Impact of 3'UTR variation patterns of the KRAS gene on the aggressiveness of pancreatobiliary tumors with the KRAS G13D mutation in a Turkish population.

OBJECTIVE: Several studies have demonstrated the importance of mutations in codons 12, 13 and 61 and variations in the 3' untranslated region (3'UTR) of the KRAS gene, frequently observed genetic events in the progression of pancreatobiliary tumors (PBT). However, limited data exist on the clinical effect of these alterations. The aim of the current study was to clarify the frequency of relevant alterations of the 3'UTR regions of the KRAS gene and the effect of KRAS 3'UTR polymorphisms on the prognosis of patients with codon 12, 13 and 61 mutations in a Turkish population with PBT. METHODS: Codons 12, 13, and 61 and 3'UTRs of the KRAS gene were screened by single-strand conformation polymorphism (SSCP) analysis and DNA sequencing in 43 patients and 10 controls. Chi-squared and independent sample T tests were used to evaluate the results of the mutation analysis and clinical features of the patients. RESULTS: We defined the c.38G > A (rs112445441, p.G13D) (39.54%) mutation and two 3'UTR variations, c.*4066delA (rs560890523) (23.26%) and c.*4065_*4066delAA (rs57698689) (6.98%), in the KRAS gene of Turkish patients. There was a statistically significant relationship between the c.*4066delA (rs560890523) and c.*4065_*4066delAA (rs57698689) variations and invasion and lymph node metastasis status of the patients (p < 0.001). Compared to patients with c.38G > A (rs112445441, p.G13D), patients with c.*4066delA (rs560890523) and c.38G > A (rs112445441, p.G13D) presented more aggressive tumors with highly invasive features. The present study contributes findings regarding the clinical effects of KRAS alterations in PBT. Based on our study, further investigations are required.

First determination of azole resistance in Aspergillus fumigatus strains carrying the TR34/L98H mutations in Turkey.

Aspergillus fumigatus is the most important etiological agent of invasive aspergillosis. Recently, an increasing number of azole-resistant A. fumigatus isolates have been described in various countries. The prevalence of azole resistance was investigated in this study using our culture collection of A. fumigatus isolates collected between 1999 and 2012 from clinical specimens. Seven hundred and forty-six A. fumigatus isolates, collected from 419 patients, were investigated. First, all isolates were screened for resistance to itraconazole by subculturing on Sabouraud dextrose agar that contained 4 mg/L itraconazole. For isolates that grew on the itraconazole containing agar, the in vitro activities of amphotericin B, itraconazole, voriconazole and posaconazole were determined using the Clinical and Laboratory Standards Institute (CLSI) M38-A reference method. After PCR amplification, the full sequence of the cyp51A gene and its promoter region was determined for all in vitro azole-resistant isolates. Itraconazole resistance was found in 10.2% of the A. fumigatus isolates. From 2000 onwards, patients were observed annually with an itraconazole-resistant isolate. According to in vitro susceptibility tests, amphotericin B exhibited good activity against all isolates whereas the azoles were resistant. Sequence analysis of the promoter region and CYP51A gene indicated the presence of TR34/L98H in 86.8% (n = 66) of isolates. This initial analysis of the resistance mechanism of A. fumigatus from Turkey revealed a common TR34/L98H mutation in the cyp51A gene.

BRCA1/2 germline mutations and their clinical importance in Turkish breast cancer patients.

BRCA1/BRCA2 genes were screened in 117 patients with breast cancer by sequencing. Fourteen percent of patients tested positive for BRCA1/BRCA2 mutations. Four frame shift mutations, four pathogenic missense mutations, and 25 different sequence variations were detected. BRCA mutation positivity was significantly associated with Ki67 (p = .001). BRCA protein expressions were decreased in the patients harboring important mutations and polymorphisms (BRCA1;P508 stop, V1740G, Q1182R, Q1756P and BRCA2;V2466A) related with disease. Our findings contribute significantly to the types of germline BRCA1/BRCA2 mutations and their biological effects in Turkish women. These data could help guide the management of BRCA1/BRCA2 mutation-carrying patients when considering breast-conserving therapy.

