Histological review

Among the 56 cases of HAS, HC was identified in all cases, TC was identified in 35 cases, and EC was identified in 15 cases. Regarding HCR, the high HCR group (≥ 80%) showed significantly worse OS than the low HCR group (< 80%) (Fig. 3). Additionally, we examined the relationship between HCR and clinicopathological factors and found that the high HCR group had a deeper tumor depth, more frequent liver metastases, and more advanced disease (Table 2a).

Fig. 3

Kaplan–Meier curve of overall survival according to HCR. Patients with a high HCR had a significantly worse OS (p = 0.0044) than those with a low HCR

Table 2 a Comparison of clinicopathological features between the high and low HCR groups. b Comparison of clinicopathological features between the high and low M2 macrophage groups in HC. c Comparison of clinicopathological features between the high and low CSF-1 expression groups in HCMolecular subtyping of HAS according to the TCGA classification

There were no EBV-positive cases, while five cases showed complete loss of MLH1 and PMS2 expressions in all tumor components. In the 51 cases, all MMR proteins were retained. Based on these, we categorized the 56 cases as follows: EBV group (EBER-positive), no cases (0%); MSI group (MLH1 and PMS2 loss), 5 cases (9%); and CIN/GS group (EBER-negative and MSS), 51 cases (91%). Our results are similar to that of a previous study [16].

Association between the number of M2 macrophages in each tumor component and clinicopathological features

There were significantly more M2 macrophages in HC than in EC or TC (HC vs EC: p = 0.008, HC vs TC: p < 0.001) (Fig. 4a). Between EC and TC, there tended to be more M2 macrophages in EC than in TC, but the difference was not significant (p = 0.078) (Fig. 4a). In each tumor component, we compared the clinicopathological features of M2 macrophages between the high and low groups. In HC, the high M2 macrophage group had a significantly worse OS than the low M2 macrophage group (Fig. 4b). Meanwhile, there was no significant difference in prognosis between EC and TC (Supplementary Fig. 1a-b). In HC, the high M2 macrophage group had a higher frequency of venous invasion, lymph node metastasis, liver metastasis, and more advanced stage than the low M2 macrophage group (Table 2b). In EC, the high M2 macrophage group had a higher proportion of older patients than the low M2 macrophage group (Supplementary Table 2a). In TC, the high M2 macrophage group had a larger tumor size than the low M2 macrophage group (Supplementary Table 2b). These suggest that the degree of infiltration of M2 macrophages in HC, but not in EC or TC, correlated with an aggressive behavior and poor prognosis in HAS.

Fig. 4

a Comparison of number of M2 macrophages among the three tumor components. There were significantly more M2 macrophages in HC than in EC or TC (HC vs EC: p = 0.008, HC vs TC: p < 0.001) (a). Between EC and TC, EC tended to have more M2 macrophages than TC, but the difference was not significant (p = 0.078). b Kaplan–Meier curve of overall survival according to the number of M2 macrophages in HC. In HC, patients with a high number of M2 macrophages had a significantly worse OS than those with a low number of M2 macrophages (p = 0.0041). c Comparison of CSF-1 immunoreactivity score among the three tumor components. HC had significantly higher CSF-1 scores than EC or TC (HC vs EC: p = 0.028, HC vs TC: p = 0.041). Comparing EC and TC, CSF-1 score tended to be higher in EC, but the difference was not significant (p = 0.560). d Kaplan–Meier curve of overall survival according to the CSF-1 score in HC. In HC, the high CSF-1 expression group had significantly worse OS than the low CSF-1 expression group (p = 0.0048). e Pearson’s correlation coefficient analysis between the degree of infiltration of M2 macrophages and CSF-1 score in HC. The degree of infiltration of M2 macrophages in HC significantly correlated with the CSF-1 score (p = 0.047)

Association between CSF-1 status in each tumor component and clinicopathological features

CSF-1 immunoreactivity scores were calculated for each tumor component. HC showed significantly higher CSF-1 scores than EC or TC (HC vs EC: p = 0.028, HC vs TC: p = 0.041) (Fig. 4c). Between EC and TC, CSF-1 scores tended to be higher in EC than TC, but the difference was not significant (p = 0.560) (Fig. 4c). In each tumor component, we compared clinicopathological features between the high and low CSF-1 expression groups. In HC, the high CSF-1 expression group had significantly worse OS (Fig. 4d) and a higher frequency of lymphatic invasion (Table 2c) than the low CSF-1 expression group. Meanwhile, the CSF-1 score in EC and TC did not correlate with prognosis (Supplementary Fig. 2a–b) and any clinicopathological feature (Supplementary Table 3a–b).

Association between the number of infiltrating M2 macrophages and CSF-1 score in each tumor component

Pearson’s correlation coefficient analysis revealed a significant correlation between the number of infiltrating M2 macrophages and the CSF-1 score in HC (p = 0.047) (Fig. 4e) but no in EC and TC (Supplementary Fig. 3a–b).

Association between the number of CD8 + TILs in each tumor component and clinicopathological features

There was no statistically significant difference in the degree of infiltration of TILs among each tumor component (Supplementary Fig. 4a). In HC and EC, the low TIL group had significantly worse OS than the high TIL group (Supplementary Fig. 4b–c). Additionally, the degree of infiltration of TILs in TC did not correlate with prognosis (Supplementary Fig. 4d). In HC, the low TIL group had a higher frequency of vascular invasion, lymph node metastasis, and liver metastasis, and had more advanced disease (Supplementary Table 4a). In EC and TC, the low TIL group had a higher frequency of liver metastasis (Supplementary Table 4b–c).

Association between AFP expression and clinicopathological features

We assessed AFP expression in each case, and compared clinicopathological features between the high and low AFP expression groups. The high AFP expression group tended to have worse OS than the low AFP expression group, but the difference was not significant (p = 0.085) (Supplementary Fig. 5a). In high AFP expression group, there were significantly less TILs than in AFP low expression group (Supplementary Fig. 5b). There was no statistically significant difference in the degree of infiltration of M2 macrophages between AFP high and low groups (Supplementary Fig. 5c). The low AFP expression group had a larger tumor size than the high AFP expression group (Supplementary Table 5). There were more AFP-positive cells in the high HCR group than in the low HCR group (Supplementary Fig. 5d).

Univariate and multivariate analysis

Univariate analysis revealed a significant association between a worse OS and pathological tumor depth, pathological stage, vascular invasion, number of M2 macrophages and TILs in HC, CSF-1 expression in HC, and HCR. Meanwhile, multivariate analysis revealed that HCR had a significant effect on OS (HR = 3.176, p = 0.006) (Table 3).

Table 3 Univariate analysis and multivariate analysis of various prognostic factors of OS in all cases