Chromophobe hepatocellular carcinoma with abrupt anaplasia: a proposal for a new subtype of hepatocellular carcinoma with unique morphological and molecular features.

Hepatocellular carcinomas exhibit heterogeneous morphologies by routine light microscopy. Although some morphologies represent insignificant variations in growth patterns, others may represent unrecognized subtypes of hepatocellular carcinoma. Identification of these subtypes could lead to separation of hepatocellular carcinomas into discrete groups with unique underlying genetic changes, prognosis, or therapeutic responses. In order to identify potential subtypes, two pathologists independently screened a cohort of 219 unselected hepatocellular carcinoma resection specimens and divided cases into potential subtypes. One of these promising candidate subtypes was further evaluated using histological and molecular techniques. This subtype was characterized by a unique and consistent set of histological features: smooth chromophobic cytoplasm, abrupt focal nuclear anaplasia (small clusters of tumor cells with marked nuclear anaplasia in a background of tumor cells with bland nuclear cytology), and scattered microscopic pseudocysts--we designate this variant as 'chromophobe hepatocellular carcinoma with abrupt anaplasia'. Thirteen cases were identified (6% of all hepatocellular carcinomas), including 6 men and 7 women with an average age of 61 years. Six cases occurred in cirrhotic livers. Serum AFP was elevated in 6 out of 10 cases. There were a variety of underlying liver diseases, but cases were enrichment for chronic hepatitis B, P=0.006. Interestingly, at the molecular level, this variant was strongly associated with the alternative lengthening of telomere (ALT) phenotype by telomere FISH. ALT is a telomerase-independent mechanism of telomere maintenance and is found in approximately 8% of unselected hepatocellular carcinomas. In contrast, 11/12 (92%) of the cases of chromophobe hepatocellular carcinoma with abrupt anaplasia were ALT-positive. In summary, we propose that chromophobe hepatocellular carcinoma with abrupt anaplasia represents a new subtype of hepatocellular carcinoma with unique morphological and molecular features.

Hepatitis B virus replication induces methylation of both host and viral DNA.

Control of viral replication is a major therapeutic goal to reduce morbidity and mortality from chronic hepatitis B virus (HBV) infection. Recently, methylation has been identified as a novel host defense mechanism, and methylation of viral DNA leads to downregulation of HBV gene expression. To better understand the mechanisms of HBV methylation, cell lines were exposed to HBV using a model system that mimics natural infection and the expression of host DNA methyltransferase genes (DNMTs) was measured. DNMT1, DNMT2, and DNMT3 were all significantly upregulated in response to HBV. DNMT3 was further studied because of its known role in the de novo methylation of DNA. Cotransfection experiments with full-length HBV and DNMT3 led to the downregulation of viral protein and pregenomic RNA production. To investigate whether the upregulation of DNMTs could also have an effect on the methylation of host DNA, cell lines were exposed to HBV in two independent model systems, one that mimics natural infection and a second model with temporary transfection. Host DNA methylation was measured by DNA microarray analysis. Increased methylation of host CpG islands was detected in both experimental systems. Two CpG islands, corresponding to genes SUFU and TIRAP, were selected, and the downregulation of these genes in hepatocellular carcinomas was confirmed. In conclusion, hepatocytes respond to HBV infection by upregulating DNMTs. The DNMTs methylate viral DNA, leading to decreased viral gene expression and decreased viral replication. However, virus-induced overexpression of DNMTs also leads to methylation of host CpG islands.

Hepatitis C-associated hepatocellular carcinomas in non-cirrhotic livers.

