Diagnostic value of the circulating tumor cells and circulating tumor-derived endothelial cells detection for non-small cell lung cancer

【Abstract】 Effective biomarkers are essential to the early diagnosis of non-small cell lung cancer (NSCLC). Herein, a retrospective study of 49 newly diagnosed and recurrent NSCLC patients, 31 patients with benign pulmonary disease and 24 healthy volunteers was conducted, to evaluate the diagnostic value of circulating rare cells for NSCLC. The expression of circulating tumor cells (CTCs) and circulating tumor-derived endothelial cells (CTECs) in peripheral blood were measured by subtraction enrichment-immunostaining-fluorescence in situ hybridization (SE-iFISH). The level of CTCs (P<0.001) and CTECs (P<0.001) was significantly higher in NSCLC group than that in benign pulmonary disease group. The proportion of small CTCs (P<0.001) and CTECs (P<0.0001) significantly increased from benign lung disease individuals to NSCLC patients. The AUC of ROC curves of total CTCs and CTECs were 0.815 (95%CI: 0.722~0.907), 0.739 (95%CI: 0.618~0.860), respectively. The cut-off values for discriminating NSCLC with benign lung disease patients were total CTCs 11.5 units/6ml and total CTECs 10.5 units/6ml, with sensitivity and specificity being 67.3% and 83.9%, 77.6% and 77.4%, respectively. When CTCs and CTECs were combined, predictive value significantly increased to 82.6% as measured by the area under the curve. Small CTCs and triploid CTCs had high positive predictive value (PPV) and positive likelihood ratio (LR+) of the diagnosis of NSCLC in early stage. CTCs and CTECs can not only be used as new biomarkers for the diagnosis of NSCLC, but can also improve diagnostic performance of the early stage NSCLC. Moreover, the combined examination of CTCs and CTECs is be superior to the single.

However, due to high heterogeneous and dynamic expression of EpCAM on tumor cells [25], as well as down-regulation of CK during epithelial-to-mesenchymal transition (EMT) [26], the clinical application of EpCAM or CK-dependent capture techniques are liable to miss detection. A comprehensive and stable detection technique should be applied in clinical practice.
In this study, subtraction enrichment-immunostaining fluorescence in situ hybridization (SE-iFISH) were performed to investigate the expression of circulating rare cells in the peripheral blood of NSCLC patients. With the use of magnetic beads coated with a variety of leukocyte surface antibodies, we performed a negative collection of circulating rare cells, which effectively avoided the heterogeneity of cell surface antigen expression and epithelial-mesenchymal transformation (EMT) interference effect on the results. At the same time, with combined chromosome 8 aneuploidy detection, we further found circulating rare aneuploidy cells, which provides a clue for the early detection and intervention of NSCLC.

Material and methods 2.1 Study subjects and specimen collection
A retrospective study was conducted on 49 patients with non-small cell lung cancer who were newly diagnosed and relapsed at the Department of Thoracic Surgery and Cancer Center in Shanghai General Hospital from August 2017 to April 2019, including 26 males and 23 females, and the average age was 61.69±11.34 years. The subject patients were diagnosed with NSCLC or benign lung diseases through clinical manifestations, medical history, and pathology. None of them received any anti-tumor treatment before detection. According to the 2015 World Health Organization (WHO) criteria for histological classification of lung tumors, there were 36 cases of lung adenocarcinoma, 8 cases of lung squamous carcinoma, 3 cases of lung adeno-squamous cell carcinoma, and 2 cases of other subtypes. According to International Association for the Study of Lung Cancer (IASLC 2009, TNM staging standard for lung cancer, 2009, 7th edition), 27 cases were stage I, 2 cases were stage II, 9 cases were stage III, and 11 cases were stage IV. Furthermore, there were 31 patients with benign lung diseases including 17 males and 14 females with average age (46 ± 11.7 years). As control，24 healthy volunteers were recruited，including 8 males and 16 females with average age (31.29±7.98 years). The study has been approved by the Ethics Committee of Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, China. All participants signed informed consent prior to blood collection. Peripheral venous blood was collected using ACD anticoagulant vacuum vessel collection tube. To avoid contamination of epithelial cells, the first 2 ml blood was discarded, 6 ml blood was then collected by ACD collection tube, and the blood were immediately mixed upside down for 8 times. It is best to store in room temperature and away from light for 24 hours, not more than 48 hours.

