Detection Of EGFR Mutations In Circulating DNA Of Lung Cancer Patients And Its Significance

Purpose: This study aims to analyze the clinical value of epidermal growth factor receptor (EGFR) mutation detection among direct sequencing, monoclonal sequencing and high resolution melting curve analysis (HRM) in the plasma circulating DNA of lung cancer patients. In addition, this study also discusses the relation between EGFR mutations and clinical pathological features with analysis of the clinical data.Method: The 96 plasma samples of lung cancer patients were from The First Affiliated Hospital of Soochow University and some upper first-class hospitals in Shanghai, 59 of which had corresponding tumor tissues. The DNA were extracted from the samples and the mutations of EGFR's exon19 and 21 were detected by direct sequencing, monoclonal sequencing and HRM. The results of HRM were compared to that of direct sequencing and monoclonal sequencing. The sensitivity of HRM was analyzed by the detection of samples containing different proportions of EGFR-mutated plasmids.Results:1. Somatic mutations of EGFR were identified in plasma from 17 of the 96 cases(17. 7%), including 15 cases (88.2%) in exon 19 and 2 cases (11.8%) in exon 21. Among all the 17 detected mutation samples, 3 homozygous mutation (2 L858R samples, 1 del E746-A750 (2) sample) and 14 heterozygous mutation samples are detected by direct sequencing. Then monoclonal sequencing is used to further determine the types of mutation in those heterozygous mutation samples mentioned above (9 del E746-A750(2) samples, 3 del E746-A750 (1) samples, 2 del L747-S752 samples).2. By means of HRM within 96 lung cancer patients, EGFR mutations were detected in 17(17/96,17.7%) cases, in which the number of cases of exon 19 and 21 mutations is 15 (15/17,88.2%) and 2 (2/17, 11.8%) respectively. The results of sequencing are consistent with it. HRM detection could identify EGFR mutation in a proportion of 5% of mutated plasmid DNA.3. Patients with adenocarcinoma had an increased frequency of mutations.The mutation was not closely related with gender and smoking history.Further analysis on 59 lung cancer patients demonstrated that the EGFR mutation in plasma was consistent with that detected in tumor tissues, suggesting that the EGFR mutations in plasma originated from the primary tumor.Conclusion1. Traditional method of direct sequencing could be applied to the detection of gene mutation. However, when sequencing the heterozygous samples, it cannot determine the types of mutations according to sequencing plots because of the heterozygous peaks. But by means of monoclonal sequencing, the plots of it have clean backgrounds and easy to determine the mutation types.2. The HRM analysis could be an optimal method for clinical screening of EGFR mutation thanks to its simpleness, swiftness and high sensitiveness to 5% mutation in the samples. Furthermore, the results of HRM are identical with that of sequencing.3. The EGFR mutations in plasma circulating DNA are in accordance with that in lung cancer tissues, demonstrating that plasma circulating DNA could be applied to the mutation detection in replace of tissue DNA.