| Part1The study of the clinicopathologic characteristics and prognosis of combined hepatocellular-cholangiocarcinomaObjective:Retrospectively analyzing the clinicopathologic information and follow-up data of the combined hepatocellular-cholangiocarcinoma (CHC), to study its clinical and pathological characteristics and evaluate its prognostic factors.Methods:Collecting the clinicopathologic information and follow-up data of38cases of combined hepatocellular-cholangiocarcinoma, who were treated by hepatectomy in the Department of general surgery of Xiangya the Second Hospital and Department of hepatobiliary surgery of Hunan People’s Hospital, and respectively comparing these data with that of40cases of hepatocellular carcinoma(HCC) and40cases of cholangiocellular carcinoma(CC), who were treated by hepatectomy in the Department of general surgery of Xiangya the Second Hospital during the same time. Results:(1) There were significant differences between CHC group and HCC group in serum CA19-9, hepatic hilar lymph node metastasis, invasion of the surrounding organs, invasion of liver capsule and pathologic differentiation.(2) There were significant differences between CHC group and CC group in gender, positive rate of serum HBsAg, serum AFP, serum CA19-9, liver cirrhosis, tumor size, portal vein tumor thrombus, invasion of liver capsule and pathologic differentiation.(3) The1-,3-,5-year survival rates of CHC group were lower than that of HCC group (71.1%vs87.5%,36.8%vs55.0%,7.9%vs15.0%, P<0.05, respectively), and were higher than that of CC group (71.1%vs57.5%,36.8%vs15.0%,7.9%vs0.0%, P<0.05, respectively).(4) The median survival time of CHC group, HCC group and CC group was25months,38months and14months, respectively.(5) The factors which had statistical significance to the prognosis of CHC were:serum CA19-9, liver cirrhosis, tumor size, hepatic hilar lymph node metastasis, portal vein tumor thrombus, invasion of liver capsule, radical excision rate and pathologic differentiation; The factors which had statistical significance to the prognosis of HCC were:liver cirrhosis, tumor size, portal vein tumor thrombus, invasion of liver capsule, radical excision rate and pathologic differentiation; The factors which had statistical significance to the prognosis of CC were:tumor size, hepatic hilar lymph node metastasis, invasion of the surrounding organs and radical excision rate.(6) Multivariate analysis showed that the independent factors influencing the prognosis of CHC were:hepatic hilar lymph node metastasis, portal vein tumor thrombus and radical excision rate; the independent factors influencing the prognosis of HCC were:liver cirrhosis, portal vein tumor thrombus and radical excision rate; the independent factors influencing the prognosis of CC were:hepatic hilar lymph node metastasis, invasion of the surrounding organs and radical excision rate.Conclusions:(1) Although the clinicopathologic characteristics of CHC were between that of HCC and that of CC, it was more inclined to HCC, and its tumor invasion force was stronger than HCC and CC.(2) The prognosis of CHC was worse than that of HCC and better than that of CC.(3) The independent factors influencing the postoperative survival rate of CHC were:hepatic hilar lymph node metastasis, portal vein tumor thrombus and radical excision rate.(4) The level of serum CA19-9may represent the cholangiocarcinoma component of CHC, but it is not the independent factors influencing the prognosis of CHC. Part2Expression of tumor-infiltrating Foxp3+regulatory T cells and its association with the prognosis of combined hepatocellular-cholangiocarcinomaObjective:Testing the infiltration of Foxp3+regulatory T cells (Tregs), CD4+T cells and CD8+T cells in the tumor microenvironment of the three types of primary liver cancer, to evaluate the prognostic significance of the infiltration of Foxp3+Tregs in intratumoral and peritumoral tissues of CHC.Methods:The infiltration of Foxp3+Tregs, CD4+T cells and CD8+T cells in the tumor microenvironment of the38cases of CHC,40cases of HCC and40cases of CC that were studied in part1, were detected respectively by immunohistochemical (IHC) method, and the differences among them were assessed. The association between Foxp3+Tregs in CHC