Subtraction Enrichment (SE)
The circulating tumor cells and circulating tumor endothelial cells in peripheral blood are firstly enriched according to the manufacture's updated protocol v5.0 (Cytelligen, San Diego, CA, USA). Blood samples were centrifuged at 200×g for 15 min at room temperature to separate plasma. Sedimented blood cells were mixed with 1×CRC buffer to 6 ml, followed by loading on the top of non-hematopoietic cell separation matrix in a 50 ml tube. Samples were subjected to centrifugation at 350×g for 6 min. Layers containing white blood cells (WBCs) and tumor cells above red blood cells (RBCs) were collected into a 50 ml tube, and subsequently incubated with 300 μl of immuno-magnetic beads coated with leukocyte surface antibodies at room temperature for 30 min. WBCs bound to immuno-beads were depleted using a 50 ml size magnetic separator (Cytelligen). The solution, free of magnetic beads, was collected into a 50 ml tube, followed by adding 1×CRC buffer to 50 ml. Samples were then spun at 500×g for 5 min at room temperature, followed by aspirating supernatants down to 100 μl. sedimented cells were gently resuspended, followed by multiple immunofluorescence staining and subsequent chromosome 8 centromere probe (CEP8) hybridization.

iFISH
Five color iFISH in this study was performed according to the manufacture's updated protocol v4.0 (Cytelligen). We chose 3 kinds of tumor-biomarker to tag different CTCs with various phenotypic, epithelial marker (EpCAM or CK18) and immunosuppressive marker: programmed death-ligand 1 (PD-L1). The resuspended sedimented cells were incubated with indicated postfluorescence labeled monoclonal antibodies at 1: 200 dilution, including Alexa Flour 594-CD45 (red), Alexa Flour 488-CD31, CK18, EpCAM or PD-L1(green), Cy5-CD31, CK18, EpCAM (gold) at room temperature for 20 min in dark [27,28]. Since the study applied a single tumor specific biomarker (CK18, EpCAM or PD-L1), different type of tumor biomarker used in each group was shown in Table 1. After washing, dry monolayer of cells mixed with the special fixative coated on the formatted CTC slides (Cytelligen). Furthermore, hybridization with Vysis chromosome 8 centromere probe (CEP8) Spectrum Orange for 4 hours was performed on the enriched cells by using a S500 ThermoBrite Slide Hybridization System. Finally, samples were mounted with mounting media containing DAPI (Vector Laboratories, Burlington, CA, USA), and subjected to automated CTC image scanning and analyses (Carl Zeiss, Oberkochen, Germany; MetaSystems, Altlussheim, Germany; and Cytelligen) [19].

Identification of lung cancer CTCs and CTECs
By high through-put scanning, obtaining and processing cell images , target cells were judged to be a circulating tumor cell or a circulating tumor endothelial cell, according to the expression of CD45, CD31, tumor markers (CK18, EpCAM, PD-L1) and karyotypic combined with the cell morphology and size [11]. Positive target cells are defined as follows: Morphological criteria: The cells are round or oval in shape, and the cell morphology and margins are intact under fluorescence. WBCs (5µm) were used as cutoff to define large or small cells. Immunofluorescence staining criteria: CTC: DAPI+/CD45-/tumor biomarker+/CD31-with chromosome 8 diploid or aneuploid; DAPI+/CD45-/tumor biomarker-/CD31-with chromosome 8 aneuploid. CTEC: DAPI+/CD45-/tumor biomarker +/CD31+. CTM: Cluster containing two or more CTCs.

Serum tumor markers detection and positive criteria
Three serum tumor markers: cancer embryonic antigen (CEA), squamous cell carcinoma antigen (SCC), and cytokeratin-19 fragment (CYFRA 21-1), associated with non-small cell lung cancer were selected for serological detection. CEA and CYFRA21-1 was detected by electrochemical luminescence immunoassay (Roche Cobas® C-601), and SCC was detected by chem-iluminescent microparticle immunoassay (Abbott Architect i2000 analyzer), respectively. The testing procedure was strictly in accordance with the kit instructions and the requirements of the testing instruments, and the normal reference values of the kits are recommended as follows: SCC< 2.5 ng/ml, CEA < 5 ng/ml, CYFRA21-1<3.3 ng/ml. Any one of the three above the normal value is considered positive.

