Insight into the molecular basis of Schistosoma haematobium-induced bladder cancer through urine proteomics.

Infection due to Schistosoma haematobium is carcinogenic. However, the cellular and molecular mechanisms underlying urogenital schistosomiasis (UGS)-induced carcinogenesis have not been well defined. Conceptually, early molecular detection of this phenomenon, through non-invasive procedures, seems feasible and is desirable. Previous analysis of urine collected during UGS suggests that estrogen metabolites, including depurinating adducts, may be useful for this purpose. Here, a new direction was pursued: the identification of molecular pathways and potential biomarkers in S. haematobium-induced bladder cancer by analyzing the proteome profiling of urine samples from UGS patients. GeLC-MS/MS followed by protein-protein interaction analysis indicated oxidative stress and immune defense systems responsible for microbicide activity are the most representative clusters in UGS patients. Proteins involved in immunity, negative regulation of endopeptidase activity, and inflammation were more prevalent in UGS patients with bladder cancer, whereas proteins with roles in renal system process, sensory perception, and gas and oxygen transport were more abundant in subjects with urothelial carcinoma not associated with UGS. These findings highlighted a Th2-type immune response induced by S. haematobium, which seems to be further modulated by tumorigenesis, resulting in high-grade bladder cancer characterized by an inflammatory response and complement activation alternative pathway. These findings established a starting point for the development of multimarker strategies for the early detection of UGS-induced bladder cancer.

CD44s and CD44v6 in diagnosis and prognosis of human bladder cancer.

UNLABELLED: The cell adhesion molecules (CAMs) CD44 standard (CD44s) and its variant 6 (CD44v6) are involved in the progression and invasion of human malignancies. However, discrepancies in the prognostic value of CD44s and CD44v6 expression need to be addressed. AIMS: To investigate the expression of CD44s and CD44v6 in bladder carcinomas and relate the results to the established prognostic factors. MATERIALS AND METHODS: 50 bladder carcinoma specimens, 30 cases with transitional cell carcinoma (TCC: 6 bilharzial and 24 nonbilharzial) and 20 cases with squamous cell carcinoma (SCC: 8 bilharzial and 12 nonbilharzial), were included. Immunohistochemical analysis for CD44s and CD44v6 was carried out using avidin-biotin peroxidase method. RESULTS: The level of both CD44s and CD44v6 in TCC was significantly higher in invasive than in preinvasive tumors and normal urothelium (p < .05). A direct association between the percentage of expression of both markers and the grade of TCC (p < .05) was observed. An inverse correlation between CD44s and SCC was seen, where metaplastic urothelium showed higher expression than invasive carcinomas. No association was observed between the expressions of both CD44s and CD44v6 and bilharzial ova, sex and age of the patient, or size of the tumor. CONCLUSIONS: The authors report statistically significant correlation between CD44s and CD44v6 expression and increasing grade and stage of TCC. No such correlation with SCC and with bilharzial cystitis, sex and age of the patient, or size of the tumor was documented.

Efficiency of diagnostic biomarkers among colonic schistosomiasis Egyptian patients.

The schistosomal parasite plays a critical role in the development of malignant lesions in different organs. The pathogenesis of cancer is currently under intense investigation to identify reliable prognostic indices for disease detection. The objective of this paper is to evaluate certain biochemical parameters as diagnostic tools to efficiently differentiate between colonic carcinoma and colonic carcinoma associated with schistosomal infection among Egyptian patients. The parameters under investigation are interleukin 2 (IL-2), tumour necrosis factor alpha (TNF-α), carcinoembryonic antigen (CEA) levels, tissue telomerase, pyruvate kinase (PK), glucose-6-phosphate dehydrogenase (G-6-PD) and lactate dehydrogenase (LDH) enzyme activities. The results revealed a significant elevation in the level of the tumour markers IL-2, TNF-α and CEA as well as the activities of LDH, telomerase and G-6-PD among non-bilharzial and bilharzial colonic cancer groups, with a more potent effect in bilharzial infection-associated colonic cancer. A significant inhibition in PK activity was recorded in the same manner as compared to normal tissues. The efficacy of this biomarker was also evaluated through detecting sensitivity, specificity, negative and positive predictive values. In conclusion, schistosomal colonic carcinoma patients displayed more drastic changes in all parameters under investigation. The combination of the selected parameters succeeded in serving as biomarkers to differentiate between the two malignant types.

