0.033 Ϯ 0.003 pmol/(min ϫ mg)] and LB831 were classified as follows: pT1 (4 of 27), pT2 (6 of 27), pT3 (16 [0.013 Ϯ 0.001 pmol/(min ϫ mg)] cell lines. In these two of 27), and pT4 (1 of 27). As shown in Table 2, no PSMA positive bladder cell lines, the amount of [3H]NAAG hydrolysis transcript was found in blood of patients presenting with TCC is 1000–2000-fold lower than the average hydrolysis obtained classified pT2 or less. In contrast, 7 of the 17 patients diagnosed measured with prostate cancer cell line LNCaP [29 Ϯ 2.6 with TCC Ն pT3 tested positive for PSMA in blood. According pmol/(min ϫ mg)]. In the PSMA negatively stained TCC- to the Tumor-Node-Metastasis classification of TCC in the latter derived cell line LB796 and prostate cancer cell lines, TSU and 7 PSMA-positive patients, 4 were Mϩ (2Nϩ Mϩ and N0 Mϩ) PC-3, no PSMA activity was detected. and 3 were N0 M0. Among the 10 PSMA-negative patients Ն These results demonstrate that a weak but consistent ex- pT3, 7 were N0 M0 and three were Nϩ M0. pression of PSMA enzymatic activity can be found in the Ten patients died during the follow-up period (range, 1–33 membrane of TCC-derived cell lines. In addition, the difference months; median, 15 months), all from metastatic TCC progres- of the amount of enzymatic activity between these cell lines and sion: 6 of 7 PSMA-positive patients versus 4 of 20 PSMA- the highly PSMA-positive prostate cancer cell line LNCAP negative patients. Conversely, 3 patients developed metastatic accurately reflects the difference of intensity staining detected disease, and 2 died in the group of PSMA-negative patients with by immunohistochemistry. TCC Ն pT3. In patients with TCC Ն pT3, 1- and 2-year Molecular Detection of PSMA mRNA in Blood Samples survival rates correlate with the detection of PSMA transcripts from Patients Treated for TCC of the Bladder. RT-PCR in the preoperative blood specimen. In patients with PSMA- amplification of PSMA mRNA transcripts in blood is reported negative assay, 1- and 2-year survival rates were 88 and 79%, to improve early detection of advanced prostate cancer (7–9). respectively, whereas positive detection was associated with a Because PSMA expression has been demonstrated in bladder, it drop of the survival rate to 75 and 29% at 1 and 2 years, was legitimate to assess whether PSMA transcripts could be respectively. Although confirmation requires larger prospective detected in bladder cancer patients. Screening for concomitant studies, these results suggest that PSMA-positive patients have prostate cancer, based on digital rectal examination and serum a worse outcome. The possibility that molecular detection of prostate-specific antigen value, was negative in all cases. Cross- PSMA transcripts could be used as a preoperative predictive sectional pathological analysis of the prostatic specimens re- marker of advanced disease, therefore, deserves further atten- vealed the existence of a concomitant prostatic adenocarcinoma tion. in 10 patients. All of the adenocarcinomas were classified pT2 The present study demonstrates that full-length, PSMA- or less. Seven of the 27 (26%) preoperative blood samples tested specific transcripts are consistently detectable in normal and positive for PSMA by RT-PCR. Only one of the PSMA-positive malignant urothelium, as well as in peripheral blood from pa- patients showed histological evidence of concomitant localized tients presenting with progressive TCC of the bladder. Although

Fig. 3 Positive CYT-351 immunostaining of PSMA protein. A, normal urothelium; B, TCC urothelium; C, TCC-derived bladder cell line LB831; D, androgen-dependent prostate cancer cell line LNCaP.

