Elsevier

Journal of Hepatology

Volume 52, Issue 3, March 2010, Pages 370-379
Journal of Hepatology

Research Article
Inflammatory tumour microenvironment is associated with superior survival in hepatocellular carcinoma patients

https://doi.org/10.1016/j.jhep.2009.07.013Get rights and content

Background & Aims

Hepatocellular carcinoma (HCC) is an aggressive malignancy with few treatment options. As the status of the tumour immune microenvironment can affect progression of established tumours, we evaluated potential immune mechanisms associated with survival in HCC.

Methods

Immune gene expression profiles were analyzed in tumour and non-tumour liver tissues from resected HCC patients using quantitative PCR and immunohistochemistry. Tumour-infiltrating leukocytes (TILs) were isolated to verify the expression of immune genes and to identify proliferating TILs. These parameters were analyzed statistically in relation with patient survival and tumour phenotype (apoptosis and proliferation).

Results

The immune microenvironment within tumours was found to be heterogeneous, although globally more inert compared to the adjacent non-tumour liver tissue. Univariate analysis in 61 patients identified a group of innate immune genes whose expression within tumours is positively associated with patient survival. TNF, IL6 and CCL2 are the most significant genes, with TNF being an independent predictor of survival in multivariate analysis. The gene set includes macrophage and NK-associated molecules such as TLR4, TLR3, CCR2, NCR3. Most of these molecules are expressed by TILs. Importantly, proliferating immune cells, predominantly NK and T cells, are present in tumours of patients with longer survival, and exclusively in areas devoid of proliferating tumour cells. NK and CD8+ T cell densities are correlated positively with tumour apoptosis, and negatively with tumour proliferation.

Conclusions

Hence, an inflammatory immune microenvironment within HCC tumours could be an important means to control tumour progression via TIL activation and proliferation.

Introduction

Hepatocellular carcinoma (HCC) is a highly aggressive cancer that claims more than 600,000 lives worldwide each year (www.who.int). Resection remains the most effective treatment for patients with early disease. However, more than 75% of patients relapse within 5 years, and the overall survival for HCC patients is poor [1]. Most cases of HCC are secondary to chronic hepatitis and cirrhosis resulting from either hepatitis B/C virus infection or from non viral-related causes such as alcohol or aflatoxin exposure. A persistent, non-specific, and ineffective activation of the immune system within the chronically inflamed liver is thought to promote carcinogenesis [2], [3], [4]. Although the importance of inflammation in liver carcinogenesis has been studied in detail, much less is known about its impact on cancer progression.

The stromal component of tumours consists of fibroblasts, endothelial cells, and a variety of immune cells. These cells play a critical role in tumour development, tumour control and response to treatment [5], [6], [7], [8], [9]. In breast, colorectal and lung cancer, the status of the stroma and the local adaptive immune response are superior prognostic factors compared to the tumour phenotype or clinical staging [7], [8], [9]. In HCC, infiltration of regulatory T cells is associated with a poor prognosis [10], while a significantly lower recurrence rate and improved survival were reported in HCC patients with marked inflammatory lymphocytic infiltrate in the tumour [11]. More recently, a predominantly Th1 signature was found in the liver tissue of HCC patients who are less likely to develop metastasis [12]. Additional gene signatures predictive of HCC patients survival have been previously identified in HCC [13], [14]. However, little attention has been given to immune-specific genes.

Based on the hypothesis that the immune phenotype of HCC tumours may be related to patient survival, we studied the type of tumour-infiltrating immune cells and their functional polarization in 61 resected HCC patient samples. Our results indicate that HCC patient survival is positively associated with higher expression of a group of inflammatory and innate immune genes within the tumours. In addition, more NK and T cells, some of which were proliferating, were found in tumours of patients with longer survival; furthermore, their presence was associated with an increased apoptosis and a reduced proliferation of tumour cells.

Section snippets

Cell lines

HCC cell lines HepG2 and HuH7 were obtained from ATCC; HCC cell lines SNU-475, SNU-387, SNU-449, SNU-386, PLC-PR5 and Hep3B from the Korean Cell Line Bank.

Patients and samples

Gene expression and/or immunohistochemistry analysis were performed retrospectively on core tumour and/or non-tumour liver tissue from a total of 68 HCC patients. Samples were obtained with Ethics Committee approval from patients who underwent curative resection at the National Cancer Centre, Singapore, between 1999 and 2006. Gene expression

The immune microenvironment in HCC tumours is more inert compared to the adjacent non-tumour liver tissue

We quantified the expression of a panel of 49 immune genes using quantitative real-time PCR in core tumour samples and/or adjacent non-tumour liver tissues from 68 resected HCC patients. The genes were selected to represent a variety of immune cell types and functions, including markers of monocyte/macrophages, neutrophils, dendritic cells and lymphocytes, inflammatory and cytotoxic molecules, Th1 and Th2 cytokines and chemokines, immunosuppressive factors, matrix metalloproteases, death

Discussion

HCC is an aggressive cancer with limited effective treatment options. This study suggests that activation of the tumour immune microenvironment can improve survival in HCC patients. More specifically, we have shown that patient survival is associated with elevated expression of inflammatory and innate immune genes mostly expressed by tumour-infiltrating immune cells. Lymphocyte recruitment and proliferation is observed in tumour areas devoid of tumour cell proliferation and rich in tumour

Acknowledgements

We thank Dr. K.B. Tan and Ms. C. Foo (Department of Pathology, National University Health System, Singapore) for providing archival HCC tissues; National University of Singapore-National University Hospital Tissue Repository for providing material for technical set-up.

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    These authors contributed equally to this work.

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