Research ArticleInflammatory tumour microenvironment is associated with superior survival in hepatocellular carcinoma patients
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.
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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.