Statistical analysis
All data were statistically analyzed by SPSS 25.0 software. Measurement data were compared between groups by Mann-Whitney U test (two groups) or Kruskal-Wallis test (multiple groups), and counting data were compared by Chi-Square test. The inspection level was α = 0.05. ROC analysis was performed to determine the cut-off values, and valuate the predictive performance of the disease progression-related biomarker signatures. For ROC analysis of the CTCs combination with CTECs, P (probability of a patient sample) was calculated for in the logistic regression by the formula: Z=logit(P) = ln(P/1-P)=b0+b1X1+b2X2+b3X3+…+bnXn ， where the bi terms were the ith regression coefficients by binary logistic regression, and the Xi terms were the relative expression levels of each biomarker. All P values were two-sided. P < 0.05 are statistically significant.

Characterization of CTCs and CTECs in NSCLC patients by SE-iFISH
Five-channel double-marker-iFISH was applied to perform phenotypic and karyotypic characterization of CTCs and CTECs enriched by SE from study subjects. Several typical cells of CTCs (CD31-/CD45-) and CTECs (CD31+/CD45-) found in NSCLC patients has been showed in Figure 1.

Comprehensive analysis of CTCs and CTECs in peripheral blood between groups
The CTCs detection rate was 97.9% (48/49) in the NSCLC group, 83.9% (26/31) and 95.8% (23/24) in the benign lung disease group and the healthy lung disease group, respectively. In the NSCLC group, only two individuals were detected with CK18+ CTCs, with a tumor marker positive detection rate of 4.2% (2/48), and the rest were tumor marker negative CTCs, 95.8% (46/48). There were no tumor-marked positive circulating tumor cells in the control group. The detection rate of CTECs was 98.0% (48/49) in NSCLC group, 87.1% (27/31) and 87.5% (21/24) in the benign group and the healthy group, respectively. One CTM was found in each of the 4 patients in the NSCLC group, and a total of 4 CTM were found, while no CTM was found in the other groups.
The results showed that the number of total CTCs or CTECs in the peripheral blood of NSCLC group was significantly higher than that of the benign lung disease group (P<0.001, P<0.01, Fig.  2A and D). Similarly, the number of CTCs or CTECs in different size in patients with NSCLC was significantly increased compared with that in patients with benign lung disease (LCTCs: P<0.05; SCTCs: P<0.001; LCTECs: P<0.01; SCTECs: P<0.001, Fig. 2B, C, E and F, respectively). In addition, no significant differences of CTCs and CTECs were found between healthy individuals and patients with benign lung disease, except for the small CTECs (SCTECs: P<0.01, Fig. 2E). Either the total CTCs and CTECs, or CTCs and CTECs in different sizes were significantly higher in all pathological subtype groups than in the benign lung disease group, except for the large CTECs (Fig.  3), while there was no statistically significant difference between lung adenocarcinoma, lung squamous cell carcinoma, and other NSCLC (Fig. 3, Supplemental Table 1). Ⅰ stage, Ⅲ stage, and Ⅳ stage NSCLC patients had a significantly higher number of CTCs than benign lung disease group, CTECs in Ⅰ and Ⅳ stage NSCLC patients is significantly higher than benign lung disease group ( Fig. 4A and D). Similar results could be obtained, when compared CTCs and CTECs in different sizes with the benign lung disease group, but there was no significant difference between TNM stages (Fig. 4B, C, E and F, Supplemental Table 1). Furthermore ,when we compared the proportions of CTCs and CTECs in different groups, we found that the proportion of small CTCs and CTECs significantly increased from benign lung disease individuals to NSCLC patients (Fig. 5).

