The clinicopathologic features of a total of 351 cases of CRC were summarized in Table 1. Briefly, the age of patients with schistosomiasis was dominantly older than that of patients without schistosomiasis. Unexpectedly, there was no significant difference in morphology between colorectal cancer associated with schistosomiasis and that of without schistosomiasis (Table 1). In the whole cohort, the median age was 69 years (range 33–91), with frequencies between women 39% (137 out of 351) and men 61% (214 out of 351). The location of the tumor in 27% of patients was rectum, in left colon was 33% and in right colon was 40%. On the basis of the AJCC Staging Manual (seventh edition), 76% cases were histologically graded as well to moderately differentiated, and 24% were poorly differentiated. The most predominant histological type was adenocarcinoma (311 patients), mucinous and signet ring cell carcinoma were 11% (34 patients). Vessel and nerve involvement were identified in 122 (35%) and 31 (1%) tumors, respectively. Lymph node metastasis was identified in 144 (40%) patients. Stage I-II cases accounted for 54% (190 out of 351), while stage III-IVa cases were 46% (161 out of 351), respectively. Schistosoma eggs could be observed under microscope in almost 39% cases.
CD4+T and CD8+T cell were observed both in cancer stroma and within cancer epithelium (i.e., intraepithelial). Representative pictures of lymphocyte infiltration are shown in Fig.1A–B. The distributions of CD4+T and CD8+T cell density are shown in Table 2 and Fig.2. The optimum cutoff value of CD4+T and CD8+T cell density were determined by X-tile program, which were 29 cell/mm2 for intraepithelial CD4 (iCD4) (Table 2 and Sup Fig.2A-C) and 145 cell/mm2 for stroma CD4 (sCD4) (Table 2 and Sup Fig.3A-C) and 77 cell/mm2 for intraepithelial CD8 (iCD8) (Table 2 and Sup Fig.4A-C) and 645 cell/mm2 for stroma CD8 (sCD8) (Table 2 and Sup Fig.5A-C). Patients were divided into 2 groups for further analysis based on their respective cutoff values. The C-reactive protein (CRP) positive staining were located in the stromal cells` and tumoral cells` cytoplasm in a diffused manner (Fig. 3A-C). The CRP positive staining was defined as positive, whereas negative staining was defined as negative.
Association of TILs density and CRP with clinicopathological features
The relationship between TILs density and patient demographics is listed in Table 3. No significant correlations were observed between iCD4+T cell density and clinical characteristics, such as age, gender, TNM stage, tumor differentiation and so on (p＞0.05). There was also no correlation between sCD4+T cell density and other clinical characteristics except for tumor budding (p=0.031), lymph node metastasis (p=0.045) and TNM stage (p=0.001). Furthermore, there were significant association between iCD8+T cell density and age (p < 0.001), tumor deposit (p=0.032). In addition, there were significant association between sCD8+T cell density and tumor deposit (p=0.004). However, there were no significant association between TILs in different tumor area and shistosomiasis (p＞0.05).
The association between sCRP and tCRP and clinical characteristic were listed in Supplementary Table 1. Results demonstrated that sCRP was inversely associated with tumor size (p=0.020) and colonic perforation (p=0.001). Besides, tCRP was also negatively associated with colonic perforation (p=0.001). Unexpectedly, there were no relationship between tCRP or sCRP and schistosomiasis as well as CD4+T cells and CD8+T cells.
Univariate and multivariate regression analysis
In the whole cohort, univariate Cox regression analysis identified clinical factors statistically significantly associated with OS (Table 4) were age (p=0.010), gender (p = 0.008), pathological T stage (p < 0.001), Lymph node metastasis (p < 0.001), TNM stage (p < 0.001), tumor differentiation (p < 0.001), lymphovascular invasion (p < 0.001), tumor deposit (p < 0.001), tumor budding (p < 0.001), schistosomiasis (p=0.044), iCD4 (P=0.015), sCD4 (p=0.002), iCD8 (p < 0.001), whereas only gender (p = 0.009), pathology T stage (p=0.035), TNM stage (p < 0.001), sCD4 (p = 0.045), iCD8 (p = 0.0089), schistosomiasis (p=0.045) and tumor deposit were identified as independent risk factors for OS (Fig.4A).
In the NSCRC set, univariate Cox regression analysis identified clinical factors statistically significantly associated with OS (Table 4) were gender (P=0.017), pathological T stage (p=0.001), Lymph node metastasis (p<0.001), TNM stage (p < 0.001), tumor differentiation (p = 0.003), lymphovascular invasion (p<0.001), tumor deposit (p < 0.001), tumor budding (p<0.001), sCD4 (p < 0.001), and iCD8 (p < 0.001) were risk factors for OS. Variables demonstrating a significant effect on OS were included in the multivariate analysis. Gender (p=0.023), pathology T stage (p=0.046), sCD4 (p=0.006), tumor budding (p=0.034), and tumor deposit(p < 0.0001) were identified as independent prognostic factors that associated with OS in the this set (Fig.4B).
In SCRC set, univariate Cox regression analysis identified clinical factors statistically significantly associated with OS (Table 4) were lymph node metastasis (p < 0.001), TNM stage (p < 0.001), tumor deposit (p < 0.001), iCD8 (p= 0.001) and sCD8 (p =0.040) were risk factors for OS, whereas only Lymph node metastasis (p < 0.001), iCD8 (p= 0.020) and tumor deposit(p < 0.001) were identified as independent prognostic factors that associated with OS in this set (Fig.4C). Unexpectedly, there were no association between stromal CRP and tumoral CRP and OS in the whole cohort and subgroups.
Kaplan-Meier Analysis of OS
Kaplan-Meier analysis, which was based on the cutoff value of the density of CD4+T cells and CD8+T cells in different tumor areas, was conducted to assess the variable in overall survival (OS) among different groups. In the whole cohort, compared to the low-density group, patients in the sCD4 and iCD8 high-density group experienced significantly higher OS (p=0.0020, p < 0.001; Fig.5A and 5B). In the NSCRC set, patients in the sCD4 high-density group gained beneficial OS compared to patients in the low-density group (p=0.0004, Fig.5C). Furthermore, patients in the iCD8 high-density also possessed favorable survival compared to patients in the low-density (p=0.0008, Fig.5D). There was no association between sCRP or tCRP and OS in any subgroups.
Comparison of TILs density and positive stained CRP in CRC patients with and without schistosomiasis
We next compared the density of CD4+ and CD8+ T cells in CRC patients with or without schistosomiasis (data was not shown). The distribution of CD4+ T cells densities in stromal or tumoral cells between NSCRC and SCRC groups were no significant differences. Similarly, there were also no significant differences in the distribution of CD8+ T cells densities in stromal or tumoral cells between the NSCRC and SCRC group. The positivity rate of stromal CRP was 22% and 30% in the NSCRC set and SCRC set, respectively. Besides, the positivity rate of tumoral CRP was 14% and 15% in the NSCRC set and SCRC set, respectively.