Zubeyde Yalniz

Promoter DNA methylation of Oncostatin M receptor-β as a novel diagnostic and therapeutic marker in colon cancer

In addition to genetic changes, the occurrence of epigenetic alterations is associated with accumulation of both genetic and epigenetic events that promote the development and progression of human cancer. Previously, we reported a set of candidate genes that comprise part of the emerging “cancer methylome”. In the present study, we first tested 23 candidate genes for promoter methylation in a small number of primary colon tumor tissues and controls. Based on these results, we then examined the methylation frequency of Oncostatin M receptor-β (OSMR) in a larger number of tissue and stool DNA samples collected from colon cancer patients and controls. We found that OSMR was frequently methylated in primary colon cancer tissues (80%, 80/100), but not in normal tissues (4%, 4/100). Methylation of OSMR was also detected in stool DNA from colorectal cancer patients (38%, 26/69) (cut-off in TaqMan-MSP, 4). Detection of other methylated markers in stool DNA improved sensitivity with little effect on specificity. Promoter methylation mediated silencing of OSMR in cell lines, and CRC cells with low OSMR expression were resistant to growth inhibition by Oncostatin M. Our data provide a biologic rationale for silencing of OSMR in colon cancer progression and highlight a new therapeutic target in this disease. Moreover, detection and quantification of OSMR promoter methylation in fecal DNA is a highly specific diagnostic biomarker for CRC.

Promoter methylation and loss of p16INK4a gene expression in head and neck cancer

Silencing of tumor suppressor genes plays a vital role in head and neck carcinogenesis. In this study we aimed to evaluate aberrant p16INK4a gene promoter methylation in patients with head and neck cancer.

Simultaneous Methylation Profiling of Tumor Suppressor Genes in Head and Neck Cancer

Head and neck cancer (HNC) is a common cancer, and its prognosis has not changed during the last decades. Detection of the disease at an early stage is crucial for successful treatment, as early diagnosis can significantly increase the survival rate. Methylation of tumor suppressor genes is an early event in cancer responsible for incorrect gene silencing. Since methylation changes are reversible, they also provide a promising target for therapy. So far, only individual genes have been analyzed for aberrant methylation in HNC. In this study, we analyzed the methylation status of 24 tumor suppressor genes simultaneously by methylation-specific multiplex ligation-dependent probe amplification in matched tumor and normal tissue samples from patients with HNC. CHFR, RARβ, DAPK1, and RASFF1 genes were the most frequently methylated genes in tumor tissue. Eight genes were not methylated in any sample. The methylation frequencies for individual genes ranged from 0% to 19%. Our results indicate that methylation of tumor suppressor genes is not high as previously reported by methylation-specific polymerase chain reaction and is confined to a smaller but significant fraction of the tumors. Whether this group represents a unique entity in the disease spectrum warrants further studies.

Assessment of microsatellite instability in head and neck cancer using consensus markers

Head and neck cancer is the sixth most common cancer in the world and one of the most lethal cancers. Microsatellite instability is an important characteristic of tumor cells and is observed both in presence and absence of mismatch repair gene mutations. The importance of microsatellite instability in head and neck cancer is not well established due to the lack of a consensus panel and selection of different markers, criteria and methodological variances. The main objective of this study was to investigate the performance of a consensus panel of microsatellite repeats by automated fragment analysis. Matched tumor and normal tissue samples from 99 patients were analyzed using five mononucleotide markers. Following PCR the amplified fragments were analyzed by capillary electrophoresis on an ABI 310 genetic analyzer. Microsatellite instability was observed in 26 patients. In 17 patients instability was detected at multiple loci. NR21 and BAT25 were the most frequently altered targets. These two mononucleotide markers could detect all samples displaying high-instability. In this study we describe a standardized fluorescent multiplex PCR combined with computerized analysis, which allows rapid and accurate analysis of a high number of samples and obviates the need to compare tumors with matching normal tissue.

Copy number profiling of tumor suppressor genes in head and neck cancer

Sensitive and reliable new biomarkers are needed in head and neck cancer to predict the outcome and for therapy that is more effective. Copy number alterations are frequent and play a critical role in cancer.