Analysis of aneuploid characteristics of CTCs and CTECs between groups
In order to further elucidate the differences of CTCs and CTECs with different chromosomal ploidy numbers among different groups in number and proportion. Three types of chromosome aneuploidy were identified: triploid, tetraploid and multiploid (CEP 8 signal ≥5). Data analysis and intergroup comparison were performed, respectively.
The results showed that CTCs and CTECs of all aneuploid subtypes in NSCLC group and NSCLC Ⅰ stage group were significantly higher than those in benign lung disease group, except for the multiploid CTCs in NSCLC Ⅰ stage group. Compared with the healthy group, the CTECs triploid and tetraploid were significantly higher in the benign lung disease group, while the number of CTCs polyploid was lower in the benign lung disease group than in the healthy group (Fig. 6A). In addition, the number of triploid and tetraploid CTECs in adenosquamous carcinoma was significantly higher than that in adenocarcinoma and squamous carcinoma, according to Mann-Whitney U test. Stage IV had significant higher number of CTECs than that in stage I (P=0.0276*, Mann-Whitney U test), shown in Supplemental Table 2. However, according to the Kruskal-Wallis test, no significant difference had been found in number of CTCs and CTECs among aneuploid subtypes and TNM stages. According to the proportion analysis, we found that the proportion of triploid/tetraploid CTCs and CTECs in benign lung disease group were significantly higher than those in healthy group. In addition, we found an increased trend that the proportion of all aneuploid subtypes of CTCs and CTECs from benign lung disease individuals to NSCLC patients (Fig. 6B).

ROC curve analysis to determine the cut-off value and assess the diagnostic performance
of NSCLC Compared with benign lung disease group, the ROC curves were established based on the results of total and different subtypes of CTCs and CTECs in NSCLC group (Fig.7 A-E). The area under ROC curve (AUC)>0.7: Total CTCs=SCTCs (0.815) > triploid CTECs (0.809) > sCTECs (0.781) > Total CTECs (0.739) > triploid CTCs (0.716), other statistical parameters of ROC analysis were shown in Table 2. Furthermore, according to morphology and aneuploid subtypes, we chose 6 items to combined whose area under ROC curve (AUC)>0.7. ROC curves were combined through the logistic regression analysis results, the combined formulas were calculated as: Z(CTCs+CTECs)=-1.499+0.148*CTCs+0.020*CTECs, Z(SCTCs+SCTECs)=-1.983+0.250*SCTCs+0.321*SCTECs, Z triploid(CTCs+CTECs)=-2.151+0.358*CTCs+0.342*CTECs. After combined, the area under the ROC curves increased, indicating that the combined items were more effective. The critical value corresponding to the maximum point of the Jordan index was used to determine the cut-off values, shown in Table 2. These cut-off values were further used as the standards to evaluate the sensitivity, specificity and other diagnostic efficiency indicators of these detection items. As a comparison, we selected three clinically common serum tumor markers associated with non-small cell lung cancer for serological detection [29], as well as CT or PET-CT imaging diagnostic results (Table 3). Results showed that the sensitivity and specificity of total CTCs and CTECs for NSCLC were 67.3% and 77.6%, 83.9% and 77.4%, respectively. Among the rest detection items shown in Table 3, small CTCs and triploid CTCs had high specificity in the diagnosis of NSCLC, which were 93.5% and 96.8% respectively. In addition, through the evaluation of the diagnostic efficiency of these items for patients with different pathological subtypes and TNM stages, the result indicated the sensitivity of total CTCs or CTECs was relatively high, which had been shown in supplemental Table 3. Especially for NSCLC patients in stage I, the sensitivity of total CTCs or CTECs was both 70.4%, which were apparently higher than those of multiple tumor markers 29.2% and imaging diagnostic results 37%, respectively. Similarly, small CTCs and triploid CTCs had high specificity in the diagnosis of NSCLC I stage, shown in Table 4. In particular, the positive predictive value (PPV) and positive likelihood ratio (LR+) of these two items were also high, which means that they can be used as ideal screening indicators for high-risk lung cancer population in early stage, such as smoking, chronic lung disease or genetic history of lung cancer.

