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EgeUn###, 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.

Aim: Triple negative breast cancer has poor prognosis requiring new combination treatments strategies. Structurally resembling the best known mTOR inhibitor of rapamycin and a calcineurin inhibitor, tacrolimus modulates mTOR in the absence of rapamycin with antiproliferative efficacy shown for cancer types like glioblastoma multiforme, hepatocellular carcinoma, and lymphoma, the aim of this study is to assess the efficacy of tacrolimus on tumor cells from an in vitro 4T1 cell line representing triple-negative breast cancer and in a cancer model in experimental animals. Method: 4T1 triple-negative breast cancer cell line was incubated with tacrolimus (1, 10, 20, 30, 40, 50, 100 µm) for 24, 48 and 72 hours. Cell viability percentages were assessed with WST-1 analysis. 14 athymic nude mice with mean weight 25 g aged 5-7 weeks were injected with 4T1 cells subcutaneously. After 150 mm3 tumor development, animals were randomized into a control group administered physiologic serum and an experimental group administered 10 mg/kg tacrolimus, on the 1st and 8th days via the intraperitoneal route. On the 14thday, animals were sacrificed and changes in tumor tissue were examined histopathologically and for apoptosis with flow cytometry with annexin-V and propidium iodide in fresh tissue suspension. Results: The LD50 dose of tacrolimus was identified as 30 µM. In the triple-negative breast cancer model in experimental animals, the targeted tumor size was reached in 8-12 days. Side effects were not observed from the two doses of tacrolimus administered at a one-week interval. After sacrifice on the 14th day, histopathologic assessment of tumor tissues observed prominent necrosis, necrobiosis and apoptosis in the tumor group compared to the control group. Annexin-V+PI flow cytometry investigation found the early apoptotic effect of tacrolimus was higher compared to late apoptosis and necrosis. Conclusion: Tacrolimus was found to be effective both in vitro and in vivo on triple-negative breast cancer triggering necrosis in addition to apoptosis. Tacrolimus is promising as a treatment choice for the clinical problem of triple-negative breast cancer.

High-dose cisplatin (CDDP) causes dose-limiting side effects in neuroblastoma (NB) treatment. Mesenchymal stem cells (MSC) are a current research area in cellular treatments due to multipotential characteristics. The aim of this study is to assess the interaction of MSC with CDDP in an athymic nude mouse NB model. Athymic male nude mice (n=28) were injected subcutaneously with C1300 NB cell line. After tumor growth to 1 cm diameter in 7-10 days, mice were randomly assigned to one of 4 experimental groups of control, CDDP treatment, MSC treatment and CDDP+MSC treatment with 7 mice in each group. Animals had basal auditory tests performed and had physiological serum or CDDP (20 mg/kg) injected into the peritoneum and were intravenously injected with 1×105 MSC once. Seven days later, hearing tests were performed again and the animals were sacrificed. Tumor tissue was assessed in terms of necrosis, apoptosis and viability. Apoptosis was evaluated with annexin V+PI flow cytometry analysis and TUNEL. Additionally, the MSC rate within the tumor was assessed with flow cytometry for triple CD34+ CD44+ and CD117-expression. Additionally, liver, kidney, brain and cochlear tissue were analyzed with light microscopy in terms of systemic side effect profile. Expression of the cochlear cell proteins of calretinin, math-1 and myosin2A were immunohistochemically assessed in ear sections. Statistical analysis used the nonparametric Kruskal Wallis and Mann Whitney U tests with p

Objective The aim of this study was to show whether local application of cadmium-impregnated bone cement can induce apoptosis and decrease the viability of residual osteosarcoma (OS) cells in nude mice. Methods K7M2 tumorigenic OS cell line was cultivated in vitro. The xenograft tumor model was formed by subcutaneously adding the tumor cells to athymic nude mice. Tumor was formed within 1 month. Then, mice were randomly assigned to five groups, each containing seven nude mice: control (group 1), wide resection (group 2), intralesional resection (group 3), intralesional resection + bone cement (group 4), and intralesional resection + cadmium embedded in bone cement (group 5). Tumor resection with 1 cm surgical margins was performed in the wide resection group. In intralesional resection groups, tumor tissue was resected with positive margins aiming to leave 15 mm3 of macroscopic tumor tissue. In group 3, the defect was left empty; groups 4 and 5 received bone cements prepared with saline and cadmium solutions, respectively. After the resection, mice were observed for 15 days and sacrificed. Next, surgical resection sites were evaluated histopathologically in each group. Results Recurrent tumor was formed in all mice in the wide resection group, and apparent progression of residual tumor was observed in groups 3 and 4. On the contrary, only a thin layer of residual tumor was observed around the bone cement in group 5. Histological evaluation revealed remarkable necrosis in group 5 and lowest viability compared to other groups. No systemic toxic effect related to cadmium was observed. Conclusion Our data suggest that local application of cadmium in bone cement has a significant potential to increase tumor necrosis and decrease the viability of residual OS cells.

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.

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.