PSMA expression is much weaker than in prostate-derived tissues, this transcript encodes a substantial amount of PSMA protein. In TCC-derived cell lines, PSMA is detectable by Table 2 Correlation between PSMA mRNA detection and 2-year survival in patients presenting with a TCC of the bladder immunohistochemical staining and is enzymatically active, pro- Correlation between molecular detection of PSMA-positive cells, ducing a level of enzymatic activity that correlates with the pathological features of 27 cystoprostatectomy specimens, and 2-year intensity of immunostaining. survival of the patients: (a) global data in patients presenting with Initially believed to be restricted to the prostatic epithe- transitional cell carcinoma patients; (b) stratification according to the lium, PSMA expression therefore appears to be more ubiqui- pathological TCC stratification; and (c) stratification according to the tous. Although this could theoretically impair prostate-specific presence or absence of concomitant prostate cancer. anti-PSMA targeting strategies, it also generates new potential 2-year applications for this biological marker (24–26). In patients PMSA No. of survival RT-PCR patients (%) presenting with invasive TCC of the bladder and no evidence of a concomitant PSMA-positive tumor, current results suggest Healthy blood donor Ϫ 200 that preoperative PSMA mRNA blood transcripts are associated ϩ 0 with advanced disease and progression after radical treatment. TCC Whether PSMA assay can be considered as an individual diag- (a) Global data nostic and prognostic marker in TCC patients, however, remains Ϫ 20 79 ϩ to be determined in a larger cohort of patients. 729 (b) Pathological stratification of TCC ՅpT2 Ϫ 10 81 ϩ 0 ACKNOWLEDGMENTS ՆpT3 Ϫ 10 81 Surgical specimens were collected by Dr. P. Degroote and Dr. A. ϩ 729 Feyaerts, Department of Urology, University of Leuven Medical (c) Concomitant prostate cancer School, Brussels, Belgium, and Drs. S. G. Benoıˆt and P. Jadin, from the No Ϫ 11 80 Department of Urology, Hoˆpital de Biceˆtre, Le Kremlin-Biceˆtre, France. ϩ 617 Ϫ Their help has been greatly appreciated. We thank France Hanon and Yes 980 ϩ 1 100 Huguette Delhez, from the Laboratory of Applied Molecular Technol-