Discussion
Since 1869, Thomas Ashworth first defined CTCs as free tumor cells shed from the primary cancer [30], a variety of technologies were developed around the world to detect them. Because the concentration of CTCs in the peripheral blood of healthy people is extremely low, and the probability of CTCs appearing is about 1 CTC per 10 9 blood cells [31], it requires high sensitivity and accuracy of the technology to detect. In recent decades, more and more researches focused on investigating the potential of CTCs in cancer diagnostic and prognostic in clinic practice.
The effectiveness of the separation and identification determines the sensitivity and specificity of CTC detection. At present, the techniques used to sort CTCs mainly include negative enrichment and positive enrichment. As a positive enrichment technology ,which were widely recognized and applied in the detection of lung cancer CTCs, the CellSearch ® system isolates and enriches CTCs based on the EpCAM expression [24]. However, the biggest limitation of such methods is that they ignore the intrinsic biological characteristics of tumor cells-high heterogeneity of EpCAM expression. The expression level of EpCAM varies in different tumors. For instance, EpCAM expression was low in bladder cancer, pancreatic cancer, lung cancer, or even not expressed in melanoma [32]. Especially in NSCLC, by down-regulating their epithelial characteristics and upregulating their stem-like characteristics, epithelial tumor cells enhance their mobility and invasiveness, thus entering the blood circulation and colonizing distant organs to form metastases [33]. Another way is based on the cell size and plasticity to sort CTCs, such as: isolation by size of epithelial tumor cells (ISET) (RareCell Diagnostics, France), Parsortix (Angle Plc, UK) and ClearCell1 (Clearbridge BioMedics Pte Ltd, Singapore). The advantage of these techniques is independent on circulating tumor cell surface antigen, to maintain the morphology and activity of circulating tumor cells. However, with low-purity of the sorting cells, and the circulating tumor cells with small size are easily lost [34]. Furthermore, due to the high expression of folic acid receptor on the surface of non-small cell lung cancer cells [35], more and more clinical laboratories in China have applied Folate receptor (FR) based polymerase chain reaction (PCR) method to detect CTCs in peripheral blood of NSCLC patients [14,[36][37][38]. However, all above methods cannot detect aneuploid circulating tumor cells.
In our research, we applied subtraction enrichment-immunostaining fluorescence in situ hybridization (SE-iFISH) to systematically detect and differentiate CTCs and CTECs in patients with non-small cell lung cancer. By using a variety of anti-leukocyte antibody coated magnetic beads, SE-iFISH ensures maximum white blood cell removal and minimal nonspecific adherence to tumor cells, well overcoming technical limitations that rely on expression of tumor cell surface markers to screen circulating tumor cells. On the other hand, compared with the use of immunofluorescence staining or FISH method alone, SE-iFISH can effectively detect single abnormal or double abnormal CTCs of chromosome or tumor biomarker, thus greatly improving the sensitivity and specificity of CTCs detection. Furthermore, the ability to detect circulating tumor endothelial cell aneuploidy gives SE-iFISH an advantage over other methods. As tumor endothelial cells (TECs) shed from solid tumor into peripheral circulating blood, tumor endothelial cells (CTECs) often exist in the form of aneuploidy, which has extremely critical significance in the process of tumor angiogenesis and metastasis [39,40].
Numerous studies have confirmed the presence of circulating tumor cells in the peripheral blood of patients with both early and late stage NSCLC [13,14,41,42]. In this study, we compared CTCs and CTECs levels in peripheral blood of non-small cell patients, benign lung diseases, and healthy individuals. Statistical analysis showed that there was no significant difference in CTCs and CTECs levels between the lung benign disease group and the healthy group. CTCs and CTECs levels of NSCLC patients was significantly higher than that of benign lung diseases and healthy group, which is consistent with previous studies [13,14,43]. Among the patients with different pathological types of NSCLC in this study, there was no statistically significant difference between pathological types, which is consistent with previous studies [13,44]. In NSCLC patients with different stages, there was no significant difference between TNM stages, which is different from results of previous researches. Their results showed that CTCs in Ⅳ stage lung cancer patients were significantly higher than Ⅰ stage patients [14,38]. There are two possible explanations for this discrepancy. On the one hand, because of the small number of cases of this study, we found only an increase trend of CTCs and CTECs between I and IV stage, but no significant difference. On the other hand, in the early stage of NSCLC patients, a considerable amount of tumor cells or tumor vascular endothelial cells may shed off into the peripheral blood circulation. These circulating rare cells are likely to be chromosomal aneuploidy that cannot be identified using folate receptor-based PCR, and in this study were detected by the more sensitive and specific SE-iFISH.