Trastuzumab, the HER2 oncogene targeting drug, shows remarkable clinical efficacy in HER2-amplified breast cancer patients. Despite of robust activity, some of the patients with HER2-positive breast cancers do not get the benefit due to trastuzumab resistance. Overexpression of p95HER2 is one of the molecular mechanisms of trastuzumab resistance. The purpose of this study was to investigate whether p95HER2 overexpressing breast cancers were resistant to trastuzumab.p95HER2 (truncated HER2) and HER2 were determined by real-time polymerase chain reaction (RT-PCR) analysis. HER2 protein expression and HER2 gene amplification were also determined by immunohistochemistry (IHC) and chromogenic in situ hybridization (CISH). Archival material from 80 formalin-fixed paraffin-embedded (FFPE) breast cancer tumor tissues was used for the study. None of the cases had metastases at the initial diagnosis. HER2-positive cases were treated with trastuzumab with/without chemotherapy.Of 80 breast cancer cases 39 (48.7%) were HER2-positive and had trastuzumab treatment. Of these 39 cases 11 (28.2%) were trastuzumab-resistant and 28 (71.8%) were not, 17 (43.6%) were recurrent cases and 22 (56.4%) were not. Three patients died during follow-up. p95HER2 mean ratio was 11.01±19.73 in 11 cases which were trastuzumab-resistant, while p95HER2 mean ratio was 1.99±1.37 in 28 cases without trastuzumab resistance. If p95HER2 ratio was low, there was no trastuzumab resistance. However, when p95HER2 ratio was high, there was trastuzumab resistance (p=0.210, Mann-Whitney U test).p95HER2 was correlated with trastuzumab resistance, but it was not an independent factor of trastuzumab resistance. We claim that p95HER2 is sensitive but not specific for the prediction of trastuzumab resistance.

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.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 values0.05 were considered statistically significant.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 (p0.05).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.AMAÇ: Multipl miyelom (MM) plazmositlerin ve bunların öncü hücrelerinin habis proliferasyonu ile kendini gösteren bir hastalıktır. T düzenleyici (regülatör) hücreler (Treg) immünsüpresyonda ve otoimmün sistemin denetiminde rol oynarlar. Ayrıca tümör hücrelerine karşı oluşan immün yanıttaki rolleri de güncel bir araştırma konusudur. Treg hücreleri MM’da otolog çevre kanı kök hücre nakli (APBSCT) ile ilişkili olarak daha önce araştırılmamıştır. Bu çalışmanın amacı, CD4+ CD25+ FoxP3+ Treg hücreleri ile, CD200 ve PD–1 ‘in APBSCT yapılmış ve yapılmamış MM hastalarındaki düzeylerini karşılaştırmaktır. YÖNTEMLER: Yaşları 41 ile 78 arasında 28 MM hastasından CD4+ CD25+ FoxP3+ Treg hücreleri ile, CD200 ve PD–1 (CD279) analizi için çevre kanı örnekleri alındı. Mononükleer hücreler dansite gradient santrifüj yöntemi ile ayrıldı. Dört renkli akış sitometri ile uygulandı. Ardışık kapılamalar (gating) ile Treg hücreleri CD4+ CD25+ FoxP3+ T hücreleri olarak tanımlandı. Sonuçlar Mann Whitney U nonparametrik testi ve Compare Means testleri ile analiz edildi. P değerleri0.05 olanlar istatistiksel açıdan anlamlı kabul edildi.Bu çalışma 28 MM hastası (10 kadın, 18 erkek) içermektedir. Otolog çevresel kan kök hücre nakli 11 hastaya uygulanmıştır. CD4+ Treg hücreler CD4+ CD25+ FoxP3+ olarak saptanmış olup APBSCT alan hastalarda daha yüksek bulunmuştur (p=0.042). CD200 ve PD-1 iki grup arasında istatistiksel anlamlı bir sonuç göstermemiştir (sırasıyla; p=0.711 ve p=0.404). APBSCT yapılan ve yapılmayan gruplar arasında CD200, PD-1 ve CD4+CD25+FOXP3+ düzeyleri karşılaştırıldığında istatistiksel anlamlı bir sonuç bulunmamıştır (p0.05). SONUÇ: Bu çalışmada Treg hücreleri APBSCT yapılmış hastalarda daha yüksek düzeyde bulunmuştur. Treg hücrelerinin bireyin kendi hücrelerine karşı periferik toleransın indüklenmesinde ve korunmasında çok önemli rolü olduğu bilinmektedir. Buna ek olarak, Treg hücreleri tümör antijenlerine karşı oluşturulacak immün yanıtı baskılayabilmektedir. Ne var ki, çalışmamızda APBSCT veya Treg hücre düzeyleri CD200 ve PD-1 ekspresyonları ile korelasyon göstermemiştir. Prognoz ile ilişkileri daha fazla sayıda olgu grupları içeren çalışmalarla aydınlatılabilir.

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 degrees 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. Copyright (C) 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc.

Breast cancer stem cells have been found to be responsible for tumorigenic potential and resistance to therapy. This study aimed at comparing gene expression profiles in breast cancer, based on the differences of stem cells in their biological characteristics.Four breast cancer cell lines with different molecular and biological characteristics were used to analyze 84 breast cancer-related gene expressions. These were the ductal human epithelial breast cancer cell line T47D (HTB-133) with metastatic origin, the invasive ductal human breast carcinoma cell line MDA-MB-231 (HTB-26), the ductal human epithelial breast cancer cell line BT-474 (HTB-20) and the human metastatic breast adenocarcinoma cell line MCF-7 (HTB-22).There were significant differences between the breast cancer cells and the stem cells, particularly in angiogenesis, migration, proliferation and the expression of the DNA repair genes.These data indicated the absence of a general cancer stem cell in breast cancer. Our study supports the use of the term "breast cancer initiating cells" instead of breast cancer stem cells. All of these genetic differences should be taken into account in the planning of final therapeutic approach.

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.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.Foxp3, CD4 and CD8 expression levels were significantly higher in peritumoral areas than in intratumoral areas in patients with gallbladder, pancreas, liver cancers (p0,05). Positivity of Foxp3, CD4 and CD8 was correlated with advanced stage (p0,05), poor differentiation, lymphovascular invasion, perineural invasion, advanced age. Patients with high positivity of Foxp3 had a shorter disease free survival (p0,05).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.