MicroRNA expression patterns of tumors in early-onset colorectal cancer patients.

BACKGROUND: The expression of microRNAs (miRNAs) may differ in tumors from patients with different ethnic origins and ages. The aims of the present study were to clarify the appropriate alterations of miRNA expression associated with the early stages of carcinogenesis in early-onset Turkish colorectal cancer (CRC) patients and to define specific biomarkers that could be used as new diagnostic and prognostic markers for this population. MATERIALS AND METHODS: The expression profiles of 38 different miRNAs associated with CRC were evaluated using miRNA polymerase chain reaction arrays in tumors and surgical margin tissue samples from 40 sporadic early-onset Turkish CRC patients. The relationships between the miRNA expression profiles and the characteristics of the tumors and patients were evaluated. RESULTS: The expression of miR-106a was found to be upregulated, and miR-143 and miR-125b levels were found to be downregulated in tumor tissues compared with the normal tissues. The high expression level of miR-106a (2.93-fold; P = 0.031) and low expression level of miR-125b (2.42-fold; P = 0.063) were observed in tumors with lymph node metastases compared with the normal colorectal mucosa samples. However, the deregulation of these miRNAs was not significantly associated with survival (log-rank P > 0.05). CONCLUSIONS: The present results implied that miR-106a and the miR-125b were associated with the formation and invasion of colorectal tumors. Thus, these miRNAs might be used as significant prognostic factors and indicators of early-stage CRC. Further studies and validations are required; these miRNAs may provide novel molecular targets for CRC treatment.

Microsatellite instability status affects gene expression profiles in early onset colorectal cancer patients.

BACKGROUND: The association between microsatellite instability (MSI) status and gene expression profiles in the early onset sporadic colorectal cancer (CRC) has not been clearly established. The aim of this study was to identify the altered gene expression patterns depending on the MSI status of early onset CRC and determine specific biomarkers that could provide novel therapeutic molecular targets in the Turkish population. MATERIALS AND METHODS: MSI markers (BAT25, BAT26, D2S123, D5S346, and D17S250) were investigated in tumors from 36 early onset sporadic CRC patients in whom gene expression profiles were analyzed previously. The relationship between the gene expression profiles depending on MSI status was evaluated. RESULTS: A total of 15 tumors (16.66%) were identified as having MSI and 21 tumors (58.33%) were identified as having microsatellite stability (MSS). CK20 and MAP3K8 upregulation, observed in MSS tumors, was significantly associated with lymph node metastasis, recurrence, and/or distant metastasis and a short median survival (P < 0.05). REG1A upregulation is also correlated with recurrence and/or distant metastasis and a short median survival in patients with MSI tumors (P < 0.05). CONCLUSIONS: High expression levels of CK20 and MAP3K8 in MSS tumors and REG1A in MSI tumors correlated with a poor prognosis in CRC patients. Further studies and validations are required; these genes may provide novel therapeutic molecular targets for the development of anticancer drugs related to MSI status for early onset CRC treatment.

Molecular approach to genetic and epigenetic pathogenesis of early-onset colorectal cancer.

Colorectal cancer (CRC) is the third most frequent cancer type and the incidence of this disease is increasing gradually per year in individuals younger than 50 years old. The current knowledge is that early-onset CRC (EOCRC) cases are heterogeneous population that includes both hereditary and sporadic forms of the CRC. Although EOCRC cases have some distinguishing clinical and pathological features than elder age CRC, the molecular mechanism underlying the EOCRC is poorly clarified. Given the significance of CRC in the world of medicine, the present review will focus on the recent knowledge in the molecular basis of genetic and epigenetic mechanism of the hereditary forms of EOCRC, which includes Lynch syndrome, Familial CRC type X, Familial adenomatous polyposis, MutYH-associated polyposis, Juvenile polyposis syndrome, Peutz-Jeghers Syndrome and sporadic forms of EOCRC. Recent findings about molecular genetics and epigenetic basis of EOCRC gave rise to new alternative therapy protocols. Although exact diagnosis of these cases still remains complicated, the present review paves way for better predictions and contributes to more accurate diagnostic and therapeutic strategies into clinical approach.