Abstract 1059: Analysis of promoter methylation levels of COX2 gene in Turkish patients with head and neck cancer

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Purpose: Methylation of CpG islands in the promoter regions of genes plays an important role in head and neck carcinogenesis. Overexpression of COX2 gene was recorded in various types of cancer in literature. In addition, the promoter region of the COX2 gene displayed high methylation in normal colorectal tissue and normal gastric tissue samples. On the other hand, it has recently been reported in two different gastrointestinal cancer studies, that low expression of COX2 was observed in some colorectal/gastric cancers and hypermethylation/related transcriptional silencing of COX2 gene is common in colorectal cancer. There is no study investigating the COX2 methylation status on head and neck cancer (HNC) in the literature. In our study we evaluated the COX2 gene promoter methylation in patients with HNC. Experimental Design: Methylation of the COX2 was investigated by bisulfite modification/methylation-specific polymerase chain reaction in tumors and matched normal tissue samples from 99 Turkish patients with HNC. Results: The promoter region of the COX2 was methylated in 53.5% and 65.6% of the primary tumors and the corresponding normal tissue samples, respectively [p = 0.082, χ2 = 3.02, OR(95%CI) = 0.602 (0.34-1.06)]. Considering the methylation levels observed in our previous studies, and published data on different cancer types, methylation levels 25%) were 30.3% and 58.6% in the tumor and normal tissues, respectively (p = 0.00006, χ2 = 19.24). The results were similar when a subgroup of 60 patients with larynx cancer from this cohort, were analyzed separately (p = 0.0006, χ2 = 14.87). Conclusions: Methylation may play an important role in HNC carcinogenesis. Although we found that there was no significant association between the methylation levels of tumor and adjacent normal tissues, high methylation levels of the COX2 gene were observed (>25%) in normal matched tissues when compared to the levels in HNC tumors. Citation Format: Semra Demokan, Cansu Ozkoklesen, Zubeyde Yalniz, Necati Enver, Murat Ulusan, Nejat Dalay. Analysis of promoter methylation levels of COX2 gene in Turkish patients with head and neck cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1059. doi:10.1158/1538-7445.AM2015-1059

Abstract A48: The K-ras let-7 miRNA binding site variant and K-ras mutations in colon cancer

The K-ras gene is one of the most commonly mutated genes in cancer. The ras proteins act as functional switches in the activation of the MAPK pathway in a complex signaling network coupling growth factor receptors to the intracellular kinases. Mutations in the K-ras gene lead to the constitutive activation of the protein and consequently of the MAPK pathway. K-ras mutations are early events in colorectal carcinogenesis and are observed in 30-40 % of the colorectal cancers. Mutations mostly occur at codons 12, 13 and 61 of the gene and result in the deregulation of the protein activity. Several studies have shown that patients with K-ras mutations do not benefit from therapies with anti-EGFR monoclonal antibodies. miRNAs are global regulators of gene expression by binding to the 39-untranslated region of the target mRNA. Recently, it has been shown that the K-ras gene is regulated at the translational level by binding of the let-7 miRNA. The let-7 family of miRNAs is known to play an important role in several cancer types. A germline SNP at the 39-UTR of the K-ras gene (rs: 61764370 T-G) has been shown to disrupt binding of the let-7 molecule to K-ras resulting in overexpression of the gene and was found to confer increased susceptibility for distinct cancer types. The let-7 miRNA variant was found to be associated with poor outcome in lunh and head and neck cancers. However, the frequency and association of this variant with other cancer types has not been investigated widely. In this study we evaluated the frequency of the K-ras gene variant in 145 patients with colon cancer and 85 healthy individuals and its possible association with the K-ras gene mutations. The K-Ras gene variant was analyzed by polymerase chan reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, K-ras gene mutations were investigated by real time-PCR. The results were evaluated using the Chi-square test. The variant allele of the K-ras gene was observed in 23 patients (15.9%) in the heterozygote form. A single patient was homozygous for the variant allele. The variant allele was detected in 12 % of the control group. K-ras gene mutations were present in 46 (32 %) of the patients. The frequency of the variant allele was 13 % among the patients harboring a K-ras gene mutation. The distribution of the variant allele is in accordance with a report on the US control population and no significant difference was observed between the patients and the controls. Citation Format: Nejat Dalay, Zubeyde Yalniz, Orkun Gurbuz. The K-ras let-7 miRNA binding site variant and K-ras mutations in colon cancer. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A48. doi: 10.1158/1557-3125.RASONC14-A48

