Protocols for Preparation and Mass Spectrometry Analysis of Clinical Urine Samples to Identify Candidate Biomarkers of Schistosoma-Associated Bladder Cancer.

Advances in mass spectrometry instrumentation have revolutionized analytical capability in clinical proteomics. In parallel, various sample preparation methods have been developed to try to address the inherent complexity and dynamic range of clinical samples, typically involving a combination of depletion of abundant proteins followed by extensive prefractionation. However, the depth of coverage routinely achieved in discovery proteomics experiments on peripheral fluids such as serum, still leaves something to be desired, especially if no depletion or prefractionation is done in order to increase the throughput of clinical samples. Remarkably, despite being an easily accessible, typically sterile and diagnostically rich clinical sample, urine is often overlooked and as such has received less development effort. As an ultrafiltrate of blood, urine contains proteins and protein fragments originating from all parts of the body which may have diagnostic or prognostic potential if accurately and reproducibly quantified. Here, we describe an efficient and simple method for the concentration of urine samples by methanol-chloroform precipitation and subsequent in-solution tryptic digestion prior to discovery or targeted mass spectrometry analysis. We exemplify this method by reference to the discovery of novel candidate urinary biomarkers of schistosomiasis. Importantly, the methods described here have been used to identify >1900 protein groups in human urine by label-free discovery proteomics, without requiring any prior depletion or prefractionation, making this approach amenable to high throughput clinical biomarker studies in many diseases.

Parasite-bacteria interrelationship.

Parasites and bacteria have co-evolved with humankind, and they interact all the time in a myriad of ways. For example, some bacterial infections result from parasite-dwelling bacteria as in the case of Salmonella infection during schistosomiasis. Other bacteria synergize with parasites in the evolution of human disease as in the case of the interplay between Wolbachia endosymbiont bacteria and filarial nematodes as well as the interaction between Gram-negative bacteria and Schistosoma haematobium in the pathogenesis of urinary bladder cancer. Moreover, secondary bacterial infections may complicate several parasitic diseases such as visceral leishmaniasis and malaria, due to immunosuppression of the host during parasitic infections. Also, bacteria may colonize the parasitic lesions; for example, hydatid cysts and skin lesions of ectoparasites. Remarkably, some parasitic helminths and arthropods exhibit antibacterial activity usually by the release of specific antimicrobial products. Lastly, some parasite-bacteria interactions are induced as when using probiotic bacteria to modulate the outcome of a variety of parasitic infections. In sum, parasite-bacteria interactions involve intricate processes that never cease to intrigue the researchers. However, understanding and exploiting these interactions could have prophylactic and curative potential for infections by both types of pathogens.

Schistosoma haematobium causing pulmonary schistosomiasis in a returning traveller.

Schistosomiasis is infrequently seen in the UK, but remains an important cause of haematuria in endemic areas. It may also be complicated by systemic illness, and can affect multiple organs, including the bladder, liver and lungs. We discuss a case of haematuria associated with lower abdominal discomfort and dry cough/wheeze in a returning traveller diagnosed as pulmonary and urinary schistosomiasis, caused by Schistosomahaematobium This case was particularly notable for the radiological findings seen on CT scan of the chest (figure 2A,B), as well as the characteristic sago nodules discovered within the bladder. It is also unusual to see pulmonary schistosomiasis associated with S. haematobium, an organism more typically characterised by bladder involvement. It is important to consider schistosomiasis and its complications, while rare in the western world, it remains an important differential diagnosis in at-risk groups with considerable morbidity if untreated.

Performance of urinary survivin as a non-invasive molecular marker of bladdercarcinoma in a schistosomiasis endemic area.

