Correlation between SSTR2 and Bcl2 expression and clinicopathological variables
Tumor tissue was defined by cell staining for SSTR2 in the cell membrane (Fig. 1A) and Bcl2 in the cytoplasm (Fig. 1B). In normal part of IHCC patient’s liver, SSTR2 expression was detected in the large bile duct, while no staining in small bile duct (Fig. 1C). Bcl2 expression was detected only in small bile duct including bile ductule and interlobular bile duct (Fig. 1D). In EHCC tumors, 32 were positive for SSTR2 but negative for Bcl2, while 5 were positive for both molecules. In these five EHCC, Bcl2 was expressed, but SSTR2 was not. Only one case stained negative for both molecules. Positive SSTR2 expression in cancer cells was present in 26 out of 52 IHCC cases (50.0%) and positive Bcl2 expression in cancer cells was present in 19 (36.5%). Of 52 total IHCC patients, 21 were categorized as Group H (40.4%) and 14 as Group P (26.9%). For the remaining 17 patients in the unclassified group (Group U), five were positive for both SSTR2 and Bcl2 (9.6% of all patients) and 12 were negative for both SSTR2 and Bcl2 (23.1% of all patients).
Table 1 presents a comparison of clinicopathological characteristics of IHCC patients categorized by SSTR2 and Bcl2 expression. Group P had a significantly better prognosis according to T classification. Group H and Group U patients had tumor infiltration into bile ducts significantly more frequently than those in Group P. Figure 2A shows that the overall survival of patients in Group P was better than that of those in Group H and Group U (p=0.098, <0.05, respectively). Similarly, Figure 2B shows that disease-free survival of patients in Group P was significantly better than that of those in Group H and Group U (p<0.05). All five cases based on gross classification that were classified as perihilar IHCC were included in Group H. However, in 47 cases gross classified as peripheral IHCC, 14 cases were included in Group P, 16 cases in Group H, and 17 cases in Group U. Comparing these 14 individuals in Group P with the 16 in Group H, those in Group H had lower curability (p=0.031), a higher T-factor (p=0.005), higher clinical stage (p=0.001) (Table 2), higher incidence of periductal infiltration (p=0.005), and worse prognosis according to disease-free survival (p=0.014) (Fig. 3).
According to typical histological findings described by Akita and Liau [6,9], perihilar IHCC consists of duct-forming and tall columnar tumor cells, as well as an abundant fibrotic stroma [6,9]. This type of tumor has a clear cytoplasm and tubular components similar to reactive bile ductules at the tumor-liver interface of the invasive front. In contrast, peripheral IHCC consists of cuboidal to low columnar cells, which form irregularly anastomosing tubular architecture with scant mucin. This type of IHCC typically has a hepatoid appearance. Figure 4 shows a representative case. According to the CT images and gross appearance, the tumor appeared to be located in the periphery of the liver (Fig. 4A-D). However, morphologically, the tumor consisted of duct-forming, abundant fibrotic stroma and little tubular architecture and cancer cells had a clear cytoplasm, which suggested it to be the perihilar type (Fig. 4E). Additionally, immunohistochemical analysis indicated that SSTR2 was expressed in the tumor, but Bcl2 was not (Group H) (Fig. 4F, G). Considering these observations, this tumor was classified as embryological perihilar immunohistologically, but as peripheral based on its gross appearance.
Relationship between the bile duct and IHCC tumor according to 3D imaging
Figure 5 shows the relationship between the bile duct and IHCC tumor according to 3D imaging. The CT image was reconstructed using SYNAPSE VINCENT® (Fujifilm, Japan) and we can identify larger vein and artery than the subsegmental branch. The distance between tumor and vessels was evaluated using 3D imaging for 17 cases of IHCC (Group H: 10 cases, Group P: 7 cases). The tumor contacted the bile duct in all cases in Group H (Fig. 5A). However, in Group P, the tumor contacted the bile duct in only four of the seven cases (Fig. 5B).
Analysis of prognostic factors
Table 3 shows the results of the univariate analysis of overall survival. The stage, curability, T-factor, lymph node metastasis, tumor thrombus in the portal vein, intrahepatic metastasis, and carbohydrate antigen 19-9 (CA19-9) were found to be significant prognostic factors for overall survival, as well as the SSTR2 and Bcl2 expression pattern (Group H and U).
Table 3 also shows the results of the multivariate analysis of overall survival. Lymph node metastasis (hazard ratio = 3.091) was identified as independent prognostic factors. However, the SSTR2 and Bcl2 expression pattern was not found to be an independent prognostic factor.