Abstract 4902: Methylation profiles in head and neck cancer

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Purpose: Among human malignancies, head and neck cancer (HNSCC) is the sixth most common cancer type in the world. Multiple genetic and epigenetic changes may contribute to the development of HNSCC. One of the epigenetic alterations, aberrant methylation in the promoter regions of multiple genes that lead to transcriptional silencing, has been shown in various cancer types including HNSCC. In this study we aimed to evaluate the epigenetic changes specific to HNSCC by investigating aberrant promoter hypermethylation of a panel of 24 tumor suppressor genes. Experimental Design: We investigated methylation of the promoter regions by methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA). The methylation status of the selected genes were analyzed in 126 patients with HNSCC. Tumor and corresponding normal tissue samples were obtained during surgical resection. Probes added to the samples hybridized with their target sequence and were amplified by PCR depending on the methylation status of the target. The amplified products were detected by sequence type capillary electrophoresis. Peak sizes and areas were normalized and the relative signal peaks were compared to determine the methylation status. Results: 18 of 24 (75 %) genes displayed methylation in the tumor samples while none of the corresponding normal tissue samples were methylated. In 67.5 % (85/126) of the tumors the promoter regions were hypermethylated in at least one of the genes. The most frequently methylated genes were RARB (retinoic acid receptor beta), CHFR (checkpoint with forkhead and ring-finger domains) and CDH13 (Cadherin 13) genes. In 50 (39.7 %) patients, methylation was observed in more than one gene. Conclusions: Methylation of the CHFR, RARB and CDH13 gene promoters is a frequent event in HNSCC, demonstrating potential for these genes as biomarkers in detection strategies. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4902.

Abstract C20: MGMT promoter methylation and analysis of microsatellite instability in head and neck cancer by consensus markers

Head and neck cancer is the sixth most common cancer in the world and traditional prognostic indices are of limited use in predicting prognosis. Therefore, there is a need for a beter understanding of the molecular pathogenesis of the disease and a great effort is being made to determine new prognostic and predictive markers that could improve stratification of the patients Polymorphic microsatellites are useful genetic markers and important tools for studying genetic alterations in tumors. Microsatellite instability refers to the impairment of the mismatch repair system resulting in gains or losses in the number of the tandem repeat units. Cells with mutated mismatch repair genes are not able to correctly repair these errors. The importance of microsatellite instability in head and neck cancer is not well established because of lack of comparable parameters, use of different markers, different criteria in classification and discrepancies concerning the loci examined. Recently it has been shown that mononucleotide markers are more sensitive and specific than dinucleotide markers for the detection of microsatelite instability. No standard approach for the study of MSI has been agreed to in HNC. In this study we analyzed the role and incidence of microsatellite instability in head and neck cancer with criteria and markers recommended by NCI which have been widely used for different types of cancer. We also analyzed methylation of the promoter region of the MGMT gene which functions in the repair pathway of alkylating nitrosocompounds. Microsatellite instability was analyzed in matching tumor and normal tissue samples from 99 patients with head and neck cancer using a panel of five (NR21, BAT25, BAT26, NR24 and MONO27) mononucletoide markers. Two pentanucleotide markers were used to correctly identify the individual tumor and normal tissue samples. The analyses were performed on an ABI Prism 310 capillary electrophoresis system. Microsatellite instability was observed in 26 patients (26.2%). The most frequently affected markers were BAT 25 and NR21 which displayed changes in repeat length in 19 and 11 patients, respectively. Among the microsatellite‐positive patients 17 tumor samples (17.1%) displayed high degrees of instability while the remaining 9 samples were instable in only one locus. In five patients instability was observed in three or more loci. Instability was more frequent in tumors with advanced stages. Significant methylation of the MGMT gene was observed in most patients. In 31 patients both alleles were methylated, while in 65 samples partial methylation was observed. Citation Information: Cancer Res 2009;69(23 Suppl):C20.