Cadherin-16 (CDH16) immunohistochemistry: a useful diagnostic tool for renal cell carcinoma and papillary carcinomas of the thyroid.

Cadherin-16 (CDH16) plays a role in the embryonal development in kidney and thyroid. Downregulation of CDH16 RNA was found in papillary carcinomas of the thyroid. To determine the expression of CDH16 in tumors and to assess the diagnostic utility a tissue microarray containing 15,584 samples from 152 different tumor types as well as 608 samples of 76 different normal tissue types was analyzed. A membranous CDH16 immunostaining was predominantly seen in thyroid, kidney, cauda epididymis, and mesonephric remnants. In the thyroid, CDH16 staining was seen in 100% of normal samples, 86% of follicular adenomas, 60% of follicular carcinomas, but only 7% of papillary carcinomas (p < 0.0001). CDH16 positivity was frequent in nephrogenic adenomas (100%), oncocytomas (98%), chromophobe (97%), clear cell (85%), and papillary (76%) renal cell carcinomas (RCCs), various subtypes of carcinoma of the ovary (16-56%), various subtyped of carcinomas of the uterus (18-40%), as well as in various subtypes of neuroendocrine neoplasms (4-26%). Nineteen further tumor entities showed a weak to moderate CDH16 staining in up to 8% of cases. Our data suggest CDH16 as a potential diagnostic marker-as a part of a panel-for the identification of papillary carcinomas of the thyroid, nephrogenic adenomas, and the distinction of renal cell tumors from other neoplasms.

Exome sequencing revealed comparable frequencies of RNF43 and BRAF mutations in Middle Eastern colorectal cancer.

Mutation-induced activation of Wnt-ß Catenin signaling pathway is frequent in CRC. The E3 ubiquitin ligase, RNF43, has been reported to negatively regulate the Wnt signaling pathway and RNF43 mutations are frequently seen in CRC. However, its role in Middle Eastern CRC remains unclear. Therefore, we employed Exome and Sanger sequencing technology to assess the frequency of RNF43 mutations and its association with other clinico-pathological features in Middle Eastern CRC. RNF43 mutations were found in 5.9% (13/220) of CRC cases and was inversely correlated to APC and TP53 mutations. A strong association of RNF43 mutations with right sided and sporadic microsatellite instable (MSI) CRC was observed. No association was identified between RNF43 mutation and other clinico-pathological features including BRAF mutation, age, tumor histological subtype, tumor grade or patients' prognosis. Multivariate logistic regression analysis revealed that MSI status and wild type APC were independent predictor of RNF43 mutation. We conclude that RNF43 mutations occur in Middle Eastern CRC at comparable frequencies with BRAF mutations and represent a distinct molecular subtype which further enhances our understanding of how different mutational subsets of Wnt tumor suppressor genes link to distinct tumor characteristics, which might be considered for treatment strategies for CRC patients.

Prevalence of germline TP53 mutation among early onset middle eastern breast cancer patients.

BACKGROUND: The data on prevalence and clinical relevance of TP53 germline mutations in early onset Middle-Eastern breast cancer (BC) is limited. METHODS: We determined TP53 germline mutations in a cohort of 464 early onset BC patients from Saudi Arabia using capture sequencing based next generation sequencing. RESULTS: Germline TP53 pathogenic mutations were found in 1.5% (7/464) of early onset Saudi BC patients. A total of six pathogenic missense mutations, one stop gain mutation and two variants of uncertain significance (VUS) were detected in our cohort. No TP53 pathogenic mutations were detected among 463 healthy controls. TP53 mutations carriers were significantly more likely to have bilateral breast cancer (p = 0.0008). At median follow-up of 41 months, TP53 mutations were an unfavorable factor for overall survival in univariate analysis. All the patients carrying TP53 mutations were negative for BRCA1 and BRCA2 mutations. Majority of patients (85.7%; 6/7) carrying TP53 mutation had no family history suggestive of Li-Fraumeni Syndrome (LFS) or personal history of multiple LFS related tumors. Only one patient had a positive family history suggestive of LFS. CONCLUSIONS: TP53 germline mutation screening detects a clinically meaningful risk of early onset BC from this ethnicity and should be considered in all early onset BC regardless of the family history of cancer, especially in young patients that are negative for BRCA mutations.

Zamzam water protects cancer cells from chemotherapy-induced apoptosis via mitogen-activated protein kinase-dependent pathway.