Expression and clinical significance of miRNAs that may be associated with the FHIT gene in breast cancer.

The dysregulation of miRNA expression has frequently been observed in breast cancer. Therefore, we investigated the expression profile of miRNAs that may be associated with expression of the FHIT gene in breast cancer and assessed their clinicopathological significance. The expression levels of miR-143, miR-663a, miR-668, miR-922 and FHIT were analyzed in normal and malignant breast tissues from 65 patients with breast cancer. We studied the correlation between the expression of miR-143, miR-663a, miR-668, miR-922 and FHIT and the clinicopathological features presented by the patients. The expression levels of the miRNAs and FHIT were downregulated in breast cancer tissue. The expression levels of miR-143, miR-663a and miR-668 were significantly reduced in FHIT downregulated tumors. miR-668 expression was also significantly altered relative to FHIT down- and up- regulated tumor tissues. Reduced miR-663a expression was statistically associated with high-grade ER/PR (+) status, benign reactive hyperplasia, lymph-node metastasis, in-situ component >25% and Ki 67>15% compared with non-tumor tissues. Additionally, reduced miR-668 expression was significantly different between tumors with and without lymph-node metastasis. miR-668 may play an important role in breast cancer development and progression by regulating the expression of FHIT. Furthermore, miR-668 and miR-663a may be potential prognostic biomarkers for breast cancer.

Association of MDR1, CYP2D6, and CYP2C19 gene polymorphisms with prophylactic migraine treatment response.

Prophylactic therapy response varies in migraine patients. The present study investigated the relationship between the resistance to the drugs commonly used in prophylactic therapy and the possible polymorphic variants of proteins involved in the metabolism of these drugs. Migraine patients with the MDR1 3435TT genotype exhibited a better treatment response to topiramate than migraine patients with the CC and CT genotypes (p=0.020). The MDR1 C3435T polymorphism was also found to be a higher risk factor for topiramate treatment failure in a comparison of the number of days with migraine (ß2=1.152, p=0.015). However, there was no significant relationship between the treatment response to topiramate and either the CYP2D6 or CYP2C19 polymorphism, and there were no significant correlations between the treatment responses to amitriptyline, propranolol, and valproic acid and the MDR1, CYP2D6 and CYP2C19 gene polymorphisms. This is the first study to investigate the effect of the polymorphic variants on prophylactic therapy response in migraine patients.

Circulating miR-195 as a Therapeutic Biomarker in Turkish Breast Cancer Patients.

BACKGROUND: Dysregulation of miRNA expression may be used as a biomarker for specific tumours because it may contribute to development of cancer. Circulating miRNA profiles have been highlighted for their potential as predictive markers in heterogeneous diseases such as breast cancer. In the literature, there is evidence that miR-195 levels are differentially expressed pre- and post-operative periods in breast cancer patients. At the same time, miRNA expression levels may vary because of ethnic origins. This study aimed to determine expression levels and potential roles of miR-195 in Turkish breast cancer patients. MATERIALS AND METHODS: The expression patterns of miR-195 were initially examined in breast cancer tissues (luminal A and B type) (n=96). Subsequently, blood samples were prospectively collected from preoperative and postoperative Turkish breast cancer patients and disease free controls. Total RNA was isolated, and the expression level of miR-195 was quantified by real-time PCR. RESULTS: We found that miR-195 level was altered in Turkish breast cancer patients, with down-regulation evident in breast cancer tissues compared to normal adjacent specimens. Furthermore, circulating levels of miR- 195 was significantly decreased in post-operative blood samples compared with pre-operative levels (p=0.01 and <0.05). However, miR-195 was significantly increased in pre-operative blood samples of the luminal B type (p= 0.04 and <0.05). CONCLUSIONS: This study represents the first report of a miR-195 expression profile in Turkish breast cancer patients. Our data suggests that miR-195 levels might be a clinically useful biomarker in the earliest stage of Turkish breast cancer patients.

Mutation analysis of the PARKIN, PINK1, DJ1, and SNCA genes in Turkish early-onset Parkinson's patients and genotype-phenotype correlations.