OBJECTIVE: To compare the sensitivity, specificity, positive predictive value, negative predictive value of urinary survivin and that of urine cytology in the diagnosis of bladder carcinoma in a schistosoma endemic area. DESIGN AND SETTING: This is a 12-month prospective study of patients with features of bladder carcinoma as study group and patients with other urologic conditions and healthy volunteers as control group. PARTICIPANTS: Patients with features of bladder carcinoma formed the study group, while patients with other urological conditions and healthy volunteers formed the control group. RESULTS: There were 52 patients in study group and 36 patients in control group. The mean ages of patients in the study and control groups were 47.17 ± 17.00 and 44.19 ± 18.89 years respectively. There were 48 males and 4 females in the study group, giving a male: female ratio of 12:1. Thirty-one (60 %) of the patients were farmers and 44 patients (85%) had history suggestive of schistosomiasis at childhood. The sensitivity of urine cytology and survivin in the study were 29.1% and 100.0% respectively. The specificity of urine cytology and survivin were 100.0% and 100.0% respectively (p= 0.05). The marker was associated with false positive (FP) results in patients with prostate cancer. CONCLUSION: Urinary survivin is highly sensitive, specific and predictive of bladder carcinoma in our environment. The marker is associated with false positive results in patients with prostate cancer. FUNDING: By authors.

Parasite-Associated Cancers (Blood Flukes/Liver Flukes).

Parasitic infection remains as a persistent public health problem and can be carcinogenic. Three helminth parasites, namely, Clonorchis sinensis (liver fluke) and Opisthorchis viverrini as well as Schistosoma haematobium (blood fluke), are classified as Group 1 carcinogens by the World Health Organization's International Agency for Research on Cancer (IARC Infection with liver flukes (Opisthorchis viverrini, Opisthorchis felineus and Clonorchis sinensis), World Health Organization, International Agency for Research on Cancer, 2011). Infection by these parasites is frequently asymptomatic and is thus rarely diagnosed at early exposure. Persistent infection can cause severe cancer complications. Until now, the cellular and molecular mechanisms linking fluke infections to cancer formation have yet to be defined, although many studies have focused on these mechanisms in recent years, and numerous findings were made in various aspects of parasite-associated cancers. Herein, we only introduce the fluke-induced cholangiocarcinoma (CCA) and bladder carcinoma and mainly focus on key findings in the last 5 years.

Development of Urinary Bladder Pre-Neoplasia by Schistosoma haematobium Eggs and Chemical Carcinogen in Mice.

Schistosoma haematobium is a biocarcinogen of human urinary bladder (UB). The present study investigated developing UB cancer mouse model by injecting S. haematobium eggs into the bladder wall and introduction of chemical carcinogens. Histopathological findings showed mild hyperplasia to epithelial vacuolar change, and high grade dysplasia. Squamous metaplasia was observed in the S. haematobium eggs+NDMA group at week 12 but not in other groups. Immunohistochemistry revealed significantly high expression of Ki-67 in urothelial epithelial cells of the S. haematobium eggs+BBN group at week 20. The qRT-PCR showed high expression of p53 gene in S. haematobium eggs group at week 4 and S. haematobium eggs+BBN group at week 20. E-cadherin and vimentin showed contrasting expression in S. haematobium eggs+BBN group. Such inverse expression of E-cadherin and vimentin may indicate epithelial mesenchymal transition in the UB tissue. In conclusion, S. haematobium eggs and nitrosamines may transform UB cells into squamous metaplasia and dysplasia in correlation with increased expression of Ki-67. Marked decrease in E-cadherin and increase in p53 and vimentin expressions may support the transformation. The present study introduces a promising modified animal model for UB cancer study using S. haematobium eggs.

The role of estradiol metabolism in urogenital schistosomiasis-induced bladder cancer.

Urogenital schistosomiasis is a neglected tropical disease that can lead to bladder cancer. How urogenital schistosomiasis induces carcinogenesis remains unclear, although there is evidence that the human blood fluke Schistosoma haematobium, the infectious agent of urogenital schistosomiasis, releases estradiol-like metabolites. These kind of compounds have been implicated in other cancers. Aiming for enhanced understanding of the pathogenesis of the urogenital schistosomiasis-induced bladder cancer, here we review, interpret, and discuss findings of estradiol-like metabolites detected in both the parasite and in the human urine during urogenital schistosomiasis. Moreover, we predict pathways and enzymes that are involved in the production of these metabolites emphasizing their potential effects on the dysregulation of the tumor suppressor gene p53 expression during urogenital schistosomiasis. Enhanced understanding of these potential carcinogens may not only shed light on urogenital schistosomiasis-induced neoplasia of the bladder, but would also facilitate development of interventions and biomarkers for this and other infection-associated cancers at large.

