Hepatocellular carcinoma (HCC) is the most common primary liver cancer, usually arising after years of chronic liver inflammation. Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections can lead to HCC, whereas non-infectious inflammatory states, such as chronic inflammation induced by alcohol consumption and hereditary iron overload can also contribute to HCC. IL-6 levels are elevated in the serum of patients with all of these chronic liver diseases 1 2 3 and could become even more elevated in those who develop HCC 4 5 6 . Recently, obesity-promoted HCC development was reported to depend on enhanced production of IL-6 which causes hepatic inflammation and activation of the oncogenic factor STAT3 7 .

Human cytomegalovirus (HCMV) is an opportunistic, species-specific herpesvirus that infects a large part of the population worldwide and causes asymptomatic latent infection in healthy subjects. However, it can cause severe disease in the absence of an effective immune response, especially in patients with AIDS and in immunocompromised solid-organ and bone marrow allograft recipients 8 . Histological and immunohistochemical studies have demonstrated the presence of infected cells in virtually all organs and the virus targets a variety of cell types in vivo, including macrophages, endothelial cells, epithelial cells, fibroblasts, stromal cells, neuronal cells smooth muscle cells, and hepatocytes 9 10 . Blood monocytes and tissue macrophages are believed to serve as target cells in infected organs, acting as viral disseminators throughout the host or as sites of HCMV latency 11 . Elevated levels of IL-6 have been reported to accompany HCMV replication in transplanted lungs and bone marrow during episodes of inflammation or rejection 12 13 . IL-6 mRNA expression is upregulated by HCMV infection in the absence of de novo expression of viral genes 14 15 16 17 . Virion binding activates multiple intracellular signal transduction pathways, including the phosphatidylinositol kinase, MAPK/ERK and protein kinase C pathways, all of which lead to the activation of nuclear factors such as NF-kB and p38 18 19 , which are known inducers of IL-6 gene transcription 20 .

In the present study, the seroprevalence of HCMV and the IL-6 production were determined in patients hospitalized who were studied on the basis of HCC, cirrhosis and the presence or not of hepatotropic viruses.

We report here that HCMV seroprevalence in patients with HCC is significantly higher than that of cirrhotic patients without HCC. We observed a positive correlation between HCMV seroprevalence and serum IL-6 levels in cirrhotic patients, but not in HCC patients. In addition serum IL-6 levels correlated positively with CRP levels. Our results question the role of HCMV in the development of HCC.

HCMV is a highly transmissible and prevalent beta herpesvirus 22 23 . This pathogen is never cleared from the body, persisting in a number of tissues via hypothesized mechanisms including chronic productive infection and/or latent infection with periodic subclinical reactivation 8 22 . Recently, HCMV has been linked to a variety of chronic diseases with an inflammatory component including cardiovascular disease 24 , cognitive decline including vascular dementia 25 , functional impairment 26 and cancer 27 . Although HCMV has been reported as involved in hepatitis 28 , its role in cancer is just starting to emerge. The involvement of HCMV in late inflammatory complications underscores its possible role in inflammatory diseases and cancer. Evidence of this involvement of HCMV in such phenomena is being accumulated (review in: 27 29 30 . Early in vitro studies suggested that HCMV was able to transform embryonic fibroblasts in culture and to induce chromosomal damages and mutations but HCMV has never been accepted as oncogenic virus 27 . Later on the concept of "oncomodulation" was proposed to explain the possible contribution of HCMV in tumour progression 31 . The oncomodulation states that HCMV infects the tumour tissue and acts as a cofactor in amplifying mechanisms of carcinogenesis without necessarily initiating tumour. Support for this idea is based on experiments showing that proteins of HCMV (or non-coding RNAs) can influence the genesis and tumour growth acting on the cell cycle, apoptosis, genetic instability, invasiveness, angiogenesis, adhesion and cell migration. These proteins have been the subject of extensive recent reviews 27 32 33 . The increased sensitivity of detection of HCMV in tumour tissues (immunohistochemistry, in situ hybridization and PCR techniques) originally proposed by Cobbs et al in 2002 served to highlight the presence of HCMV proteins and DNA in tumour cells but not in adjacent cells of several cancers such as glioma 34 , colon cancer 35 , prostate cancer 36 , and some skin cancers 37 . Interestingly, the presence of HCMV was also highlighted in the pre-cancerous lesions such as colorectal polyps 35 , and prostatic intraepithelial neoplasia 36 .

