Rajesh Kannangai

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.

mTOR and P70 S6 Kinase Expression in Primary Liver Neoplasms

Purpose: mTOR and P70 S6 kinase (S6K) play a key role in regulating protein translation. The role of mTOR and S6K in hepatocellular carcinoma has not been investigated, but this pathway is of particular interest because an effective inhibitor, rapamycin, is available. This study was undertaken to determine the prevalence and clinicopathological correlates of mTOR pathway activation in hepatocellular carcinoma and to determine whether rapamycin inhibits the pathway in cell culture. Experimental Design: Total and phosphorylated mTOR and S6K protein expression were studied by immunohistochemistry in hepatocellular carcinomas (n = 73), fibrolamellar carcinomas (n = 13), and hepatic adenomas (n = 15). Results were correlated with tumor growth pattern as defined by the WHO (trabecular, pseudoglandular/acinar, compact, and scirrhous), tumor size, Ki-67 proliferation index, and the modified Edmondson nuclear grade, which has a scale of 1 to 4. HepG2 and Hep3B cell lines were treated with rapamycin to see the effect on proliferation and S6K phosphorylation. Results: Increased expression of total mTOR was seen in 5% of hepatocellular carcinoma, whereas overexpression of phospho-mTOR was evident in 15% of hepatocellular carcinoma. Phospho-mTOR positivity correlated with increased expression of total S6K, which was found in 45% of cases. Total S6K overexpression was positively correlated with tumor nuclear grade, inversely with tumor size, and was unassociated with the proliferation index or WHO growth pattern. Rapamycin treatment of HepG2 and Hep3B cell lines markedly inhibited cell proliferation and reduced S6K phosphorylation in both cell lines. Conclusions: The mTOR pathway is activated in a subset of hepatocellular carcinoma. Rapamycin can inhibit proliferation of neoplastic hepatocytes in cell culture.

High prevalence of occult hepatitis B in Baltimore injection drug users

Occult hepatitis B is defined by the presence of hepatitis B virus (HBV) DNA in a serum or liver in the absence of hepatitis B surface antigen (HBsAg). The prevalence and clinical correlates of occult hepatitis B remain incompletely defined. A cross‐sectional study was performed to determine the prevalence of occult hepatitis B in a high‐risk cohort composed of 188 injection drug users in Baltimore, Maryland. All individuals had chronic hepatitis C viral infections confirmed by RNA detection and liver biopsy. Serologic assays for HBsAg and core antibody (HBcAb) were performed. Serum HBV DNA was detected using the COBAS HBV AMPLICOR monitor assay (lower limit of detection, 200 HBV copies per milliliter) and a semi‐nested polymerase chain reaction (PCR) assay (lower limit of detection, 15 HBV copies per milliliter). Although almost all individuals (96%) were anti‐HBC positive, only 8 of 188 (4%) were HBsAg positive. Occult hepatitis B was not identified using the COBAS assay, but was found in 81 of 180 (45%) of individuals using semi‐nested PCR. Of the 8 HBsAg positive individuals, HBV DNA was found in 1/8 using the COBAS assay and 6/8 using the nested PCR assay. Overall, liver disease was mild, with a median serum alanine aminotransferase (ALT) of 38 IU/L, median activity grade of 3/18, and median fibrosis stage of 1/6. No association was found between the serum AST (aspartate aminotransferase), activity grade, or stage of liver disease and the presence of occult hepatitis B. Serum ALT levels were slightly higher in patients without occult hepatitis B (46 vs. 35 IU/L), and the median years since first injection drug use was somewhat longer in those without occult hepatitis B (24 vs. 20 years). In conclusion, although further research is needed to assess its clinical significance, there is a high prevalence of occult HBV infection in this cohort of HCV‐infected injection drug users. (HEPATOLOGY 2004;39:51–57.)

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.

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.