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.

LYMPHOEPITHELIOMA-LIKE CARCINOMA OF THE URINARY BLADDER ASSOCIATED WITH SCHISTOSOMIASIS: A CASE REPORT AND REVIEW OF LITERATURE.

Lymphoepithelioma-like carcinoma is an undifferentiated carcinoma with histological features similar to undifferentiated, non-keratinizing carcinoma of the nasopharynx. Lymphoepithelioma-like carcinoma of the urinary bladder is uncommon with a reported. incidence of 0.4% -1.3% of all bladder cancers. This case describes an 80 years old Egyptian male patient presented with recurrent hematuria and necroturia. Cystoscopy revealed a tumor involving the left lateral and the posterior wall of the urinary bladder. The patient underwent transurethral resection of the bladder tumor. Pathological examination showed muscle invasive lymphoepithelioma-like carcinoma associated with schistosomiasis of the urinary bladder. To the best of our knowledge the association of schistosomiasis with lymphoepithelioma-like bladder cancer was not described in the literature before this case report.

Molecular markers and Schistosoma-associated bladder carcinoma: A systematic review and meta-analysis.

BACKGROUND: Molecular mechanisms and pathogenesis of schistosomal-associated bladder cancer (SABC), one of the most common malignancies in Africa and parts of the Middle East, is still unclear. Identification of host molecular markers involved in schistosomal related bladder carcinogenesis is of value in prediction of high-risk group, early detection and timely intervention. METHODS: PubMed, Scopus, Google Scholar, Cochrane Library and African Journals Online databases were systematically searched and reviewed. A total of 63 articles reporting 41 host molecular factors were included in the meta-analysis. RESULTS: Pooled odds ratio demonstrated associations of p53 expression, telomerase activity and sFas with SABC as compared to other schistosomal patients (p53 expression: OR=9.46, 95%CI=1.14-78.55, p=0.04; telomerase by TERT: OR=37.38, 95%CI=4.17-334.85, p=0.001; telomerase by TRAP: OR=10.36, 95%CI=6.08-17.64, p<0.00001; sFas: OR=34.37, 95%CI=3.32-355.51, p=0.003). In comparison to bladder cancers of other etiology, positive associations were found between SABC and p15 deletion, p16 deletion, telomerase activity and sFas (p15 deletion: OR=4.20, 95%CI=2.58-6.82, p<0.00001; p16 deletion: OR=4.93, 95%CI=2.52-9.65, p<0.00001; telomerase by TERT: OR=3.01, 95%CI=1.51-5.97, p=0.002; telomerase by TRAP: OR=2.66, 95%CI=1.18-6.01, p=0.02; sFas: OR=4.50, 95%CI=1.78-11.40, p=0.001). Other identified associations were reported by few numbers of studies to enable reliable interpretation. CONCLUSIONS: Variations in gene expression or genomic alterations of some molecular markers in SABC as compared to non-SABC or other schistosomal patients were identified. These suggest minute differences in the pathogenesis and physiological profile of SABC, in relation to non-SABC.

[Histological aspect of urinary schistosomiasis in Togo: results of a cohort of 192 cases]. / Aspect histologique des séquelles de la bilharziose urinaire au Togo: résultats d'une série de 192 cas.

The high prevalence of urinary schistosomiasis in Togo and its late management often lead to sequels. This study takes stock of these cancerous and non-cancerous lesions observed on histological pieces in the pathology laboratory of the Teaching Hospital of Lome in 10 years. We find 28.1 % of malignant tumor lesions, dominated by urothelial carcinoma.

Recurrence patterns after open and robot-assisted radical cystectomy for bladder cancer.

