DETECTING EGFR MUTATIONS IN PATIENTS WITH NON-SMALL CELL LUNG CANCER
Hammoudeh ZA, Antonova O, Staneva R, Nikolova D, Kyuchukov Y, Penev A, Mintchev T, Koleva V, Hadjidekova S, Toncheva D
*Corresponding Author: Zora A. Hammoudeh, Molecular Biologist, Department of Medical Genetics, Medical University Sofia, 2 Zdrave Str., 1431 Sofia, Bulgaria. Tel: +359-2-917-2735. Mobile: +359-88-943-0505. E-mail: zorahammoudeh@yahoo.com
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MATERIALS AND METHODS

Tumor samples from 551 Bulgarian patients diagnosed with NSCLC have been collected for the last 5 years. The patients were aged between 24 and 84 years old and the average age was 63. The gender ratio in the tested group was 407 (73.9%) males and 144 females (26.1%). All procedures were supervised and approved by the hospitalís Ethics Committee. For examination of the samples, the patients provided a signed written informed consent. DNA was extracted from FFPE NSCLC samples from primary tumors by QIAamp DNA FFPE Tissue Kit (Qiagen Ltd.) for genomic DNA purification. The concentrations of the DNA samples were measured by NanoDrop Lite spectrophotometer system (Thermo Fisher Scientific, Waltham, MA, USA). The therascreen EGFR RGQ PCR Kit (Qiagen Ltd.) was custom-made for detecting 29 somatic mutations in exons 18-21 of the EGFR gene using a RT-PCR technology. The amplification reaction was done on the Rotor-Gene Q instrument (Qiagen Ltd.). For detecting the mutations with RT-PCR, the assay used two technologies, ARMS and Scorpions (Qiagen Ltd.). Mutation-specific amplification was performed using the ARMS technique, meanwhile detection of amplification was performed using Scorpions PCR primers linked to a probe. After completing the PCR run, each DNA sample was analyzed for the presence of EGFR gene mutations, or not, by Rotor-Gene Q Series (Qiagen Ltd.). If there was amplification on one or more mutation reaction in the sample, the difference between the mutation cycle threshold (CT) and control (CT) from the same sample was used to calculate CT values. Then we compare the CT value for the sample with the cutoff point for the assay in a table according to the manufacturerís guideline (Qiagen Ltd.). We used the χ2 tests to evaluate the association between EGFR gene mutations and clinicopathological features, such as gender, smoking status and histological type. The statistical analysis was performed between the positive EGFR patientís groups, divided into males vs. females, smokers vs. non smokers and adenocarcinomas and other histological types (squamous cell carcinoma and large cell carcinoma). All the statistical analyses were performed using the R Core Team (2013) software (http:// www.R-project.org/.) The results were considered statistically significant at a p value of less than 0.05.



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