OBJECTIVE: Variations in PARK genes (PRKN, PINK1, DJ-1, and SNCA) cause early-onset Parkinson's disease (EOPD) in different populations. In the current study, we aimed to evaluate the frequencies of variations in PARK genes and the effects of these variations on the phenotypes of Turkish EOPD patients. METHODS: All coding regions and exon-intron boundaries of the PRKN, PINK1, DJ-1, and SNCA genes were screened by heteroduplex analysis followed by direct sequencing of the detected variants in 50 Turkish EOPD patients. These variants were evaluated using SIFT, PolyPhen, HSF, and LOVD web-based programs. RESULTS: The frequency of EOPD-associated variations in the PRKN gene was 34%. Among these variations, p.A82E in exon 3 and p.Q409X in exon 11 was determined to be pathogenic. We also defined previously unknown cryptic variations, including c.872-35 G>A and c.872-28T>G in exon 8 of PRKN and c.252+30 T>G and c.322+4 A>G in exons 4 and 5 of DJ1, respectively, that were associated with EOPD. Although no significant association was observed between the PARK gene mutations and clinical features (P>0.05), the alterations were related to the clinical symptoms in each patient. CONCLUSION: An increasing number of studies report that PRKN, PINK1, DJ1 and SNCA mutations are associated with early-onset Parkinson's disease; however, a limited number of studies have been conducted in Turkey. Additionally, our study is the first to evaluate the frequency of SNCA mutations in a Turkish population. The aim of this study was determine the frequency distributions of the PRKN, PINK1, DJ1, and SNCA gene mutations and to analyze the relationships between these genetic variations and the clinical phenotype of EOPD in Turkish patients.

miR-216b Targets FGFR1 and Confers Sensitivity to Radiotherapy in Pancreatic Ductal Adenocarcinoma Patients Without EGFR or KRAS Mutation.

OBJECTIVES: The success of gemcitabine plus radiotherapy is dependent on the mutation status of pancreatic ductal adenocarcinoma (PDAC) tumors in the EGFR and KRAS genes; however, radiotherapy resistance may also be modulated epigenetically by microRNA (miRNA) regulation. In this study, we examined the potential effect of miRNAs on the resistance to radiotherapy in cases without EGFR or KRAS mutation. METHODS: The association of EGFR and KRAS mutation status and different expression patterns of 6 selected miRNAs related to the EGFR/KRAS signaling pathway were evaluated in the tumors of 42 patients with PDAC. RESULTS: Reduced miR-216b and miR-217 expression was associated with aggressive tumor characteristics and shortened disease-free survival. In addition, miR-216b expression was reduced 2.7-fold in the cases that did not benefit from therapy, although they did not demonstrate EGFR or KRAS expression (P = 0.0316). A negative correlation between FGFR1 and miR-216b expression (r = -0.355) was found in the tumors of these cases. CONCLUSIONS: Further studies and validations are required; in the tumors of patients with PDAC without activating mutations and induced expression of EGFR/KRAS genes, down-regulated miR-216b expression may be associated with a poor response to radiotherapy via deregulation of another signaling pathway related to FGFR1 signaling.

Evaluation of genetic variations in miRNA-binding sites of BRCA1 and BRCA2 genes as risk factors for the development of early-onset and/or familial breast cancer.