Genomic imbalances in Schistosoma-associated and non-Schistosoma-associated bladder carcinoma. An array comparative genomic hybridization analysis.

Carcinoma of the urinary bladder is the most common malignancy in many tropical and subtropical countries due to endemic infection by Schistosoma hematobium (bilharzia). In the current study, we performed a high-resolution analysis of gene copy number amplifications using array comparative genomic hybridization to compare DNA copy number changes in pools of Schistosoma-associated (SA) and non-Schistosoma-associated (NSA) bladder cancer (BC). Many DNA copy number changes were detected in all studies, with multiple gains and losses of genetic material. The most frequent alterations were gains on 5p15.2 approximately p15.33, 8q13.1, and 11q13, and losses on 8p21.3 approximately p22 and 22q13. Even when SA pools showed no Schistosoma-specific gene copy number profiling as compared to NSA pools, some genes seemed to be gained (ELN on 7q11.23) and some lost (PRKAG3 on 2q35 and PRDM6 on 5q23.2) in SA-SCC. The following genes were gained in all histopathologic categories: SRC (20q11.23), CEBPB (20q13.13), and GPR9 (Xq13.1). Our study did not provide clear evidence of differences in carcinogenesis of SA-BC and NSA-BC.

Can bladder adenocarcinomas be distinguished from schistosomiasis-associated bladder cancers by using array comparative genomic hybridization analysis?

Bladder cancer is the most common malignancy in many tropical and subtropical areas, correlating well with the endemicity of schistostomiasis. The majority of schistostomiasis-associated (SA) bladder cancers are squamous cell cancers, whereas the majority of non-SA cases in the Western world are transitional cell cancers, suggesting different carcinogenetic mechanisms. Approximately 6% of SA and 1% of non-SA cases are adenocarcinomas. To achieve fine-resolution information of DNA copy number changes in SA adenocarcinomas, 10 tumor samples were analyzed on an oligonucleotide-based CGH array. The frequency of aberrations ranged from 2 to 17, with an average of 10 alterations per sample. The most frequently gained regions were 20q and 8q (in 70 and 60% of the cases, respectively), whereas the most frequently lost regions were 5q and 8p (both in 40% of the cases). In addition, six regions of amplification were found in three samples, containing both well characterized and novel regions. Comparison of the DNA copy number profiles to previously reported profiles of SA transitional cell carcinoma and squamous cell carcinoma revealed similarities (e.g., gains at 5p and 8q), as well as differences (e.g., TCC- and SCC-associated losses at 18p and 20p, and adenocarcinoma-associated gains at 20q). The results suggest that although SA cancers share genetic features, there also exist histology-specific regions of gain and loss.

Chronic inflammation and bladder cancer.

The relation between Schistosomiasis and bladder cancer is well-established and accounts for the high rates of bladder cancer in a number of developing countries, including Egypt. In developed countries, transitional cell carcinoma is the predominant type of bladder cancer, whereas in Schistosomiasis-endemic regions, squamous cell carcinoma is the most common type. In this review, experimental and observational data on infection, inflammation, and bladder cancer are summarized with special emphasis on transitional cell carcinoma. Findings from numerous studies suggest that inflammation is likely to have an important role in bladder carcinogenesis in developed countries. Future studies need to focus in greater detail on risk factors that increase inflammation of the bladder, examine genetic susceptibility to inflammatory pathways, and include markers of inflammation measured prior to cancer diagnosis. Understanding the role of inflammation on transitional cell carcinogenesis may provide important insights on how to prevent the sixth most common cancer in the United States.

Human papilloma virus and p53 expression in bladder cancer in Egypt: relationship to schistosomiasis and clinicopathologic factors.

