Sahsine Tolunay

Aven blocks DNA damage-induced apoptosis by stabilising Bcl-xL

Induction of apoptosis by DNA-damaging agents involves the activation of mitochondrial apoptotic pathway. Aven has been identified as an antiapoptotic protein and has been shown to activate ATM in response to DNA damage. In this study, we demonstrated that enforced expression of Aven blocks UV-irradiation-, SN-38- or cisplatin-induced apoptosis upstream of mitochondria by stabilising Bcl-xL protein levels in breast cancer cells. Aven silencing by RNA interference markedly enhanced apoptotic response following treatment with DNA-damaging agents. Aven is complexed with Bcl-xL in untreated breast cancer cells and treatment with DNA-damaging agents led to decreased Aven/Bcl-xL interaction. Importantly, Bcl-xL was necessary for the prosurvival activity of Aven and depletion of Bcl-xL abrogated Aven-mediated protection against DNA damage-induced apoptosis. Analysis of breast cancer tissue microarrays revealed decreased Aven nuclear expression in breast cancer tissues compared with non-neoplastic breast tissues. In particular, we detected reduced nuclear expression of Aven in infiltrating ductal carcinoma and papillary carcinoma breast cancer subtypes compared with non-neoplastic breast tissues and infiltrating lobular breast cancer tissues. Our results suggest that Aven is an important mediator in DNA damage-induced apoptotic signalling in breast cancer cells and its nuclear expression is altered in breast cancer tissues, which may contribute to genomic instability in breast cancer tumours.

Pathologic characteristics of pediatric intracranial pilocytic astrocytomas and their impact on outcome in 3 countries: a multi-institutional study.

Pilocytic astrocytoma (PA) is one of the most common glial neoplasms in the pediatric population, and its gross total resection can be curative. Treatment of partially resected or recurrent tumors is challenging, and the factors associated with increased recurrence risk are not well defined. Identification of favorable and unfavorable factors can lead to a better understanding and management of patients with PA. We studied the pathologic characteristics of 116 intracranial PAs from 4 institutions representing 3 distinct geographic regions to identify factors that may be associated with biological behavior. The study included 65 boys and 51 girls with a median age of 6 years. Median follow-up for all patients was 101 months, during which time 38 patients had recurrence. Progression-free and overall survivals were better in patients who underwent gross total resection. We were not able to identify any differences in pathologic and molecular markers among the 4 institutions from 3 different countries. However, progression-free survival varied significantly among institutions. Sox-2 was the most prevalent stem cell marker in PA, and many tumors showed synaptophysin positivity. BRAF immunostaining was not useful in determining BRAF duplication. BRAF duplication was more typical of posterior fossa tumors. There was a strong correlation between BRAF duplication and pERK immunostaining, suggesting that the RAF/MEK/ERK pathway is active in these tumors. This finding has significant implications given its role in oncogen-induced senescence and possible influence on treatment decisions of subtotally resected tumors.

The Promoter Hypermethylation Status of GATA6, MGMT, and FHIT in Glioblastoma

Glioblastoma (GBM) is an aggressive and lethal cancer, accounting for the majority of primary brain tumors in adults. GBMs are characterized by large and small alterations in genes that control cell growth, apoptosis, angiogenesis, and invasion. Epigenetic alterations also affect the expression of cancer genes, either alone or in combination with genetic mechanisms. The current evidence suggests that hypermethylation of promoter CpG islands is a common epigenetic event in a variety of human cancers. A subset of GBMs is also characterized by a locus-specific and genome-wide decrease in DNA methylation. Epigenetic alterations are important in the molecular pathology of GBM. However, there are very limited data about these epigenetic alterations in GBM. Alterations in promoter methylations are important to understand because histone deacetylases are targets for drugs that are in clinical trial for GBMs. The aim of the current study was to investigate whether the promoter hypermethylation of putative tumor suppressor genes was involved in GBM. We examined the methylation status at the promoter regions of GATA6, MGMT, and FHIT using the methylation-specific polymerase chain reaction in 61 primary GBMs. Our results reveal that there is no promoter hypermethylation of FHIT in the examined GBM tissue specimens. In contrast, the promoter hypermethylation of GATA6 and MGMT was detected in 42.8 and 11.11% of GBMs, respectively. The frequency of MGMT promoter hypermethylation was low in the group of patients we evaluated. In conclusion, our study demonstrates that promoter hypermethylation of MGMT is a common event in GBMs, whereas GATA6 is epigenetically affected in GBMs. Furthermore, inactivation of FHIT by epigenetic mechanisms in GBM may not be associated with brain tumorigenesis.

