Balkan Journal of Medical Genetics

EVALUATION OF METHYLATION PROFILES OF AN EPIDERMAL GROWTH FACTOR RECEPTOR GENE IN A HEAD AND NECK SQUAMOUS CELL CARCINOMA PATIENT GROUP
Mutlu M, Mutlu P, Azarkan S, Bayır O, Ocal B, Saylam G, Korkmaz MH
*Corresponding Author: Associate Professor Murad Mutlu, M.D., Department of Otorhinolaringology, Health Sciences University, Ministry of Health, Dışkapı Yildirim Beyazit Training and Research Hopsital, Sehit Ömer Halisdemir Street, No. 20, 06110, Dışkapı, Ankara, Turkey. Tel.: +90-312-516-2000. Fax: +90-312-318-6690. E-mail: muradmutlu78@yahoo.com
page: 65

MATERIALS AND METHODS

Study Population. Forty-seven unrelated Turkish HNSCC patients who were clinically diagnosed at the Department of Otorhinolaryngology, Dışkapı Yıldırım Beyazıt Training and Research Hospital, Ankara, Turkey, and 48 unrelated healthy volunteers from different geographic regions of Turkey, were included in this study. The control group was selected to match the patients in terms of demographic data including age and gender. All individuals in the study groups gave informed consent and approval of the local ethics committee was obtained from Dışkapı Yıldırım Beyazıt Training and Research Hospital [2018.10.15; #55/18]. The study was conducted in accordance with the guidelines of the Declaration of Helsinki. Clinicopathological parameters of patients were determined by both tumor type and tumor stage. Tumor stage 1 (T1) represents the primary tumor (<2 cm) and at this stage no cancer cells are present in nearby structures such as lymph nodes or distant sites. Tumor stage 2 (T2) shows the tumors that measure between 2-4 cm with no cancer cells in nearby structures, lymph nodes or distant sites. Tumor stage 3 (T3) shows either tumors larger than 4 cm across with no cancer cells present in nearby structures, lymph nodes or distant sites, or any size but with cancer cells that present in one lymph node that is located on the same side of the head or neck as the primary tumor. Finally, tumor stage 4 (T4) represents the head and neck cancer tumor in any size but is spreading to nearby structures, lymph nodes, invaded deeper tissues or distant sites (Table 1). DNA Isolation and Bisulfite DNA Modification. Genomic DNA was isolated from both HNSCC patient and control group’s peripheral blood samples using QIAamp® DNA Blood Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions. Bisulfite DNA modification was performed using EZ DNA Methylation Gold™ Kit (ZymoResearch, Irvine, CA, USA). Bisulfite modified DNAs were stored at –80 °C until they were used for methylation-specific-polymerase chain reaction (MS-PCR) analyses. Methylation-Specific-Polymerase Chain Reaction. Promoter methylation of EGFR gene was detected by MSPCR. Bisulfite modified DNAs were used as the template for this analysis. As a positive methylation control, bisulfite converted universal methylated human DNA standard (ZymoResearch) was used. Specific primers for both methylated and unmethylated DNA sequences were obtained for the EGFR gene (Table 2) [29]. Two sets of primers were specific for nucleotides –130 to –300 in the 5’ untranslated region (5’UTR) of the human EGFR promoter. The MS-PCR mixture contained 10 × LightCycler®480 SYBR Green I Master Mix (Roche Diagnostics International AG, Rotkreuz, Switzerland), 10 × PCR primers and Bisulfite-modified DNA (500 ng) in a final volume of 20 μL. Control DNA was used for each set of PCR. The MSPCR reaction was performed using the LightCycler®480 Instrument (Roche Diagnostics International AG). The MS-PCR conditions were as follows: pre incubation at 95 °C for 5 min., amplification for 45 cycles at 95 °C for 10 seconds, 60 °C for 10 seconds and 72 °C for 10 seconds with single acquisition mode at 72 °C. Melting curve analysis was performed at 95 °C for 5 seconds, 65 °C for 1 min. and continuous acquisition mode at 97 °C. At the end of the methylated and unmethylated MSPCR reactions, cycle threshold (Ct) values were evaluated. In general, Ct values smaller than 35 represents the true positive cases. Thus, Ct values that were equal to or lower than 35 were considered to be positive. The values between 35-40 can be false positive so we did not accept Ct values over 35 as positive. Statistical Analyses. All statistical analyses were performed using the Statistical Package for Social Science (SPSS), version 21.0 (IBM Inc., Armonk, NY, USA). The correlation between EGFR promoter methylation profile and clinical characteristics were examined using the χ2 test. A two-sided test was considered statistically to be significant at p <0.05.

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