Immunohistochemical Detection of Prostate-specific Antigen ...

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ANTICANCER RESEARCH 28: 4149-4154 (2008)

Immunohistochemical Detection of Prostate-specific Antigen Expression in Primary Urothelial Carcinoma of the Urinary Bladder JUNG-CHIN CHEN1,2, CHUNG-LIANG HO1,3, HUNG-WEN TSAI1,3, TZONG-SHIN TZAI4, HSIAO-SHENG LIU5, NAN-HAW CHOW1,3, WEN-HORNG YANG4 and HONG-LIN CHENG4 1Department

of Pathology, National Cheng Kung University Hospital, Tainan 704; Departments of 2Medical Laboratory Science and Biotechnology, 3Pathology, 4Urology, and 5Microbiology and Immunology, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan, R.O.C.

Abstract. Background: Prostate-specific antigen (PSA) is

a 33-kDa serine protease that is secreted by prostatic epithelium and non-prostate tissues. Ectopic expression of PSA has also been reported in a variety of non-prostatic epithelial tumors. Patients and Methods: Expression of PSA and its clinical implication were examined in a total of 422 cases of primary urothelial carcinoma of the bladder. Results: Cytoplasmic reactivity to PSA was observed in 6 cases (1.4% ), with the staining being to a low degree in 3 and a high degree in the remaining 3 cases. Expression of PSA was positively related to multiplicity, large tumors (≥3 cm) and muscle invasion (≥pT2) (p=0.0008, 0.004 and 0.03, respectively). Patients with tumors of low PSA expression had a better survival than those with tumors of high PSA expression (p=0.03). Conclusion: Focal expression of PSA can occasionally be detected in some large, invasive urothelial cancer cells. This phenotypic change should be considered in the differential diagnosis of poorly differentiated carcinoma of the lower urinary tract. Prostate-specific antigen (PSA) is a 33-kDa serine protease that is secreted by prostatic epithelium and non-prostate tissues, such as epithelial lining of the periurethral and perianal glands, urachus remnant and cowper’s glands (1). It functions in the liquefaction of seminal coagulum to allow the release of spermatozoa. Because of its role in the detection and prognosis of prostate cancer, PSA remains the

Correspondence to: Dr. Hong-Lin Cheng, Department of Urology, National Cheng Kung University Hospital, 138 Sheng-Li Road, Tainan 70428, Taiwan, R.O.C. Tel: +886 6 2353535 ext. 5271, Fax: +886 6 2383678, e-mail: [email protected] Key Words: Prostate-specific antigen, urothelial carcinoma, invasion, prognosis, PSA.

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only serum biomarker recommended by the American Cancer Society for use in the screening of malignancies (2). In addition, immunohistochemical analysis of PSA expression in tumor tissue is generally successfully applied in the identification of the prostatic origin of metastatic carcinoma (3) and the distinction between poorly differentiated prostatic cancer and urothelial carcinoma in the bladder neck region (3-5). Despite fact that PSA may be a specific marker for prostatic differentiation (3-5), ectopic expression of PSA has been reported in a variety of non-prostatic tumors (6-16), most commonly in breast cancer (6-9) and salivary gland neoplasms (10-12). Other non-prostatic tumors reported to express PSA include adenocarcinoma of the urinary bladder (13), melanomas (14), acinar cell carcinoma of the pancreas (15) and lung cancer (17, 18). Interestingly, Mai et al. demonstrated a phenotypic alteration in the urothelial carcinoma of the bladder when it spreads into the prostate. All ten primary urothelial tumors of urinary bladder were negative for PSA, whereas six tumors invasive to the prostate were focally positive (19). Urothelial cancer cells thus might exhibit PSA expression through mesenchymal−epithelial interaction in the prostatic stroma. However, small amounts of PSA mRNA were also detected in the normal human bladder and in the cancerous tissue by reverse transcriptase polymerase chain reaction (RT-PCR) (20). We thus performed this cohort study to examine the expression of PSA and its potential clinical implication in conventional urothelial carcinoma of the bladder.

Patients and Methods

Cell lines and culture. Immortalized E6 human uroepithelial cells and five human bladder cancer cell lines (RT4, TSGH-8301, TCC-SUP, J82, and T24) were propagated as described elsewhere (21). The localized established cell lines UB09, UB37, UB40 and UB47 were derived from transitional cell bladder cancer of grade II pT2, grade III

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ANTICANCER RESEARCH 28: 4149-4154 (2008)

pT3, grade III pT1 and grade III pT3, respectively. They were maintained in Dulbecco’s modified Eagle’s medium (Gibco BRL, Grand Island, NY, USA) supplemented with 10% fetal calf serum (FCS; Gibco BRL), L-glutamine (2 mmol/l), sodium pyruvate (110 mg/l), penicillin (100 U/ml), and streptomycin (50 g/ml) at 37˚C in a 5% CO2-humidified atmosphere. The androgen-sensitive, PSAproducing human prostate cancer cell line, LNCaP, was chosen as positive control for experiments in vitro. Cells were grown in RPMI1640 supplemented with L-glutamine, non-essential amino acids, 10% FCS (Life Technologies), and antibiotics (200 U/ml penicillin and 100 mg/ml streptomycin).

