The combination of CTCs and CEA can help guide the management of patients with SPNs suspected of being lung cancer

We recruited 161 patients with SPNs who undergoing treatment were from January 2017 to September 2018 for enrolment in the study. The diagnosis of SPNs was based on assessments of low-radiation computed tomography scans by two radiologists. All patients received established diagnoses by pathologic examination. The inclusion criteria were the presence of one solid SPN, which is defined as a nodule with at least a solid component > 80% of the total volume, and available CT scan encompassing the lungs and a definitive diagnosis by means of tissue biopsy or imaging follow-up, as suggested by guidelines [26]. The exclusion criteria were the presence of visible nodule calcifications and the presence of more than one nodule in the same patient. No patient underwent preoperative radiotherapy, chemotherapy or any other treatment. Disease stages were based on the eighth edition of the American Joint Committee on Cancer staging manual. Surgery was performed under the supervision of the three thoracic surgeons at the thoracic center, and all operations were performed as two-hole thoracoscopic lung nodule wedge resections or lobectomy. The demographics and diagnoses of the patients are shown in Table 1.

This retrospective trial was performed at a clinical center in the Department of Respiratory and Critical Care Medicine and Thoracic Surgery in our hospital to assess the number of CTCs, the concentration of CEA and the nodule characteristics on chest CT to predict the likelihood that SPNs were cancerous. The primary inclusion criteria were measurable nodules on the imaging examinations and the availability of data regarding CEA and CTCs. The Institutional Review Board at Shanghai General Hospital, Shanghai Jiaotong University, approved the current retrospective study and informed written consent was obtained from all subjects before the study commenced.

The detection of CTCs was performed as described previously by Liu et al. and Ge et al. [27, 28] In briefly, CTC enrichment and identification was performed according to the instructions of the Cytelligen CTC Enrichment Kit and Human Tumor Cell Identification Kit (Cytelligen, San Diego, CA, USA). Blood samples were collected in 6-ml ACD-containing tubes from an antecubital vein in all patients before surgery. The technicians who performed the CTC detection tests were blinded to the sample identification. All samples had been de-identified before the technicians received them. After the blood samples were centrifuged, the white buffy was collected and mixed with a tumor-associated antigen CK18 (showing that the captured cells were epithelial cells) and the CD45 antibody (demonstrating that the captured cells derived from non-leukocytes). After shaken, the mixture was subjected to magnetic separation. The resulting cell pellet was mixed with 100 μl of fixative and used to coat CTC slides. A Centromere Probe (CEP 8) Spectrum Orange (Vysis, Abbott Laboratories, Abbott Park, IL, USA) was added to the slides, denatured at 76 °C for 10 min and hybridized at 37 °C for 3 h. The slides were incubated for 2 h in the dark at room temperature. After being washed twice, 5 μl of mounting medium(Ultra Cruz, Santa Cruz Biotechnology, CA, USA) staining with 4′, 6-diamidino-2-phenylindole (DAPI, showing the karyotypes of the captured cells) was added, and then, the cell number was counted under a Carl Zeiss Axioplan 2 imaging fluorescence microscope (ZEISS Company, Germany). The identification of CTC was DAPI+/CK18+/CD45−/CEP8 ≥ 3pots. The count was repeated 3 times, and the mean was selected as the final value for each patient.

Peripheral venous blood samples (3.0 ml) were collected from patients prior to surgery. The serum was separated by centrifugation at 4000 rpm for 10 min within 2 h. The concentration of CEA was measured by an automated electrochemiluminescence analyzer (Roche Diagnostics, Bavaria, Germany). All tests were performed according to the protocols in the instrument operating manual. CEA values obtained within ±7 days of the date of a given CTC measurement were included.

Computed tomography imaging scans of the chest were first performed to identify lung nodules. The images were assessed by two certified radiologists, including nodule margin, nodule diameter, nodule density, burr sign, pleural traction, and vascular bundle sign. Finally, the thoracic surgeon decided whether to perform an operation.