BACKGROUND: Concerns remain whether robot-assisted radical cystectomy (RARC) compromises survival because of inadequate oncologic resection or alteration of recurrence patterns. OBJECTIVE: To describe recurrence patterns following open radical cystectomy (ORC) and RARC. DESIGN, SETTING, AND PARTICIPANTS: Retrospective review of 383 consecutive patients who underwent ORC (n=120) or RARC (n=263) at an academic institution from July 2001 to February 2014. INTERVENTION: ORC and RARC. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Recurrence-free survival estimates were illustrated using the Kaplan-Meier method. Recurrence patterns (local vs distant and anatomic locations) within 2 yr of surgery were tabulated. Cox regression models were built to evaluate the effect of surgical technique on the risk of recurrence. RESULTS AND LIMITATIONS: The median follow-up time for patients without recurrence was 30 mo (interquartile range [IQR] 5-72) for ORC and 23 mo (IQR 9-48) for RARC (p=0.6). Within 2 yr of surgery, there was no large difference in the number of local recurrences between ORC and RARC patients (15/65 [23%] vs 24/136 [18%]), and the distribution of local recurrences was similar between the two groups. Similarly, the number of distant recurrences did not differ between the groups (26/73 [36%] vs 43/147 [29%]). However, there were distinct patterns of distant recurrence. Extrapelvic lymph node locations were more frequent for RARC than ORC (10/43 [23%] vs 4/26 [15%]). Furthermore, peritoneal carcinomatosis was found in 9/43 (21%) RARC patients compared to 2/26 (8%) ORC patients. In multivariable analyses, RARC was not a predictor of recurrence. Limitations of the study include selection bias and a limited sample size. CONCLUSIONS: Within limitations, we found that RARC is not an independent predictor of recurrence after surgery. Interestingly, extrapelvic lymph node locations and peritoneal carcinomatosis were more frequent in RARC than in ORC patients. Further validation is warranted to better understand the oncologic implications of RARC. PATIENT SUMMARY: In this study, the locations of bladder cancer recurrences following conventional and robotic techniques for removal of the bladder are described. Although the numbers are small, the results show that the distribution of distant recurrences differs between the two techniques.

Estrogen-like metabolites and DNA-adducts in urogenital schistosomiasis-associated bladder cancer.

An estrogen-DNA adduct mediated pathway may be involved in the pathogenesis of the squamous cell carcinoma of the bladder associated with infection with the blood fluke Schistosoma haematobium. Extracts from developmental stages of S. haematobium, including eggs, induce tumor-like phenotypes in cultured cells. In addition, estrogen-derived, reactive metabolites occur in this pathogen and in sera of infected persons. Liquid chromatography-mass spectrometry analysis was performed on urine from 40 Angolans diagnosed with urogenital schistosomiasis (UGS), half of who also presented UGS-associated squamous cell carcinoma and/or urothelial cell carcinoma. The analysis revealed numerous estrogen-like metabolites, including seven specifically identified in UGS cases, but not reported in the database of metabolites in urine of healthy humans. These schistosome infection-associated metabolites included catechol estrogen quinones (CEQ) and CEQ-DNA-adducts, two of which had been identified previously in S. haematobium. In addition, novel metabolites derived directly from 8-oxo-7, 8-dihydro-2'-deoxyguanosine (8-oxodG) were identified in urine of all 40 cases of UGS. These metabolites can be expected to provide deeper insights into the carcinogenesis UGS-induced bladder cancer, and as biomarkers for diagnosis and/or prognosis of this neglected tropical disease-linked cancer.

P53 and cancer-associated sialylated glycans are surrogate markers of cancerization of the bladder associated with Schistosoma haematobium infection.

