AJCP / Original Article

Reduced Glucocorticoid Receptor Expression Predicts Bladder Tumor Recurrence and Progression Hitoshi Ishiguro, PhD,1,2 Takashi Kawahara, MD,1,2 Yichun Zheng, MD, PhD,1,2 George J. Netto, MD,1 and Hiroshi Miyamoto, MD, PhD1,2 From the 1Departments of Pathology and Urology, The Johns Hopkins University School of Medicine, Baltimore, MD, and 2Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, Rochester, NY. Key Words: Bladder cancer; Glucocorticoid receptor; Immunohistochemistry; Progression; Recurrence

CME/SAM

Am J Clin Pathol August 2014;142:157-164 DOI: 10.1309/AJCPU8UCEZYG4WTV

ABSTRACT Objectives: To assess the levels of glucocorticoid receptor (GR) expression in bladder tumors because the status and its prognostic value remain largely unknown. Methods: We immunohistochemically stained for GR in bladder tumor and matched non-neoplastic bladder tissue specimens. Results: Overall, GR was positive in 129 (87%) of 149 urothelial tumors, which was significantly (P = .026) lower than in non-neoplastic urothelium (90 [96%] of 94). Forty-two (79%) of 53 low-grade tumors vs 45 (47%) of 96 high-grade carcinomas (P < .001) and 61 (73%) of 84 non– muscle-invasive (NMI) tumors vs 26 (40%) of 65 muscleinvasive (MI) carcinomas (P < .001) were moderately to strongly immunoreactive for GR. Kaplan-Meier and log-rank tests revealed that loss or weak positivity of GR significantly or marginally correlated with recurrence of NMI tumors (P = .025), progression of MI tumors (P = .082), and cancer-specific survival of MI tumors (P = .067). Multivariate analysis identified low GR expression as a strong predictor for recurrence of NMI tumors (P = .034). Conclusions: GR expression was downregulated in bladder tumors compared with nonneoplastic bladder tumors and in high-grade/MI tumors compared with low-grade/NMI tumors. Decreased expression of GR, as an independent prognosticator, predicted recurrence of NMI tumors. These results support experimental evidence suggesting an inhibitory role of GR signals in bladder cancer outgrowth.

© American Society for Clinical Pathology

Upon completion of this activity you will be able to: • correlate the status of glucocorticoid receptor (GR) expression in bladder tumors with patient outcomes. • discuss the role of GR signaling in bladder cancer progression. The ASCP is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity for a maximum of 1 AMA PRA Category 1 Credit ™ per article. Physicians should claim only the credit commensurate with the extent of their participation in the activity. This activity qualifies as an American Board of Pathology Maintenance of Certification Part II Self-Assessment Module. The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. Questions appear on p 273. Exam is located at www.ascp.org/ajcpcme.

Bladder cancer is the second most common genitourinary malignancy, leading to significant morbidity and mortality.1-3 Two-thirds to three-fourths of patients with bladder tumor initially present with non–muscle-invasive (NMI; pTa or pT1) tumors that can often be treated with conservative approaches, but they experience recurrences, occasionally with grade and/or stage progression. Patients with muscle-invasive (MI; ≥pT2) bladder cancer have high risks of disease progression and metastasis in spite of available aggressive treatment modalities. However, current molecular markers remain insufficient to precisely predict the potential for tumor recurrence and progression. Glucocorticoid receptor (GR) belongs to the steroid hormone receptor superfamily. As is the case with other steroid receptors, glucocorticoid (GC)-bound GR translocates into the nucleus and binds to GC response elements, which in turn transactivates various genes.4,5 The activated GC-GR complex also interacts with transcription factors, such as nuclear