In order to determine the role of HCMV as a potential oncomodulator of HCC, we measured HCMV seroprevalence in patients with HCC, in cirrhotic patients and HCC-/Cir- subjects. Our results clearly indicate that HCMV seroprevalence is increased in patients with HCC. Using multimodal statistical analysis, the HCMV seropositivity was 3.45-fold more frequent in HCC+ patients and was independent on the presence of cirrhosis or on infection with HCV or HBV. Also, HCC was positively associated with HCMV seropositivity and with infection with HCV or HBV. These results suggest that HCMV could be involved in the appearance of HCC, as it is the case for HCV and HBV. To confirm this hypothesis future studies will determine whether HCMV genome and/or HCMV antigens can be detected in hepatocytes of HCC patients. It is also possible that high HCMV seroprevalence observed in HCC patients and not in cirrhotic patients could result from HCMV infection of vessels of the liver and/or from infected circulating monocytes arriving at sites of liver inflammation 9 38 .

We also observed a positive correlation between HCMV seroprevalence and serum IL-6 levels in cirrhotic patients, but not in HCC patients. Increased serum IL-6 levels have been reported in cirrhotic patients and HCC patients infected with HBV and HCV 39 40 41 . Nevertheless, serum IL-6 levels in HCC patients depend on the virus involved (HBV versus HCV), the genotype of the virus especially for HCV and ultimately from the IL-6 gene polymorphism 42 43 44 45 . Elevated levels of IL-6 have been reported to accompany HCMV replication in transplanted lungs and bone marrow during episodes of inflammation or rejection 12 13 . IL-6 mRNA expression is up-regulated by HCMV infection 15 16 17 and increased IL-6 levels are a critical factor involved in inflammation and carcinogenesis, especially in liver 7 . IL-6 production has been detected in many cell types, however the primary sources of the cytokine are monocytes and macrophages at sites of inflammation during acute inflammation, as well as T cells in chronic inflammation. In homeostatic conditions, IL-6 levels are low, whereas under stress conditions, amounts of IL-6 rise quickly in the serum. Production of IL-6 depends on several transcription factors, primarily NF-kB, C/EBPβ (formerly NF-IL6) and AP-1 46 . The stimuli that trigger activation of these transcription factors include TNFα and IL-1, bacterial products (LPS) or viral infections such as HIV, HCV and HCMV 47 48 49 . After IL-6 is secreted from the activated monocyte or macrophage, it can act on other cells locally or systemically. In classical IL-6 signaling, the cytokine engages its receptor IL-6R at the cell surface. IL-6R is a non-signaling receptor normally present only on hepatocytes and certain leukocytes 50 . A homodimer of the signal-transducing receptor gp130 is recruited to the IL-6-IL-6R complex, followed by activation of Janus kinase (JAK) which is recruited to the intracellular portion of gp130. JAK in turns activates the transcription factor STAT3 (signal transducer and activator of transcription) 48 51 by phosphorylation. Phosphorylated STAT3 dimerizes and travels to the nucleus, there initiating a transcriptional program 50 . This program's primary function is to promote growth and differentiation and prevent apoptosis, and includes among others the induction of genes encoding cyclin D1, survivin and Bcl-2 52 . In addition, STAT3 regulates genes that promote angiogenesis through regulation of vascular endothelial growth factor (VEGF) 53 . The IL-6 signal also activates the mitogen-activated protein kinase (MAPK) pathway, specifically extracellular signal-related kinase (ERK) through JAK activation of SHP2 (a protein-tyrosine phosphatase), which eventually activates the proto-oncogene Ras, a GTPase found mutated in many human cancers 50 , especially in HCC 54 . In addition, IL-6 antagonizes TGF-β-induced apoptosis in human hepatoma cell line Hep3B 55 , and this could be critical during cirrhosis favoring the transformation of hepatocytes parallel to the development of liver fibrosis. In addition, proinflammatory cytokines are part of the host response to HCMV infection and participate in viral clearance. The finding that HCMV seropositivity correlates with serum IL-6 levels in HCC-/Cir+ patients and not in HCC+ patients suggest that IL-10 which is produced during HCMV infection (vIL-10) does not impair the control of HCMV, but rather may inhibit local production of proinflammatory cytokines such as IL-6 by infiltrating lymphocytes in target organs. Our results are in accord with the observation of Cheeran et al. 56 , who reported similar MCMV viral loads in IL-10 deficient mice and in immunocompetent mice, C57BL/6 (which is a model simulated by the IL-10 KO mice repleted with mrIL-10).

