The population of the study
Bladder cancer patients
Eighty four (84) patients (63 men and 21 women) with bladder cancer, confirmed by histopathology, were included in this study in the period from March 2007 to May 2008. The patients with bladder cancer were retrieved, examined, interviewed, and sampled in the region of The Middle East (Jordan, Syria and Iraq). The investigational study was conducted in the University Putra Malaysia (UPM) in Malaysia. The patients' age ranged 38–72 years old with mean age 59.49 ± 5.7 years. The involved patients were selected from 3 central teaching hospitals without bias to age, sex, or cancer pathology. The involved patients were sampled before the beginning of anti-cancer therapy. The diagnosis of bladder cancer was established by doing urine cytology and diagnostic cyctoscopy where the histopathology of biopsies confirmed the diagnosis of bladder cancer and determined the tumor grade, local invasiveness, and the histopathological type of cancer. The tumor spread and metastasis was assessed by CT scans and cystoscopy.
Moreover, past schistosomal infection was monitored by retrieving the previous medical records. The current diagnosis of schistosomiasis was done by cystoscopy through finding bilharzial granuloma or egg in histopathological sections. Accordingly, patients with bladder cancer were categorized into 45 patients with SBT and 39 patients with NSBT. The involved patients with bladder cancer did not show extra-bladder tumors. The stages of the retrieved patients ranged from I to IV.
Moreover, cancer patients were categorized accordingly into muscle invasive (T2, T3, and T4) and non invasive tumors (Ta, T1, and CIS). For comparative purposes with previous reports, the 1973 WHO grading system (papilloma, G1, G2 or G3) was used in this study which is still the most commonly used system despite being superseded by the 2004 WHO. The retrieved tumors were histologically categorized as low grade (grades 1–2) and high grade (grade 3). Moreover, the tumor morphology was categorized by cystoscopy into 71 cases papillary, 12 cases sessile and 1 case nodular. Written consents were granted by the involved subjects for sampling. The handling with human subjects was done under the permission of the regional committee of Ethics for biomedical research.
The group of benign bladder lesions
This group encompassed 44 untreated cases of chronic cystitis patients (29 men and 15 women) with mean age 57.62 ± 3.78 years. The patients with chronic cystitis were retrieved and sampled from the same geographical region of cancer patients, the Middle East. Patients with chronic cystitis were diagnosed by urine cytology and diagnostic cystoscopy coupled with histopathological examination. There were no signs of premalignant lesions (squamous metaplasia, dysplastic changes, or leukoplakia) nor were signs of prostatic enlargement found. Under the same diagnostic protocols done for bladder cancer patients, the chronic cystitis patients were grouped into 16 schistosomal cystitis (SC) patients and 28 non-schistosomal cystitis (NSC) patients.
Control group
Twenty age- and sex- matched individuals (12 men and 8 women) at mean age 58.3 ± 6.1 years old were involved from the Middle East region. Their bladders were investigated by routine cystoscopy and biopsies were taken. They were found free of bladder cancer or any other bladder disease or inflammation, therefore, they were considered as control group (CTL).
Processing of biopsies
The bladder cancer patients, the chronic cystitis patients, and CTL subjects underwent transurethral resection of bladder tumor (TUR-BT), cystitis tissues, and normal mucosal tissues respectively. The retrieved specimens were composed of multiple pieces, 2–5 mm in thickness. Specimens were immersed in 10% formalin in order to make a paraffin block. The histopathological paraffin blocks of biopsies were sectioned into 4 um thick sections. Hematoxylin and Eosin slides were prepared and examined by a histopathologist for confirming the histopathological diagnosis, the grade, and the invasiveness of the tumor. A set of steps were pursued under the supervision of a pathologist to minimize as could as possible the fixation-related loss of target proteins. These steps were: minimal prefixation time of 1 hour, the use of cold 4% paraformaldehyde and cold fixation at 4°C, and short duration of fixation up to 5 hours [18]. Moreover, the paraffin-embedded sections processed for immunohistochemistry (IHC) assay were examined in a period not more than 3 days. It was stated that insignificant loss of nucleic acids or proteins was observed within the first 3 days of fixation-paraffin embedding [18].