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factor (NF)-kB, that are involved in cell proliferation/survival and cell cycle regulation.5,6 Thus, GC-regulated genes via the GR pathway play a vital role in maintaining various cellular, molecular, and physiologic networks. GCs are frequently used, for instance, in the treatment of inflammatory and autoimmune disorders. In cancer patients, GCs are also used for alleviating the adverse effects of chemotherapy or radiotherapy.7 In addition, antitumor activities of GCs have been observed, and several GCs are indeed being used clinically as cytotoxic drugs, with or without other chemotherapeutic agents, mainly for hematologic malignancies and castration-resistant prostate cancer.8-10 In bladder cancer, using cell line models, GC treatment was shown to induce resistance to cytotoxic effects of cisplatin.11,12 We found that GR activation by a synthetic GC dexamethasone strongly inhibited cell invasion and metastasis of bladder cancer in vitro and in vivo.13 Nonetheless, the functional role of GR signaling in bladder cancer progression needs to be further investigated. It appears that GR expression status had never been examined in human bladder cancer. In our previous study with 24 cystectomy cases,13 GR expression tended to be weaker in tumor cells than in nonneoplastic urothelial cells, and strong GR positivity tended to correlate with a better prognosis. However, the findings were unlikely conclusive, presumably because of the relatively small number of cases studied, with no lower grade tumors. In the current study, we aim to validate our earlier results in a larger patient cohort with a wider range of histopathologic features and further elucidate the potential role of GR expression as a biomarker in bladder cancer.

Materials and Methods We retrieved 152 bladder tissue specimens obtained through transurethral resection or cystectomy performed at the Johns Hopkins Hospital (Baltimore, MD) or the University of Rochester Medical Center (Rochester, NY). All the sections were reviewed for confirmation of original diagnoses according to the 2004 World Health Organization/ International Society of Urological Pathology classification system for urothelial neoplasms1 (149 urothelial tumors and three squamous cell carcinomas). Appropriate approval was obtained from the institutional review board at each institution before construction and use of the tissue microarray (TMA). Bladder TMAs, including 152 tumor tissues and 94 benign-appearing tissues from bladders of patients with tumors, were constructed from formalin-fixed paraffin-embedded specimens, as described previously.14 The tumors included 10 papillary urothelial neoplasms of low malignant potential (PUNLMPs), 43 noninvasive (pTa) lowgrade urothelial carcinomas, 31 NMI high-grade urothelial 158 158

Am J Clin Pathol 2014;142:157-164 DOI: 10.1309/AJCPU8UCEZYG4WTV

carcinomas, and 65 MI high-grade urothelial carcinomas, in addition to three pT2N0 squamous cell carcinomas. All 65 patients with MI urothelial carcinoma underwent cystectomy. None of the patients had received radiotherapy or systemic chemotherapy preoperatively, whereas 17 cases had intravesical bacillus Calmette-Guérin treatment before radical cystectomy. All 149 cases with urothelial tumor were included in our prior study analyzing 188 cases for the expression of androgen receptor (AR), estrogen receptor (ER)-α, and ERβ.14 Immunohistochemical staining was performed using the primary antibody to GR (H-300, 1:200 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), which recognizes both main human isoforms, GRa and GRb, as described previously.13,14 All the stains were manually scored by one pathologist (H.M.) blinded to patient identity. The German immunoreactive score was calculated by multiplying the percentage of immunoreactive cells (0% = 0; 1%-10% = 1; 11%-50% = 2; 51%-80% = 3; 81%-100% = 4) by staining intensity (negative = 0; weak = 1; moderate = 2; strong = 3). The immunohistochemical scores (ranging from 0-12) were considered negative (0; 0 or 1), weakly positive (1+; 2, 3 or 4), moderately positive (2+; 6 or 8), and strongly positive (3+; 9 or 12) for GR expression. Statistical analyses were performed using PASW statistics 18 software (IBM, Chicago, IL). The Fisher exact test was used to evaluate the association between categorized variables. Survival rates in 149 patients with urothelial tumor were calculated with the Kaplan-Meier method and comparison was made with the log-rank test. Tumor recurrence was evaluated in patients with NMI tumor. Tumor progression was separately evaluated in patients with NMI tumor (development of high-grade carcinoma [in those with initial PUNLMP or low-grade carcinoma], pT1-pT4 tumor [in those with initial pTa tumor], or pT2-pT4 tumor [in those with initial pT1 tumor]) and in patients with MI tumor (development of local recurrence or metastatic tumor after cystectomy). Disease-specific survival was evaluated in patients with MI tumor. In addition, the Cox proportional hazards regression analysis was used to determine statistical significance of predictors in a multivariate setting. P values less than .05 were considered to be statistically significant.

