The role of cancer stem cells in immunotherapy for bladder cancer: An in vitro study.

OBJECTIVE: Bladder cancer is characterized by frequent recurrence and progression. CD44+ cancer stem cells (CSCs) might be one of the main reasons for recurrence. Although Bacillus Calmette Guerin (BCG) has become a gold standard immunotherapy, after treatment recurrence frequently occur. Based on this knowledge, the aim of this study was to evaluate the changes in cytokine and chemokine expressions in bladder cancer and CSCs cultures in vitro with BCG only and in combination with IL2 and lymphocyte (MNCs) applications. MATERIAL AND METHODS: In this study, 3 cell lines of human bladder cancer cells with different characteristics (T24, 5637, and JMSU-1) and CD44+ bladder CSCs isolated by magnetic bead isolation (Miltenyl Magtech) were used. Bladder cancer cell lines and bladder CSCs in complete medium were cultured under humidified conditions of 37°C temperature in 5% CO2. BCG only and its combination with IL2 and MNCs were applied to bladder cancer cell lines and bladder CSCs for 24, 48, and 72 hours. Annexin V-PI was used to detect the percentages of apoptotic and necrotic cells in treatment groups and control groups. After treatments, total RNAs were isolated and converted to cDNA for each group and controls. Quantitative fold changes in terms of gene expression were measured by RT2-PCR array and fold changes for expression levels of genes were compared among groups. Eighty-four genes were analyzed in standard array of chemokines and cytokines (Biorad). RESULTS: BCG treatment with 7.32 µg/ml dose alone and in combination with IL2 (1000 IU/ml) and MNCs (1000 cells/ml) were found to be most effective on bladder cancer cells. When BCG and its combinations were applied to CSCs of the 3 cell lines, BCG treatment showed cytotoxic effect on CSCs as well as cancer cells. CSCs of 3 cell lines over expressed CXCL5, CCL8, CNTF, and CSF2 compared with cancer cells. Cancer cells over expressed IL6, TNSFF11, FASLG, and CXCL9 compared with CSCs. In all 3 cell lines, BCG application increased expression of CXCL5 and LTB and also decreased CCL20 and IL6. When BCG was combined with IL2 and MNCs, CXCL10, CXCL5, and IFNG were increased and CXCL12, IL6, and TNSF11 were decreased. BCG treatment of CSCs caused increases in ADIPOQ, CXCL10, and XCL1 and a decrease in CCL8. When IL2 and MNCs were combined with BCG, the expression of many cytokines and chemokines decreased. CONCLUSION: BCG treatment changes the expression of many cytokines and chemokines in bladder cancer. The expression differs in 3 different cell lines and their CSCs. Immune modulation of each case differs from each other. The effectivity of BCG-based immunotherapy in bladder cancer on CSCs might decrease in combination with IL2. Our results indicate that recurrence after BCG treatment for bladder cancer may not occur mainly based on the CSCs hypothesis considering bladder cancer occurs at different loci of surface epithelium.

MicroRNA-17, MicroRNA-19b, MicroRNA-146a, MicroRNA-302d Expressions in Hepatoblastoma and Clinical Importance.

Hepatoblastoma (HB) is the most common liver malignancy in children. The prognosis changes according to the histologic subtypes of HB. In the present study, we aimed to characterize the expression level of selected microRNAs (miRNAs) in HB as well as in histologic subtypes, and to consider the association with the prognosis. A total of 22 HB tumor samples, subtyped as fetal (n=16) and embryonal (n=6), and 10 nontumorous surrounding liver samples were evaluated in this study. Expressions of miR-17, miR-146a, miR-302d, and miR-19b were analyzed in 22 HB tumor samples and 10 nontumorous surrounding liver samples by quantitative real-time polymerase chain reaction. Lower miRNA-17 expression levels were obtained in tumor samples in comparison with nontumorous surrounding liver samples (P=0.028). Lower miRNA-17 expression was significant for predicting prognosis in HB patients (area under receiver-operator characteristic curve=0.875, P=0.044). A higher-level of miR-19b was found in embryonal samples (P=0.008). Overall and event-free survival was not found to correlate with miRNA expression levels (P>0.05). This research finds miRNA-17 and miRNA-19b expression levels can provide important data on diagnosis and prognosis in HB showing different clinical behaviors.

Analysis of genetic and non genetic risk factors for cisplatin ototoxicity in pediatric patients.

OBJECTIVE: The aim of this study was to analyse the genetic and non genetic risk factors for cisplatin ototoxicity. METHODS: This study was conducted on 72 children who received cisplatin based chemotherapy. Brock and Muenster classifications were used to evaluate ototoxicity seen in these children. 6 single nucleotide polymorphisms (SNP); ERCC1 rs 11615, GSTP1 rs1138272, GSTP1 rs1695, LRP2 rs 2075252, TPMT rs 12201199, COMT rs 9332377, were evaluated as genetic factors by real time PCR. Non genetic factors such as cranial irradiation, cumulative doses of cisplatin, age, gender, administration of other ototoxic drugs were analysed as well. By using Chi-square test, risk factors were matched with the ototoxicity classifications. Significant risk factors were reevaluated using logistic regression modelling. RESULTS: According to univariate analyses, male gender, co-treatment with aminoglycosides and mutant genotype of GSTP1 rs1695 were significantly related with cisplatin ototoxicity. Logistic regression modelling analyses also showed that male gender, co-treatment with aminoglycosides were found to be significantly related with cisplatin ototoxicity. Mutant genotype of GSTP1 rs1695 was not found to be significant, but close to the level of statistical significance. CONCLUSION: Male gender, co-treatment with aminoglycosides are significant risk factors for cisplatin ototoxicity in pediatric patients. Mutant genotype of GSTP1 rs1695 seems to be a genetic risk factor in univariate analyses, although not confirmed by multivariate analyses. Therefore, GSTP1 rs1695 SNP needs to be studied in larger series.

