Quantitative Multiplex Immunohistochemistry Analyzes The Immune Microenvironment And Clinical Significance In Cholangiocarcinoma

Backgrond:Cholangiocarcinoma(CCA)patients often present in an unresectable state.After excision,the recurrence rate is high and it is not sensitive to chemoradiotherapy.Immunotherapy has been successful in a variety of malignancies and is becoming new direction for CCA.It is very important to explore the immune microenvironment of CAA and find valuable targets for immunotherapy.Currently,the standard method for detecting in-situ proteins is immunohistochemistry.However,traditional immunohistochemistry is difficult to evaluate the co-localization expression of two or more antigens on the same cell.Multiplex immunohistochemistry can stain many times on the same pathological section and perform quantitative multiparameter analysis on the same cell.Objective:To analyze the immune microenvironment of CAA and its relationship with clinicopathological variables and overall survival.So,it is more comprehensively to analyze the immune microenvironment and explore potential immunotherapy targets.Methods:Clinicopathological variables and follow-up data on 31 patients of distal cholangiocarcinoma(DCC)and 28 patientes of intrahepatic cholangiocarcinoma(ICC)were retrospectively analyzed.Multiplex immunohistochemistry was used to stain 22 antibodies on the same pathological sections repeatedly,and multiparameter analysis was performed on the same cell via computer.Furthermore,the immunological factors associated with clinicopathology and prognosis in the immune microenvironment of CCA was explored.Results:22 antibodies were analyzed multiply.Lymphoid cells include CD8+T cells,Thl cells,Th2 cells,Th17 cells,regulatory T cells,CD20+B cells,and CD56+NK cells.Myeloid cells include macrophages(M1TAM and M2TAM),dendritic cells(DC-SIGN+DC and DC-LAMP+DC),CD66b+Granulocytes(CD66b+Gr)and Tryptase+ Mast cell.Four functional phenotypes include PD-1,PD-L1,Granzyme B and Ki67.The median age of the DCC was 66(45-89)years and the median survival time was 718(105-3287)days.High density CD8+T cells(P= 0.041),Th17 cells(P=0.000),Thl cells(P=0.001),B cells(P=0.008),and NK cells(P=0.016)were associated with increased survival.The higher Thl/Th2 was associated with increased survival(P=0.010).There was no statistical difference between M1TAM/M2TAM and the overall survival(OS)(P=0.775).GranzymeB+CD8+T cells was associated with favorable prognosis(P=0.020).On COX multivariate analysis,GranzymeB+CD8+T cells(HR=0.805,P=0.000)and Thl/Th2(HR=0.297,P=0.049)were correlated with increased OS.Treg(HRF=1.649,P=0.033)was a risk factor for prognosis.The higher PD-L1 of M1TAM was correlated with short OS(P=0.011).The PD-L1 of T cells was significantly negatively correlated with GranzymeB+CD8+T cells(R=0.360,P=0.047).The PD-L1 of M1TAM was significantly negatively correlated with GranzymeB+CD8+T cells(R=0.365 P=0.043).The median age of the ICC was 65(51-81)years and the median survival time of 1048(105-3177)days.High density of Tregs was associated with increased patient survival(p=0.007).High density M2TAM cell was associated with poor prognosis(p=0.027).The higher Th1/Th2 was associated with increased survival(P=0.001)and the higher M1TAM/M2TAM was associated with increased survival(P=0.010).GranzymeB+CD8+T cells was associated with favorable prognosis(P=0.020).On COX multivariate analysis,Granzyme B+CD8+ T cells(HR=0.868,P=0,037),NK cells(HR=0.866,P=0.0.008)and M1TAM/M2TAM(HR=0.753,P=0.026)were correlated with increased OS.The higher PD-L1 of M1TAM(P= 0.028)and M2TAM(P=0.031)were correlated with short OS.The PD-L1 of T cells was significantly negatively correlated with Granzyme B+CD8+T cells(R=0.379,P=0.047)and the PD-L1 of M2TAM was significantly negatively correlated with GranzymeB+CD 8+T cells(P=0.204).Conclusions:1.This continuous optimized multiplex immunohistochemtry can effectively evaluate the co-localization expression and perform quantitative multiparameter analysis on the same cell.It can more comprehensively and accurately analyze immune microenvironment.2.Lymphoid and myeloid cells are associated with overall survival of DCC and ICC.3.The infiltrated CD8+ T cells in the tumor may be low-efficiency T cells,which have no effect on survival.High density of GranzymeB+CD8+T cells are associated with favorable prognosis,and it is a valuable phenotype for evaluating the efficiency of cytotoxic T cells.4.The expression of PD-L1 on myeloid cells is more than on lymphoid lines,which is correlated with poor prognosis,and is a target for immunotherapy.