Construction of CTC-ALK gene fusion detection system based on the multi-site magnetic separation in lung cancer and its clinical verification.

Abstract

Detected in a variety of solid tumors, including lung cancer, the EML4-ALK fusion gene plays an important role in promoting the occurrence and development of cancer. The existing detection methods for EML4-ALK fusion gene are all targeted at surgical or post-sampling tumor tissues, which cannot achieve early detection and real-time monitoring; therefore, a minimally invasive ALK gene fusion detection system is explored and constructed. Vimentin, EpCAM, and EGFR antibodies were grafted, respectively, to prepare multi-site immunoliposome magnetic beads, so as to capture CTC in blood for RT-PCR detection, and then the feasibility of this method was verified by detecting the positive rate of the EML4-ALK fusion gene and clinical information in combination with WB and IHC. The prepared multi-site immunoliposome magnetic beads showed high specificity and stability, and the average proliferation rate and capture rate of cells were 95% and 85%, respectively. In clinical blood samples, the CTC level of the grade I (G1) patients before the operation was lower than grade 2 (G2), and that of grade II (G2) was significantly lower than grade III (G3), but the difference was not significant after the operation. The RT-PCR results of CTC and the RT-PCR, WB, and IHC results of tissues were highly consistent in the fusion gene detection, and the positive rate of ALK gene fusion in 60 lung cancer patients was 31.67% and 28.33% before and after the operation, mostly EML4-ALK (V3) gene fusion. The CTC-ALK gene fusion detection system constructed successfully could avoid the problem of difficult sampling and post-sampling complications, and truly achieve the minimally invasive biopsy, so it was of important clinical significance for the diagnosis and efficacy evaluation of lung.