Chronic hepatitis C viral infection can lead to cirrhosis and hepatocellular carcinoma. It is generally believed that hepatitis C infection is not oncogeneic per se, but that the presence of cirrhosis determines the increased risk for hepatocellular carcinoma. However, a search of surgical pathology files from two large tertiary care centers for the years 2001-2008 identified a total of 18 hepatocellular carcinomas in non-cirrhotic livers with chronic hepatitis C infection. In six cases the background livers showed bridging fibrosis, while the remainder showed lower stages of fibrosis. Cases were negative for clinical and serological evidence of hepatitis B co-infection, and occult hepatitis B test was negative by PCR of formalin-fixed, paraffin embedded tissues. The tumors were also negative for TP53, exon 7, codon 249 mutations, a characteristic mutation strongly linked to aflatoxin exposure. The hepatocellular carcinomas had typical histology with no enrichment for unusual growth patterns or histological features. Among all resected hepatocellular carcinomas in non-cirrhotic livers over this time period, the prevalence of 16% with HCV infection was significantly greater than that expected by chance. In conclusion, these results demonstrate that hepatocellular carcinomas can arise in livers chronically infected with hepatitis C but without cirrhosis. These findings raise the possibility that in some cases hepatitis C infection and inflammation can be directly oncogeneic. It is also possible that established cirrhosis may have regressed in some cases. Regardless of the mechanism, these findings highlight an important and previously under-recognized risk for hepatocellular carcinoma in HCV-infected individuals who do not have cirrhosis.

Clinicopathological features and genotype distribution in patients with hepatitis C virus chronic liver disease.

BACKGROUND AND OBJECTIVE: Hepatitis C virus (HCV) genotype influences the severity of disease and response to therapy. This retrospective study examined the clinical and histological features and the genotype distribution in biopsied patients with HCV related chronic liver disease. METHODS: Of 105 biopsies from patients with HCV infection, 96 from patients with chronic liver disease were reviewed. The Ishak scoring system was used for histological analysis. RESULTS: Genotype 3 was most common accounting for 77.1%, and genotype 1 for 9.4% of cases. There was no significant association of transaminase levels, viral load or necro-inflammatory activity score with genotype. A severe degree of fibrosis was seen in 77.8% cases of genotype 1 and in 63.5% of genotype 3 (p=0.76). Variable degrees of steatosis were noted in 68.8% of cases. However, severe steatosis was noted only in genotype 3 (7 cases). Serum transaminase levels did not correlate with either histological activity (p=0.43) or degree of fibrosis (p=0.72). Severe fibrosis / cirrhosis was seen in 74.24% of patients above 40 years of age as compared to 33.3% of patients below 40 years (p=0.001). The frequency of Mallory hyaline was significantly different between genotypes 1 and 3 infection (P<0.001). CONCLUSIONS: This study confirms the preponderance of genotype 3 in Indian patients with HCV related chronic liver disease. Severe steatosis was seen only in genotype 3 and Mallory hyaline was very common in genotype 1. The small numbers of patients in non genotype 3 could be a reason for the apparent lack of histological differences between different HCV genotypes. Severe fibrosis seen in older age groups confirms that HCV infection is progressive and major acceleration of the disease process occurs after 40 years of age.

Quantitation of hepatitis B virus DNA in plasma using a sensitive cost-effective "in-house" real-time PCR assay.

BACKGROUND: Sensitive nucleic acid testing for the detection and accurate quantitation of hepatitis B virus (HBV) is necessary to reduce transmission through blood and blood products and for monitoring patients on antiviral therapy. The aim of this study is to standardize an "in-house" real-time HBV polymerase chain reaction (PCR) for accurate quantitation and screening of HBV. MATERIALS AND METHODS: The "in-house" real-time assay was compared with a commercial assay using 30 chronically infected individuals and 70 blood donors who are negative for hepatitis B surface antigen, hepatitis C virus (HCV) antibody and human immunodeficiency virus (HIV) antibody. Further, 30 HBV-genotyped samples were tested to evaluate the "in-house" assay's capacity to detect genotypes prevalent among individuals attending this tertiary care hospital. RESULTS: The lower limit of detection of this "in-house" HBV real-time PCR was assessed against the WHO international standard and found to be 50 IU/mL. The interassay and intra-assay coefficient of variation (CV) of this "in-house" assay ranged from 1.4% to 9.4% and 0.0% to 2.3%, respectively. Virus loads as estimated with this "in-house" HBV real-time assay correlated well with the commercial artus HBV RG PCR assay ( r = 0.95, P < 0.0001). CONCLUSION: This assay can be used for the detection and accurate quantitation of HBV viral loads in plasma samples. This assay can be employed for the screening of blood donations and can potentially be adapted to a multiplex format for simultaneous detection of HBV, HIV and HCV to reduce the cost of testing in blood banks.