The aim of the current study was to compare the role of p53 and human papillomavirus (HPV) in schistosomiasis-related and schistosomiasis-unrelated carcinoma of the urinary bladder. To achieve this aim, we investigated 114 bladder carcinomas for p53 oncoprotein expression by immunohistochemistry and for human papillomavirus by in situ hybridization technique. The results revealed that 64 tumors (56.1%) were schistosomiasis-associated. Sixty seven (58.8%) were transitional cell carcinomas and 32 (28%) were squamous cell carcinomas. The remaining 15 tumors (13.2%) included adenocarcinomas and sarcomatoid carcinomas. In both schistosomiasis-associated and non-associated carcinomas, p53 oncoprotein expression was significantly higher in poorly differentiated tumors. However, it was significantly higher in locally more invasive tumors in the schistosomal carcinomas only. HPV types 16/18 could be detected in 1 of the 114 bladder carcinomas (0.95%), which was schistosomiasis-related squamous cell carcinoma in situ. These results suggest that p53 immunohistochemistry can be a prognostic factor in both schistosomal and nonschistosomal bladder cancer. More importantly, HPV does not seem to play a role in the pathogenesis of either type of bladder cancer in our country.

The CYP2D6 extensive metabolizer genotype is associated with increased risk for bladder cancer.

Inheritance of certain polymorphic metabolizing genes is associated with the development of a number of environmental cancers and may also influence the clinicopathological tumor outcome. We have investigated the association between the inheritance of the polymorphic cytochrome P-450 2D6 (CYP2D6) gene and the development of transitional and squamous cell carcinomas (TCC and SCC) of the bladder in 37 Egyptian cancer patients and 27 matched controls. Genotypic analysis using the polymerase chain reaction (PCR) and the restriction fragment length polymorphism (RFLP) assays revealed that the CYP2D6 extensive metabolizer genotype (CYP2D6*1A) is over represented in bladder cancer patients compared to controls (79 versus 44%, respectively) and is significantly associated with increased risk for bladder cancer (odds ratio (OR) = 4.5, 95% confidence limit (CL) = 1.3-15.7, P = 0.006). Our results also indicate that individuals who have inherited this genotype are more likely to develop TCC (OR = 5.9, 95% CL = 1.4-27.9, P = 0.006) rather than SCC (OR = 3.1, 95% CL = 0.7-15.9; P = 0.09). When the relative risk associated with this genotype was estimated among subjects who were smokers or schistosoma infected, the same tendency towards the development of TCC was observed. These data suggest that the predisposing CYP2D6 gene may not only increase the risk for bladder cancer among Egyptians, but may also influence the clinicopathological tumor outcome.

Patterns of Schistosoma haematobium egg distribution in the human lower urinary tract. III. Cancerous lower urinary tracts.

The distribution of S. haematobium eggs in urinary bladders containing tumors and removed at surgery has been studied; the majority of these tumors are well differentiated squamous cell carcinomas. The same three anatomic patterns of egg accumulation described in part I of this series (noncancerous lower urinary tracts) were found in these cancerous bladders, but, in addition, most of the tumors were surrounded by a collar of heavy S. haematobium egg deposition. The egg burdens in these collars were, on the average, twice the average egg burden in the remainder of the urinary bladder. These collars do not seem to be artifacts created by growth of the tumor and subsequent displacement of the adjacent normal tissue, creating a region of heavy egg concentrations; rather, these heavy S. haematobium egg concentrations seem to act as promoters of urothelial carcinogenesis.

Chromosomal aberrations in Cis and Ta bilharzial bladder cancer: a theory of pathogenesis.