BACKGROUND: Many Muslims believe that water from the Zamzam well is the cure for every disease. Zamzam water (ZW) is naturally alkaline water consumed by millions of people worldwide. The current study investigated the effect of ZW on cell viability and apoptosis in breast, colorectal and ovarian cancer cell lines and compared the effect with that of drinking water (DW). METHODS: Three different ZW samples collected from different sources at different periods were used. To balance the tonicity, ZW and DW were buffered using PBS and the pH was adjusted to 7.4. For the treatment, ZW and DW were diluted to 50% with RPMI medium (10% FBS). Cancer cell lines were treated with ZW or DW, with and without chemotherapeutic agents, for 24 h. Apoptosis was measured using flow cytometry whilst the level of protein expression was determined by Western blotting. RESULTS: The results showed that treatment with ZW significantly increased cell proliferation compared to DW control. Treatment with ZW significantly suppressed the effect of chemotherapeutic agents on decreasing cell viability and inducing apoptosis in all the cancer cell lines compared to chemotherapeutic agents alone treated in DW. Furthermore, ZW treatment increased the phosphorylation of CRAF, MEK1/2, ERK1/2 and P38 proteins in these cell lines. Notably, treatment with ZW suppressed the effect of chemotherapy-induced reduction of CRAF, MEK1/2, ERK1/2 and P38 phosphorylation in breast and ovarian cancer cell lines. We also showed that silencing of ERK1/2 significantly increased the chemotherapy-induced apoptosis in breast and colorectal cancer cell lines. These data suggest that MAPK proteins; especially activated ERK1/2 may play a role in ZW mediated suppression of chemotherapy-induced cell death. CONCLUSIONS: These findings clearly demonstrate that ZW protects cancer cells from chemotherapy-induced apoptosis through activation of the ERK1/2-MAPK signaling pathway.

FoxM1 is an independent poor prognostic marker and therapeutic target for advanced Middle Eastern breast cancer.

Breast cancer (BC) is the most common cause of cancer-related death in females in Saudi Arabia. BC in Saudi women tend to behave more aggressively than breast cancer in the West. Therefore, identification of new molecular targets and treatment strategies are highly warranted to improve patient outcome. FoxM1 has been shown to play a critical role in pathogenesis of various malignancies. In this study, we explored the prevalence and clinical implication of FoxM1 overexpression in Saudi breast cancer. FoxM1 protein overexpression was seen in 79% (770/975) of BC tissues and was associated with aggressive clinical parameters such as younger age (< 30 yrs) (p = 0.0172), high grade (p < 0.0001), mucinous histology (p < 0.0001) and triple negative phenotype (p < 0.0001). Overexpression of FoxM1 was significantly associated with activated AKT (p < 0.0001), Ki67 expression (p < 0.0001), VEGF (p < 0.0001), MMP-9 (p < 0.0001), XIAP (p < 0.0001) and Bcl-xL (p = 0.0300). Importantly, FoxM1 overexpression is found to be an independent prognostic marker in multivariate analysis in advanced stage (Stage III and IV) breast cancer (p = 0.0298). In vitro data using BC cell lines showed that down-regulation of FoxM1 using specific inhibitor, thiostrepton or siRNA inhibited cell migration, invasion and angiogenesis. In addition, treatment of BC cell lines with thiostrepton resulted in inhibition of proliferation and induction of apoptosis in a dose-dependent manner. In vivo, thiostrepton treatment regressed MDA-MB-231 cells generated xenografts via down-regulation of FoxM1 and its downstream targets. Our results suggest that FoxM1 may be a potential therapeutic target for the treatment of aggressive breast cancers.

Overexpression of PARP is an independent prognostic marker for poor survival in Middle Eastern breast cancer and its inhibition can be enhanced with embelin co-treatment.

Patients with aggressive breast cancer (BC) subtypes usually don't have favorable prognosis despite the improvement in treatment modalities. These cancers still remain a major cause of morbidity and mortality in females. This has fostered a major effort to discover actionable molecular targets to treat these patients. Poly ADP ribose polymerase (PARP) is one of these molecular targets that are under comprehensive investigation for treatment of such tumors. However, its role in the pathogenesis of BC from Middle Eastern ethnicity has not yet been explored. Therefore, we examined the expression of PARP protein in a large cohort of over 1000 Middle Eastern BC cases by immunohistochemistry. Correlation with clinico-pathological parameters were performed. Nuclear PARP overexpression was observed in 44.7% of all BC cases and was significantly associated with aggressive clinico-pathological markers. Interestingly, nuclear PARP overexpression was an independent predictor of poor prognosis. PARP overexpression was also directly associated with XIAP overexpression, with PARP and XIAP co-expression in 15.8% (159/1008) of our cases. We showed that combined inhibition of PARP by olaparib and XIAP by embelin significantly and synergistically inhibited cell growth and induced apoptosis in BC cell lines. Finally, co-treatment of olaparib and embelin regressed BC xenograft tumor growth in nude mice. Our results revealed the role of PARP in Middle Eastern BC pathogenesis and prognosis. Furthermore, our data support the potential clinical development of combined inhibition of PARP and XIAP, which eventually could extend the utility of olaparib beyond BRCA deficient cancer.

XIAP over-expression is an independent poor prognostic marker in Middle Eastern breast cancer and can be targeted to induce efficient apoptosis.

