Detection of EGFR Mutations in the Circulating Tumor Cells (CTC)
|School||Beijing Union Medical College|
|Keywords||CTC EGFR immunomagnetic negative enrichment Immuno-fluorescence staining LCM WGA single cell nest PCR sequencing|
Objective:We aim to establish a method for detecting EGFR mutations in CTC, to guide EGFR-TKI treatment decisions for the NSCLC patients.Method:Firstly, we mixed H1975cell with whole blood from healthy subject to mimic the CTC. The tumor cells were then enriched by negative selection using immunomagnetic beads, and identified from blood cells by immunofluorescence staining. FISH was also tested as an alternative method to identify tumor cells. Secondly, we compared Laser Capture Microdissection (LCM) with other methods to isolate the single cell. Third, we contrasted single cell PCR with PCR of several cells. Fourth, we amplified the single cell DNA with or without Whole-genome Amplification (WGA) before Nest PCR. We then detected the mutatiois in the PCR preparation using sequencing.Results:Through the immunomagnetic and immunofluorescence staining, the H1975cell line had a recovery rate of77.2±9.5%. LCM performed better for single cell isolation. The positive rate for PCR of several cells is only50%, with no apparent advantage over single cell PCR. WGA didn’t affect the PCR positive rate, but it enabled us to detect other gene mutation states in the same cell sample. Through these methods, the sensitivity of sequencing is sufficient for detecting EGFR mutations.Conclusions:The results of EGFR mutation detection in single cell were fully confirmed in H1975cell line, and it could be used in NSCLC patients. This method can be also used for detecting multiple genes locus on single cell in any kind of solid tumor.