Hubert Darius Daniel

Inactivating mutations of the chromatin remodeling gene ARID2 in hepatocellular carcinoma

Through exomic sequencing of ten hepatitis C virus (HCV)-associated hepatocellular carcinomas (HCC) and subsequent evaluation of additional affected individuals, we discovered novel inactivating mutations of ARID2 in four major subtypes of HCC (HCV-associated HCC, hepatitis B virus (HBV)-associated HCC, alcohol-associated HCC and HCC with no known etiology). Notably, 18.2% of individuals with HCV-associated HCC in the United States and Europe harbored ARID2 inactivation mutations, suggesting that ARID2 is a tumor suppressor gene that is relatively commonly mutated in this tumor subtype.

Incidence and prognostic impact of KRAS and BRAF mutation in patients undergoing liver surgery for colorectal metastases.

Molecular biomarkers offer the potential for refining prognostic determinants in patients undergoing cancer surgery. Among patients with colorectal cancer, KRAS and BRAF are important biomarkers, but their role in patients undergoing surgical therapy for liver metastases is unknown. In this study, the incidence and prognostic significance of KRAS and BRAF mutations were determined in patients undergoing surgical therapy of colorectal liver metastases (CRLM).

Hepatitis B Virus Replication Induces Methylation of both Host and Viral DNA

ABSTRACT 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.

Distribution of Hepatitis B Virus Genotypes in Blood Donors and Chronically Infected Patients in a Tertiary Care Hospital in Southern India

Hepatitis B virus (HBV) genotypes differ in their potential for causing disease. Consecutive patients with chronic HBV infection (CHBV) (n=122) and blood donors (n=67) positive for hepatitis B surface antigen and HBV DNA were genotyped using polymerase chain reaction-restriction fragment-length polymorphism. The ratio of male to female subjects was significantly higher in the blood donor group than in the group of patients with CHBV (P=.0004). Among patients with CHBV, genotype D was detected in 57.3%, genotype A was detected in 18%, and genotype C was detected in 11.5%. Only genotypes D and A were detected in blood donors. The difference between the detection rate of genotype C in patients with CHBV and in blood donors was significant (11.5% vs. 0%; P=.009). Patients with CHBV who had genotype C had higher alanine transaminase (ALT) levels than those who had genotype A (P=.044) or genotype D (P=.014). Detection of genotype C in patients with CHBV and the association of genotype C with higher ALT levels may predict that this genotype has a greater potential for causing disease than other genotypes.

Fibrolamellar carcinomas are positive for CD68

Fibrolamellar carcinomas are a unique type of liver carcinoma that arise in non-cirrhotic livers of young individuals. Despite their distinctive appearance, recent studies have demonstrated a lack of consistency in how fibrolamellar carcinomas are diagnosed by pathologists. As a potential aide in diagnosis, we investigated the staining properties of CD68. The CD68 gene encodes for a transmembrane glycoprotein located within lysosomes and endosomes. Macrophages as well as other cell types rich in lysosomes/endosomes are CD68 positive. Cases of fibrolamellar carcinoma were collected from four academic centers. Control groups included hepatocellular carcinomas arising in both non-cirrhotic livers and cirrhotic livers. A group of cholangiocarcinomas were also stained. CD68 immunostaining was scored for both intensity and distribution on a scale of 0 to 3+. Twenty-three primary fibrolamellar carcinomas and 9 metastases (total of 24 individuals) were immunostained and showed a distinctive granular, dot-like or stippled pattern of cytoplasmic staining in nearly all cases (31/32), with a median distribution and intensity score of 3+. In control hepatocellular carcinomas that arose in non-cirrhotic livers, 10/39 showed CD68 staining with a median distribution and intensity score of 2+. In hepatocellular carcinomas arising in cirrhotic livers, 3/27 cases showed CD68 positivity, all with stippled dot-like cytoplasmic staining similar to that of fibrolamellar carcinomas. All five cholangiocarcinomas were negative. Overall, CD68 positivity was strongly associated with fibrolamellar carcinomas, P<0.001 and had a sensitivity of 96%, a specificity of 80%, and a negative predictive value of 98%. In sum, tumor positivity for CD68 staining was highly sensitive for fibrolamellar carcinoma and a lack of CD68 staining should suggest caution in making a diagnosis of fibrolamellar carcinoma.

Mitochondrial Mutations in Hepatocellular Carcinomas and Fibrolamellar Carcinomas

Mitochondrial mutations are well documented in hepatocellular carcinoma, but their role in carcinogenesis remains unclear. To clarify their significance, a comprehensive analysis was performed of hepatocellular carcinomas (N=24), including quantifying the total mitochondrial DNA levels, quantifying the levels of mitochondrial DNA with the common deletion, and complete sequencing of the mitochondrial control region. In addition, these studies were expanded and reinforced by analysis of fibrolamellar carcinomas (N=15), a unique type of liver carcinoma that has increased numbers of mitochondria on electron microscopy. Overall, approximately 50% of hepatocellular carcinomas had lower levels of total mitochondrial DNA than paired non-neoplastic tissues. Interestingly, despite their increased numbers of mitochondria, primary fibrolamellar carcinomas had lower levels of total mitochondrial DNA. In contrast, metastatic fibrolamellar carcinomas had greatly increased mitochondrial DNA levels. Overall, deletions in the control region were associated with lower total DNA levels in typical hepatocellular carcinoma, but somatic single base pair mutations were not. In fact, almost all single base pair mutations were either reversions to the wild-type sequence or known population polymorphisms, strongly suggesting they are not directly oncogenic. Complete sequencing of the entire mitochondrial genome in fibrolamellar carcinomas identified several somatic mutations, but no consistent pattern of mutations was found. Overall, the levels of the common deletion were highest in tissues with lower total mitochondrial DNA. In conclusion, control region deletions, but not somatic mutations, may influence total DNA copy numbers. Somatic control region mutations in hepatocellular carcinoma are not directly oncogenic but instead may be adaptive.

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.

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

ABSTRACT 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.

An evaluation of saliva as an alternative to plasma for the detection of hepatitis C virus antibodies.

PURPOSE Seroepidemiological studies on the prevalence of Hepatitis C virus (HCV) in India have been hampered by reluctance of subjects to provide blood samples for testing. We evaluated the use of saliva as an alternate specimen to blood for the detection of antibodies to HCV. METHODS Chronic liver disease (CLD) patients attending the liver clinic were recruited for this study. A saliva and plasma sample (sample pair) was collected from each patient included in the study. Saliva samples were collected using a commercially available collection device--OmniSal. Sample pairs were tested with an in-use ELISA for the detection of antibodies to HCV (HCV-Ab), with a minor modification in the manufacturers protocol while testing saliva. The cut-off absorbance value for declaring a sample as positive was determined by receiver operating curve (ROC) analysis. HCV-Ab positivity in saliva was compared with that in plasma as well as with viral load in plasma and infecting genotype of the virus. Sensitivity, specificity, positive and negative predictive values, and correlation coefficients were calculated using Medcalc statistical software. RESULTS The optimal accuracy indices were: sensitivity-81.6%; specificity-92.5%; PPV-85.1% and NPV-90.5%. No correlation was found between salivary positivity and HCV viral load in plasma or infecting genotype. CONCLUSIONS The accuracy indices indicate that the assay must be optimized further before it can be recommended for routine use in epidemiological surveys for HCV-Ab.