The level of CTCs was correlated with malignant SPNs.
There are amounts of studies detected CTCs in advanced cancer patients, including lung cancer. However, whether CTCs could be detected from the peripheral venous blood of SPNs patients is unknown. We first enriched the CTCs from patients diagnosed as SPNs by CT examination. We collected the histopathologic diagnosis of the SPNs if the patient underwent surgery. We also examined the serum tumor markers before the surgery. Fifty-three patients which successfully completed CTCs, CT, serum markers, and histopathology examinations were included for the subsequent statistically analysis. The demographic and clinic pathologic characteristics of the patients were displayed in Supplementary table S1.
We found that 41/53 patients were lung cancer, and most of which (38/41) were adenocarcinomas. Since the invasiveness of adenocarcinomas provide critical guidelines for the resection strategy and is correlated with the prognosis, we classified the patients into 3 groups, AIS, MIA, and IA. We also divided the patients into two groups (≤Ⅰa and >Ⅰa) based on the pTNM stage, given that ≤Ⅰa stage was recommended for limited surgical resection, such as segmentectomy, sublobar or lobectomy resection, which would both increase the survival rate and maximally preserve the lung function [22–25]. Twelve out of 53 patients were benign pulmonary diseases, such as fibrosis, hyperplasia, and tuberculosis. The representative hematoxylin & eosin (H&E) staining of these tissues were shown in Fig. 1.
Then, we analyzed the correlation between CTCs level and malignant SPNs. CEP8+/ CD45−/ DAPI + were determined as CTCs. Representative staining was shown in Fig. 2A. The level of CTCs was presented by the number of CTCs in each sample. We found that the CTCs level was significantly higher in malignant SPNs than benign SPNs by Mann-Whitey U test (p < 0.05) (Fig. 2B). Similarly, the average CTCs numbers of malignant SPNs were significantly higher than benign SPNs by the student t test (Supplementary figure S1A). Moreover, the CTCs levels were significantly lower in >Ⅰa stages (Ⅰb/Ⅱ/Ⅲ) of lung cancer than ≤Ⅰa stage (0/Ⅰa) of lung cancer (p < 0.05) (Fig. 2C). Since 38/41 malignant SPNs were adenocarcinomas (Supplementary figure S1B), we analyzed the correlation between the CTCs levels and cancer invasiveness. However, no significant difference of the CTC levels was observed among AIS, MIA and IA groups (Fig. 2D). Considering the limited sample size, we pooled MIA and AIS to make up noninvasive group compare with invasive group (IA), since MIA was considered as noninvasiveness pathologically. We did not observe any statistical difference in the CTCs levels (Supplementary figure S1C).
Furthermore, the ROC analysis showed that the sensitivity and specificity for the CTCs as a diagnostic marker to distinguish malignant SPNs from benign SPNs were 92.7% and 50%, respectively (Fig. 2E, Table 1). The area under curve (AUC) of the CTC levels was 0.713 with a statistical significance, indicating it would be an independent diagnostic marker for malignancy (p < 0.05). However, the CTCs levels showed no significant difference to distinguish ≤Ⅰa stage lung cancer from >Ⅰa stage lung cancer (Fig. 2F), as well as to distinguish invasive from non-invasive SPNs (Supplementary figure S1D) and adenocarcinoma from other types (Supplementary figure S1B and S1E). These findings suggest that the levels of CTCs were increased in malignant SPNs compared to benign SPNs, albeit very early stage of lung cancer released more CTCs than late stage disease. The level of CTCs would serve as a potential liquid biopsy marker for the diagnosis of malignant SPNs.
Table 1
results of ROC analysis (malignant versus benign)
| AUC (95% CI) | p value | Cut-off value | Sensitivity (%) | Specificity (%) |
CTCs | 0.713 (0.525, 0.902) | 0.026 | 0.5 | 92.7 | 50.0 |
Size | 0.462 (0.274, 0.651) | 0.694 | 0.5 | 34.1 | 58.3 |
Density | 0.702 (0.551, 0.853) | 0.034 | 0.5 | 48.8 | 91.7 |
CTCs + Density | 0.726 (0.537, 0.914) | 0.018 | 0.5 | 95.1 | 50.0 |
Combined use of CTCs level and density features of the CT examination identified malignant SPNs.
Currently, with the growing use of CT examination, especially thin-section CT scan, millions of SPNs were detected annually; however, only 30% were finally identified as malignant. Thus, more adjuvant diagnostic methods were urgently needed. We firstly analyzed the correlation between the CT morphology and the clinicopathological characteristics of SPNs, based on the most frequently used three criteria, such assize (the largest diameter), number, and density of the nodules. As shown in Table 1, no significant correlation was observed between size and malignancy of the nodules, neither between the nodule number and malignacy. However, the density of the nodules showed significant positive correlation with the malignancy (Table 2). Statistical analysis also showed that there had no correlation between size, number, or density of the nodules and the cancer stages, neither any correlation with cancer types were observed (Supplementary table S2). Moreover, the number of the nodules displayed significant correlation with invasiveness of adenocarcinomas, but no correlation exhibited between the size and the invasiveness (Supplementary table S3). Furthermore, no obvious correlation was observed between the density and the invasiveness (Supplementary table S3). Considering this may be caused by limited sample size, we pooled AIS and MIA together and observed a significant positive correlation between density and invasiveness (Supplementary table S4). These findings were consistent with other reports that the high density of the nodules was an important indicator for malignancy.
