The current study included 71 patients, (47 males and 24 females) with histologically proven MIBR-SCC of the bladder (Stage pT2a-T3N0-N3M0) of different grades (grade 1 to 3).
All patients underwent radical cystectomy for male patients and anterior pelvic exenteration for female patients in the period from January 2005 to December 2006 in the South Egypt Cancer Institute and the department of urology, faculty of medicine, Assuit University. The presence of Schistosoma haematobium eggs in the cystectomy specimens was an inclusion criterion.
Every patient was offered the information about the effects, benefits and possible side effect of adjuvant radiotherapy. The patients were free to choose to be treated according to the international routine treatment of bladder SCC which is radical cystectomy alone, or to be subjected to the routine of our institute, which is radical cystectomy followed by radiotherapy.
Among those, 38 patients (group 1) agreed to receive AR to the pelvis 3 weeks after cystectomy. AR was given in the dose of 50Gy/25 fractions/5 weeks using linear accelerator (Siemens Mevatron) 6 - 15 MV Photons at 100 cm source axis distance. The upper border of the target was the junction of L5 - S1 vertebrae, the lower border was the lower margin of the obturator foramen. The lateral borders were 1.5 cm lateral to the pelvic brim, the anterior border was the anterior of the symphysis pubis and the posterior border included the anterior one third of the rectum. Three fields' technique (one anterior and 2 lateral wedged fields) was used. This arrangement is supposed to give a homogenous distribution to the target volume. The remaining 33 patients (group 2) refused AR based on different social and cultural backgrounds, such as how far they lived from the institute (sometimes more than 500 KM) and some prevalent exaggerated concerns about the side effects of radiotherapy, sometimes potentiated by personal previous experience of a relative of the patient.
Study scientific approval was obtained from the urology department scientific board and ethically approved by the ethics committee of the Faculty of Medicine, Assiut University (that conforms to the provisions of the Declaration of Helsinki). Informed written consents were obtained from all patients before enrollment in the study with guarantee of confidentiality.
Formalin fixed, paraffin embedded tissue from parts of the cystectomy specimens from all 71 patients were used for ordinary histopathological studies. Immunohistochemical characterization for Bcl-XL expression was done by the indirect immuno-peroxidase technique using the kit produced by LAB VISION Corporation. It is supplied as total IgG purified from rabbit anti-serum by protein A chromatography. It is prepared at 1 mg/ml in 10 mM PBS (Phosphate-buffered saline), ph 7.4.
Tissue sections (4 μm thick) were cut immediately before staining and were heated to 56°C for 20 minutes. Protein blocking was accomplished through application of Ultra V block for 5 minutes and application of 5% normal Goat serum for 30 minutes. The primary antibody was applied in a concentration of 1:400 and was incubated in a moist humidity chamber overnight. Biotinylated Goat Anti-polyvalent was then applied and incubated for 10 minutes at room temperature. Streptavidin Peroxidase was then applied and incubated for 10 minutes at room temperature. One drop (40 μl) of Diaminobenzidine Chromogen was added to 1 ml of Diaminobenzidine Substrate (mixed by swirling) and the mixture was then applied to the tissue sections and incubated for 5 minutes. The sections were counterstained with Mayer's hematoxylin for few seconds.
Tissue samples with known expression for the marker (colon carcinoma stained with anti- Bcl-XL) served as positive control. Negative controls were sections treated as described above, but with the primary antibody replaced with pooled non-immune mouse IgG of the same concentration.
All sections were analyzed with a BX-40 bright field microscope under X10-20 objectives. When questions arouse concerning tissue morphology, H&E-stained sections were reviewed for confirmation. Staining was classified as negative when no or weak (less than 20%) expression of the anti-apoptotic marker Bcl-XL was observed within the cytoplasm of tumor cells. Staining was classified as positive when significant uniform cytoplasmic expression of the anti-apoptotic marker Bcl-XL (greater than 20%) was observed within the cytoplasm of tumor cells.
Follow up of all patients for 3 years after cystectomy or until recurrence had occurred was done every 3 months and included clinical examination, serum creatinine, alkaline phosphatase, abdominal ultrasonography and chest x-ray. Abdominal computerized tomography and bone scans were done when findings suggested disease progression, defined as any emerging local or distant tumor.
All data were analyzed using SPSS (Statistical Program for Social Sciences version 16 for windows, 2001, SPSS Inc., Chicago, IL, USA). Univariate analysis was done using chi-square test with Fisher's exact correction and independent T test. Multivariate analysis was done using Logistic regression analysis to identify the most important risk factor for recurrence, the Kaplan Meier survival analysis with log rank p value was done to compare the effect of radiotherapy on recurrence. The effect of radiotherapy was adjusted to age group (older or younger than 50 years), sex, tumor size (≥ or <3 cm), stage (≥ or < stage III), grade, lymph node involvement and Bcl-XL expression as univariate predictors. Cox regression was done to test the significant factors as multivariate predictors of the efficacy of AR. P value < 0.05 was considered to be significant.