Patient characteristics
A total of 901 LARC patients were eligible for our analysis. Among them, 75 (8.3%) were assigned to the young group and 826 (91.7%) patients were assigned to the old groups. The median ages in the two groups were 34.3 and 58.1 years, respectively. Additionally, the old group was associated with a higher American Society of Anaesthesiology (ASA) grade (P < 0.05). As shown in Table 1, there were no significant differences between the two groups in terms of sex, the interval between NCRT and surgery, distance from the anal verge, clinical T stage, clinical N stage, pre-NCRT CA19-9 level, pre-NCRT CEA level, post-NCRT CA19-9 level, and post-NCRT CEA level (all P > 0.05),
Perioperative, pathological and survival outcomes
No significant differences were observed between the two groups in terms of estimated blood loss, operation time, surgical approach, peri-NCRT complication rates, and preserve organ procedure (all P > 0.05, Table 2). There were no significant differences between the two groups in terms of postoperative hospital stay and postoperative complications (P = 0.124, P = 0.736, respectively). Similarly, the chemotherapy regimen did not differ between the two groups (P = 0.461). Compared to the young group, mucinous or signet ring cell carcinoma, (17.3% vs. 7.6%, P = 0.008) or poorly differentiated tumors (24.3% vs. 9.0%, P < 0.001) were more common in the old group. Moreover, the young group was associated with a higher TRG (P = 0.003), as well as a higher rate of perineural invasion (17.3% vs. 6.5%, P = 0.002). Pathological TNM stage and pathological type were similar in both groups (P = 0.957, P = 0.936). Similarly, there were no differences between the two groups in terms of vascular invasion and tumor size did (P = 0.346, P = 0.069). A positive circumferential resection margin (CRM) was observed in one patient (1.3%) in the young group, 10 patients (1.2%) in the old group, with no significant difference between the two groups (P = 1.000). Moreover, the Kaplan-Meier curve analysis demonstrated that young patients were associated with poorer prognosis in LARC patients following NCRT. The 3-year OS rate in the old group (≥40 years) was significantly higher than that in the young group (<40 years) (88.3% vs. 71.6%; P = 0.01, Fig. 3M). Moreover, the 3-year DFS rate for the old group (≥40 years) was higher than that in the young group (<40 years) (83.8% vs. 68.6%; P = 0.204, Fig. 3L).
Association between young age and pCR
To explore the association between young age and treatment response to NCRT, we identified predictive factors for pCR by logistic regression analysis. In the univariate analysis, tumor size (≤1.8cm vs. 1.9–3.1 cm OR = 6.764, P < 0.001; vs. ≥3.2 cm OR = 2.022, P = 0.007), age (≥40 years vs. <40 years, OR = 2.087, P = 0.044), pre-NCRT clinical T stage (OR = 0.731, P = 0.031), pre-NCRT clinical N stage (OR = 0.550, P = 0.016), distance from the anal verge (OR = 0.919, P = 0.018), and post-NCRT CEA (OR = 0.381, P < 0.001) were significantly associated with pCR in LARC patients. In the multivariate analysis, tumor size (≤1.8 cm vs. 1.9–3.1 cm OR = 6.764, P < 0.001; vs. ≥3.2 cm OR = 2.022, P = 0.007), age (≥40 years vs. <40 years, OR = 2.382, P = 0.027), pre-NCRT clinical N stage (OR = 0.560, P = 0.029), and post-NCRT CEA (OR = 0.873, P = 0.001) were independent predictive factors for pCR in LARC patients (Table 3).
Predictive nomogram for pCR and decision curve analysis
By incorporating the significant determinants in the logistic regression analysis, we developed a predictive nomogram for pCR in LARC patients after NCRT as shown in Figure 2A. The c-index of the nomogram was 0.70 (95% CI 0.70–0.71). The calibration curve (Figure 2B) presented good statistical performance upon internal validation between predicted and actual pCR rates. DCA was used to evaluate the performance of the nomogram. As shown in Figure 2C, the model including age group provided more predictive power than either the pCR scheme or the non-pCR scheme. The clinical impact curve (Fig. 2D) shows the prediction of risk stratification of 1,000 patients using a resampling bootstrap method.
The association of CD133 expression with survival, pCR rate, and young age
We further investigated the reason for the lower pCR rates in young LARC patients by examining CSC frequency (based on CD133 expression, Fig. 3E, F, G and H). A total of 169 LARC patients were eligible for immunohistochemical analysis. Since the CD133 expression scores were continuous variables, the X-tile program was utilized to identify the optimal cut-off points for determining the greatest actuarial survival difference. Using this method 11 was identified as the cut-off value for CD133 expression (Fig. 3A and 3B). Based on these cut-off points for CD133 expression, we divided the cohort into low (n = 127) and high (n = 42) subgroups in terms of OS and DFS.
Higher CD133 scores were associated with poorer prognosis in LARC patients following NCRT. The 3-year OS rate for the low CD133 group was significantly higher than that in the high CD133 group (95.1% vs. 62.9%; P < 0.01, Fig. 3D). Moreover, lower CD133 scores were correlated with improved DFS (Fig. 3C). The 3-year DFS rate for the low CD133 group was significantly higher than that in the high CD133 group (92.8% vs. 50.7%; P < 0.01). As shown in Figure 3I, young patients had a higher CD133 expression score compared to that of old patients (P < 0.01). Moreover, the correlation analysis demonstrated that CD133 expression was associated with patient age (P < 0.01, Fig. 3J). Moreover, we examined the CD133 expression in the pCR and non-pCR groups, the results demonstrated that the CD133 expression value in the pCR group was significantly lower than in the non-pCR group (P < 0.01, Fig. 3K). Taken together, these findings indicated that young LARC patients were associated with a higher CD133+ CSC burden, which might contribute to the lower pCR rates.