| Objective: To compare the survival difference between patients with spontaneous rupture of HCC(sr HCC)and those without rupture(nr HCC).Clinical independent risk factors for sr HCC were analyzed.Immunohistochemistry and immunofluorescence were used to investigate the causes of spontaneous rupture.The differential proteins between sr HCC and nr HCC were searched to explore the potential mechanism of spontaneous rupture,and hope to find potential molecular therapeutic targets to improve the prognosis of patients with sr HCC.Methods: Complete clinical and prognostic follow-up data of patients with HCC who underwent surgical treatment in the First Affiliated Hospital of Anhui Medical University from 2002 to 2019 were collected.Propensity score matching(PSM)was used to balance the differences between the sr HCC and nr HCC groups,and the prognostic differences between sr HCC and nr HCC groups were compared via Kaplan Meier method.Cox regression analysis was used to determine the clinical independent risk factors for sr HCC,and a prognostic model was established.1-year and 3-year survival probabilities of patients with sr HCC was forecasted with the nomogram.Calibration curves were used to evaluate the model.In addition,20 cases of sr HCC and nr HCC paraffin-embedded tissues were collected from the Department of Pathology for making pathological sections.Immunohistochemistry and immunofluorescence were used to locate the vascular walls in HCC,and the differences between sr HCC and nr HCC vascular lesions were observed and compared.Finally,9 pairs of fresh tissues in tumor and paracancer from both sr HCC and nr HCC were collected to extract the corresponding proteins.Label free quantitative proteomics experiments based on liquid chromatography-tandem mass spectrometry(LC-MS / MS)were performed to analyze the differential proteins between sr HCC and nr HCC.The GO functional annotation and KEGG pathway enrichment analysis of the differentially expressed proteins were performed with bioinformatics methods.Results: In the first part of this study,a clinical retrospective analysis was conducted for the HCC patients.A total of 273 sr HCC and 1092 nr HCC patients were included after1:4 propensity matching,with a good balance of clinicopathological variables between these two groups(P>0.05).The median survival of patients with sr HCC and nr HCC was16 months and 25 months,respectively.The overall survival(OS)and disease-free survival(DFS)of patients with sr HCC were significantly lower than those of patients with nr HCC(P=0.004 and P<0.001,respectively),and Cox regression analysis showed that spontaneous rupture was an independent risk factor for the prognosis of patients with HCC.Further analysis of the sr HCC subgroup showed that the overall survival(OS)and disease-free survival(DFS)of the sr HCC patients underwent primary hepatectomy alone were significantly lower than those accepted primary TAE plus secondary hepatectomy(both P<0.001).Independent risk factors for overall survival(OS)in patients with sr HCC were tumor maximum diameter,microvascular invasion,Child-Pugh B grade,and HBs Ag infection,while the only independent risk factor for disease-free survival(DFS)was the number of tumor.Finally,the 1-year and 3-year survival probability of patients with sr HCC could be predicted accurately by using the nomogram constructed with independent risk factors,and calibration showed that the prediction effect was good.In the second part of this study,it was found that arteriole rupture was common in sr HCC tumor by immunohistochemical and fluorescence experiments and combining with the previous research results of our group.The phagocytic function of macrophages was found to be down-regulated in sr HCC,immune complex deposition was observed in the vascular wall(especially the arteriole wall).The rupture of elastic membrane and disintegration of collagen fiber were observed at the deposition site.Together,these results may be one of the causes of blood vessel rupture and hemorrhage in HCC.In the third part of this study,a total of 261 differential proteins were identified in tumor tissues,of which 70 were significantly up-regulated and 191 were significantly down-regulated in sr HCC.While a total of 110 differential proteins were screened in paracancerous tissues,of which 91 were significantly high expressed and 19 were low expressed in sr HCC.The main functions of differential proteins were cellular-cellular adhesion,protein transport,regulation of macroautophagy,protein binding,cadherin binding hydrolase activity and other functions,which involved in cell metabolism.KEGG pathway enrichment analysis showed that the differential proteins in tumor tissues were mainly involved in Endocytosis,fatty acid synthesis and metabolic pathways,platelet activation and other signaling pathways,while the differential proteins in paracancer tissues were mainly involved in extracellular matrix receptor interaction,spliceosome,phagocyte and alanine,aspartic acid and glutamate metabolism and other pathways.TBC1D9 B protein was found to be significantly down-regulated in the ruptured group,and pathway enrichment analysis revealed that enrichment pathways included endocytosis pathways.As a member of the TBC1 D family,TBC1D9 B has the function of lipid and protein transport.It may transport the CD16,surface receptor of macrophage,through endocytosis,the phagocytic function of macrophages could be indirectly reflect by TBC1D9 B expression.Conclusion: The prognosis of patients with sr HCC was very poor.The curative effect of TAE+ secondary hepatectomy in patients with sr HCC was better than that of surgery alone.The risk factors of the prognosis of sr HCC included tumor maximum diameter,HBs Ag infection,Child-Pugh grade B and microvascular invasion.In sr HCC with hepatitis B infection,large amounts of hepatitis B virus bind to immunoglobulin to form antigen-antibody immune complexes.However,in sr HCC patients,the function of macrophage was significantly down-regulated,and the immune complexes could not be cleared in time,resulting in deposition of the immune complexes on the vascular wall and activation of inflammatory response to damage the vascular wall,inducing vascular rupture and bleeding.These results confirmed the first part of the retrospective analysis of HBs Ag infection and microvascular invasion as prognostic risk factors for sr HCC.Because TAE mainly blocks intrahepatic arterioles in clinic,it explains the good therapeutic effect of TAE.TBC1D9B may be involved in the membrane transport of macrophage surface receptor CD16.The down-regulated of TBC1D9 B in sr HCC patients may lead to a decrease in the efficiency of CD16 transport.As a result,phagocytosis function of macrophage was down-regulated and immune complex deposition damages blood vessels,especially arterioles.In addition,dysregulation of fatty acid synthesis and metabolism in tumors may be one of the causes of vascular injury and rupture in tumors.However,the results were just based on the bioinformatics,so the internal interaction mechanism needs further verification. |
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