BACKGROUND: Bladder cancer manifests many different forms ranging from superficial to aggressive muscle invasive stages, which suggests that various genetic alterations are involved. Several attempts have been made to establish correlations between specific genetic alterations and various stages of the disease. At the National Cancer Institute (NCI), Cairo, bladder cancer constitutes 30.3% of all cancers. Bladder cancer observed among Egyptians has a clinico-pathological profile that differs from transitional cell carcinoma (TCC) seen in the western world. PATIENTS AND METHODS: We used fluorescence in situ hybridization to detect numerical chromosome changes in 25 patients presenting with carcinoma in situ and Ta lesions. Twenty-four cases had transitional cell carcinoma and one case had squamous cell carcinoma. RESULTS: Five out of 24 TCC cases had diploid chromosome count with all the probes. Numerical chromosome aberrations were detected in 19 cases (79%). In eight cases, a loss of chromosome 9 was observed. In one case, an additional loss of chromosome 17 was detected. One case demonstrated a loss of chromosome 17, whereas another three cases showed a gain of chromosome 7. Loss of chromosome Y was observed in nine of the 22 male cases studied (40.9%). The only case with SCC had normal diploid chromosome count with all the probes used. CONCLUSION: When the genetic basis of bilharzial related bladder cancer is fully understood, new diagnostic and therapeutic strategies will be developed, which in turn may promote better clinical management by pathologists and urologists. A theory of bilharzial related bladder cancer pathogenesis is suggested.

The incidence of squamous and transitional cell carcinomas of the urinary bladder in northern Tanzania in areas of high and low levels of endemic Schistosoma haematobium infection.

Squamous cell carcinoma is the commonest type of bladder malignancy in most areas of northern Tanzania. Of 172 cases of bladder cancer recorded in 9 years, 72% were squamous cell carcinomas. Of these, 46% had Schistosoma haematobium eggs in sections taken from tumour tissue. The geographical distribution of this tumour closely corresponded to the prevalence of S. haematobium infection. The Mt Kilimanjaro area is free of schistosomiasis and virtually lacks squamous cell carcinoma. Although transitional cell carcinoma is rare in all regions of northern Tanzania, the relative frequency of bladder cancer in the Mt Kilimanjaro area was only one-third of that seen in other regions; population-based incidence rates were also very low in this area.

[Radiography and ultrasonography in the management of bladder tumors: 71 cases at the National Hospital Center of Yalgado Ouedraogo (Burkina Faso)]. / Radiographie et échographie dans la prise en charge des tumeurs de la vessie: à propos de 71 cas au Centre hospitalier national Yalgado Ouedraogo (Burkina Faso).

The bladder's cancer is frequent in West Africa. Urinary schistosomiasis endemicity helps to explain this high incidence. It is a pathology of late diagnosis, little or badly explored by imaging. Through a retrospective survey of 71 patients' files aged in average of 51.7 years, all having a bladder's tumour which is clinically shown by an haematuria, the major symptom, often by a pelvic volume, and who have all gone through an abdominal echography and/or intravenous urography (IVU) and/or retrograde urethrocystography (UCR), we have tried to point out the role of imaging in the caring of this pathology in our working context. Imaging, with a 98.5% sensibility for sonography and 100% for IVU, took part in all the cases to the diagnosis, to the search of urinary signs of reflux, associated signs authorizing a diagnostic orientation, but was excluded from the evolutive follow-up due to the poverty of our populations. So, despite some limits specific to the survey, particularly the absence of historadiological comparison for all the files, echography and IVU with cystography have always led to malignancy diagnosis. Therefore they should be requested for any patient consulting for haematuria.

TGF-B1 pathway as biological marker of bladder carcinoma schistosomal and non-schistosomal.

OBJECTIVES: Study TGF-ß1 pathway in bladder carcinoma. DESIGN AND METHODS: Eighty-one patients were enrolled: 16 chronic cystitis and 60 malignant bladder lesions; 15 schistosomal squamous cell carcinoma (SQCC), 45 transitional cell carcinoma (TCC). Five healthy individuals served as controls. mTGF-ß1, protein, and its receptor expression in urine and bladder tissue were measured using in situ hybridization and immunohistochemical techniques, respectively. RESULTS: Overexpression of TGF-mRNA in invasive TCC group was compared with superficial TCC, high grade TCC was compared with low grade, and SQCC was compared with TCC. TGF-ß1 protein and its receptor I (TGF-ßR1) were overexpressed in urine samples in malignant group compared with chronic cystitis and in SQCC group compared with TCC group. TGF-ß1 protein and its receptor were significantly increased in schistosomal malignant group compared with non-schistosomal group. CONCLUSION: Expression of TGF-ß1 and TGF-ßR1 could be used as biological markers of bladder carcinoma.

