St Cheung

Liver Transplantation in Asian Patients With Chronic Hepatitis B Using Lamivudine Prophylaxis

Objective nTo report the results of liver transplantation in 31 Asian patients with chronic hepatitis B using lamivudine prophylaxis in an open-label study.

An integrated data analysis approach to characterize genes highly expressed in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the major causes of cancer deaths worldwide. New diagnostic and therapeutic options are needed for more effective and early detection and treatment of this malignancy. We identified 703 genes that are highly expressed in HCC using DNA microarrays, and further characterized them in order to uncover novel tumor markers, oncogenes, and therapeutic targets for HCC. Using Gene Ontology annotations, genes with functions related to cell proliferation and cell cycle, chromatin, repair, and transcription were found to be significantly enriched in this list of highly expressed genes. We also identified a set of genes that encode secreted (e.g. GPC3, LCN2, and DKK1) or membrane-bound proteins (e.g. GPC3, IGSF1, and PSK-1), which may be attractive candidates for the diagnosis of HCC. A significant enrichment of genes highly expressed in HCC was found on chromosomes 1q, 6p, 8q, and 20q, and we also identified chromosomal clusters of genes highly expressed in HCC. The microarray analyses were validated by RT–PCR and PCR. This approach of integrating other biological information with gene expression in the analysis helps select aberrantly expressed genes in HCC that may be further studied for their diagnostic or therapeutic utility.

Hedgehog signaling in human hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the fourth most common malignancy and one of the leading causes of death world wide. Signaling pathways important for tumor initiation and progression in HCC are poorly understood. Hedgehog signaling (Hh) has been implicated in multiple events during development and has also been proposed to play important roles in several tumor types. However, it remains unclear whether this pathway is activated in HCC. Here, we report the detection of transcripts for hedgehog pathway signaling molecules in both HCC cell lines and tumor samples. Quantitative real-time RT-PCR also revealed the decreased expression of Hip1 and increased expression of Gli1 and smo in HCC samples compared with non-tumor liver tissues. Blocking the hedgehog pathway with cyclopamine inhibited proliferation, induced apoptosis and repressed c-Myc and cyclin D expression in a subset of HCC cell lines. The study therefore, for the first time, provides evidence that hedgehog signaling may be activated in some HCC tumors. The results also indicate that the hedgehog pathway may be a new candidate for therapeutic targeting in HCC.

Novel endothelial cell markers in hepatocellular carcinoma

Hepatocellular carcinoma is characterized by hypervascularity and a propensity for vascular invasion. Detailed analysis of complementary DNA (cDNA) microarray global gene expression data and further validation on a smaller independent sample set by reverse transcription-polymerase chain reaction established the presence of two endothelial gene clusters in hepatocellular carcinoma. Cluster I, consists of 20 cDNA clones, representing 15 unique genes. Cluster II consists of nine unique genes. The expression of the cluster I genes appeared to be significantly upregulated in hepatocellular carcinoma compared with normal liver, cirrhotic liver, or nontumor liver tissues adjacent to the hepatocellular carcinoma. The pattern of gene expression of cluster I genes correlated positively with the ‘proliferation gene cluster’ and ‘stromal cells cluster 2’. Expression of cluster II genes, in contrast, was not significantly different between hepatocellular carcinoma and non-neoplastic liver tissues. Studies conducted to localize the protein products of these genes by immunohistochemical staining of tissue arrays with up to 350 cores of tissues, and by in situ hybridization led to the discovery of novel sinusoidal endothelial cell markers in hepatocellular carcinoma: podocalyxin-like and regulator of G protein signaling-5. Our results underscore fundamental differences not only between neoplastic vs non-neoplastic liver cells but also between the hepatic sinusoidal endothelium of hepatocellular carcinoma and normal liver.