Chromosomal aneuploidy is a common biological feature of tumor cells. About 90% solid malignant tumors and 75% blood tumors have chromosome aneuploidy of tumor cells, especially in tumor cells of lung cancer, stomach cancer, colon cancer, liver cancer, esophageal cancer and other solid tumors [11]. In addition, there were also aneuploidy of chromosome 8 in CTCs and CTECs in peripheral blood of tumor patients [19,20]. Among all CTCs detected in this study, only five tumor-marked positive CTCs were found in NSCLC patients, and the rest were all tumormarked negative expression of chromosome 8 aneuploidy, indicating that most circulating tumor cells in peripheral blood were tumor-marked negative. In addition, these non-hematologic aneuploid cells were not only present in the peripheral blood of patients with NSCLC, but also in small amounts in healthy people, and the number of these abnormal cells in the peripheral blood of NSCLC patients was significantly higher than that of healthy people. Similarly, most of the CTECs in the form of aneuploidy in the peripheral blood of NSCLC patients were also found in the peripheral blood of healthy people, and the level was significantly lower than that of patients with NSCLC, which was the same as the previous research results of P. Lin's team [19]. In order to characterize a full spectrum of aneuploid CTCs and CTECs in NSCLC patients, CTCs and CTECs were analyzed in more detail according to the chromosome ploidy number. Results showed that there were statistically significant differences in the number of triploids and tetraploids of CTCs, as well as in the number of triploids, tetraploids and multiploids of CTECs between the lung benign disease group and the stage I lung cancer group. However, compare to the amount analysis, no significant differences in the proportion of karyotype in CTCs or CTECs between the above two groups were revealed. Therefore, we believe that CTC aneuploids can be used as an auxiliary method for clinical differentiation of benign lung diseases and early lung cancer. Surprisingly, between healthy and lung benign disease group, significant differences were found not only in the number of cells but also in the proportion of karyotypes in CTCs or CTECs. As for the source of non-hematologic aneuploid cells in the peripheral blood in normal people, some research suggested that occasional CD31+ aneuploid circulating endothelial cells in the blood of normal people are mainly from stromal cell trans-differentiation or cell aging, which is unrelated to tumor and can be cleared by the body's immune system [43]. But why did these non-hematologic aneuploid cells in the peripheral blood increased, when suffered benign lung disease such as pulmonary nodules, remains further research.
ROC curve analysis showed that sensitivity and specificity of total CTCs and CTECs being 67.3% and 83.9%, 77.6% and 77.4%, respectively for differential diagnosis of NSCLC, which are obviously superior to serological tumor marker and imaging diagnosis. Furthermore, for the NSCLC I stage, the sensitivity of total CTCs and CTECs are both 70.4%, which means both of them can be used as an important basis for early diagnosis of NSCLC. Surprisingly, small CTCs and triploid CTCs had been found to have high positive predictive value (PPV) and positive likelihood ratio (LR+) in the diagnosis of early stage NSCLC, which has obvious diagnostic advantages in screening high-risk lung cancer population in early stage compared with other routine clinical examinations.
Many studies have confirmed that aneuploid CTECs and CTCs in peripheral blood has important clinical significance in the prognosis and treatment of cancer. For example, CTCs with high ploidy have been shown to be associated with tumor resistance and relapse [28]. Jingyao Li et al. found that patients in stage IIIA lung cancer with CTCs≥0.5 had significantly lower DFS than those with CTCs<0.5 [13]. In addition, with the concept of precision medicine emerging, more and more attention has been paid to the research of tumor immunotherapy. In these studies, PD1/PD-L1 is the most popular target in immunotherapy of lung cancer. Many studies have shown that the expression level of PD-1/PD-L1 in NSCLC tissues is significantly correlated with the prognosis of patients [45][46][47][48]. However, few studies on the expression of PD-L1 on circulating rare cells in peripheral blood and the significance for the prognosis of NSCLC immunotherapy have been reported. Recent study indicated that, PD-L1+ CTCs and CTECs were associated with poor prognosis [20,49]. In our research, we found two PD-L1+ CTECs existed in the peripheral blood of a patient with stage I lung adenocarcinoma, which means PD-L1+ CTECs are not only exist in patients with advanced lung cancer, but also in early stage lung cancer. However, due to our small sample size, we were unable to obtain the positive rate of PD-L1+ CTECs in patients with early lung cancer, which needs further study.

Conclusions
In conclusion, the liquid biopsy technique of SE-iFISH to detect CTCs and CTECs has good sensitivity and specificity for the diagnosis of NSCLC, which are ideal biomarkers for NSCLC. In the context of accurate diagnosis and individualized treatment, this highly specific, sensitive and non-invasive examination is of great significance for the early diagnosis of NSCLC, and will have greater application space in the early diagnosis and treatment of other cancers in the future.