Comprehensive Genetic and Epigenetic Analysis of Occult Hepatitis B from Liver Tissue Samples

BACKGROUND Occult infection with hepatitis B virus (HBV) is a type of chronic HBV infection that is characterized by the absence of a detectable hepatitis B surface antigen in the blood and by very low levels of HBV DNA in the blood and liver. The mechanisms leading to occult HBV infection remain poorly understood but include possible genetic mutations and deletions. Recently, it has been shown that HBV has CpG islands that are methylated, raising the possibility that epigenetic changes may also be important. METHODS The full-length genomes of isolates from 5 cases of occult HBV infection were cloned and analyzed for mutations and deletions. Additional studies were performed to examine for APOBEC3G (1 member of a family of deaminating proteins that are part of the innate immune systems defense against viral infection) hyperediting and methylation of viral DNA. RESULTS Numerous mutations and deletions were found in the genomes of occult HBV. However, similar types and locations of polymorphisms were also noted in the genome sequences of HBV isolated from control liver tissue samples obtained from individuals with nonoccult HBV infection. Evidence of APOBEC3G hyperediting was found in 1 case of occult HBV infection, but hyperedited sequences made up only a small proportion of the viral sequences. Methylation of HBV CpG islands 1 and 2 was evident in both occult and nonoccult HBV sequences, with island 2 more densely methylated in occult HBV sequences and island 1 more densely methylated in nonoccult HBV sequences. CONCLUSION Deletions and mutations are common in occult HBV but are also found in control nonoccult HBV, and no unique genetic signature for occult HBV was found. Methylation patterns differ between cases of occult and nonoccult HBV infection, suggesting that epigenetic changes may be relevant to occult HBV. Together, these findings suggest that multiple mechanisms can contribute to occult HBV infection.

Survivin overexpression in hepatocellular carcinoma is associated with p53 dysregulation

AbstractBackground: Survivin is a recently described anti-apoptotic protein that is suppressed by wild-type p53 and is overexpressed in 41–70% of hepatocellular carcinomas from Asia. Two alternatively spliced transcripts have also been described: anti-apoptotic survivin-ΔEx3 and non—anti-apoptotic survivin-2B. Survivin splice variant expression has not been studied in HCC, and little is known about survivin expression in hepatocellular carcinomas arising in other parts of the world, where risk factors are often different than they are in Asia. Aim of the Study: We studied survivin mRNA and protein expression in a United States cohort of hepatocellular carcinomas and correlated the findings with p53 immunopositivity. Methods: RT-PCR was performed for survivin, survivin-2B, and survivin-ΔEx3 in 20 HCCs and one intrahepatic cholangiocarcinoma. Expression levels of total survivin were evaluated with real-time PCR. Protein expression was examined by immunohistochemistry. Results: Survivin was the major transcript, and all transcripts were present in all normal and neoplastic tissues; 11/20 (55%) HCCs and the one cholangiocarcinoma showed twofold or greater overexpression of survivin. Next, we examined survivin and p53 protein expression by immunohistochemistry on a separate series of 79 HCC, 13 fibrolamellar carcinomas, and 15 hepatic adenomas; 14/79 (17%) HCC, but none of the fibrolamellar carcinomas or hepatic adenomas, showed survivin protein overexpression, and 25/79 HCC (32%) showed abnormal nuclear accumulation of p53, which correlated with increased survivin expression. Conclusions: All three survivin transcripts are present in normal liver and HCC. Survivin is the dominant transcript in HCC and is overexpressed in 55% of cases. Survivin protein overexpression is associated with aberrant p53 nuclear positivity.

