Human Leukucyte Antigen-G Protein Expression And Its Relationship With The Prognosis Of Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC) is a frequently diagnosied malignancy worldwide. HCC is most prevalent in China and ranks as the second leading cause of cancer-related death in this region.Due to the rising incidence of HCC in western countries,it catches much attention worldwide now.Although surgery provides a potential opportunity to cure this disease,the major hurdle for patients' outcome is tumor relapse.The mechanism of HCC recurrence and metastasis has been the key to overcome HCC.Recently,the impact of microenviroment on tumor recurrence and metastasis attracted much attention from researchers.Tumor local immune state is determined by tumor and the host.From the host's perspective,local immune response comprises two arms,anti-tumor immunity such as CD8~+ T cells,natural killer(NK) cells and NK T cells and pro-tumor factors network including tumor-derived repressive factors and regulatory cells(mainly T regulatory cells,Tregs).The study of the balance of antiand pro-tumor factors is crucial to tumor progression,a more accurate molecular classification,a precise prognosticator and the exploration of more effective therapeutic regimens.From the perspective of tumor,it can affect local immune status by producing a lot of immune regulatory factors.Human leukocyte antigen-G(HLA-G) is a nonclassical major histocompatibility complex class I(MHC-I) molecule,initially found expressed at the fetal-maternal interface,protecting the fetus from attacting by materai NK cells. Expressed as seven isoforms,including four embrane-bound(HLA-G1 to -G4) and three soluble(HLA-G5 to -G7) forms,it has emerged as a tumor-derived immunosuppressive molecule with the ablility to dampen almost all components of innate and adaptive immunity including NK cells,CD8~+T lymphocytes,antigen presenting cells,etc.As a result,HLA-G is regared as a molecule with panoply of immune repression.Overexpression of HLA-G protein in colorectal cancer,lung cancer and gastric cancer were reported associated with poor prognosis.However,controversy still exists for contrast results observed in same tumor types,such as gastric cancer and B-cell chronic lymphocytic leukemia.Regarding the role HLA-G playing in HCC.no related studies have been carried out yet. The host and tumor interacts at diverse levels and through different machenisms.A comprehensive study of factors derived from the host and tumor will add our knowledge about their interactions and lead to a more comprehensive prognosticator. To this end,immunohistochemical staining of HLA-G expression as well as tumor infiltrating FoxP3~+ regulatory and CD8~+ cytotoxic T cells were carried out on tissue microarrays containing 173 HCC tissue specimens.Meanwhile,the potential prognostic value of HLA-G and Tregs/CD8~+ ratio and their combination were studied, representing both the tumor intrinsic,extrinsic factors and their combination. Membrane-bound HLA-G1 protein expression in five human HCC cell lines was detected by Western blot.PartⅠHuman Leukucyte Antigen-G Protein Expression and Its Relationship with HCC PrognosisThis part of study aimed at exploring the relationship between intratumoral HLA-G protein expression and HCC prognosis,and also the relationship with clinicopathologic parameters.The clinical relevance of HLA-G protein in HCC was elucidated.180 patients received curative resection at Zhongshan Hospital,Fudan University from Janunary 2002 to Sepetember 2006 were randomly selected as study cohort. Immunohistochemical staining was carried out and it was evaluated by quantified methods.Intratumoral HLA-G expression and its relevance with HCC prognosis and clinicopathoiogic parameters was analysized.Membrane-bound HLA-G1 protein expression in five human HCC cell lines was detected by Western blot.Results On univariate analysis,high HLA-G group had a significant shorter overall survival(OS)(P=0.024) and a tendency to shorter time to recurrence(TTR)(P=0.070) than low expressers.Among