Ex vivo evaluation of the effect of regulatory T cells on the anti-tumor activity of bortezomib in multiple myeloma.

Multiple myeloma (MM) is a hematologic cancer characterized by malignant proliferation of plasma cells and their precursors. Immunosuppressive CD4+CD25+Foxp3+ regulatory T (Treg) cells are increased in the peripheral blood of patients with MM. On the basis of this finding, we sought to evaluate the ex vivo effect of CD4+CD25+Foxp3+ Treg cells on the anti-tumor effect of the proteosome inhibitor bortezomib on MM cells. We collected peripheral blood and bone marrow aspiration samples from 20 patients with newly diagnosed MM and isolated CD4+CD25+Foxp3+ Treg cells from peripheral blood mononuclear cells. The bone marrow mononuclear cells were cultivated in RPMI at 37°C and 5% CO2 for 72 hours. The LD50 doses of bortezomib, isolated Treg cells, and their combination were added. After 24 hours, the viability of CD138+ myeloma cells was evaluated by WST-1. We compared the anti-tumor effect of bortezomib alone and in combination with Treg expansion and statistically analyzed the measured differences with respect to the clinical parameters of the patients. Treg cells had varied effects on bortezomib, increasing, decreasing, or not changing its anti-tumor effect. The increased in vitro anti-tumor effect of bortezomib after Treg cell expansion was correlated in patients who did not develop bortezomib resistance in vivo (p = 0.022). These patients with in vivo non-bortezomib-resistant MM also responded to Treg expansion with decreased cell viability (p = 0.024). Our data indicate that the ex vivo expansion of Treg cells increased the cytotoxic effect of bortezomib in clinically sensitive cases.

Regulatory T cells and their prognostic value in hepatopancreatobiliary tumours.

BACKGROUND/AIMS: The aim of this study was to determine the prognostic values of Foxp3+ Treg cells, CD4+ Tcells and CD8+ T cells in cancer cases of gallbladder, pancreas and liver. METHODOLOGY: This study included 20 patients with gallbladder cancer, 25 patients with pancreatic cancer and 8 patients with liver cancer. Foxp3, CD4 and CD8 were immunohistochemically evaluated and compared with histopathological and clinical prognostic parameters. RESULTS: Foxp3, CD4 and CD8 expression levels were significantly higher in peritumoral areas than in intratumoral areas in patients with gallbladder, pancreas, liver cancers (p<0,05). Positivity of Foxp3, CD4 and CD8 was correlated with advanced stage (p<0,05), poor differentiation, lymphovascular invasion, perineural invasion, advanced age. Patients with high positivity of Foxp3 had a shorter disease free survival (p<0,05). CONCLUSION: Our results indicate that the ratio of Tregs/T helper cells (Foxp3+/CD4+) cells was higher in intratumoral area in hepatopancreatobiliary tumors. We conclude that intratumoral inlamatory cells might work for cancer cells, besides peritumoral cells work against cancer cells.

The correlation between T regulatory cells and autologous peripheral blood stem cell transplantation in multiple myeloma.

OBJECTIVE: Multiple myeloma (MM) is characterized by malignant proliferation of plasmocytes and their precursors. T regulatory cells (Tregs) have a role in immunosuppression and control of autoimmunity, and are currently an important topic in the study of immune response to tumor cells. The correlation between Tregs and autologous peripheral blood stem cell transplantation (APBSCT) in MM has not been studied. The aim of this study was to compare CD4+CD25+FOXP3+ Treg, CD200, and PD-1 levels in MM patients that did and did not undergo APBSCT. METHODS: Peripheral blood samples were collected from 28 MM patients ranging in age from 41 to 78 years for analysis of CD4CD25+ FOXP3+ Tregs, PD-1 (CD279), and CD200. Peripheral blood mononuclear cells were isolated via density gradient centrifugation. Four-color flow cytometry was performed. Using a sequential gating strategy, Tregs were identified as CD4+CD25+FOXP3+ T-cells. Results were analyzed using the Mann Whitney U non-parametric test and a compare means test. p values <0.05 were considered statistically significant. RESULTS: The study included 28 MM patients (10 female and 18 male). In all, 11 of the patients underwent APBSCT. The level of Tregs identified as CD4+CD25+FOXP3+ T-cells was higher in the patients that underwent APBSCT (p=0.042). CD200 and PD-1 levels did not significantly differ between the 2 groups (p=0.711 and p=0.404, respectively). There weren't any statistically significant differences in CD200, PD-1, or CD4+CD25+FOXP3+ T-cell levels between the patients that did and did not undergo APBSCT (p>0.05). CONCLUSION: Treg levels were higher in the patients that underwent APBSCT. Tregs are crucial for the induction and maintenance of peripheral tolerance to self-antigens. In addition, Tregs can suppress immune responses to tumor antigens; however, APBSCT and Treg levels were not correlated with CD200 or PD-1 expression. Relationship of Tregs with prognosis needs to be determined by studies that include larger cohorts.