and CD4+T cells, CD8+T cells, the clinicopathologic characteristics of CHC were analyzed, respectively. The prognostic significance of lymphocytes infiltration in tumoral microenviroment to the three types of primary liver cancer was evaluated, respectively. Results(1) In the three types of primary liver cancer, the lymphocytes infiltration in intratumoral tissue presented in a diffuse pattern and that in peritumoral tissue was dense distribution and was inclined to form lymphoid aggregates, and the numbers of Foxp3+Tregs, CD4+T cells and CD8+T cells in peritumoral tissue were higher than those in intratumoral tissue correspondently (P＜0.001). In CHC group, the Foxp3+Tregs/CD8+T cells ratio infiltrating in intratumoral tissue was significantly higher than that in peritumoral tissue (P＜0.001). In CC group, the Foxp3+Tregs/CD4+T cells ratio infiltrating in intratumoral tissue was also obviously higher than that in peritumoral tissue (P＜0.001).(2) There were statistical differences between CHC group and HCC group in Foxp3+Tregs/CD4+T cells ratio (P=0.002), Foxp3+Tregs/CD8+T cells ratio (P=0.005) in intratumoral tissue and the number of Foxp3+Tregs (P=0.003), Foxp3+Tregs/CD4+T cells ratio (P=0.004), Foxp3+Tregs/CD8+T cells ratio (P=0.0005) in peritumoral tissue.(3) There were significant differences between CHC group and CC group in the number of CD8+T cells (P=0.017), Foxp3+Tregs/CD8+T cells ratio (P=0.020) in peritumoral tissue and the number of Foxp3+ Tregs (P=0.028) in intratumoral tissue.(4) In CHC, the number of Foxp3+Tregs infiltrating in peritumoral tissue was negatively correlated with the number of CD8+T cells (rs=-0.587, P<0.001), but had no correlation with the number of CD4+T cells. However, the Foxp3+Tregs count infiltrating in intratumoral tissue had no significant association with CD4+T cells and CD8+T cells. Only the Foxp3+Tregs/CD8+T cells ratio in peritumoral tissue was obviously related with the portal vein tumor thrombus and pathologic differentiation of CHC.(5) The factors which had statistical significance to the prognosis of CHC were:the number of Foxp3+Tregs, Foxp3+Tregs/CD4+T cells ratio in intratumoral tissue and Foxp3+Tregs count, CD8+T cell count, Foxp3+Tregs/CD8+T cells ratio in peritumoral tissue.The factors which had statistical significance to the prognosis of HCC were:Foxp3+Tregs count, Foxp3+Tregs/CD4+T cells ratio in intratumoral tissue and Foxp3+Tregs/CD8+T cells ratio in peritumoral tissue.The factors which had statistical significance to the prognosis of CC were:Foxp3+Tregs count in intratumoral tissue and Foxp3+Tregs count, CD8+T cell count, Foxp3+Tregs/CD8+T cells ratio in peritumoral tissue.(6) Multivariate analysis showed that the independent factors influencing the prognosis of CHC were:Foxp3+Tregs count in intratumoral tissue and Foxp3+Tregs count, Foxp3+Tregs/CD8+T cells ratio in peritumoral tissue; the independent factors influencing the prognosis of HCC were:Foxp3+Tregs count in intratumoral tissue and Foxp3+Tregs/CD8+T cells ratio in peritumoral tissue; the independent factors influencing the prognosis of CC were:Foxp3+Tregs count, Foxp3+Tregs/CD8+T cells ratio in peritumoral tissue.Conclusions:(1) In the three types of primary liver cancer, the lymphocytes infiltration in intratumoral tissue presented in a diffuse pattern and that in peritumoral tissue was dense distribution, and the numbers of Foxp3+Tregs, CD4+T cells and CD8+T cells in peritumoral tissue were higher than those in intratumoral tissue correspondently.(2) Although the immunological characteristics of CHC were between that of HCC and CC, it was more inclined to CC.(3) In CHC, the number of Foxp3+Tregs infiltrating in peritumoral tissue was negatively correlated with the number of CD8+T cells, and the Foxp3+Tregs/CD8+T cells ratio in peritumoral tissue was obviously related with the portal vein tumor thrombus and pathologic differentiation of CHC, and the higher the ratio, the lower the degree of pathologic differentiation and the higher the portal vein tumor thrombus formation rate.(4) The independent factors influencing the prognosis of CHC were: Foxp3+Tregs count in intratumoral tissue and Foxp3+Tregs count, Foxp3+Tregs/CD8+T cells ratio in peritumoral tissue, these factors were all negatively related to the postoperative survival rate of CHC significangtly.