A Modified Extraction Method of Circulating Free DNA for Epidermal Growth Factor Receptor Mutation Analysis

PURPOSE: Circulating free DNA (cfDNA) in plasma is promising to be a surrogate for tumor tissue DNA. However, not all epidermal growth factor receptor (EGFR) mutations in tumor tissue DNA has been detected in matched cfDNA, at least partly due to inefficient cfDNA extraction method. The purpose of this study was to establish an efficient plasma cfDNA extraction protocol. MATERIALS AND METHODS: The yield of plasma cfDNA extracted by our modified phenol-chloroform (MPC) method from non-small-cell lung cancer (NSCLC) patients was compared with that by QIAamp MinElute Virus Spin kit (Qiagen kit) as control, using the Wilcoxon rank-sum test. TaqMan quantitative polymerase chain reaction (qPCR) assays were used to quantify the plasma cfDNA extracted. Both Mutant-enriched PCR (ME-PCR) coupled sequencing and DxS EGFR mutation test kit were used to evaluate the impact of extraction method on EGFR mutation analysis. RESULTS: MPC method extracted more plasma cfDNA than Qiagen kit method (p=0.011). The proportion of longer fragment (> or =202 bp) in cfDNA extracted by MPC method was significantly higher than by Qiagen kit method (p=0.002). In the sequencing maps of ME-PCR products, a higher mutant peak was observed on plasma cfDNA extracted by MPC method than by Qiagen kit method. In DxS EGFR mutation test kit results, plasma cfDNA extracted by MPC method contained more tumor-origin DNA than by Qiagen kit method. CONCLUSION: An improved plasma cfDNA extraction method of MPC is provided, which will be beneficial for EGFR mutation analysis for patients with NSCLC.

Research advancement on EGFR mutation detection in peripheral blood of patients with non-small cell lung cancer / 肿瘤研究与临床

The main therapies of NSCLC are surgery, chemotherapy, radiotherapy and targeted therapy. With development of targeted therapy, it was found that tyrosine kinase inhibitor(TKI) for patients with mutations in the EGFR gene was effective. Screening of such drugs before treatment is a premise of individualized treatment, and tissue samples are the current gold standard for genetic testing. However, it is hard to acquire the tumor tissues of patients with advanced NSCLC, so EGFR mutations detection of free DNA in peripheral blood had become an option. This article reviews the detections of TKIs-sensitive mutation,exon 19/21 mutation, and TKIs-acquired resistant mutation, T790M mutation at exon20, in serum or plasma of NSCLC patients.

Effect of mesenchymal stem cells on subcutaneous xenograft tumors in mice with Lewis lung cancer / 上海交通大学学报（医学版）

Objective To investigate the effect of mesenchymal stem cells (MSCs) on subcutaneous xenograft tumors in mice with Lewis lung cancer. Methods MSCs isolated from bone marrow of C57BL/6 mice were made into single cell suspension and were cultured in vitro. The cells of the 4th to 5th passage were used for the subsequent experiments. Fifty six C57BL/6 mice were inoculated subcutaneously with Lewis lung cancer cells, and were grouped into Group D0 (MSCs were given simultaneously with inoculation)and Group D10(MSCs were given 10 d after inoculation). Group D0 included three subgroups (n=8): Group 1 with inoculation of tumor cells, Group 2 with inoculation of tumor cells and MSCs, and Group 3 with inoculation of tumor cells and tail intravenous injection of MSCs. Group D10 included four groups: Group 4 with inoculation of tumor cells and injection of MSCs in tumors, Group 5 with equivalent PBS (the control of Group 4), Group 6 with inoculation of tumor cells and tail intravenous injection of MSCs, and Group 7 with equivalent PBS (the control of Group 6). The time of tumor formation and the volume of tumor were observed and compared among the groups. ResultsIn Group D0, earlier onset of tumor development was observed in Group 2 as compared to Group 1 and Group 3 (P<0.05), while there was no significant difference on the volume of tumor in the three groups (P>0.05). In Group D10, the volume of tumors were larger in Group 4 compared to the control (P<0.05), while there was no significant difference on the volume of tumors between Group 6 and the control (P>0.05). Conclusion Inoculating mixture of MSCs and Lewis lung cancer cells accelerates tumor formation,and injection of MSCs in tumors stimulates the growth of tumors.