RNA preparation and RT-PCR. The RT-PCR assay and the primer sets for PSA have been described previously (22). Briefly, the upstream primer sequence was: 5’-GATGACTCCAGCCACGACCT3’; and the downstream sequence was: 5’-CACAGACACCC CATCCTATC-3’. The PCR reaction conditions included 2 μM dig-11-dUTP and 0.4 μM PSA primers. The PCR was performed for 35 cycles consisting of the following steps: denaturation at 94˚C for 1 min; annealing at 66˚C for 1 min; and extension at 72˚C for 2 min. Gel analysis was then examined for PCR product spanning 710 bp, and the glyceraldehyde-3-phosphate dehydrogenase housekeeping gene was used to assess the mRNA integrity. LNCaP was used to evaluate the sensitivity of PCR experiments. Internal negative control for RT-PCR was performed using all of the reagents in the absence of cDNA.

Clinicopathological characteristics. As approved by the institutional review board, patients who underwent transurethral resection of bladder tumor or radical cystectomy for urothelial carcinoma of the bladder at National Cheng Kung University Hospital, Tanian, Taiwan, ROC were identified using a database. Paraffin-embedded specimens between 1990 and 1999 were retrieved from the pathology archives. Those patients with primary carcinoma in situ, urothelial crcinoma with glandular lumen or prostatic involvement, primary urothelial carcinoma of the prostate, and mixed carcinoma were excluded. A total of 422 patients with conventional urothelial carcinoma of the bladder were recruited for this investigation. There were 256 men and 166 women, ranging in age from 23 to 93 years old (mean age, 64.2±10.4 years). All cases were reviewed for histological grading according to the World Health Organization classification (1973). Clinical staging was determined according to the Tumor-Node-Metastasis System of the Union International Contre Cancer (2002) using a survey of the clinical details, image studies and pathological data. The treatment and follow-up of patients were conducted according to the standard strategy previously described in detail (21). Briefly, all patients with superficial bladder cancer (n=371) received transurethral resection and postoperative intravesical chemotherapeutic agent instillation with either thiotepa (30 mg in 30 ml of normal saline for 70 patients) or epirubicin (40 mg in 40 ml of normal saline for 34 patients) weekly for 8 consecutive weeks. Those patients who received intravesical bacillus Calmette-Guerin therapy or neoadjuvant chemotherapy were excluded from this study to preclude the potential bias in prognostic correlation (e.g. recurrence rate or tumor progression). The patients were followed up every 3 months for the first 2 years, every 6 months for an additional 2 years, and yearly thereafter. Recurrent tumours were confirmed by biopsy and the patient was given a transurethral operation. This was followed by another 8-week course of intravesical chemotherapeutic instillation

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therapy or by a radical or partial cystectomy if there was disease progression. Disease progression at recurrence was defined as being at a higher stage than the previous result. Fifty-one patients with muscle-invasive tumors were given a radical cystectomy. Forty-five patients received systemic chemotherapy with methotrexate, cisplatin, doxorubicin, and vinblastine. Patient survival status was determined by checking outpatient clinic records, interviewing patients’ families, or both. Clinical follow-up ranged from 8 to 17 years (median, 10 years).

Immunohistochemistry (IHC) of PSA expression. To preclude the possibility of sampling bias, histological sectioning, rather than tissue array, of surgical specimens was chosen for this investigation. Immunostaining procedures were previously described (21). Briefly, tissue sections were incubated at room temperature for 2 h with monoclonal anti-PSA antibody (Dako Corp., Carpenteria, CA, USA) (19). The optimal dilution (1/25) was determined using normal human prostate as a control. The StrAviGen Super Sensitive MultiLink kit (BioGenex Laboratories, Inc., San Ramon, CA, USA) was used to detect the resulting immune complex. Peroxidase activity was visualized by an aminoethyl carbazole substrate kit (Zymed Laboratories, Inc., San Francisco, CA, USA). Sections were finally counterstained with hematoxylin. For negative control, non-immune mouse immunoglobulin was substituted for primary antibody in the incubation. To evaluate the PSA staining, both reviewers (N-H.C. and H-L.C.) were blinded to the clinical outcomes. Since there was no apparent difference of staining intensity, a three-category scoring system was modified from previous reports (19, 23). "High expression (++)" indicates that more than 10% of the tumor cells exhibited PSA staining; "low expression (+)", between 0% and 10% reactivity; and "negative (−)", no immunoreactivity for PSA was detected. For negative control, different types of non-prostatic tissue were tested. There was no staining in positive controls when treated with non-immune sera in place of the PSA primary antibody.

Statistics. Associations of PSA expression and clinicopathological indicators of bladder cancer were examined using Fisher’s exact or χ2 test. The difference of survival in relation to PSA expression was examined by t-test. All measures of association were calculated by two-tail test. Only those variables with a p-value