| Abstract: | Analysis of hepatitis C virus (HCV) replication and quasispecies distribution within the tumor of patients with HCV-associated hepatocellular carcinoma (HCC) can provide insight into the role of HCV in hepatocarcinogenesis and, conversely, the effect of HCC on the HCV lifecycle. In a comprehensive study of serum and multiple liver specimens from patients with HCC who underwent liver transplantation, we found a sharp and significant decrease in HCV RNA in the tumor compared with surrounding nontumorous tissues, but found no differences in multiple areas of control non-HCC cirrhotic livers. Diminished HCV replication was not associated with changes in miR-122 expression. HCV genetic diversity was significantly higher in livers containing HCC compared with control non-HCC cirrhotic livers. Tracking of individual variants demonstrated changes in the viral population between tumorous and nontumorous areas, the extent of which correlated with the decline in HCV RNA, suggesting HCV compartmentalization within the tumor. In contrast, compartmentalization was not observed between nontumorous areas and serum, or in controls between different areas of the cirrhotic liver or between liver and serum. Our findings indicate that HCV replication within the tumor is restricted and compartmentalized, suggesting segregation of specific viral variants in malignant hepatocytes.Hepatitis C virus (HCV) is a hepatotropic, single-stranded RNA virus that replicates in the cytoplasm of hepatocytes and does not integrate into the host genome (1). HCV circulates in vivo as a dynamic distribution of closely related viral variants that are commonly referred to as “quasispecies” (2). Such diversity confers a remarkable advantage to the virus under host selective constraints (3–5). One of the most important features of HCV is its extraordinary ability to persist in up to 80% of infected individuals (6). Approximately 20–30% of chronic HCV carriers develop cirrhosis and its long-term sequelae, including hepatic decompensation and hepatocellular carcinoma (HCC), leading to orthotopic liver transplantation (OLT) or liver-related death (6). The incidence of HCC in the United States has more than tripled over the past 3 decades (7), an alarming trend due primarily, if not exclusively, to HCV infection (8).Although epidemiologic evidence has linked chronic HCV infection to a significantly elevated risk of developing HCC (9), the mechanisms whereby HCV promotes hepatocarcinogenesis remain to be elucidated (10). Whether HCV elicits liver cancer indirectly, through chronic inflammation, fibrosis, and continuous liver regeneration (11), or directly, through the expression of tumor-promoting viral proteins, in a manner analogous to other oncogenic viruses, such as human papillomaviruses and Epstein–Barr virus (12, 13), remains unknown. The major challenges in defining the role of HCV in the pathogenesis of HCC include the inherent limitations of available experimental systems, the low number of infected hepatocytes, the low level of HCV antigen expression, and the limited availability and size of infected liver samples (14).Insights into the levels of HCV replication within the tumor of patients with HCV-associated HCC may shed light on the role of HCV in hepatocarcinogenesis. This remains controversial, however, in part because of the difficulty in obtaining multiple paired liver specimens from the tumor and adjacent nontumorous tissue. Although some previous investigations have shown no differences in the presence and levels of HCV RNA between the tumor and nontumorous liver tissues (15–17), others have reported low to undetectable levels of HCV RNA within the tumor (18–20). Several reports have indicated that miR-122 is an essential cellular host factor for HCV replication (21, 22). Initial studies performed in liver cancer, regardless of the etiology, have shown a reduced expression of miR-122 (23, 24), whereas recent reports have demonstrated that its expression in HCV-associated HCC is maintained (25, 26) or even increased (27). Thus, the central questions of whether HCV actively replicates in malignant hepatocytes, its relationship with miR-122 expression, and whether viral replication is directly involved in hepatocarcinogenesis remain to be fully elucidated. A related issue is whether changes in viral replication within the tumor are associated with selection of specific viral variants; however, very limited information is available on the composition of the HCV quasispecies and the possible compartmentalization between the tumor and the surrounding nontumorous areas or serum from the same individuals with HCV-associated HCC (20, 28–30).To investigate the role of HCV in hepatocarcinogenesis, we took advantage of a unique collection of multiple liver specimens from patients with HCV-associated HCC who underwent OLT or partial hepatectomy to simultaneously study the level of viral replication, its correlation with the intrahepatic expression of miR-122, and the composition and distribution of the viral population both within and outside the tumor. |
