Patients
Enrolled were 31 patients diagnosed with stage I to III esophageal squamous cell carcinoma (neoadjuvant arm: 12, surgery arm: 19). The clinicopathological characteristics of the patients are illustrated in Table 1 and Figure 3.
Longitudinal Evaluation of CTCs Detected in Patient Samples
The longitudinal evolution of CTC numbers was depicted in Figure 4. Prior to neoadjuvant therapy, CTCs were found in 6 out of 12 patients (50%, median CTC count 0.5; range 0–5). Before surgery, CTCs were found in 14 out of 31 patients (45.16%, median CTC count 0; range 0–19). On postoperative day 2, CTCs were found in 22 out of 31 patie.0nts (70.97%, median CTC count 3; range 0–37). At one week postoperative, CTCs were found in 16 out of 31 patients (51.6%, median CTC count 1; range 0–14). Three months postoperative, CTCs were found in 5 out of 31 patients (16.1%, median CTC count 0; range 0–4).
A comparative analysis of CTC numbers at various time points was conducted. Patients were categorized into three groups: all patients (31), the surgery arm (19), and the neoadjuvant arm (12). The results revealed statistical differences in CTC numbers between preoperative and postoperative day 2 (p=0.05), preoperative and postoperative 3 months (p=0), postoperative day 2 and postoperative 1 week (p=0.008), postoperative day 2 and postoperative 3 months (p=0), and postoperative 1 week and postoperative 3 months (p=0) among all 31 patients. In the surgery arm, statistical significance was observed in preoperative vs postoperative 3 months (p=0) and postoperative 1 week vs postoperative 3 months (p=0). In the neoadjuvant arm, with the exception of preoperative vs postoperative 1 week (p=0.231), pre-neoadjuvant chemotherapy vs preoperative (p=0.212), pre-neoadjuvant chemotherapy vs postoperative 1 week (p=0.832), all other comparative analyses exhibited statistical significance (Table 2).
Number of CTCs change after neoadjuvant chemotherapy or surgery
The number of circulating tumor cells (CTCs) was computed at two distinct time points for 31 patients through subtraction. The outcomes were categorized into two groups: a reduced or equal group and an increased group. The findings indicated that, in the comparison of CTCs numbers between patients undergoing neoadjuvant chemotherapy (pre-neoadjuvant minus preoperative), 66.70% exhibited a decrease or remained unchanged, while 33.3% showed an increase (Figure 5a). In the analysis of CTCs numbers for patients undergoing surgery (postoperative day 2 minus pre-treatment), 58% experienced an increase, whereas 42% demonstrated a reduction or remained unchanged (Figure 5b). Similarly, for the interval between post-operative 1 week and preoperative, 61.3% displayed a decrease or remained equal, and 38.7% manifested an increase (Figure 5c).
Association between CTCs and survival time
The median survival time for the cohort of 31 patients was 36 months. Among patients initially detected with circulating tumor cells (CTCs+, n=13), the neoadjuvant arm exhibited an extended progression-free survival (PFS) (p=0.05, Figure 6a) and overall survival (OS) (p=0.04, Figure 6b) compared to the surgical arm. The ROC curve (Figure 7) demonstrated that the number of CTCs on postoperative day 2 provided a superior diagnostic value for predicting patient prognosis (AUC=0.758, p=0.014, 95% CI 0.579-0.938).
The determined cutoff value for the number of CTCs was 3.5. Consequently, a cutoff value of 4 was selected for subsequent analysis. On postoperative day 2, 17 patients had CTCs<4, while 14 patients had CTCs≥4. Patients with CTCs<4 exhibited significantly prolonged PFS (p<0.01, HR=6.26, 95% CI 1.96-19.96, Figure 6c) and OS (p<0.01, HR=6.63, 95% CI 2.08-21.13, Figure 6d) compared to those with CTCs≥4.
Longitudinal Evaluation of CTCs and survival time
In the comparison of CTCs numbers between postoperative day 2 and pre-treatment, 20 patients exhibited an increased quantity of CTCs. Among them, 10 patients experienced an increase exceeding 3, while the remaining 10 patients showed an increase of less than 3. Notably, patients with a CTCs increase of ≤3 demonstrated significantly prolonged progression-free survival (median, 59 months vs. 20 months, p=0.035, HR=3.29, 95% CI 1-10.9, Figure 6e) and overall survival (median, 61 months vs. 21 months, p=0.035, HR=3.78, 95% CI 1-14.3, Figure 6f) compared to those whose CTCs increased by >3.
Association between CTM and survival time
CTM was detected in 14 out of 31 patients (45.16%) at all time points, as previously indicated. The majority of CTM instances were identified post-initial treatment, with only 3 (21.4%) CTM+ patients detected pre-treatment. The CTM detection rates were 26.3% (5/19) and 75% (9/12) in the surgery arm and neoadjuvant arm, respectively. Survival analysis revealed that CTM-negative patients exhibited an extended progression-free survival (PFS) (p=0.029, HR=2.92, 95% CI 1.05-8.07, Figure 6g) and overall survival (OS) (p=0.03, HR=2.92, 95% CI 1.06-8.09, Figure 6h) compared to CTM-positive patients. Further analysis of CTM-positive patients indicated that, when comparing the difference in CTCs numbers between postoperative day 2 and preoperative, patients with a decreased or unchanged number of CTCs demonstrated superior survival (no statistical difference, Figure 6i-j) compared to those with an increased number.