| Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and also the third most common cause of cancer-related death, in China, mortality rate had rised second. It has an extremely poor prognosis, attributed to the high frequency of intrahepatic metastatic recurrence. To date, the only effective approaches for patients with HCC are resection or liver transplantation. But even to small HCC, the recurrence rate still was up to 60-70% in 5 years. It is most important to find effective mechanism of metastasis and recurrence aimed to improving the prognosis of HCC. Base on traditional staging system, the patients were hard to predict who had samillar clinical character. How to identify stratify subgroups with different outcome still were great difficulties.Immunological/ inflammatory mechanisms are important in the surveillance of malignancy and control of tumor progression. In this study, we focused on HCC noncancerous tissue immune microenvironment to further validate and refine a gene or protein signature of cytokine which can predict prognosis of HCC after curative treatment.Part APurpose: Hepatocellular carcinoma (HCC) is a common malignancy, but the prognosis remains poor due to metastasis and recurrence after curative hepatectomy. A 17-cytokine gene expression signature in non-cancerous hepatic tissue from patients with active metastatic HCC was recently found to predict HCC venous metastases. We examined whether a more refined cytokine gene expression profile of noncancerous liver tissue could predict survival of early stage HCC after curative hepatectomy.Methods: We analyzed a 20-cytokine gene expression profile in noncancerous liver tissue from 121 Chinese patients with TNM stage I～II of HCC using TaqMan@ low density arrays. We then used supervised hierarchical clustering and log-rank test to analyze the prognostic ability of the cytokine gene profile. Using methods of bioinformatics, build a predictical model, then refined it to explore optimal factor.Results: Clinicopathologic characteristics Cox analysed hadn't find signifinant effection on recurrence except tumor size (HR = 1.793, 95%CI: 1.056-3.045 ;P = 0.03). Cox analyzed effection on recurrence IL-10, IL-2, IL-15 were ranked in the first three sites. The expression of other Th2 cytokines, including IL-4, IL-6 and IL-8, were decreased in most samples tested compared to normal pool tissuse, but no remarkable relativity was observed with the survival and HCC recurrence. The mRNA-based predictor with logistic regression method, which employed all 20 cytokine gene expression, was excellent recurrence predictor in gene dataset(P= 4.28E-5, the area under the curve=0. 81, accuracy=0.76 on tranining dataset. After refined the model, the expression levels of IL-2 (P = 0. 001) and IL-15 (P=0. 0002) correlated inversely and significantly with intrahepatic microvascular invasion. In the univariate analysis, only the mRNA levels of IL-15 and IL-2, preferable than the traditional clinical characteristics, were significantly associated with HCC recurrence. In the multivariate analysis, the mRNA levels of both IL-2 (HR=2. 0; P = 0.015) and IL-15 (HR=2.0; P = 0.011) were independent predictors of HCC recurrence. Higher mRNA levels of IL-2 (P = 0. 016) or IL-15 (P = 0. 001) were associated with a significantly better relapse-free survival of HCC patients compared to those with lower levels of these cytokines. IL-2 and IL-15 had significant correlation in the noncancerous liver tissues using the Spearman test (Spearman'sÏ= . 487; P = 1.88×10-7).Conclusions: IL-2 or IL-15 of immunity-associated genes signature should allow a sensitive and specific monitoring of survival in HCC.Part BPurpose: By analyzing the gene expression profiles of noncancerous hepatic tissue from patients with hepatocellular carcinoma (HCC), we recently identified a 17-cytokine signature that can predict HCC metastasis. In this study, we further validated and refined this signature in protein level for tumor recurrence over a long follow-up period in a much larger and specific cohort of patients with early HCC.Methods: The protein expression levels of 20 cytokines in the noncancerous hepatic tissues from 187 patients with early stage of HCC were determined using ELISA assays. Univariate and multivariable Cox proportional hazards model and Kaplan-Meier method were used for analyzing the association of their expression profiles with tumor recurrence and survival of patients with HCC. To determinate the threshold values by ROC analyze.Results: Ranked 20 cytokines by a univariate Cox model for recurrence (Table 1). Of all 20 proteins, IL-2 (P = 0.013), IL-15 (P = 0.049) were the top two significant cytokines for recurrent classification. The protein expression levels of IL-2 (P = 2. 68×10-5) and IL-15 (P = 0. 005) correlated strongly with tumor recurrence, and these correlations had been validated in mRNA levels. The patients with higher level (s) of IL-2 and/or IL-15 had a significantly better relapse-free survival compared with those with lower level (s) of IL-2 (P = 1.02×10-5) and/or IL-15 (P = 0.004). The expression levels of IL-2 and IL-15 correlated with each other. Furthermore, the expression levels of IL-2 and IL-15 correlated inversely and significantly with intrahepatic microvascular invasion.Conclusion: The IL-2 and IL-15 are useful markers for stratifying the patients with early stage of HCC into subgroups with different probabilities of tumor recurrence, and predicting the patients' prognosis after surgical treatment.Part CPurpose: By analyzing the CD molecular expression profiles ofnoncancerous hepatic tissue from patients with hepatocellular carcinoma,we want to identify signature of tumor infiltrating lymphocytes whichmaybe affect the cytokines expression between different outcome HCCpatients. In this study, we identified TIL subtype by some lymphocyticdifferentiation antigen and lymphocyte surface mark.Methods: The lymphocyte surface marks of tumor infiltrating lymphocytesin the noncancerous hepatic tissues from 146 HCC patients were determinedusing IHC. Univariate and multivariable Cox proportional hazards model and Kaplan-Meier method were used for analyzing the association of their expression levels with tumor recurrence and survival of patients with HCC. Results: The phenotype and cell numbers of tumor-infiltrating lymphocytes (TILs) in noncancerous hepatic tissues of HCC were analyzed via immunohistochemistry of sections from patients undergoing surgery for HCC and via flow cytometry of peripheral blood mononuclear cells. Tumor-infiltrating T-cell function and activation status were assessed via CD80, CD86. More than 86. 4% patients of TILs were quiescent as measured via CD4+ or Foxp3 expression, while more than 90% of CD3(+) T cells expressed CD8+. The proportion of Th cell was quite low compared with the ratio of CD8+ T cell. The proportion in distant nontumor tissue CD19 and CD20 was zero. The proportion T cells subgroup isolated from HCC circulating whole blood didn't show significant shift comepared with normal control, CD4+ T:CD8+ T=1.167±1.04,CD+8 T:CD3+T=0. 288±0.116; CD4+T: CD3+T0.429±0.178. The proportion of CD8+ cells in noncancerous hepatic tissues was higher than in blood (P = 0.000). The expression of HLA-I,HLA-II,CD80,CD86 in noncancerous tissues didn't show significant different between different prognostic group, only showed some tendency for outcome. There had no definite proof about the increasing IL-2 or IL-15 only form activation of Th1, maybe they can be secreted by hepatic cell. Conclusion: TILs in HCC noncancerous hepatic tissues are increased and contain a subpopulation of CD3+CD8+ T cells. Activation functional deletion of tumor-infiltrating T cells could damage tumor-specific immunotherapy. |
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