Changing patterns (age, incidence, and pathologic types) of schistosoma-associated bladder cancer in Egypt in the past decade.

OBJECTIVE: To assess the patterns of schistosomiasis-associated bladder cancer in Egypt from 2001 to 2010 in a retrospective study. Bilharzial bladder carcinoma is the most common cancer, particularly in Egyptian men. Classically, carcinoma in a bilharzial bladder is most commonly of the squamous cell type. During the past decade, certain changes have occurred in the features in Schistosomiasis-associated carcinoma in Egypt with a decline in the frequency of squamous cell carcinoma and increase in the frequency of transitional cell carcinoma. METHODS: This was a retrospective study of 1932 patients treated at Kasr Al Aini Hospital, Cairo University, from 2001 to 2010. Two groups were selected: group 1 included 1002 patients from 2001 to 2005 and group 2 included 930 patients from 2006 to 2010. RESULTS: The mean patient age increased from 41±11.2 years to 52±8.6 years, and the male/female ratio changed from 5.6:1 to 4.2:1. The incidence of associated bilharziasis decreased from 80% to 50%. A significant increased occurred in transitional cell carcinoma from 20% to 66%, with a significant decrease in squamous cell carcinoma from 73% to 25%. No difference was observed in the tumor stage or grade or incidence of lymph node metastases between the 2 groups. CONCLUSION: The pattern of incidence of the various histologic types of bladder cancer have changed, with most cases now transitional cell carcinoma, in contrast to the findings in the earlier Egyptian series. Additional studies are encouraged to explain the factors explaining these changes.

Urinary retinoic acid receptor-ß2 gene promoter methylation and hyaluronidase activity as noninvasive tests for diagnosis of bladder cancer.

OBJECTIVES: We evaluated the significance of urinary retinoic acid receptor-ß2 (RAR-ß2) gene promoter methylation and hyaluronidase activity in comparison with voided urine cytology (VUC) in diagnosis of bladder cancer. DESIGN AND METHODS: This study included 100 patients diagnosed with bladder cancer, 65 patients with benign urological disorders and 51 healthy volunteers. Urine supernatant was used for determining hyaluronidase activity by zymography while urine sediment was used for cytology and detection of methylated RAR-ß2 gene promoter by methylation specific nested PCR. RESULTS: The sensitivity and specificity were 53% and 90.5% for VUC, 65% and 89.7% for percent methylation fraction of RAR-ß2 gene promoter, and 89% and 90.5% for hyaluronidase activity; combination of the three parameters increased sensitivity to 95%. A significant association was observed between investigated markers and advanced grade tumor. CONCLUSIONS: Combined use of RAR-ß2 gene promoter methylation, hyaluronidase activity and VUC is promising non-invasive tool for bladder cancer detection.

Tumor markers of bladder cancer: the schistosomal bladder tumors versus non-schistosomal bladder tumors.