BCLC early stage patients,the nagetive impact of HLA-G on prognosis is more significant,with P values being 0.012 and 0.038 for OS and TTR,respectively.Multivariate analysis further verified HLA-G expression as an independent prognosis factor of OS(HR,1.987;95%CI,1.241-3.180;P=0.004). Among BCLC early stage patients,it was also an independent prognosis factor of both OS(HR,3.145;95%CI,1.051-9.418;P=0.041) and TTR(HR,3.208;95%CI, 1.176-8.750;P=0.023).Membrane-bound HLA-Gl protein expression in human HCC cell lines was in line with their metastatic potentials,while in situ HLA-G protein levels revealed no relations with clinicopathologic parameters.Conclusions HLA-G was a prognosticator in HCC,especially in early stage disease which was valuable to refine the prognosis of early stage patients.HLA-G expression possibly augmented tumor invasiveness through indirect ways.PartⅡTumor Infiltrating T Regulatory and CD8~+ Cells Ratio and Its Relationship with HCC PrognosisThis part of the study aimed at analysizing the relationship of Tregs,CD8~+ cells and its ratio with HCC prognosis.The most powerful predictor was further analysized among different disease stages.The relationship with clinicopatholgic variables was also studies.Immunohistochemical analysis and manual count of Tregs and CD8~+ cells was carried out on the already constructed tissue microarray.Results Tregs,CD8~+ cells and Tregs/CD8~+ ratio were all independent prognosticators of OS and TTR,with high Tregs,low CD8~+ and high ratio indicating poor prognosis.Among them,Tregs/CD8~+ ratio was the most comprehensive variable for prognosis prediction.By subgroup analysis stratified according to disease stages, Tregs/CD8~+ ratio was an independent prognosticator for both OS(P=0.017) and TTR (P=0.003) among advanced stage HCC,while it was only a significant variable for OS (P=0.007) in early to intermediate patients.As to the relations with clinicpathologic parameters,Tregs/CD8~+ ratio was found prominently relavant to tumor size(P=0.012), vascular invasion(P=0.002),AFP level(P=0.003) and BCLC stage(P=0.001 ).Conclusions Tregs,CD8~+ cells and Tregs/CD8~+ ratio were all independent prognosticators,among which Tregs/CD8~+ ratio was the most comprehensive indicator.Tregs/CD8~+ ratio was more efficient when predicting the prognosis of advanced stage patients.Tregs/CD8~+ ratio was relevant to biological behavior of HCC.PartⅢCombined Analysis of Human Leukucyte Antigen-G and Tregs/CD8~+ Ratio and Its Prognostic Value in HCCThis part aimed at illustrating the relationship between tumor intrinsic factor (HLA-G) and extrinsic microenvironmental immune factor(Tregs/CD8~+ ratio) and the prognostic value of their combination in HCC.Results Tregs/CD8~+ ratio was found positively associated with HLA-G level (P=0.012,r=0.190),indicating high HLA-G expressors having a concordant high Tregs/CD8~+ ratio.Tregs/CD8~+ ratio between HLA-G groups displayed significant difference(P=0.013).After combining Tregs/CD8~+ ratio and HLA-G expression(3 groups,concurrent low levels,either high levels,concurrent high levels),patients with concurrent high levels of both variables were at more than 3 times of risk of death and recurrence than concurrent low levels ones(both P＜0.001).The prognostic power of combined groups outperformed either of them alone through ROC analysis.Conclusions The positive relevance between HLA-G and Tregs/CD8~+ ratio indicated that HLA-G might involve in the regulation of immune suppressive or effective cells. The combination of HLA-G and Tregs/CD8~+ ratio resulted in a more precise predictor of HCC prognosis.Taken together,the comprehensive study of tumor intrinsic and extrinsic(host) immune factors could result in a more precise prediction of prognosis, meanwhile conferring a new stragety of combined tumor-stroma targeted immunotherapy.Potential ApplicationsThis study offered a novel prognosticator of HCC and indicated a new strategy of combined tumor-stroma targeted immunotherapy.NoveltyThis study elucidated the prognostic value of HLA-G in HCC for the first time.