The study included a total of 131 renal cancer patients with TNM stage T1-3N0M0. The clinicopathological features of the study cohort were summarized in Table 1, including age, sex, histology, differentiation, T stage, surgery types and renal score. We obtained blood samples from 131 patients and enriched for CTCs using CanpatrolR technique. Based on the EMT markers, the detected CTCs could be classified into three phenotypes: epithelial, bi-phenotypic and mesenchymal (Fig. 1a). In the present study, whereas the majority of CTCs were single, we also detected CTC-WBC clusters. Three types of CTCs and CTC-WBC clusters were depicted in Fig 1. No CTC was detected in patients with benign renal disease. Baseline CTCs were detected (≥ 1/5 mL) in 125 out of 131 renal cancer patients (95.4%) with a median of 6 CTCs/5 mL blood, ranging from 0/5 mL to 53/5 mL. The positive rate of mesenchymal CTCs (MCTC) before surgery in all patients was 61.1% (80/131), ranging from 0/5 mL to 15/5 mL. CTC-WBC clusters were present in 11.5% (15/131) patients, ranging from 0/5 mL to 2/5 mL.
For better deciphering the potential application of CTCs in clinical practice, patients were divided into two parts according to the median number of total CTC counts (CTC<6 or CTC ≥6), MCTCs (negative/positive) and CTC-WBCs (negative/positive), respectively. Of the 131 patients included in the CTC analysis, 58 (44%) had CTCs<6 and 73 (56%) had CTC ≥6 at baseline. The preoperative serum CTC level showed no association with clinicopathological features, while CTC level after surgery was significantly correlated with types of surgery, P=0.007 (Table 1). Similarly, the preoperative and postoperative MCTC showed little correlation with patients’ parameters. The positive rate of CTC-WBC clusters was 11.5% (15/131) before operation, and 13.0% (17/131) after operation. Similarly, no association between the existence of CTC-WBC and clinicopathological characteristics was found.
The follow-up duration of all patients was 6 to 61 months, with a median of 24 months. In total, 129 (98.5%) patients were alive and 111 (84.7%) patients were in the disease-free status at the end of follow-up. The Kaplan–Meier’s survival curves revealed that patients with CTCs ≥6 after surgery had significantly poorer PFS (P < 0.001) than subgroup with CTC< 6, while the CTC level at baseline was found to have no significant correlation with PFS (P = 0.459, Fig. 2A and B). Similarly, patients with the presence of MCTCs per 5 mL blood after surgery was more likely to have unfavourable PFS compared with those without MCTCs(P = 0.002, Fig. 2D). Likewise, the survival curves revealed that patients with/without CTC-WBC clusters before surgery exhibited no remarkable differences in PFS, while the negative of CTC-WBC after surgery was significantly associated with longer PFS (P = 0.916 and 0.017, respectively, Fig. 2E and F).
CTC counts after operation were available from all patients. Patients with CTCs post-operation increased when compared with CTCs pre-operation were categorized as CTC increased, the other patients were categorized as CTC decreased or no change. Similarly, these patients were also categorized according to the variations of MCTC during operation. The increase of CTC or MCTC were significantly associated with poorer PFS of renal cancer patients, P=0.006 and 0.012, respectively (Figure 3A and 3B).
In univariate analysis, clinical factors significant for survival were T stage and operation methods. CTC related univariate analyses revealed significant association between post-operative CTC/MCTC/CTC-WBC, CTC/MCTC changes and PFS (all, P<0.05) (Table 2). In multivariate Cox regression analysis, after adjusting for the clinically significant univariate factors, CTC level (HR 7.521, 95%CI 2.065-27.397, P=0.001), existence of MCTC (HR 8.232, 95%CI 1.826-37.820, P=0.006) and CTC-WBC (HR4.108, 95%CI 1.507-11.199, P=0.006) after operation remained as highly significant predictors of PFS. Patients with increased CTC (HR 2.784, 95%CI 1.001-7.743, P=0.05) or MCTC (HR 2.329, 95%CI 0.901-6.021, P=0.081) had slightly higher risk of progression compared with those in the CTC or MCTC decreased/unchanged group.