Immunohistochemistry assay
Antibodies
IHC staining was conducted using a set of mouse monoclonal antibodies; anti-p53, anti-p16, anti bcl-2, and anti-c-myc (InnoGenex, USA) and anti Ki-67, anti-Rb-1, and anti-EGFR (DakoCytomation). Secondary biotinylated goat anti-mouse antibodies were used (DakoCytomation). Antibodies were diluted in the recommended antibody diluting buffer (Dako). The working dilutions and the final concentrations of the primary antibodies were 1:100 and 0.005 mg/mL for anti-p53, 1:120 and 0.008 mg/mL for anti-p16, 1:75 and 0.006 mg/mL for anti-bcl-2, 1:100 and 0.01 mg/mL for c-myc, 1: 50 and 0.01 mg/mL for anti-Rb-1, 1:200 and 0.005 mg/mL for anti-ki67, and 1:120 and 0.008 mg/mL for anti-EGFR antibodies. The used dilution and concentration of the biotinylated goat anti-mouse antibodies were 1:800 at final concentration 0.0025 mg/mL.
Immunohistochemistry procedure
The procedure of IHC was conducted according to the manufacturer instructions (LSAB2 Universal Dakocytomation strepavidin-biotin detection system). After baking slides in oven at 65°C overnight, slides were deparaffinized by applying sequential immersion for 5 min in xylene, 95% ethanol, 70% ethanol, and distilled water (DW). Autoclave-based antigen retrieval was standardized for each target protein. Slides were placed in a jar containing antigen retrieval solution (0.1 M citrate buffer from BDH at pH 6) and left in autoclave, for 0.5–8 min (variable time for each target protein) at 121°C. 100 μL of the diluted primary antibodies were then applied onto the sections and the slides were incubated in a humid chamber overnight at 4°C.
The next day, slides were rinsed gently with PBS (Merck)-Tween (Sigma) and placed in fresh PBS-Tween bath for 1 min. One-two drops of the diluted biotinylated secondary goat anti-mouse antibodies (DakoCytomation) were applied onto the sections and the slides were incubated in a humid chamber for 1 h at 37°C. After rinsing step, One-two drops of streptavidin-Horseradish peroxidase reagent (DakoCytomation) was applied onto the sections, slides were incubated for 30 min at 37°C. The prepared DAB-substrate chromogen solution was applied onto sections, Slides were incubated in dark at room temperature for 20 min. Mayer's hematoxylin stain was used as counterstain, then slides were dehydrated and mounted with DPX mounting fluid. In every run, two negative controls were used. The first negative control was antibody diluting buffer added alone without primary antibodies. This is essential for measuring the non-specific noise of staining. The second negative control was a known normal urothelium section devoid of any positive staining of the corresponding target molecule. On the other hand, a strong and consistently stained section was used as a positive control for each target. The detected staining noise, if any, was subtracted from the corresponding test section.
Staining analysis
The tumor cell staining, membranous, cytoplasmic, and nuclear compartments were taken into consideration. Furthermore, staining of the stromal cells dispersed between tumor epithelial cells (not more than 5% of the total cells in the section) was taken into account as these cells reflected the same mutational abnormality of the epithelial cells. However, other stromal cells scattered throughout the section were not taken into account. The pattern of staining was dominantly nuclear for p53, p16, Rb, and bcl-2, nuclear and cytoplasmic for ki-67, cytoplasmic and membranous for EGFR, and mainly cytoplasmic for c-myc. Since differences in the staining intensity of the studied proteins were slight, the qualitative positive/negative system was used. The immunostained cells at moderate to intense dark brown color were considered positive while other cells were considered negative (Figure. 1). Under light microscope, the mean percentage of the positively stained cells in each section was calculated from 3 dense, medium, and light staining areas. In each area, the percentage of brown stained cells was calculated out of total countable cells in 5 high power fields. Due to the numerous, sometimes contradicting, scoring systems of the target proteins, the mean percentage of the positively stained cells was quantitively compared among the different groups of this study. To keep the scientific fidelity and to ensure the impartial evaluation, the immunostained slides were examined blindly by two scientists, one from the research team and a consultant histopathologist outside the research team.
Statistical Analysis
Statistical analysis was conducted using SPSS software version 10 and MS Excel 2000. Chi-square test of independence was used for evaluating the significant association of histopathology type, tumor grade, tumor invasiveness, disease staging, and disease recurrence with SBT and NSBT groups. After proving that the studied groups obey the normal distribution pattern by using Kolmogorov and Semirnov normalization tests, parametric tests were used. Accordingly, student t test was used to measure the significant difference of the mean percentage of the positively stained cells for p53, p16, Rb, bcl-2, ki-67, c-myc, and EGFR proteins among the different groups of the study. Moreover, Pearson's correlation coefficient (r) was used to measure the correlating behavior of the studied markers with each other. P value less than 0.05 was considered as significant.