Table 2
the correlation between CT morphology and histology of SPNs
CT parameters | n | Histology | X2 value | p value |
| | Malignant | Benign |
Size | | | | | |
<15 mm | 34 | 27 | 7 | 0.018 | 0.892 |
≥15 mm | 19 | 14 | 5 | | |
Number | | | | | |
Single | 30 | 23 | 7 | 0.019 | 0.891 |
Multiple | 23 | 18 | 5 | | |
Density | | | | | |
Solid | 32 | 21 | 11 | 4.77 | 0.029 a |
Non-solid | 21 | 20 | 1 | | |
Note: a, adjust chi-square test, 1 < T < 5. |
Then, we analyzed the correlation between the CTCs levels and CT examination. We did not observe any significant difference of the CTCs level between the small (< 15 mm) and big (≥ 15 mm) SPNs group based on the CT examination (Fig. 3A). We investigated the prediction of SPNs malignancy by considering both the CTCs level and the size of CT morphology. As shown in Fig. 3B, the CTCs level was significantly higher in malignant than in benign SPNs in big SPNs group, but not in small group. Moreover, although no significant difference in CTCs level was exhibited between the number of single and multiple groups, the level of CTCs showed a significant increase in malignancy compared with benign SPNs when only single SPNs are considered (Fig. 3C and D). No difference of the CTCs level was observed between solid and non-solid group, albeit the level of CTCs was obviously higher in malignancy than benign SPNs when only solid SPNs are considered (Fig. 3E and F). We did not analyze non-solid group for the reason of lacking enough sample number in benign SPNs group.
Next, we analyzed the sensitivity and specificity of the CTCs level combined with CT characteristics for prediction of malignancy of SPNs by ROC analysis. The AUC of the density, the CTCs levels, but not the size, showed statistical significance (Fig. 3G). Moreover, combined use of the CTCs levels and the density features exhibited higher AUC than each single item. As shown in Table 1, the CTCs showed the highest sensitivity (92.7%) and the density showed the highest specificity (91.7%), when they were solely used for identifying malignancy. Combined use of these two features did further increase the sensitivity but not the specificity.
Additionally, we analyzed the CTCs level, size and density features of the nodules in differentiating the ≤ Ⅰa stages from > Ⅰa stages of lung cancer. The ROC curve showed that the AUC of each feature did not display any statistical significance; however, combined use of CTCs level and the density increased the AUC to 0.735 compared to each single one (0.61 for CTCs and 0.648 for density) (Fig. 3H). As shown in Table 3, combined use of CTCs level and density significantly correlates with SPNs stage (p = 0.041) and, with a sensitivity of 97% and specificity of 50%, this combined diagnostic strategy improved the overall diagnostic effect compared with when solely CTCs level or density was considered.
Table 3
results of ROC analysis (≤ Ⅰa stages versus > Ⅰa stages)
| AUC (95% CI) | p value | Cut-off value | Sensitivity (%) | Specificity (%) |
CTCs | 0.610 (0.369, 0.851) | 0.34 | 0.5 | 97.0 | 25.0 |
Size | 0.324 (0.128, 0.540) | 0.126 | 0.5 | 27.3 | 37.5 |
Density | 0.648 (0.441, 0.855) | 0.199 | 0.5 | 54.5 | 75.0 |
CTCs + Density | 0.735 (0.369, 0.851) | 0.041 | 0.5 | 97.0 | 50.0 |
These findings suggest that combined use of the density feature of the CT examination and the CTCs level could be developed as a potent adjuvant quality and stage diagnosis of SPNs.
The serum tumor markers did not show any correlation with malignant SPNs, but the CEA levels were correlated with the CTCs levels.
We also examined the serum tumor makers of the SPNs patients. To our surprise, most of the patients showed negative results based on the normal criteria (< 5.0 ng/mL for CEA, < 7.0 ng/mL for CYFRA 21 − 1, < 7.29 ng/mL for AFP, and < 27.0 Unit/mL for CA 19 − 9), possible due to most of the subjects in this study are early lung cancer or benign diseases. Moreover, the average concentrations of the each marker did not show any significant difference between malignant and benign SPNs groups (Fig. 4A). We analyzed these markers between the two groups in CTCs positive or negative patients. As shown in Fig. 4B, among the 4 serum markers, only the CEA concentrations exhibited difference between CTC + and CTC- groups. These findings indicate that the concentration of CEA would be different if the patient has CTCs.
The comparison the CEP8, EpCAM, and pan CKs for CTCs identification.
Currently, the specific markers of CTCs have not been well identified. Even the same population could show opposite results for CTCs levels when different methods were used [26, 27]. In this study, CEP8 positive cells by FISH assay was defined as the CTCs. We then compared other frequently used markers such as EpCAM and pan CKs for the detection of CTCs using immunofluescence staining in the same slides following CEP8 examine (Fig. 2A). We found that among the 22 samples that successfully completed all three markers staining, only 5 patients (1 slide for each patients) had dual positive cells EpCAM+/pan CKs + with a detection rate of 22.7%, which was consistent with others report [28]. And there were some cells showed triple positive staining. The level of CTCs showed no significant difference between pan CKs and EpCAM positive cells. However, CEP8 positive cell numbers were higher than EpCAM or pan CKs with a statistical significance (Supplementary figure S2). These findings indicated that different markers detected different subpopulations of CTCs due to the heterogeneity of cancer cells.