Between January 2017 and July 2018, 161 patients were enrolled, 131 of whom met the inclusion and exclusion criteria and were assessable for the objectives pertaining to lung cancer or benign nodules. At the time of these analyses, the two groups had similar demographic characteristics (age, gender, and smoking). Patient characteristics are listed in Table 1.

The aim of the study was to facilitate decision-making for patients with SPNs suspected of being lung cancer. As presented in Fig. 1a, the median numbers of CTC units were 5.5 (4.13–7.28) and 12.09 (7.11–15.88) in benign nodules and patients with NSCLC, respectively. Compared with patients with benign nodules, patients with NSCLC had significantly higher levels of CTC (p = 0.016). The CTC level was related to age (p = 0.041) and but not gender (p = 0.842). Of the 131 evaluable patients, all (100%) underwent imaging studies. The CTC level was related to nodule position (p = 0.0064), especially in the upper lobe than other lobes, but not nodule size (p = 0.074), regardless of the nodule type (Fig. 1). Table 2 summarizes the comparison of clinical characteristics and imaging results among patient with different CTC unit levels, including age at diagnosis (≤60 years vs. > 60 years), smoking, gender (male vs. female), maximum tumour diameter (MTD, < 8 mm vs. ≥8 mm), nodule site, and type of nodules (ground-glass opacity (GGO) vs. subsolid vs. solid).

To investigate the significance of CTC levels in patients with NSCLC, the imaging and pathologic characteristics of individuals, including tumor grade (ACIS vs. MIA vs. ADI) and nodule site (upper lobe vs. others), were also tested for their associations with CTC levels. The CTC levels of patients with nodules in the upper lobe of the lung were higher than those in patients with nodules in other lobes (p = 0.011; Fig. 2).

To further analyze the diagnostic efficiency of CTCs in patients with SPNs, we compared CTCs with CEA. With the cutoff value of 6 CTC units, the sensitivity was 67.1%, and the specificity was 56.5% in the diagnosis of NSCLC in patients with SPNs. The sensitivity of the existing clinical tumor biomarker (CEA, cutoff value 2.09 ng/ml) (54.1%) was lower than that of CTCs, but the specificity of CEA was higher (73.9%) than that of CTCs. As shown in Fig. 3a and Table 3, the CTC units had a higher AUC (0.78; 95% CI 0.70–0.86) and Youden index value (0.2358) than CEA (AUC 0.626; 95% CI 0.526–0.725). With a cutoff point for nodule size of 8 mm, the sensitivity was 88.2%, and the specificity was 73.9% in the diagnosis of NSCLC in patients with SPNs; the AUC was 0.572 (95% CI 0.466–0.677). Furthermore, with regard to the diagnosis of NSCLC in patients with SPNs, nodule type (nodule density − 600 HU) and nodule site had sensitivities of 70.6 and 58.8% and specificities of 45.7 and 47.8%, respectively.

The AUC of CTC combined with CEA and nodule type was 0.827, and the 95% confidence interval was 0.752–0.901. In addition, the AUC of CTC combined with CEA, nodule type, nodule size, and nodule site was 0.841, and the 95% confidence interval was 0.769–0.914, which indicates a satisfactory discrimination of patients with early-stage NSCLC from those with benign lung nodules (Fig. 3a and Table 3). Nodule type, CEA and CTC are independent prognostic factors for lung cancer.

We also analyzed outcomes based on the number of CTCs (≥12 CTCs/6 ml) and CEA values (> 1.78 ng/ml) in the diagnosis of nodules in the upper lobe of the lung, nodules ≥8 mm, and subsolid nodules. As expected, with the cutoff values of 12 CTC units and 1.78 ng/ml CEA, the sensitivities were 51.85 and 62.96%, and the specificities were 90 and 80%, respectively. The AUCs were 0.70 (95% CI, 0.527–0.839) and 0.68 (95% CI, 0.508–0.825), respectively. The AUC for CTC combined with CEA was 0.73, and the 95% confidence interval was 0.566–0.900 (Fig. 3b and Table 4). It is noteworthy that among the patients with upper lobe, subsolid and ≥ 8-mm nodules, the combination of the number of CTCs with the CEA value resulted in a distinct improvement of the sensitivity and specificity in the diagnosis of lung cancer.