At the end of the 19th century, PAGET put forward the "seed and soil" theory of tumor metastasis. It is undeniable that the number of CTC in peripheral blood is very small, and some patients are even difficult to obtain in clinic. In the process of continuous adaptation to the microenvironment of peripheral blood, some CTCs undergo epithelial-mesenchymal transition (EMT). Some show apoptosis or reduced antigen expression . In order to overcome the difficulty of CTC detection caused by the above problems, in 2018, Zhenlong Ye et al  studied the clinical significance of aneuploidy of CTC in patients with various malignant tumors. 594 blood samples from 479 cases of 19 different cancers and 30 healthy samples were collected for CTC detection and analysis by SE-iFISH. The results of this study show that no positive CTC was found in all 30 healthy samples. In the diagnosis of malignant tumors, the overall positive rate of CTC was 89. 0% (75. 0% -100. 0%). The results were basically consistent with the positive rate of CTC detection in this study (95. 83%).
Other studies [6, 26–29] have shown that an average of 11, 5, 8, and 4 CTCs per 7.5 mL were observed in lung, liver, kidney, and colorectal cancers, respectively. In this study, the average number of CTC detected per 6 mL blood of each case was 12. 4 (285/23), which was similar to the results of previous studies, but slightly better. In addition, more than 95% of the positive rate is also closely related to the clinical stage and pathological type of the patients enrolled in this study. All patients were in stage IV except one in stage IIIB, and CTC was more easily detected in patients with metastasis. On the contrary, if the patient cannot complete the clinical staging evaluation, CTC measurement can also effectively predict whether the patient has metastasis, which has a certain value for the prognosis of patients. At the same time, patients with pathological type distribution: adenocarcinoma 70.83% (17 cases), small cell carcinoma 12.50% (3 cases), squamous cell carcinoma 16.67% (4 cases). It is well known that adenocarcinoma and small cell lung cancer are more likely to metastasize, and the proportion of adenocarcinoma and small cell lung cancer patients in this study is high (83.33%) and all of them were advanced lung cancer. Due to the high sensitivity of SE-iFISH, we got a high positive rate of 95.83% CTC detection results.
There was no significant correlation between the positive rate of CTC and the clinical characteristics of lung cancer patients, including gender, age, ECOG score, histopathological type and serum CEA. The results are basically consistent with those of other clinical studies (gender, age, histopathological type). However, some other studies [30–32] have pointed out that the detection rate of CTC in patients with lung cancer in clinical stage III ~ IV is higher than that in patients with lung cancer in clinical stage I ~ II, but this study did not find that there is a significant correlation between the detection rate of CTC and the clinical stage of lung cancer. The possible reason of this result is that the number of patients enrolled in this study is small, and all of them are stage IV metastatic lung cancer (only 1 case of stage IIIB), and there is a lack of early stage (stage I, stage II) and stage IIIA patients. But the results of this study showed that there was a significant difference between the detection rate of CTC and whether the patients smoked (p = 0.014), that is, the detection rate of CTC in smoking patients was lower than that in non-smoking patients. Only one patient in the whole sample was negative for CTC, and this patient had a history of smoking, so this conclusion needs to be further confirmed.
We also found that there was no significant correlation between the number of CTCs and the clinical characteristics of patients (including gender, age, ECOG score, serum CEA), but there was a certain correlation between the number of CTCs and the histopathological type of patients. There was a difference in the number of CTCs between adenocarcinoma and squamous cell carcinoma (p = 0.02), but there was no difference between adenocarcinoma and small cell carcinoma (p = 0.151) or between squamous cell carcinoma and small cell cancer (p = 0.102). To sum up, we can conclude that the three main pathological types of lung cancer (adenocarcinoma, squamous cell carcinoma, small cell carcinoma) have no significant difference in the detection rate of CTC in peripheral blood, but there are some differences in the number of CTC. The difference between adenocarcinoma and squamous cell carcinoma was statistically significant. In NSCLC, it has been found that CTC count is correlated with tumor metastasis [33, 34], while adenocarcinoma is a pathological type prone to hematogenous metastasis, squamous cell carcinoma is relatively inert, ana its growth and development is slow, not easy to early invasion and metastasis. So it is not difficult to understand, compared with squamous cell carcinoma, peripheral blood CTC count of adenocarcinoma patients is more. In theory, small cell lung cancer has the highest degree of malignancy and is most likely to undergo hematogenous metastasis and dissemination in the early stage. However, there was no significant difference in CTC count between small cell carcinoma and adenocarcinoma, small cell carcinoma and squamous cell carcinoma, which may be due to the small number of cases of small cell cancer and squamous cell carcinoma selected in this project. There were only 3 cases of small cell carcinoma and 4 cases of squamous cell carcinoma. The median CTC counts (25% -75%) in lung cancer, 9 (6.5–13) of adenocarcinoma, 2.5 (2 ~ 4.5) of squamous cell carcinoma, 7 (3 ~.) of small cell carcinoma. If the sample size is expanded, it is expected that there may be some statistical difference in CTC count between small cell carcinoma and squamous cell carcinoma.
