Balkan Journal of Medical Genetics

THE FREQUENCY OF EGFR AND KRAS MUTATIONS IN THE TURKISH POPULATION WITH NON-SMALL CELL LUNG CANCER AND THEIR RESPONSE TO ERLOTINIB THERAPY
Demiray A, Yaren A, Karagenç N, Bir F, Demiray AG, Karagür ER, Tokgün O, Elmas L, Akça H, https://orcid.org/0000-0002-3343-0184.
*Corresponding Author: Professor Dr. Hakan Akça, Cancer Research Center, Pamukkale University, 11 University Street, Denizli Turkey. E-mail: hakca@pau.edu.tr (primary), hakanakca@yahoo.com
page: 21

MATERIALS AND METHODS

Patients. This study was performed in paraffin-embedded tissues of 300 NSCLC patients who attended the Medical Oncology Department, Pamukkale University, Denizli, Turkey, Medical Oncology Department, Antalya Education Research Hospital, Antalya, Turkey and the Medical Oncology Department, Afyon Kocatepe University, Afyon, Turkey, between 2011 and 2014. DNA was extracted from paraffin-embedded tissues using QIamp DNA FFPE Tissue kit (Qiagen GmbH, Hilden, Germany, its purity was checked spectrophotometrically and stored at –20 °C. Clinical features of patients are given in Table 1. Pyrosequencing Method. The extracted DNA was sequenced by pyrosequencing, a method which gives effective with as little as 15 ng DNA. This method is based on a real time non electrophoretic system. Pyrosequencing is more cost-effective when compared to the dideoxy Sanger method. Pyrosequencing is more reliable and effective at determining EGFR mutations than the classic sequencing method [9]. Polymerase chain reaction (PCR) reaction composed of 20 pmol of each primer, 0.2 mmol each of deoxy-nucleotide triphosphates, 1.5 mmol/L MgCl2 and 1.25 U of FastStart Taq DNA polymerase (Qiagen GmbH) as total final volume of 50 μL. The PCR method consisted of an initial denaturation step at 95 °C for 15 min., followed by 42 cycles at 95 °C for 20 seconds, 54 °C for 30 seconds, 72 °C for 20 seconds, and a final extension step at 72 °C for 5 min. The PCR products (10 μL) were analyzed by electrophoresis on a 3.0% agarose gel to confirm successful amplification. After validation of the PCR products on the gel, 40 μL of the products were bound to streptavidin Sepharose HP (GE Healthcare, Waukesha, WI, USA), purified, washed, and denatured using a 0.2 mol/L sodium hydroxide solution, and washed again. Subsequently, 0.3 μmol/L pyrose-quencing primers were annealed to the purified single-stranded PCR products, and the pyrosequencing was performed on a PyroMark ID system (Qiagen GmbH), according to the manufacturer’s instructions and analyzed in the AQ mode of the PyroMark (Qiagen GmbH) software. Statistical Analyses. All statistical analyses were done using the Statistical Package for the Social Sciences (SPSS) version 17.0 (SPSS® IBM Corporation, Armonk, NY, USA). For descriptive analyses, the χ2 and t-tests were performed. Progression-free survival and overall survival analyses, according to EGFR and KRAS mutations’ status were done using the Kaplan Meier Method and estimating the survival difference with the use of long rank test. A value of p <0.05 was accepted to be statistically significant.

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