Cyclooxygenase-2 and B-cell lymphoma-2 expression in cystitis glandularis and primary vesicle adenocarcinoma

Tissues from 75 patients, including 60 cases of CG, 11 cases of chronic cystitis (CC), and 4 cases of primary vesicle adenocarcinoma (ADC), were obtained from the Surgical Pathology Archives of the No. 2 People’s Hospital of Zhenjiang, affiliated with Jiangsu University between 1989 and 2009. Normal bladder specimens from nine subjects who underwent cystectomy for benign causes were used as controls. The Institutional Review Board of Nanjing Medical University (Nanjing, China) approved this study. At the time of patient recruitment, written informed consent was obtained from all participants. We classified CG into CGTP and CGIT based on routine hematoxylin and eosin-stained sections. One of the ADC patients had the intestinal type of CG and interrupted use of antibiotics rather than intravesical instillation of the anticancer agent. Another patient had neurogenic bladder with suprapubic cystostomy for fifteen years. The other two patients had classic bladder exstrophy with an unsuccessful initial closure.

Sections (5 μm thick) were cut from formalin-fixed, paraffin-embedded tissue blocks and stained with hematoxylin and eosin. Additional sections from appropriately selected blocks were cut for use in immunohistochemical analyses as described previously [13, 14]. Two primary antibodies were used for immunochemical staining: monoclonal antibodies against COX-2 (monoclonal mouse anti-human D12; Santa Cruz Biotechnology, USA) and Bcl-2 (monoclonal mouse anti-human; Dako, Carpinteria, USA). Briefly, sections were baked for 2 hour at 72°C and deparaffinized by sequential immersion in xylene, 95% ethanol, 80% ethanol, and distilled water for 5 min each. Next, slides were placed in an autoclave containing antigen retrieval solution (0.1 M citrate buffer from BDH at pH 6.0) for 2 min at 121°C. Diluted primary antibodies (100 μl) were applied to the sections and slides were incubated in a humid chamber for 2 h at 37°C. Slides were rinsed gently with PBS and placed in a fresh PBS bath for 5 min. Next, one or two drops of diluted biotinylated secondary goat anti-mouse antibodies (Dako Cytomation) were applied to the sections and the slides were incubated in a humid chamber for 2 h at 37°C. After rinsing, one or two drops of streptavidin-horseradish peroxidase reagent (Dako Cytomation) was added to the sections and slides were incubated for 30 min at 37°C. Next, the prepared DAB substrate chromogen solution was applied to the sections and slides were incubated in the dark at room temperature for 5 min. Mayer's hematoxylin stain was used as a counterstain, and slides were dehydrated and mounted.

Staining was evaluated as described previously [14, 15]. Briefly, two pathologists who were unaware of the clinical data scored immunohistochemical expression in a semi-quantitative fashion. Expression levels were assessed by evaluating the percentage of the cell that was stained, and recorded as absent, weakly, moderately, or markedly positive. (5-25% indicated weakly, 25-50% indicated moderately, >50% indicated markedly) Using light microscopy, the mean percentage of \ positively stained cells in each section was calculated from three dense, medium, and light staining areas. In each area, the percentage of brown stained cells was calculated from the total number of countable cells in five high power fields. Therefore, expression scoring was determined to be discernible and reproducible.

Kruskal-Wallis H tests were employed to evaluate differences in the amount of COX-2 and Bcl-2 expression among control, CC, CGTP, CGIT and ADC specimens. To further compare the expression of two groups, we performed Mann–Whitney U tests. Spearman’s tests were used to analyze the correlation between COX-2 and Bcl-2 expression in CG specimens. P values less than 0.05 were considered significant, and all P values are two-sided. All analyses were performed using SPSS version 13.0 (SPSS, USA).