BACKGROUND: Bladder cancer is a significant health problem in rural areas of Africa and the Middle East where Schistosoma haematobium is prevalent, supporting an association between malignant transformation and infection by this blood fluke. Nevertheless, the molecular mechanisms linking these events are poorly understood. Bladder cancers in infected populations are generally diagnosed at a late stage since there is a lack of non-invasive diagnostic tools, hence enforcing the need for early carcinogenesis markers. METHODOLOGY/PRINCIPAL FINDINGS: Forty-three formalin-fixed paraffin-embedded bladder biopsies of S. haematobium-infected patients, consisting of bladder tumours, tumour adjacent mucosa and pre-malignant/malignant urothelial lesions, were screened for bladder cancer biomarkers. These included the oncoprotein p53, the tumour proliferation rate (Ki-67>17%), cell-surface cancer-associated glycan sialyl-Tn (sTn) and sialyl-Lewisa/x (sLea/sLex), involved in immune escape and metastasis. Bladder tumours of non-S. haematobium etiology and normal urothelium were used as controls. S. haematobium-associated benign/pre-malignant lesions present alterations in p53 and sLex that were also found in bladder tumors. Similar results were observed in non-S. haematobium associated tumours, irrespectively of their histological nature, denoting some common molecular pathways. In addition, most benign/pre-malignant lesions also expressed sLea. However, proliferative phenotypes were more prevalent in lesions adjacent to bladder tumors while sLea was characteristic of sole benign/pre-malignant lesions, suggesting it may be a biomarker of early carcionogenesis associated with the parasite. A correlation was observed between the frequency of the biomarkers in the tumor and adjacent mucosa, with the exception of Ki-67. Most S. haematobium eggs embedded in the urothelium were also positive for sLea and sLex. Reinforcing the pathologic nature of the studied biomarkers, none was observed in the healthy urothelium. CONCLUSION/SIGNIFICANCE: This preliminary study suggests that p53 and sialylated glycans are surrogate biomarkers of bladder cancerization associated with S. haematobium, highlighting a missing link between infection and cancer development. Eggs of S. haematobium express sLea and sLex antigens in mimicry of human leukocytes glycosylation, which may play a role in the colonization and disease dissemination. These observations may help the early identification of infected patients at a higher risk of developing bladder cancer and guide the future development of non-invasive diagnostic tests.

The biochemical value of urinary metalloproteinases 3 and 9 in diagnosis and prognosis of bladder cancer in Egypt.

BACKGROUND: Matrix metalloproteinases (MMPs) have long been associated with cancer-cell invasion and metastasis. Few studies are available that describe this association with bladder cancer either related or unrelated to schistosoma infection.Evaluating the urinary levels of MMP3 and MMP9 as diagnostic and prognostic biomarkers in different stages of schistosomal and non schistosomal bladder cancer was the aim of the present study.Urine samples were collected from 70 patients with schistosomal and non schistosomal bladder cancer at early and advanced stages and also from 12 healthy volunteers as controls. Urinary levels of MMP-3 and MMP-9 was measured by ELISA technique. Sensitivity and specificity of both markers were determined. RESULTS: Urinary levels of both MMP-3 and MMP-9 were significantly elevated in all bladder cancer patients compared with controls. MMP-3 started to elevate in early stages of schistosomal bladder cancer ( 0.173 ng/ml) and non-schistosomal bladder cancer patients (0.308 ng/ml) compared to control (0.016 ng/ml) and remained elevated in advanced stages (0.166, 0.235 ng/ml) of both types of bladder cancer patients. In contrast, MMP-9 showed a significant elevation in advanced stages only of both schistosomal and non schistosomal bladder cancer patients (10.33, 21.22 ng/ml) compared to control (0.409 ng/ml) and this elevation of both markers was much higher in non schistosomal bladder cancer. Both Metalloproteinases were specific for the diagnosis of the disease but MMP-3 was more sensitive and this sensitivity was evident in the early stage (84.85% for MMP3, 27.28% for MMP9). CONCLUSIONS: MMP3 may be the recommended urinary metalloproteinases as early diagnostic biomarker in the early stages of both types of bladder cancer although both MMP9 and MMP3 can be used in the diagnosis of advanced stages. Further studies are required on large number of urine samples to confirm these results.

Genetic polymorphisms in NQO1 and SOD2: interactions with smoking, schistosoma infection, and bladder cancer risk in Egypt.