(5) Foxp3+Tregs/CD8+T cells ratio infiltrating in peritumoral tissue was a common independent prognostic factor of the three types of primary liver cancer, which played a key role to the postoperative survival rate of primary liver cancer. Part3Study of the origin of combined hepatocellular-cholangiocarcinomaObjective:Exploring the relationship between hepatic oval cells and the origin of CHC, to provide new ideas for the treatment of CHC.Methods:The expression of AFP, CK7, CK19, OV-6and C-kit in the tumor tissues of the38cases of CHC,40cases of HCC and40cases of CC that were studied in part1, were detected by immunohistochemical (IHC) and immunofluorescence methods, and differences of the expression between CHC group and HCC group, CC group were analyzed respectively.Results:(1) CK7positive rate of CHC group was significantly higher than that of HCC group (χ2=20.53, P＜0.05), but had no significant difference with CC group (P>0.05).(2) CK19positive rate of CHC group was significantly higher than that of HCC group (χ2=34.78, P＜0.05), but had no significant difference with CC group (P>0.05).(3) CK7or CK19positive rate of CHC group was significantly higher than that of HCC group (χ2=31.39, P＜0.05), but had no significant difference with CC group (P>0.05).(4) AFP positive rate of CHC group was significantly higher than that of CC group (χ2=25.97, P＜0.05), but had no significant difference with HCC group (P>0.05).(5) C-kit positive rate in CHC group, HCC group and CC group was5.26%,0.00%and0.00%, respectively.(6) OV-6positive rate of CHC group was significantly higher than that of HCC group (χ2=34.64, P＜0.05) and CC group (χ2=43.49, P＜0.05).(7) CK7or CK19and AFP positive rate of CHC group was significantly higher than that of HCC group (χ2=15.00, P＜0.05) and CC group (χ2=23.93,P<0.05).(8) In CHC group,24/31(77.42%) cases with OV-6positive expression simultaneously expressed cholangiocarcinoma molecular phenotypic marker (CK7or CK19) and hepatocellular carcinoma molecular phenotypic marker (AFP); in HCC group,6/6(100.00%) cases with OV-6positive expression simultaneously expressed cholangiocarcinoma molecular phenotypic marker (CK7or CK19) and hepatocellular carcinoma molecular phenotypic marker (AFP); in CC group,3/3(100.00%) cases with OV-6positive expression simultaneously expressed cholangiocarcinoma molecular phenotypic marker (CK7or CK19) and hepatocellular carcinoma molecular phenotypic marker (AFP).(9) In CHC group,2/2(100.00%) cases with C-kit positive expression simultaneously expressed cholangiocarcinoma molecular phenotypic marker (CK7or CK19), hepatocellular carcinoma molecular phenotypic marker (AFP) and OV-6.(10) In the HCC component of CHC group, AFP positive rate was65.79%, which was obviously higher than that of CC group (10.00%) and had no significant difference with HCC group (57.50%); however, its cholangiocarcinoma molecular phenotypic marker (CK7or CK19) positive rate (86.84%) was obviously higher than that of HCC group (30.00%) and had no significant difference with CC group (87.50%).(11) In the CC component of CHC group, hepatocellular carcinoma molecular phenotypic marker (AFP) positive rate was60.53%, which was obviously higher than that of CC group (10.00%) and had no significant difference with HCC group (57.50%); however, its cholangiocarcinoma molecular phenotypic marker (CK7or CK19) positive rate (92.11%) was obviously higher than that of HCC group (30.00%) and had no significant difference with CC group (87.50%). Conclusions:(1) In the molecular phenotypic features, CHC had both the characteristics of HCC and the characteristics of CC.(2) OV-6may be a specific molecular marker which expressed in the hepatic oval cells.(3) Most of the CHC may originate from the arrested differentiation of hepatic oval cells, but only a few may arise from the dedifferentiation of mature hepatocytes, and only a handful of hepatic oval cells could be derived from hematopoietic stem cell system.(4) Most of the HCC and CC may arise from the dedifferentiation of mature hepatocytes, but only a few may originate from the arrested differentiation of hepatic oval cells. |
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