Results Using immunohistochemistry, we investigated the expression of GR in 149 bladder urothelial neoplasm specimens and corresponding 94 non-neoplastic bladder tissues. Positive signals were detected predominantly in the nuclei of benign and malignant epithelial cells ❚Image 1A❚, ❚Image 1B❚, ❚Image 1C❚, and ❚Image 1D❚. Correlations of the © American Society for Clinical Pathology

AJCP / Original Article

expression status with different non-neoplastic and neoplastic bladder tissues are summarized in ❚Table 1❚. GR was positive in 90 (96%; 32 were 1+, 33 were 2+, and 25 were 3+) of 94 non-neoplastic urothelial tissues and 129 (87%; 42 were 1+, 51 were 2+, and 36 were 3+) of 149 urothelial tumors. Of the 94 cases in which both benign and tumor tissues were stained, only 20 (21%) cases showed higher scores of GR expression in tumor, compared with benign urothelium (P = .046 with the sign test). Overall, the rate of GR positivity was significantly lower in tumors than in non-neoplastic tissues (P = .026). On the other hand, GR was negative in one of three pure squamous cell carcinomas, whereas the remaining two cases showed moderate positivity (2+).

Next we evaluated the correlation of GR expression levels in urothelial tumors with the clinicopathologic profile available for our patient cohort. There was no significant difference in expression pattern between male and female cases. Fifty-one (96%) of 53 lower grade tumors (PUNLMPs + low-grade carcinomas) were GR-positive, whereas 78 (81%) of 96 high-grade carcinomas were GR-positive (P = .011). Similarly, 81 (96%) of 84 NMI tumors expressed GR compared with 48 (74%) of 65 MI tumors (P < .001). Differences in GR expression (0/1+ vs 2+/3+) remained significant by grade (lower: 79% vs high: 47%, P < .001) and stage (NMI: 73% vs MI: 40%, P < .001). Even higher rates of increased GR expression (2+/3+) were seen in lower grade (P = .042) or NMI (P = .151) tumors than in

A

B

C

D

❚Image 1❚ Immunohistochemistry showing glucocorticoid receptor expression (A-D). A, strong staining in non-neoplastic urothelium; B, strong staining in non–muscle-invasive tumor; C, strong staining in muscle-invasive (MI) tumor; and D, weak staining in MI tumor. © American Society for Clinical Pathology

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❚Table 1❚ Correlation of Glucocorticoid Receptor Expression with Clinicopathologic Profile of the Patients Negative Expression, No. (%)

No.

0

P Value

Positive Expression, No. (%) 1+

2+

3+

0 vs 1+/2+/3+

0/1+ vs 2+/3+

0/1+/2+ vs 3+

Tissue .026 .687 .761 Non-neoplastic urothelium 94 4 (4.3) 32 (34.0) 33 (35.1) 25 (26.6) Urothelial neoplasm 149 20 (13.4) 42 (28.2) 51 (34.2) 36 (24.2) Sex .785 .847 1.000 Male 114 16 (14.0) 32 (28.1) 38 (33.3) 28 (24.6) Female 35 4 (11.4) 10 (28.6) 13 (37.1) 8 (22.9) Tumor grade .011

Reduced glucocorticoid receptor expression predicts bladder tumor recurrence and progression.

To assess the levels of glucocorticoid receptor (GR) expression in bladder tumors because the status and its prognostic value remain largely unknown...
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