Although genetic markers identifying women at an increased risk of developing breast cancer exist, the majority of inherited risk factors remain elusive. Mutations in the BRCA1/BRCA2 gene confer a substantial increase in breast cancer risk, yet routine clinical genetic screening is limited to the coding regions and intron- exon boundaries, precluding the identification of mutations in noncoding and untranslated regions. Because 3' untranslated region (3'UTR) polymorphisms disrupting microRNA (miRNA) binding can be functional and can act as genetic markers of cancer risk, we aimed to determine genetic variation in the 3'UTR of BRCA1/BRCA2 in familial and early-onset breast cancer patients with and without mutations in the coding regions of BRCA1/ BRCA2 and to identify specific 3'UTR variants that may be risk factors for cancer development. The 3'UTRs of the BRCA1 and BRCA2 genes were screened by heteroduplex analysis and DNA sequencing in 100 patients from 46 BRCA1/2 families, 54 non-BRCA1/2 families, and 47 geographically matched controls. Two polymorphisms were identified. SNPs c.*1287C>T (rs12516) (BRCA1) and c.*105A>C (rs15869) (BRCA2) were identified in 27% and 24% of patients, respectively. These 2 variants were also identified in controls with no family history of cancer (23.4% and 23.4%, respectively). In comparison to variations in the 3'UTR region of the BRCA1/2 genes and the BRCA1/2 mutational status in patients, there was a statistically significant relationship between the BRCA1 gene polymorphism c.*1287C>T (rs12516) and BRCA1 mutations (p=0.035) by Fisher's Exact Test. SNP c.*1287C>T (rs12516) of the BRCA1 gene may have potential use as a genetic marker of an increased risk of developing breast cancer and likely represents a non-coding sequence variation in BRCA1 that impacts BRCA1 function and leads to increased early-onset and/or familial breast cancer risk in the Turkish population.

Overexpression of CK20, MAP3K8 and EIF5A correlates with poor prognosis in early-onset colorectal cancer patients.

PURPOSE: Due to ethnic, genetic and environmental factors, the clinical and molecular characteristics of Turkish colorectal cancer (CRC) patients are different from those of Western populations. The aim of this study was to clarify the relevant alterations of gene expression associated with colorectal carcinogenesis in early-onset patients and to identify specific biomarkers that could provide novel therapeutic molecular targets in this population. METHODS: The expression profiles of 114 different genes were evaluated using mRNA PCR arrays in 39 tumors and 20 surgical margin tissue samples from 39 sporadic CRC patients diagnosed at less than 50 years of age. RESULTS: The expression levels of IMPDH2, CK20, MAP3K8 and EIF5A were strongly up-regulated in CRC tissues compared with normal colorectal tissues (p < 0.05). The highly significant expression ratios of CK20, MAP3K8 and EIF5A observed in the colorectal tumors of patients predicted recurrence (p < 0.05). The expression of IMPDH2, CK20, MAP3K8 and EIF5A was significantly higher in the tumors of patients with short median survival (log-rank p value < 0.05). Progression-free survival was also significantly increased in patients with low expression of the EIF5A gene compared with those who exhibited high expression of this gene (log-rank p value < 0.05). CONCLUSION: We demonstrated that high CK20, MAP3K8 and EIF5A expression levels were significant prognostic factors for poor overall survival in CRC patients. Further studies and validations are required; these genes may provide novel therapeutic molecular targets for CRC treatment, as well as new directions for the development of anticancer drugs.

Olea europaea leaf extract alters microRNA expression in human glioblastoma cells.

PURPOSE: Glioblastoma multiforme (GBM) is the most common and the most lethal form of primary malignant tumors in the central nervous system. There is an increasing need for the development of more efficient therapeutic approaches for the treatment of these patients. One of the most attractive cancer therapy methods to date is the induction of tumor cell death by certain phytochemicals. Interestingly, bioactive compounds have been shown to alter micro RNA (miRNA) expression involved in several biological processes at the posttranscriptional level. The present study aimed to evaluate whether Olea europaea leaf extract (OLE) has an anticancer effect and modulates miRNA expression in GBM. MATERIALS AND METHODS: Firstly, the anti-proliferative activity of OLE and the nature of the interaction with temozolomide (TMZ) of OLE were tested in human glioblastoma cell line T98G cells by trypan blue and WST-1 assays and than realized miRNA PCR array analysis. Potential mRNA targets were analyzed bioinformatically. RESULTS: OLE exhibited anti-proliferative effects on T98G cell lines. Cells were treated with temozolomide (TMZ) in the presence OLE, and changes to miRNA expression levels were identified by PCR array analysis. miRNA target genes are involved in cell cycle and apoptotic pathways. Specifically, miR-181b, miR-153, miR-145, miR-137, and let-7d were significantly upregulated after treatment with both TMZ and OLE. CONCLUSION: Our results suggest that OLE modulates the expression of some miRNAs related to anticancer activity in GBM and the response to TMZ. Further studies and validations are needed, but we suggest that OLE might be used for in vivo studies and future medical drug studies.

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.