Localization of HCV RNA and capsid protein in human hepatocellular carcinoma

Abstract

Key Words: hepatitis C virus, liver neoplasms; carcinoma, hepatocellular; in situ hybridization; immunohistochemistry

INTRODUCTION

The relation of HCV to hepatocytic carcinoma (HCC) has been emphasized recently in low HBV infection countries and regions. The distribution patterns of HCV in liver tissues are not well understood although studies on HCV infection in blood and hepatocytes have been conducted by PCR. In this study, 42 liver cancers and surrounding liver tissues were detected for HCV RNA and HCAg using photosensitive biotin-labeled HCV cDNA probe and Immuno-gold-silver stain (IGSS) method.

MATERIALS AND METHODS

Human HCC and paratumor tissues

Fourty-two samples including cancers and their surrounding tissues were collect ed and processed into formalin-fixed, paraffin-embedded blocks.

Probe preparation

NS5 cDNA probe PCR product from NS₅ region was inserted into charomid 9-42. This resulted in recombinant HCV plasmid which could be spliced into specific cDNA fragment with DNA length 534 bp[1].

HCV cDNA probe HCVC cDNA probe was the PCR product of C region.

The preparation of photosensitive biotin-labelled probe was as described in literature[2].

In situ hybridization

5 μm sections were dewaxed, rehydrated in buffered solutions, then digested in proteinase K, and fixed in polyformaldehyte. After dehydration, prehybridization was performed and then the sections were denatured (steaming bath at 100 °C, 10 min, then rapidly cooling down in ice bath), and hybridized again (42 °C, overnight). After rinse, the sections were conjugated with gold-labeled strepto-biotin, stained with AgNO₃ buffered solution, then restained in hemoxylin and eosin or eosin. Positive stain should be present as intracytoplasmic or intranuclear black granules.

Immunohistochemical detection of HCAg PAP method was used. The specific human anti-HCV antibody was from Laboratory of Immunology at Tongji Medical University.

Negative control Sections were treated with RNase (0.1 g/L), 1 h, at 37 °C.

RESULTS

HCV RNA in liver tissues

HCV RNA positivity was detected in 8 cancers and their surrounding tissues including 2 positivity in cancers, 1 in both cancer and their surrounding tissue, 5 in surrounding liver tissues. Positive cells appeared diffuse, clustered, or discrete in cancer and its surrounding tissues. Three types of positive granules were noted: ① cytoplasmic, diffuse granules in cytoplasm with prominent perinuclear staining (Figure 1); ② nuclear, evenly diffuse granules in nucleus; ③ nuclear-cytoplasmic, positive granules present in both cytoplasm and nucleus. Detection by NS₅ cDNA probe revealed that 7 out of 42 were positive; detection by C cDNA probe showed one nuclear positive in 2 cases.

Figure 1 HCV-ISH (cytoplasmic type), biotin marker, ABC methods, HCCII, × 132

HCAg expression in liver tissues

Ten out of 42 stained positive for HCAg and positive cells were distributed in cancers and its surrounding tissues. HCAg positive cells appeared diffuse, clustered or discrete. Two distributing patterns of intracellular positive granules: ① cytoplasmic type: diffuse positive granules in cytoplasm; ② inclusion type: positive granules present as inclusions in cytoplasm (Figure 2).

Figure 2 HCAg (inclusion pattern), cancerous surrounding liver, PAP method, × 200

DISCUSSION

HCV is a single-stranded RNA virus which reproduces itself on the template of negative strand and its copies are rarely present in tissues. It has been established that replication of HCV RNA occurs mainly in liver tissues. How it infects liver cells and at which point it starts duplication remains unknown.

Since 1992, there have been some reports on detection of HCV RNA in hepatic tissues using ISH, and HCV RNA was present in cytoplasm or nucleus[3-8]. However, it is still unclear whether HCV RNA is present in hepatocytic carcinoma. Using ISH, we found that HCV RNA did infected cancers and their surrounding tissues, which was in good agreement with other authrs’ reports. HCV infect ed cells appeared diffuse, clustered or discrete in cancers and their surrounding tissues.

There is a viewpoint that the reverse transcriptive form of HCV RNA does not exist at all during its replication. Therefore, it is disputable on the significance of its presence in nucleus. Kayhan[8] reported that HCV RNA was present in nucleus and nucleoli, and suggested that HCV duplication might use its host’s genes or intranuclear apparatus, or HCV granules accumulated in infected cells like HBV. Our results were in accordance with the above findings. The studies on yellow fever virus also supported the above viewpoints: NS5 protein could be detected in nucleus and nucleoli, indicating that viral replication could occur in nucleus[9].

In 42 liver cancers, 10 were positive for HCAg, while only 8 expressed HCV RNA by ISH, indicating that positivity of viral genes was lower than that of viral antigen. It is suggested that this phenomenon could be accounted by the variations of viral genes, and the fixing time by formalin. HCV is a variable virus, it may differ individually and may vary at different stages of infection. Therefore, the variation of gene sequence may result in less sensitivity of ISH detection. In addition, longer fixation may cause loss of viral genes or false negativity. The above defects could be overcome by using probes of different fragments of the same gene. Two cases with HCAg(+), NS5 cDNA(-) were hybridized again and revealed positive signals of NS₅ cDNA in one case. This agreed well with Haruna’s[7] findings. Although ISH is not as sensitive as PCR, it is a promising technique in the study of relation of HCV RNA and infected cells[10-12].

The significance and mechanism of continual variation of HCV gene in vitro remain to be resolved in recent years. It was reported that HCVs continual variation in vivo may be related to virus escape from host immune deletion and virus configurative change brought about by immune injury introduced by the host[13-15].