In this study, SE-iFISH was used to detect the expression of CK18 and Vimentin on the surface of lung cancer cells in peripheral blood, and combined with the ploidy of CEP 8 to determine CTC. Among the 23 patients in whom CTCs were detected, only one CK18 + triploid CTC on chromosome 8 was detected in one patient, and in the remaining 22 patients, all CTCs were CK18 -. The positive rate of CK18 + CTC was 4.35% (1/23). 5 of 7 patients had both Vimentin + and Vimentin -, only one had Vimentin + CTC, the others only had Vimentin - CTC, the detection rate of Vimentin + CTC was 85. 71% (6/7). It is noteworthy that chromosome 8 of Vimentin + CTC is mostly diploid and monosomic. Vimentin - CTC showed more heteroploid diversity of chromosome 8, including monosomy, triploid, tetraploid and polyploid, especially polyploid was more common. These results showed the complexity and complementarity of the expression of CTC surface protein markers and chromosome 8 aneuploidy in peripheral blood of patients with advanced lung cancer, and the detection sensitivity of chromosome 8 aneuploidy was the highest, reaching 100%. Vimentin + was the second with 85.71%, while CK18 + was the worst with 4.35%.
In Addition, small cell (≤ 5 um) CTC was detected in 6 of 7 patients (85.71%). Another method for enriching CTCs is based on the tumor cell size separation method (ISET), which can only capture CTCs with larger diameter than the filter pore (> 8 um) from the blood sample and is independent of the surface antigen of CTCs. But there are also a small number of CTCs with smaller diameter in peripheral blood. Similar to blood cells or even smaller, this method is easy to lose this part of the small size of CTCs. In this study, Vimentin expression and CEP 8 aneuploid were used to detect 85.71% (6/7) of patients with primary lung cancer who had small cell CTC in peripheral blood, which avoided the missed detection of small cell CTC ≤ 5 um.
Therefore, SE-iFISH platform improves the sensitivity of CTC detection, avoids the limitations of tumor heterogeneity, CK downregulation, deletion and small size easy to miss detection.
Chromosome aneuploidy exists in various tumor cells. Many studies have shown that aneuploid tumor cells are associated with poor prognosis. Polyploid tumor cells are genetically unstable and not sensitive to radiotherapy and chemotherapy. It is generally believed that the prognosis of patients with polyploid subcloned tumor cells is worse .
In this study, 23 of 24 patients were positive for CTC in peripheral blood, and all of them were detected with chromosome 8 heteroploid CTC, and the ploidy of chromosome 8 was obviously heterogeneous. The number of polyploid CTC was the largest, followed by triploid and tetraploid CTC. In this study, we compared the correlation between the cutoff value of different ploidy number of chromosome 8 and PFS of the first-line treatment or OS in order to find a reference method to predict the efficacy and poor prognosis. The results showed that a tetraploid CTC count ≥ 2 was an unfavorable predictor of response, it can predict poor PFS in patients with advanced lung cancer. A tetraploid CTC count ≥ 1 is an unfavorable prognostic predictor of poor OS in patients with advanced lung cancer. It is suggested that quantitative analysis of chromosome 8 tetraploid in CTCs by SE-iFISH may be a useful tool for evaluating the therapeutic effect and judging the prognosis.
Studies have found that lung cancer metastasis is likely to have occurred in the early stage of lung cancer, and there is dissemination of lung cancer cells. A large number of CTCs aggregate and form CTM with an high metastatic potential, this stage is called micrometastasis, which is also known as occult metastasis. Therefore, CTM has stronger ability of invasion and metastasis than CTC. In This study, CTM was found in 2 of 7 lung cancer patients (28.57%, 2/7). Both of the two cases were stage IVB adenocarcinoma patients, and their serum CEA levels were 33.80ng/ml and 57.54ng/ml, respectively. After the first-line treatment, the PFS was only 1 month and 3 months respectively, and the OS was 2 months and 4 months respectively. It is suggested that the presence of CTM in peripheral blood may predict the later clinical stage, the worse therapeutic effect and the poor prognosis of lung cancer patients.
In summary, this study further confirms that SE-iFISH can effectively enrich CTC in the peripheral blood of patients with primary lung cancer. According to the specific needs of clinic or research, the combination of immunofluorescence staining with freely selected tumor biomarkers and chromosome FISH further improves the sensitivity and specificity of CTC detection. CTCs effectively detected by SE-iFISH can be used as a preliminary predictor of immediate efficacy and prognosis of patients with metastatic primary lung cancer. Quantification of chromosome 8 aneuploidy also shows potential as a predictive biomarker for disease progression and prognosis. Due to the small sample size, there are still some limitations in this study, which needs to be verified by a larger sample.