BACKGROUND: Breast cancer is the most common cancer in females and is ranked second in cancer-related deaths all over the world in women. Despite improvement in diagnosis, the survival rate of this disease has still not improved. X-linked Inhibitor of Apoptosis (XIAP) has been shown to be over-expressed in various cancers leading to poor overall survival. However, the role of XIAP in breast cancer from Middle Eastern region has not been fully explored. METHODS: We examined the expression of XIAP in more than 1000 Middle Eastern breast cancer cases by immunohistochemistry. Apoptosis was measured by flow cytometry. Protein expression was determined by western blotting. Finally, in vivo studies were performed on nude mice following xenografting and treatment with inhibitors. RESULTS: XIAP was found to be over-expressed in 29.5% of cases and directly associated with clinical parameters such as tumor size, extra nodal extension, triple negative breast cancer and poorly differentiated breast cancer subtype. In addition, XIAP over-expression was also significantly associated with PI3-kinase pathway protein; p-AKT, proliferative marker; Ki-67 and anti-apoptotic marker; PARP. XIAP over-expression in our cohort of breast cancer was an independent poor prognostic marker in multivariate analysis. Next, we investigated inhibition of XIAP using a specific inhibitor; embelin and found that embelin treatment led to inhibition of cell viability and induction of apoptosis in breast cancer cells. Finally, breast cancer cells treated with combination of embelin and PI3-kinase inhibitor; LY294002 synergistically induced apoptosis and caused tumor growth regression in vivo. CONCLUSION: These data suggest that XIAP may be playing an important role in the pathogenesis of breast cancer and can be therapeutically targeted either alone or in combination with PI3-kinase inhibition to induce efficient apoptosis in breast cancer cells.

High throughput tissue microarray analysis of FHIT expression in diffuse large cell B-cell lymphoma from Saudi Arabia.

Recent studies have suggested a potential prognostic role of alterations of the fragile histidine triad (FHIT) gene in diffuse large B-cell lymphoma. To evaluate possible mechanisms of FHIT inactivation and to further clarify its potential prognostic relevance, we analyzed a set of 114 diffuse large B-cell lymphoma with clinical follow-up information. Tissue microarrays were analyzed by immunohistochemistry for protein expression, and corresponding DNA samples were analyzed for FHIT promotor hypermethlyation. Reduced or absent FHIT expression was found in 75 of 114 diffuse large B-cell lymphoma (66%), but was unrelated to clinical tumor stage or patient prognosis. FHIT promotor hypermethylation was observed in 29 of 93 (23%) interpretable diffuse large B-cell lymphoma. Hypermethylation was not significantly correlated to protein expression loss, which could be explained by competing mechanisms for FHIT inactivation in a substantial fraction of non FHIT hypermethylated diffuse large B-cell lymphoma. Hypermethylation was significantly associated with poor prognosis of diffuse large B-cell lymphoma patients and predominantly seen in nongerminal center diffuse large B-cell lymphoma (27%), but less frequent (13%) in germinal center diffuse large B-cell lymphoma. In summary, these data suggest that promotor hypermethylation is responsible for reduced FHIT expression in a substantial subset of diffuse large B-cell lymphoma, which is primarily composed of nongerminal center subtype with poor patient prognosis.

High frequency and strong prognostic relevance of O6-methylguanine DNA methyltransferase silencing in diffuse large B-cell lymphomas from the Middle East.

Several clinically relevant molecular classifiers of diffuse large B-cell lymphoma (DLBCL) have recently been demonstrated in Western populations. However, substantial molecular differences have recently been shown between tumors derived from different ethnic groups. To investigate prevalence and interrelationship of recently suggested molecular prognostic markers in Middle East DLBCL, we analyzed coexpression of CD10/Bcl6 (by immunohistochemistry), t(14;18) translocations (by fluorescence in situ hybridization), and methylation of the gene encoding the DNA repair enzyme O(6)-methylguanine DNA methyltransferase (MGMT) in a series of 190 DLBCL patients from Saudi Arabia with clinical follow-up data. Coexpression of CD10/Bcl6 (germinal center-like immunophenotype) was found in 13%, t(14;18) translocations in 17.9%, and MGMT methylation in 75.9% of cases. There was a trend toward better prognosis (although statistically insignificant) in tumors with coexpression of CD10/Bcl6. MGMT methylation were significantly related to good prognosis. The combined analysis of both parameters revealed that MGMT methylation was independent of immunophenotype and remained a significant predictor of prognosis in nongerminal center-like DLBCL subgroup. t(14;18) was significantly associated with CD10/Bcl6 coexpression (46.7%) but infrequent in CD10-/Bcl6-negative lymphomas (9.4%; P = .0073). However, t(14;18) was unrelated to clinical outcome. In summary, our data suggest a strong prognostic importance of MGMT methylation independent of DLBCL immunophenotype. Based on previous data from Western patients, the rate of MGMT hypermethylation was higher, and the portion of germinal center-like DLBCL was lower than expected. These results provide evidence for molecular differences between Saudi Arabian and Western DLBCL.