CTCs refers to tumor cells released into the peripheral blood by primary tumors and/or metastatic lesions. Because CTCs are important to the formation of metastasis, and they are highly implicated in tumor-related deaths. Therefore, the detection of CTCs in peripheral blood is important for early diagnosis and for efficacy and prognosis evaluation[8-10, 16]. however, due to the very limited number of CTCs in peripheral blood circulation, the heterogeneity of CTCs subtypes, and the easy aggregation into micro-plugs etc., the sensitivity, specificity, and efficiency of CTCs detection technology are extremely challenged.
The key steps for CTCs detection are enrichment and identification. Currently, CTCs are sorted from other cells in the blood mainly through physical characteristics (such as the size, density, chargeability and deformability of CTCs, etc.) and biological characteristics (such as the cell surface antigen) . Sorting CTCs according to physics characteristics is simple in operation and relatively low in cost, but cannot avoid the interference of individual heterogeneity, while sorting CTCs according to biological characteristics ensures the accuracy, but is limited by the types of cell surface-expressed antigen. CTCs identification techniques include cell counting which is based on flow cytometry and nucleic acid detection which is based on a reverse transcriptase-polymerase chain reaction. Cell counting method can quantitatively detect the number of CTCs and analyze various parameters of the CTCs (such as the size, morphology, intracellular and extracellular biomarkers, as well as the genomic mutations), but the detection sensitivity is low and requires a large volume of blood sample; The advantages of the nucleic acid detection method are time-saving, highly specific and requiring fewer blood samples, but this process inevitably destroys cell morphology and function, making further analysis impossible. In addition, due to the easy degradation of mRNA and the influence of non-specific amplification, the false positive rate increases[18-21]. The CellSearch system is currently widely recognized and used in the detection of lung cancer CTCs, which consists mainly of automated immunomagnetic separation systems and immunofluorescence analysis systems. The CTCs are isolated and enriched based on the EpCAM expression, but mesenchymal CTCs that had undergone epithelial-mesenchymal transformation could not be detected. Therefore, currently, there is no ideal method for detecting CTCs in the peripheral blood of NSCLC patients.
The CanPatrolTM technology used in this study combined nanomembrane filtration technology and multiple RNA in situ analysis techniques to sort and identify CTCs. CanpatrolTM CTC detection technology (CanpatrolTM, Surexam) effectively overcomes the limitations of only isolating a specific epithelial phenotype of CTC and missing the detection of leukocyte-CTC cell clusters. CTCs are retained by nano-membrane filtration and analyzed the specific genes by highly sensitive multiple RNA in situ analysis (MRIA). Accurate classification of human peripheral blood CTCs was achieved. It contains five types including epithelial CTCs, mesenchymal CTCs, epithelial-mesenchymal CTCs, cluster CTCs, and leukocyte-CTCs cluster. We used nanomembrane with a self-optimized pore size of 8um to filter peripheral blood so that the tumor cells in the peripheral blood were highly enriched. Previous studies have shown that the enrichment rate was as high as 89%, and the leukocyte removal rate was as high as 99.98% . The advantage of this method is that it can completely sort all types of CTCs (epithelial, epithelial-mesenchymal and mesenchymal CTCs) without relying on specific biomarkers, and could be applied to enrich most of the solid tumors’ CTCs. In addition, CanpatrolTM adopts a novel multiple mRNAs in situ analysis method to hybridized the specific probes to the target gene and further enhance the sensitivity and specificity of the detection through the fluorescence signal cascade amplification system. In this study, we compared CTCs in peripheral blood of patients with NSCLC and benign lung diseases. Statistical analysis showed that there were differences in the number of three subtypes of CTCs and total CTCs between the two groups. ROC curve analysis showed that the sensitivity and the specificity of CanPatrolTM technology for the detection of peripheral blood CTCs in NSCLC was 81.6% and 86.8%, respectively. It can be concluded that this method has better diagnostic accuracy for NSCLC and has obvious diagnostic advantages compared with other methods. Additionally, as a non-specific physical enrichment technology, CanpatrolTM reduces the damage of tumor cells in peripheral blood preserving the original cellular information, such as morphology, cell function, molecular biology information, etc. Therefore, CanpatrolTM technology is beneficial for subsequent immunofluorescence, fluorescence in situ hybridization (FISH), gene expression, gene mutation detection, and microdissection based single-cell sequencing analysis of CTCs. Moreover, this technology can also be used for cell culture and animal models to develop new drugs and conduct the drug susceptibility testing, which would comprehensively and dynamically reveal tumor molecular information and guide the individualized treatment for cancer patients.
In this study, there was no statistically significant difference in the number of CTCs between lung adenocarcinoma, lung squamous cell carcinoma, and other NSCLCs which is consistent with previous studies[23, 24]. CTC is mainly to predict the risk of recurrence and metastasis and to evaluate the efficacy. There is not much correlation with the pathological type. This conclusion is in accordance with others studies[25, 26]. As for whether there is a difference, is it because the number of cases is not enough to obtain an accurate conclusion, more studies are needed to confirm the correlation between staging and CTC. There was no statistical difference in the number of subtype CTCs and total CTCs between different ages (≦ 60 years or > 60 years), indicating that age is not a factor influencing CTCs, and our result is consistent with previous studies [23, 24, 27]. Through COX proportional hazard regression analysis of the follow-up data, we found that pathological stage is a risk factor for recurrence and metastasis which indicating that it is more scientific to plot the survival curve after risk screening and stratification. The results of 63 follow-up patients showed that the number of metastases in CTC-positive patients accounted for most of the total number of metastases. Therefore, we believe that CTC can be used as an auxiliary method for clinical prognosis of lung cancer. According to the ROC curve analysis and the cut-off value, the number of CTCs ≥ 0.5 was judged as positive. After a survival analysis of 14 patients with stage IIIA, we concluded that patients with NSCLC with a total number of CTCs ≥ 0.5 have significantly lower DFS than patients with number < 1, which is consistent with previous reports [23, 28]. Our data suggest that the number of total CTCs ≥ 0.5 in peripheral blood (5 ml) of NSCLC patients could predict the prognosis. However, it is necessary to expand the number of cases and extend the follow-up time to verify this conclusion.