HCMV is not an uncommon feature in patients with liver diseases and may be a cause of systemic inflammation. Plasma cytokine interleukin-6 (IL-6) is mainly produced by circulating and peripheral cells and induces the hepatic synthesis of C-reactive protein (CRP), which is the main acute phase reactant. CRP expression in vivo is assumed to be restricted mainly to the liver 57 where CRP is produced under the control of various proinflammatory cytokines such as interleukin 1 (IL-1), IL-6 and tumor necrosis factor α (TNFα) 58 . Since IL-6 has been reported to accompany HCMV replication 12 13 , IL-6 could favor CRP production in the liver of HCMV-infected patients. Our results are in agreement with this observation, since serum IL-6 correlated positively with that of CRP. Nevertheless, we cannot exclude that CRP production is increased in these patients by bacterial infections that could also favor IL-6 production.

To determine whether HCMV DNA was specifically present in liver tumour area, we further performed a nested PCR for HCMV UL82 (pp71) gene. Interestingly, HCMV DNA was specifically detected in the tumour area but not in safe area for one patient among 3 considered patients as previously described for glioma 34 , prostate cancers 36 , colon cancers 35 , and skin cancers 37 . However, HCMV DNA was either detected in both tumour and safe areas or not detected for the other two patients, indicating that the detection of HCMV DNA in tumour area is not an invariant result during hepatocellular carcinoma with our experimental conditions. One limitation of this experiment might be the use of fixed and paraffin embedded biopsies, as such treatment was described to impair DNA 59 . Histological evaluation of more liver biopsies might further be considered to confirm these results.

Some limitations of the current study should be considered. First, the diagnosis of HCMV infection was based on an indirect test and the absence of anti-HCMV-IgG with the detection of HCMV DNA can occur, especially in immunocompromised patients. Second, the measurement of soluble receptor IL-6 might have been helpful as an indirect marker of cell hepatic responsiveness to IL-6 and therefore of CRP expression. Third, the patients were characterized with only one measurement of IL-6 and CRP.

Our results indicate that HCMV seroprevalence in patients with HCC is significantly higher than in patients without HCC and is positively correlated with serum IL-6 levels in cirrhotic patients. If HCMV infection plays a significant role in the etiology of HCC, the elimination of HCMV infection via the development and administration of treatments or vaccines 60 may reduce HCC mortality rates. Colugnati et al. predicted that a vaccination against HCMV would not need to have high efficacy nor wide-spread coverage to make a substantial impact on HCMV transmission, and elimination of HCMV from the population has the potential to greatly reduce the incidence of disease attributable to HCMV infection 61 . Therefore, elimination of HCMV infection is a potentially feasible and important avenue of study for preventing diseases linked to HCMV infection. Future studies will be needed to further define the role of HCMV in cancers including HCC.

HCC: hepatocellular carcinoma; HBV: hepatitis B virus; HCV: hepatitic C virus; HCMV: human cytomegalovirus; HIV: human immunodeficiency virus; Cir: cirrhosis; CRP: C-reactive protein; IL-6: interleukin-6; JAK: Janus kinase; STAT3: signal transducer and activator of transcription 3; MAPK: mitogen-activated protein kinase; ERK: extracellular signal-related kinase; NF-kB: nuclear factor kappa b; TNF-α: tumor necrosis factor α; TGF-β: transforming growth factor β; LPS: lipopolysaccharide; SD: standard deviation.

The authors declare that they have no competing interests.

Conceived and design the experiments: QL, VDM, BK, GH. Performed the experiments: QL, MKT. Analysed the data: QL, MKT, GH. Wrote the paper: GH. All the authors have read and approved the final manuscript.