ogy, Department of Clinical Biochemistry, Universite´ Catholique de 14. Chomczynski, P., and Sacchi, N. Single-step method of RNA iso- Louvain, Brussels, Belgium, for the quality of their technical assistance. lation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem., 43: 156–159, 1987. REFERENCES 15. Gala, J. L., Heusterspreute, M., Loric, S., Hanon, F., Tombal, B., Van Cangh, P., De Nayer, P., and Philippe, M. Illegitimate transcription 1. Johansson, S. L., and Cohen, S. M. Epidemiology and etiology of of the prostate-specific antigen and prostate-specific membrane antigen bladder cancer. Semin. Surg. Oncol., 13: 291–298, 1997. in blood cells: implications for the detection of hematogenous prostate 2. Foresman, W. H., and Messing, E. M. Bladder cancer: natural his- cells and standardization. Clin. Chem., 3: 472–481, 1998. tory, tumor markers, and early detection strategy. Semin. Surg. Oncol., 16. Gala, J. L., Chenut, F., Bui Thi Hong, K., Rodhain, J., Camby, P., 13: 299–306, 1997. Philippe, M., and Scheiff, J. M. A panel of monoclonal antibodies for 3. Lapham, R. L., Grignon, D., and Ro, J. Y. Pathologic prognostic the immunostaining of Bouin’s fixed bone marrow trephine biopsies. parameters in bladder urothelial biopsy, transurethral resection, and J. Clin. Pathol. (Lond.), 6: 521–524, 1997. cystectomy specimens. Semin. Diagn. Pathol., 14: 109–122, 1997. 17. Gala, J. L., Vermylen, C., Cornu, G., Ferrant, A., Michaux, J. L., 4. Skinner, D. G., Stein, J. P., Lieskovsky, G., Skinner, E. C., Boyd, Philippe, M., and Martiat, P. High expression of bcl-2 is the rule in S. D., Figueroa, A., Cote, R., and Groshen, S. 25-year experience in the acute lymphoblastic leukemia except in Burkitt subtype at presenta- management of invasive bladder cancer by radical cystectomy. Eur. tion, and is not correlated with the prognosis. Ann. Hematol., 69: Urol., 33: 25–26, 1998. 17–24, 1994. 5. Horoszewicz, J. S., Leong, S. S., Kawinski, E., Karr, J. P., Rosenthal, 18. Pinto, J. T., Suffoletto, B. P., Berzin, T. M., Hong Qiao, C., Lin, S., H., Chu, T. M., Mirand, E. A., and Murphy, G. P. LNCaP model of Tong, W. P., May, F., Mukherjee, B., and Heston, W. D. W. Prostate- human prostatic carcinoma. Cancer Res., 43: 1809–1818, 1983. specific membrane antigen: a novel folate hydrolase in human prostatic 6. Horoszewicz, J. S., Kawinski, E., and Murphy, G. P. Monoclonal carcinoma cells. Clin. Cancer Res., 2: 1445–1451, 1996. antibodies to a new antigenic marker in epithelial prostatic cells and 19. Carter, R. F., Feldman, A. R., and Coyle, J. T. Prostate-specific serum of prostatic cancer patients. Anticancer Res., 7: 927–936, membrane antigen is a hydrolase with substrate and pharmacological 1987. characteristics of a neuropeptidase. Proc. Natl. Acad. Sci. USA, 93: 7. Fair, W. R., Israeli, R. S., and Heston, W. D. W. Prostate-specific 749–753, 1996. membrane antigen. Prostate, 32: 140–148, 1997. 20. Rhee, M. S., Ryan, T. J., and Galivan, J. H. ␥-Glutamyl hydrolase 8. Israeli, R. S., Miller, W. H., Su, S. L., Powell, C. T., Fair, W. R., secreted from human tumour cell lines. Cell. Pharmacol., 2: 289–292, Samadi, D. S., and Heston W. D. W. Sensitive nested reverse transcrip- 1995. tion polymerase chain reaction detection of circulating prostatic tumor 21. Tiffany, C. W., Lapidus, R. G., Merion, A., Calvin, D. C., and cells: comparison of prostate-specific membrane antigen and prostate- Slusher, B. S. Characterization of the enzymatic activity of PSM: specific antigen based assays. Cancer Res., 54: 6306–6310, 1994. comparison with brain NAALADase. Prostate, 39: 28–35, 1999. 9. Loric, S., Dumas, F., Eschwe`ge, P., Blanchet, P., Benoit, G., Jardin, 22. Jackson, P. F., Cole, D. K., Slusher, B. S., Stetz, S. L., Ross, L. E., A., and Lacour, B. Enhanced detection of hematogenous circulating Donzanti, B. A., and Trainor, D. A. Design, synthesis, and biological prostatic cells in patients with prostatic adenocarcinoma by using nested activity of a potent inhibitor of the neuropeptidase N-acetylated ␣-linked reverse transcription polymerase chain reaction assay based on prostate- acidic dipeptidases. J. Med. Chem., 39: 619–622, 1996. specific membrane antigen. Clin. Chem., 41: 1698–1704, 1995. 23. Olsson, C. A. Reverse transcriptase polymerase chain reaction in 10. Uria, J. A., Velasco, G., Santamaria, I., Ferrando, A., and Lopez- prostate cancer. In: G. Murphy, K. Griffiths, L. Denis, S. Khoury, C. Otin, C. Prostate-specific membrane antigen in breast carcinoma. Lan- Chatelain, and A. T. Cockett (eds.), Proceedings of the First Interna- cet, 349: 1601, 1997. tional Consultation in Prostate Cancer, pp. 121–132. Jersey, United 11. Maraj, B. H., Aldersley, M. A., and Markham, A. F. Prostate- Kingdom: Science Communication International, 1997. specific membrane antigen expression in the duodenum: implications in 24. Gala, J. L., and Loric, S. Sensitivity or specificity of RT-PCR coeliac disease and immunotherapy for prostate cancer. Lancet, 351: assays: the real challenge for molecular staging of prostatic carcinomas. 1559–1560, 1998. Int. J. Cancer, 77: 161–163, 1998. 12. Dumas, J., Gala, J. L., Berteau, P., Brasseur, F., Eschwege, P., 25. Babain, R. J., Sayer, J., Podoloff, D. A., Steelhammer, L. C., Paradis, V., Philippe, M., and Loric, S. Molecular expression of PSMA Bhadkamkar, V. A., and Gulfo, J. V. Radioimmunoscintigraphy of mRNA and protein in primary renal tumors. Int. J. Cancer, 80: 799– pelvic lymph nodes with 111-indium-labeled monoclonal antibody 803, 1999. CYT-356. J. Urol., 152: 1952–1955, 1994. 13. Gueguen, M., Patard, J. J., Brasseur, F., Renauld, J. C., Van Cangh, 26. Chengazi, V. U., Feneley, M. R., Ellison, D., Stalteri, M., P. J., Boon, T., and Van den Eynde, B. J. An antigen recognized by Granowski, A., Granowska, M., Nimmon, C. C., Mather, S. J., Kirby, autologous CTLs on a human bladder carcinoma. J. Immunol., 160: R. S., and Britton, K. E. Imaging prostate cancer with technetium-99m– 6188–6194, 1998. 7E11-C5.3 (CYT-351). J. Nucl. Med., 38: 675–682, 1997.

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Jean-Luc Gala, Sylvain Loric, Yves Guiot, et al.

Clin Cancer Res 2000;6:4049-4054.

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