SPARC and Hevin expression correlate with tumour angiogenesis in hepatocellular carcinoma

Both Secreted Protein Acidic and Rich in Cysteine (SPARC) and Hevin are multifunctional matricellular glycoproteins. Recent experimental studies suggested that Hevin and SPARC together diminish angiogenesis, but their significance in hepatocellular carcinoma (HCC) remains unclear. This study aimed to correlate SPARC and Hevin expression with angiogenesis and clinicopathological features in HCC. SPARC and Hevin protein and mRNA expression in HCC specimens were assessed by immunostaining, immunoblotting, and quantitative reverse transcriptase‐polymerase chain reaction. Tumour microvessel density (MVD) was assessed by CD34 immunostaining. The role of SPARC and Hevin in HCC was further assessed in an in vivo nude mice xenograft model. Both SPARC and Hevin mRNA levels were significantly higher in tumours than in non‐tumourous livers. A significant correlation between tumour SPARC and Hevin mRNA levels was found. Moreover, SPARC protein localized in the tumour sinusoidal area correlated significantly with Hevin protein localized in HCC cells. Truncated forms of SPARC and Hevin proteins were detected in clinical samples. Truncated SPARC protein localized in the tumour sinusoidal area correlated significantly with tumour MVD. On the other hand, overexpression of full‐length SPARC in tumour xenografts in athymic nude mice significantly delayed tumour growth, and this delay was related to a decrease in tumour angiogenesis. Expression of Hevin protein within HCC cells was related to the presence of tumour encapsulation and the absence of hepatitis B surface antigen in clinical samples. Overexpression of Hevin in tumour xenografts also significantly delayed tumour growth. In conclusion, this study has shown that SPARC and Hevin are upregulated in HCC compared with non‐tumourous liver, and that they are inter‐related at both mRNA and protein levels. Moreover, both SPARC and Hevin were related to HCC angiogenesis and tumour progression. Copyright

Fibrosing cholestatic hepatitis secondary to precore/core promoter hepatitis B variant with lamivudine resistance: successful retransplantation with combination adefovir dipivoxil and hepatitis B immunoglobulin.

Fibrosing cholestatic hepatitis (FCH) is a peculiar variant of hepatitis B virus (HBV) infection in immunocompromised patients characterized by rapid viral replication. Posttransplant patients receiving lamivudine for prophylaxis or treatment of HBV infection may develop drug resistance due to viral mutants, but FCH is rare because escape mutants are usually replication deficient. We report the development of FCH due to lamivudine‐resistant HBV mutants in 2 patients at 12 and 13 months after liver transplantation. Rapidly progressive graft failure, accompanied by an escalating HBV DNA level, developed within weeks of onset. Analysis of gene sequence variation by polymerase chain reaction (PCR) and direct sequencing showed that both had a core promoter variant A1762T/G1764A and 1 had a concomitant precore stop codon G1896A variant in prelamivudine and postrecurrence serum samples. Comparison of the HBV polymerase gene in the 2 serum samples revealed a single mutation with methionine‐to‐isoleucine substitution at codon 552 (M552I) in both patients. “Add‐in” treatment with adefovir dipivoxil resulted in a more than 2 to 3 log10 reduction in HBV DNA level within 2 weeks and retransplantation was performed with adefovir dipivoxil and hepatitis B immunoglobulin (HBIG) prophylaxis. Both patients were alive at 15 months and 48 months after retransplantation, with normal graft function and serum negative for HBsAg and HBV DNA by quantitative PCR (< 200 copies/mL). The current report demonstrates that recurrent graft infection by precore/core promoter variant with lamivudine‐resistant escape mutation may result in FCH. With combination of adefovir and high‐dose HBIG, however, long‐term survival can be achieved after retransplantation. (Liver Transpl 2004;10:557–563.)

Complete genomic sequence of Epstein-Barr virus in nasopharyngeal carcinoma cell line C666-1

BackgroundNasopharyngeal carcinoma is a distinct type of head and neck cancer which is consistently associated with Epstein-Barr virus (EBV). The C666-1 cell line is the only in vitro native EBV-infected NPC cell model commonly used for study of the viral-host interaction. Nevertheless, the complete EBV genome sequence in this in vitro EBV-infected NPC model has not been characterized.ObjectiveTo determine the complete EBV genome sequence in C666-1 cells.MethodsThe C666-1 genome was sequenced by 100-bases pair-end massive parallel sequencing. Bioinformatics analysis was performed to extract the EBV sequences and construct an EBV consensus sequence map. PCR amplification and Sanger DNA sequencing were used for sequence validation and gap filling. A phylogenetic analysis of EBV strain in C666-1 cells and other reported EBV strains was performed.ResultsA 171,317 bp complete EBV genome of C666-1 was successfully constructed (GenBank accession number: KC617875). Phylogenetic analysis of EBV genome in C666-1 revealed that the C666-1 EBV strain is closely related to the reported strains in NPC primary tumors.ConclusionC666-1 contains a representative NPC-associated EBV genome and might serve as an important model for studying the roles or function of viral proteins in NPC tumorigenesis.