Occult hepatitis B viral DNA in liver carcinomas from a region with a low prevalence of chronic hepatitis B infection

Summary.  Occult hepatitis B is defined by the presence of hepatitis B viral (HBV) DNA in the serum or liver in persons lacking hepatitis B surface antigen (HBsAg) in the serum. A high prevalence of occult HBV has been reported in hepatocellular carcinoma (HCC) from Asia, but little information is available on the prevalence of occult HBV in HCC from regions with a low prevalence of typical chronic hepatitis B infection. In a retrospective study, 19 cases of primary liver cancer were investigated for the presence of occult HBV DNA by amplification of the surface, core, and X gene. In addition, HBV copy numbers were quantitated by real time polymerase chain reaction, genotyped, and samples tested for covalently closed circular HBV DNA, which is a marker of active viral replication. Occult HBV was found in three of 19 cases (16%). Genotyping was successful in two cases, both of which were genotype A. HBV DNA copy numbers were low, all less than 10 copies/μg liver DNA. No closed circular HBV DNA was detected. Thus, in this study occult HBV was of genotype A and was found in a low percentage of cases of HCC and was associated with low tissue HBV DNA copy numbers and no detectable evidence for viral replication.

Colonic spirochetosis in children and adults.

We undertook a retrospective analysis of colonic spirochetosis in 14 cases: females, 3; males, 11; children, 4; adults, 10. Two men had HIV infections. All children and both HIV-infected men had abdominal complaints, diarrhea, or both. Most other adults underwent colonoscopy for polyp screening (n = 4) or follow-up of Crohn disease (n = 1) or had other indications (n = 2) or diarrhea (n = 1). Histologically, spirochetosis was identified in all parts of the colon and was not strongly associated with active inflammation, mucosal injury, or changes of chronicity. Genotype analysis of 13 cases showed that 11 resulted from Brachyspira aalborgi and 2 from Brachyspira pilosicoli infections. Only 2 patients were treated specifically with antibiotics, with complete resolution of abdominal symptoms in 1 patient with follow-up. Follow-up biopsy result were available for 2 patients who did not receive treatment; one showed persistent spirochetosis, and the other was negative. Spirochetosis in this series had a male predominance, was generally caused by B aalborgi, and occurred in 2 distinct clinical settings: children who often have abdominal symptoms and adults who typically are asymptomatic. While treatment information remains limited, treatment can lead to resolution of symptoms in some cases.

EGFR is phosphorylated at Ty845 in hepatocellular carcinoma

Epidermal growth factor receptor (EGFR) is overexpressed in a significant proportion of hepatocellular carcinomas. Recent studies of EGFR inhibitors to treat hepatocellular carcinoma have been encouraging and better understanding of EGFR signaling may lead to more effective strategies for inhibiting this key pathway. The EGFR can be phosphorylated at different tyrosine sites, leading to subsequent activation of different pathways. Cell line and animal studies have shown that MAPK and STAT-3 are important mediators of the EGFR signal in liver cells. However, little is known about EGFR phosphorylation and subsequent signaling in primary hepatocellular carcinoma. We investigated the site of EGFR phosphorylation by Western blot in 18 hepatocellular carcinomas. Fourteen of 18 hepatocellular carcinomas had detectable EGFR by Western blotting and 13 of 14 showed phosphorylation at tyrosine 845. In contrast, no EGFR phosphorylation was detected at tyrosine 998, tyrosine 1045, or tyrosine 1068, which signal through other pathways including STAT-3 and MAPK. These findings were further explored by examination of EGFR expression and signaling pathway activation in tissue arrays comprised of 73 hepatocellular carcinomas using antibodies that recognize phosphorylated (or activated) proteins. Tissue array studies also found no correlation between EGFR expression (29% of cases) and STAT-3 nuclear positivity (16%), AKT (4%), MAPK (3%), or STAT-5 (3%) positivity, all P>0.05. EGFR expression was correlated with hepatitis B infection, but not with tumor size, nuclear grade, or proliferative rate. We conclude that EGFR is phosphorylated at tyrosine 845 in most hepatocellular carcinomas and that EGFR expression by immunohistochemistry does not correlate well with STAT-3, STAT-5, MAPK, or AKT immunostaining.