Whole blood as an alternative to plasma for detection of hepatitis C virus RNA.

Peripheral blood mononuclear cells are reported to be one of the extrahepatic replication sites contributing to the persistence of hepatitis C virus (HCV) infection. Whole-blood and plasma samples from 61 individuals were compared as sources for the detection of HCV RNA. Forty-four of the individuals were receiving antiviral therapy, while 17 were treatment naïve. The quantitation of HCV RNA was done by a sensitive in-house real-time reverse transcription-PCR. When the viral loads in the two types of samples were compared, a correlation coefficient of 0.858 (P < 0.001) was found, indicating that plasma and whole blood are equally acceptable sources for testing for HCV RNA.

Quantitation of hepatitis C virus using an in-house real-time reverse transcriptase polymerase chain reaction in plasma samples.

Even with the most advanced 3rd-generation assays, the serologic window period of hepatitis C virus (HCV) is approximately 74 days. HCV RNA detection would reduce the risk of transmission during this period. Furthermore, quantitation of HCV RNA is necessary for proper planning of treatment, monitoring disease progression, and assessing response to antiviral therapy. We have standardized an in-house HCV real-time reverse transcriptase polymerase chain reaction (RT-PCR) for screening and accurate quantitation and detection of HCV RNA in plasma samples. The in-house real-time assay was compared with a commercial assay using 100 chronically infected individuals and 70 blood donors who are negative for hepatitis B surface antigen, HCV antibody, and HIV antibody. The lower limit of detection of this in-house HCV real-time RT-PCR as assessed against the World Health Organization (WHO) standard was 50 IU/mL. Interassay and intraassay coefficient of variation ranged from 1.3% to 6.4% and 0.0% to 2.3% respectively. Virus loads as estimated with this in-house HCV real-time assay correlated with the commercial artus HCV RG RT-PCR assay (r = 0.59, P < 0.0001). This assay could be used in screening and monitoring individuals on therapy, showing no genotype-dependent differences in detection.

Evaluation of a rapid assay as an alternative to conventional enzyme immunoassays for detection of hepatitis C virus-specific antibodies.

A rapid membrane flow-through immunoassay to detect antibodies to hepatitis C virus was compared with a commercial enzyme immunoassay (EIA) and microparticle enzyme immunoassay (MEIA) using 2,590 serum samples. Sensitivity and specificity of the "rapid assay" in comparison to the EIA/MEIA were 99.3 and 99.0%; the correlation coefficient being 0.91. This assay is suitable where infrastructure and laboratory expertise are limited.

Age-wise exposure rates to hepatitis e virus in a southern Indian patient population without liver disease.

To determine the age-stratified exposure to hepatitis E virus (HEV) in patients attending a tertiary care hospital in southern India, serum samples from 600 individuals were tested using a commercial HEV IgG enzyme-linked immunosorbent assay. Subjects were composed of blood donors, antenatal women, and pre-operative individuals who were negative for hepatitis B surface antigen and antibody to hepatitis C virus with no evidence of liver disease; 200 each were 1-5 and 6-15 years old and 100 each were 16-40 and > or = 41 years old. One (0.5%) sample was positive in those 1-5 years old, two (1.0%) in those 6-15 years old, eight (8%) in those 16-40 years old, and 13 (13%) in those > or = 41 years old. The overall seropositivity rate was 4%. However, there was an age-related increase in exposure to HEV that was statistically significant (P < 0.001), with a higher HEV seropositivity in urban individuals.