Impact of novel PTEN mutations in Turkish patients with glioblastoma multiforme

Glioblastoma multiforme (GBM) represents the most common and aggressive type of primary neoplasms of the central nervous system. The PTEN (phosphatase, tensin homologue, deleted on chromosome TEN; MIM # 601728) tumor suppressor gene has an essential biological role in the formation of glioblastomas. It is known that there are variations in genetic alterations in tumors that develop in patients with different ethnic backgrounds and because there is no study evaluating PTEN mutation in Turkish patients with GBM, we aimed to realize the present study. We investigated 62 GBM tumors for mutations of the PTEN gene using single strand conformational polymorphism (SSCP) method followed by DNA sequencing. As a result of our investigation, PTEN mutations were detected in 15 of 62 tumors (24.19%). Nine different sequence variants were identified: one novel promoter site mutation (5′UTR −9C→T), one novel intronic mutation (IVS2-2delA), four novel point mutations (61A→G, 105T→G, 248C→G, and 364C→G), two novel frameshift mutations (213delC) and 378delGATA) and one previously reported global exonic transition type mutation (129G→A). Since the majority of PTEN mutations identified in the present study are novel, we believe that these alterations may be specific to Turkish population. Furthermore, though no significant correlation was found between PTEN mutations and histopathological properties of GBM tumors, our findings indicate that localizations of mutations in PTEN gene may have an effect on clinical aggressiveness of GBM tumors.

Phase II Study of Gemcitabine Plus Paclitaxel in Metastatic Breast Cancer Patients with Prior Anthracycline Exposure

Chemotherapy provides palliation and modest prolongation of symptom-free survival in metastatic breast cancer. Taxane containing regimens are commonly considered to be among the initials in metastatic setting due to earlier use of anthracyclines in the course of breast cancer. Therefore, we conducted this Phase II study to assess efficacy and safety of gemcitabine plus paclitaxel (GT) combination therapy in anthracycline pretreated metastatic first-line setting. Patients and Methods: The study enrolled 26 women with pathologically confirmed and measurable metastatic breast cancer who were previously treated with anthracycline but no prior chemotherapy for metastatic disease. Twenty six and twenty four patients were eligible for toxicity and efficacy evaluations respectively. Mean age was 47.3 years and median ECOG performance status was 0. Twenty patients (76.9 percent) had visceral metastases, most commonly located in liver and lung. Treatment schedule was as follows: paclitaxel 175 mg/m2 was administered intravenously in 3 hours on Day 1 and gemcitabine 1000 mg/m2 was administered intravenously in 30 minutes on Day 1 after paclitaxel application, and on Day 8 every 21 days. Results: Objective response rate was 41.7 percent (95 percent CI: 21.9–61.4) with 16.7 percent (95 percent CI: 1.7–31.6 percent) CR, and 25.0 percent (95 percent CI: 7.6–42.3 percent) PR. Median time to progression and overall survival were 9.6 and 14.5 months, respectively. Grade 3–4 toxicity was observed in 34.6 percent (9) patients. Treatment of two patients was discontinued due to toxicity, consisting of Grade 3 hypersensitivity reactions and Grade 4 infections in one patient each. Dose reductions due to myelotoxicity were performed in 4 (15.3 percent) patients. Hematologic toxicities were generally manageable with appropriate dose modifications and supportive care. Conclusion: Gemcitabine and paclitaxel combination regimen is effective and has manageable toxicity profile as first line metastatic setting.

BRCA mutations cause reduction in miR-200c expression in triple negative breast cancer.

Triple negative breast cancer (TNBC) is the most aggressive and poorly understood subclass of breast cancer (BC). Over the recent years, miRNA expression studies have been providing certain detailed overview that aberrant expression of miRNAs is associated with TNBC. Although TNBC tumors are strongly connected with loss of function of BRCA genes, there is no knowledge about the effect of BRCA mutation status on miRNA expressions in TNBC cases. The aims of this study were to evaluate the expression profile of miRNAs that plays role in TNBC progression and the role of BRCA mutations in their regulation. The expression level of BC associated 13 miRNAs was analyzed in 7 BRCA mutations positive, 6 BRCA mutations negative TNBC cases and 20 non-tumoral tissues using RT-PCR. According to RT2 Profiler PCR Array Data Analysis, let-7a expression was 4.67 fold reduced in TNBCs as compared to normal tissues (P=0.031). In addition, miR-200c expression was 5.75 fold reduced in BRCA mutation positive TNBC tumors (P=0.005). Analysis revealed a negative correlation between miR-200c and VEGFA expressions (r=-468). Thus, miR-200c may be involved in invasion and metastasis in TNBC cases with BRCA mutation. In this study we provide the knowledge on the first report of association between microRNA-200c and BRCA mutations in TNBC. Further studies and evaluations are required, but this miRNA may provide novel therapeutic molecular targets for TNBC treatment and new directions for the development of anticancer drugs.

CK19, CK20, EGFR and HER2 status of circulating tumor cells in patients with breast cancer.

