Expression Of DAP12 In Hepatocellular Carcinoma And Relevant Clinical Significance

BackgroundHepatocellular carcinoma(HCC)is a common malignancy all over the world which can be seriously harmful to health and threaten to people's life.The occurrence of HCC is closely related to inflammation and could be known as a typical inflammation-connected tumor.The tumor microenvironment is closely related to the occurrence and malignant progression of HCC.Previous studies have testified that tumor-associated macrophages(TAMs)located in tumor microenvironment are major tumor-related inflammatory cells which can exhibit multiple biological behaviors such as tumor growth,invasion,and metastasis.The bone marrow-derived cell line membrane DNAX-activating protein of 12kDa(DAP12)binds to the immunoreceptor tyrosine-based activation motif(ITAM)signaling pathway which can bind to DAP12 associated receptors such as the triggering receptor expressed on myeloid cells(TREM-1,TREM-2)and Siglecs-15 to exert immune pathogen clearance,immunosuppression,macrophage polarization,secretion of various pro-tumor cytokines,etc.Macrophage polarization and secretion of pro-tumor factors exert multiple biological effects,which are closely relative to the occurrence and development of tumors.However,the role of DAP12 in HCC and its role in the polarization of liver cancer-associated macrophages has not been reported.Therefore,we designed a series of research methods to preliminarily study the expression and regulation of DAP12 in HCC,and to explore the relationship between DAP12 and relative clinical indicators to further explicate its clinical significance in HCC.ObjectivesTo explore the expression of DAP12 and analyze its relationship with clinicopathological features and prognosis of patients,and to explicate its clinical significance.MethodsFirstly,liver cancer tissues(paraffin and frozen sections)were examined throuth immunohistochemistry and immunofluorescence,respectively.The relationship between DAP12-positive cells and CD163-positive cells in the same specimen was clearly identified.DAP12-positive cells were identified to be associated with tumor-associated macrophages.Subsequently,the sample size was increased,and tissue microarray immunohistochemical staining was used to analyze the expression characteristics of DAP12 in liver cancer and paracancer tissues,statistical analysis of the relationship between DAP12 and CD163 positive expression in liver cancer and paracancer tissues to clarify the quantitative relationship and correlation.In the second part,we collected detailed clinical indicators and prognostic survival data of 94 HCC cases to analyze the correlation between DAP12 expression and clinical data of liver cancer patients by statistical methods.Positive factors obtained via single factor analysis were collected to perform further single or multiple factor survival analysis to identify the independent risk factors impacting the prognosis of HCC patients.ResultsImmunohistochemistry and immunofluorescence examination of serial sections revealed that DAP12 was mainly expressed in the membrane or cytoplasm of hepatoma stromal cells,and the location of DAP12-positive cells and CD163-positive cells in the same specimen substantially overlapped.In the analysis of tissue microarray immunohistochemical staining,it was found that the distribution of DAP12 and CD163 positive cells in HCC tissue was uneven,and there was a relative clustering distribution around the cancer cell mass.In the paracancer,positive cells distributed in the sinusoidal area.The median number of DAP12 in hepatocellular carcinoma was 10(×400 magnification),paraneoplastic tissue was 13,CD163 was 8 in HCC,and paraneoplastic tissue was 10.DAP12 and CD163 expression was higher in the adjacent tissue(P <0.001,P =0.002).There was no significant difference in the positive expression number of DAP12 and CD163 in HCC(P=0.310).Nevertheless,the expressions of DAP12 and CD163 showed significant relevance(r=0.775).Comparing the expression of CD163,we indirectly testified that the stromal cells expressing DAP12 in liver cancer tissues were mainly CD163-positive tumor-associated macrophage populations which was mainly M2 tumor-associated macrophage populations.The correlation analysis between the density of DAP12 macrophage infiltration in HCC tissue and the clinical data of HCC revealed that the expression of DAP12 was associated with tumor diameter,pathological grade,and TNM staging(P=0.007,0.036,0.013).There was no statistical difference in age,HBsAg,serum AFP,number of tumors,clinical data on vascular invasion,and regional lymph node metastasis.Cox proportional hazards regression analysis and Kaplan-Meier survival analysis showed that the later TNM stage and higher cancer tissue DAP12-positive macrophage infiltration density were independent risk factors impacting the prognosis of HCC patients.TNM stage HR was 2.441,The infiltration density of DAP12-positive macrophage HR was 2.036.Higher infiltration density of DAP12 in HCC tissues could lead to lower survival rate and worse prognosis.