BACKGROUND: The aim of this study is to comparatively elucidate the underlying molecular pathways and clinicopathological criteria in schistosomal bladder tumor (SBT) versus non-schistosomal bladder tumor (NSBT). METHODS: This study explored the role of p53, p16, bcl-2, ki-67, c-myc, Rb and EGFR, by using Immunohistochemistry assay, in 45 SBT and 39 NSBT patients in comparison with 16 schistosomal chronic cystitis (SC), 28 non-schistosomal chronic cystitis (NSC), and 20 normal control (CTL) subjects. The studied markers in SBT and NSBT were correlated with different clinicopathological criteria namely, tumor histopathology, grading, invasiveness, stage, and presentation of the disease. RESULTS: SBT was associated with high grade invasive squamous cell carcinoma (SCC) while NSBT was associated with lower grade less invasive transitional cell carcinoma (TCC). The expression of p53, bcl-2, c-myc, and EGFR was higher in SBT than in NSBT while Rb was higher in NSBT than in SBT. However, p16 and ki-67 were not different between SBT and NSBT. The profile of molecular markers in SC was similar to NSC except for EGFR which was higher in SC than in NSC. Both SC and NSC showed higher level of p53, bcl-2, ki-67, and EGFR than in CTL group while p16, Rb, and c-myc were not different. p53 was associated with high grade SCC in both SBT and NSBT. Bcl-2 was associated with high grade invasive tumors in SBT and NSBT. P16 was associated with low grade, late stage, and recurrent SBT and high grade, invasive, late stage, and recurrent NSBT. Rb was associated with SCC in SBT, invasive tumors in NSBT, and late stage and recurrent presentation in both SBT and NSBT. C-myc was associated with high grade, invasive, and late stage SBT and SCC, high grade, invasive, and late stage NSBT. EGFR was associated with invasive SCC in SBT and invasive, high grade, and late stage TCC in NSBT. ki-67 was associated with invasive SBT and high grade late stage NSBT. CONCLUSION: SBT and NSBT showed distinct molecular profile of tumor development and progression which can be taken into consideration in fine adjusting the anti-cancer therapy for SBT and NSBT.

Time-trend in epidemiological and pathological features of schistosoma-associated bladder cancer.

OBJECTIVE: To investigate the different emerging trends in the features of bladder cancer along 17 years. PATIENTS AND METHODS: During a 17-year period (1988- 2004), 5071 epithelial bladder cancer patients underwent radical cystectomy at the National Cancer Institute (NCI), Cairo University, Egypt. The time was divided into 3 time periods to detect changes of the clinicopathologic features of patients in these periods. RESULTS: There was a significant progressive increase in the patients' age with time and decrease in squamous/transitional ratio, with transient increase in male predominance during the 2nd time period. Moreover, there was a decrease in the well differentiated (grade 1) tumor (p<0.001) and an increase in the frequency of pelvic nodal involvement (p<0.001). Transitional cell carcinoma (TCC) patients were significantly older than those with squamous cell carcinoma (SCC) (p<0.001). Progressive increase of age with time was evident in TCC, SCC and adenocarcinoma patients. Male to female ratio changed significantly in TCC and SCC. CONCLUSION: Time trend was confirmed with relative decrease in frequency of SCC and increase of TCC with changes in their pathological details. The differences between their characteristics and that of the Western countries are decreasing.

Nitric oxide synthase immunoreactivity in human bladder carcinoma.

AIMS: To study the expression of the endothelial and inducible isoforms of nitric oxide synthase (eNOS and iNOS, respectively) in human bladder carcinoma and schistosomal bladder disease, and to compare it with normal adult and fetal urothelium. Nitric oxide is thought to play a complex role in human carcinogenesis, but has only recently been investigated in bladder cancer. METHODS: Immunohistochemistry was performed on paraffin wax embedded sections of 33 human bladder carcinomas and five bladder carcinoma cell lines; in addition, seven schistosomal bladder cases and normal and fetal urothelium were investigated. In the cell lines enzymatic activity was examined by the NADPH diaphorase reaction. RESULTS: Immunoreactivity for eNOS was present in most cells of all 31 cases examined. Immunoreactivity for iNOS was less abundant and was seen in 23 of 25 cases. Similar findings were noted in schistosomal bladder cancer. In the normal bladder mucosa, eNOS immunoreactivity was found only in the superficial cell layer and iNOS was not expressed, whereas in the fetal urothelium immunoreactivity for both isoforms was seen in all cell layers. Enzymatic activity and immunoreactivity for eNOS and iNOS were evident in the five bladder carcinoma cell lines. CONCLUSIONS: It is possible that NOS plays a role in the differentiation of the transitional epithelium in fetal life, has a biological function in the adult bladder mucosa, and is involved in bladder carcinogenesis. eNOS and iNOS immunoreactivity do not differ in schistosomal and non-schistosomal bladder carcinoma, but resemble the pattern of expression typical of fetal urothelium.