AIMS AND BACKGROUND The major cause of death in breast cancer patients is metastasis. Various biomarkers have been used for the early detection of circulating tumor cells in the peripheral blood of breast cancer patients. The aims of the current study were to analyze circulating tumor cells in the blood of breast cancer patients by investigating EGFR, CK19, CK20 and HER2 expression profiles and to evaluate their prognostic importance. METHODS CK19, CK20 and EGFR gene expression profiles were evaluated in the blood samples of 84 female patients with primary invasive ductal breast cancer and 20 healthy female volunteers using SYBR green-based real-time qPCR assays. HER2 expression analyses were conducted in 46 patients who had an HER2-positive primary tumor and in 30 healthy women to determine the cutoff level of positivity. RESULTS The positive rates of CK20, EGFR, CK19 and HER2 mRNA expression in the peripheral blood were 28.57% (24/84), 20.23% (17/84), 5.95% (5/84) and 2.17% (1/46), respectively. The high positive ratio of CK20 mRNA expression in the peripheral blood of breast cancer was identified for the first time in the current study. Significant differences were identified in CK20 expression status and several clinical parameters related with aggressiveness of tumors using a binary logistic regression analysis. Higher CK20-positive levels were observed in patients who had lymph node metastasis and advanced-grade primary tumors, which were estrogen receptor-negative. We have demonstrated that CK20 may be a novel biomarker that is useful to identify circulating tumor cells and predict breast cancer progression. CONCLUSIONS The results suggest that the investigation of CK20 mRNA with other biomarkers in the peripheral blood of breast cancer patients may be useful to monitor the presence of disseminated tumor cells in the blood circulation and to predict the prognosis of breast cancer.

Primary small cell carcinoma of the breast: report of seven cases and review of the literature

AIMS AND BACKGROUND The aim of the study was to analyze the clinicopathological characteristics, treatment modalities, and clinical outcome of patients with primary small cell carcinoma of the breast. METHODS Fifty-three cases of primary small cell carcinoma of the breast were identified; 7 cases in this series and 46 from the English-language medical literature. RESULTS There were 52 females and 1 male. The mean age was 53 years. Tumor size ranged from 1 to 18 cm (mean, 4.53). Axillary node metastasis was present in 61.7%. Only one patient had distant metastases at presentation. The presence of hormone receptors was reported in 24.5% of the tumors. Modified radical mastectomy was the most common surgical procedure and was performed in 50.9% of the patients. Adjuvant radiotherapy was administered to 39.6% of the patients, and 69.8% underwent chemotherapy. Thirteen percent of patients received adjuvant tamoxifen therapy. The mean follow-up was 20.75 months (range, 3-60), and 10 of 53 cases (18.9%) died of metastatic disease. CONCLUSIONS The prognosis of primary small cell carcinoma of the breast largely depends on the initial stage of the disease. Multimodality treatment including surgery, radiotherapy and chemotherapy seems to be the most appropriate strategy for early disease. Chemotherapy is usually unsuccessful in treating metastatic disease.

FHIT Gene Sequence Variants and Reduced Fhit Protein Expression in Glioblastoma Multiforme

Molecular studies have an important role in the elucidation of the mechanisms involved in Glioblastoma multiforme (GBM) development. The occurrence of FHIT gene alterations, which has an important role in different cancers, has not yet been studied well in GBM. We aimed to investigate the occurrence of alterations of FHIT gene sequence and protein expression in the GBMs. Sequence alterations in exons 5–9 of the FHIT gene were screened in 63 GBMs using the single-strand conformational polymorphism method, followed by DNA sequencing. Additionally, the level of Fhit protein expression in tissues of 48 tumors was assessed by immunohistochemistry (IHC). In our investigation, FHIT gene alterations in the coding region were detected in 11 of the 63 GBM cases (17.5%). Two different sequence variants were determined: one novel missense variant (G→C transition at codon 49) and one previously described silent alteration (C→T transition at codon 88). Using web-based programs, such as SIFT and ESEfinder, it was determined that both alterations might have caused significant modification on protein function. In addition, we identified a previously reported an intronic polymorphism (T→A transition at IVS8-17) in 47.5% of cases as a similar rate (45%) in the control group. Moreover, it was observed that Fhit protein expression was reduced in 87.5% of tumors. In conclusion, the reduction or loss of Fhit protein expression by genetic alterations or epigenetic mechanisms in GBM might be associated with brain tumorigenesis.

Investigation of MMAC/PTEN Gene Mutations and Protein Expression in Low Grade Gliomas

The MMAC/PTEN tumor suppressor gene has an essential biological role in the formation of glioblastomas. It is known that there are variations in genetic alterations in tumors that develop in patients with different ethnic backgrounds; thus, we aimed to evaluate the incidence of MMAC/PTEN mutations and protein expression among various low grade gliomas of Turkish patients. We investigated 28 low grade gliomas for mutations of the MMAC/PTEN gene using single strand conformational polymorphism method followed by DNA sequencing. Additionally, the level of MMAC/PTEN protein expression in the tissues of 26 tumors was assessed by immunohistochemistry. In our investigation, MMAC/PTEN mutations were detected in 2 of 28 tumors (7.14%). One novel sequence variant G → A transition at codon 159 was identified. This missense variation was a result of an alteration from AGG (Arginine) to AAG (Lysine). Moreover, it was observed that MMAC/PTEN protein expression was reduced to 73.08% of tumors. In conclusion, reduced MMAC/PTEN expression by genetic and/or epigenetic mechanisms in low grade gliomas might be associated with glioma tumorigenesis.