BACKGROUND: Bladder cancer is the most prevalent form of cancer in men among Egyptians, for whom tobacco smoke exposure and Schistosoma haematobium (SH) infection are the major risk factors. We hypothesized that functional polymorphisms in NAD(P)H: quinone oxidoreductase 1 (NQO1) and superoxide dismutase 2 (SOD2), modulators of the effects of reactive oxidative species, can influence an individual's susceptibility to these carcinogenic exposures and hence the risk of bladder cancer. METHODS: We assessed the effects of potential interactions between functional polymorphisms in the NQO1 and SOD2 genes and exposure to smoking and SH infection on bladder cancer risk among 902 cases and 804 population-based controls in Egypt. We used unconditional logistic regression to estimate the odds ratios (OR) and confidence intervals (CI) 95%. RESULTS: Water pipe and cigarette smoking were more strongly associated with cancer risk among individuals with the TT genotype for SOD2 (OR [CI 95%] = 4.41 [1.86-10.42]) as compared with those with the CC genotype (OR [CI 95%] = 2.26 [0.97-6.74]). Conversely, the risk associated with SH infection was higher among the latter (OR [CI 95%] = 3.59 [2.21-5.84]) than among the former (OR [CI 95%] = 1.86 [1.33-2.60]). Polymorphisms in NQO1 genotype showed a similar pattern, but to a much lesser extent. The highest odds for having bladder cancer following SH infection were observed among individuals with the CC genotypes for both NQO1 and SOD2 (OR [CI 95%] = 4.41 [2.32-8.38]). CONCLUSION: Our findings suggest that genetic polymorphisms in NQO1 and SOD2 play important roles in the etiology of bladder cancer by modulating the effects of known contributing factors such as smoking and SH infection.

Urothelial carcinoma of the urinary bladder mixed with squamous differentiation or squamous cell carcinoma in areas with schistosomiasis is showing high risk of recurrence and poor survival.

In schistosomiasis haematobium areas endemic, bladder cancer is the first cause of malignancy in men and fourth in women. The chronic schistosomiasis would lead to variant histologic patterns which manifest in squamous cell carcinoma (SCC) or squamous differentiation (SqD). This study evaluated the clinical outcome after radical cystectomy (RC) in patients with urothelial carcinoma (UC) mixed with SCC or SqD, Comparison was done with two arms of pure UC and pure SCC, indication for RC was muscle-invasive-disease, and evaluation included recurrence, metastases, and overall survival. The data of patients treated with RC for muscle-invasive-disease, selection was revised for 127 patients with urothelial carcinoma mixed with SCC/SqD, two comparative arms were 100 patients with pure UC, and 100 patients had pure SCC. Follow up was on 8 months, 3 years, and 5 years to detect recurrence, metastasis, and overall survival in the three groups. The results showed that by comparison of disease aggressiveness in the three groups regarding recurrence, metastasis, and overall survival was analysed. Overall survival with mixed tumours was significantly lower than pure UC or SCC, recurrence and metastases were higher in mixed tumour which was an independent factor for poor prognosis and low survival.

c-KIT positive schistosomal urinary bladder carcinoma are frequent but lack KIT gene mutations.

Urinary bladder squamous cell carcinoma (SCC), one of the most common neoplasms in Egypt, is attributed to chronic urinary infection with Schistosoma haematobium (Schistosomiasis). The proto-oncogene c-KIT, encoding a tyrosine kinase receptor and implicated in the development of a number of human malignancies, has not been studied so far in schistosomal urinary bladder SCCs. We therefore determined immunohistochemical (IHC) expression of c-KIT in paraffin sections from 120 radical cystectomies of SCCs originally obtained from the Pathology Department of Suez Canal University (Ismailia, Egypt). Each slide was evaluated for staining intensity where the staining extent of >10% of cells was considered positive. c-KIT overexpression was detected in 78.3% (94/120) of the patients, the staining extents in the tumor cells were 11-50% and >50% in 40 (42.6%) and 54 (57.4%) respectively. The positive cases had 14.9%, 63.8%, 21.3% as weak, moderate and strong intensity respectively. Patients with positive bilharzial ova had significantly higher c-KIT expression than patients without (95.2% vs. 38.9%, P=0.000). Mutation analysis of exons 9-13 was negative in thirty KIT positive cases. The high rate of positivity in SBSCC was one of the striking findings; However, CD117 may be a potential target for site specific immunotherapy to improve the outcome of this tumor.