Genomic and proteomic biomarkers for diagnosis and prognosis of hepatocellular carcinoma

Hepatocellular carcinoma is one of the most deadly liver malignancies found worldwide, with hepatitis virus infection being the prominent risk factor for this lesion. Patients with hepatocellular carcinoma are usually first diagnosed when in the advanced stage; thus, long-term clinical outcomes are poor and patients have limited treatment options. Currently, surveillance of hepatocellular carcinoma relies upon serological testing of alpha-fetoprotein levels and hepatic ultrasonography, which have low sensitivity and specificity, and are sometimes operator-dependent, respectively. Therefore, discovery of new biomarkers for early and accurate detection of hepatocellular carcinoma would be of great clinical value. Genomic and proteomic approaches are two major laboratory platforms for the identification of candidate hepatocellular carcinoma biomarkers based on profiling and validating with tumor and nontumor clinical samples. Frequently, these diagnostic markers have been found in association with genetic aberrations, protein-level alterations, post-translational modifications and immune functions. With the discovery of these biomarkers, earlier detection of hepatocellular carcinoma in high-risk subjects (e.g., cirrhosis and hepatitis carriers) becomes possible, which will enable clinicians to offer patients better clinical management and more effective treatment modalities.

De novo sarcoma of donor origin in a liver allograft determined by microsatellite analysis: A short report

This case report describes a patient who underwent liver transplantation for HCV cirrhosis for hepatocellular carcinoma. At 3.5 years post transplant, 6 cm tumor was found with CT scanning. With microsatellite analysis it was determined that the tumor was of donor origin. The patient underwent successful right hepatectomy of the tumor that proved to be sarcoma. The patient is doing well without recurrence 1.5 years after resection and 5 years post transplant. (Liver Transpl 2004;10:320–323.)

Abstract 3425: Whole-transcriptome analyses of EBV-associated nasopharyngeal carcinoma using next-generation transcriptome sequencing

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CAnnNasopharyngeal carcinoma (NPC) is unique among epithelial cancers arising from the head and neck regions. This specific cancer type is consistently associated with EBV infection. Using RNA-seq, we have conducted a comprehensive assessment of EBV and cellular gene expression in six NPC cell line and patient-derived xenografts (PDXs). In these tumor lines, expression of EBV latent genes including EBNA1, LMP1, LMP2A and BamH1 A transcripts was quantitated. Among the viral genes, the BamH1 A transcripts were detected as the major transcripts with various splicing variants. Expression of these transcripts in NPC was further validated by qRT-PCR analysis. Active lytic gene expression and lytic viral replication was not observed in these tumor lines. Analysis of the RNA-seq data has identified multiple fusion transcripts. A total of 18 candidate fusions were validated in these tumor lines. A novel fusion transcript, UBR5-ZNF423 was detected in C666-1 cells and 8.3% of primary tumors. The transcript produces a chimeric fusion protein which might alter the function of EBF transcription factor. A significant growth inhibitory effect was observed in UBR5-ZNF423 knockdown C666-1 cells. Constitutive expression of UBR5-ZNF423 fusion in NIH3T3 cells induced in vivo tumor formation in nude mice model. The findings strongly suggest that the fusion gene play a causative role in NPC tumorigenesis. Using the expression profile of immortalized nasopharyngeal epithelial cells as control, our study also revealed the differential expression of cellular genes and specific alternative spliced transcripts in NPC. Significant upregulation of genes involved in cytokine-cytokine receptor interaction (e.g. CCL5, CCL20, CXCL10, CXCR4, IL2RG) was consistently detected in the tumor lines. Most of them were targets of the NF-kappaB signaling pathways, which are constitutively activated in EBV-associated NPC. In addition, the analyses also discovered several potential druggable targets (e.g. CD74) and oncogenic non-coding RNAs (e.g. AFAP1-AS1) in NPC, which may contribute to the development of new therapeutic strategies for this cancer. The whole-transcriptome analyses greatly enhance our understanding of the molecular basis of EBV-infected NPC.nnCitation Format: Cathy Ka-Yan Mak, Grace Tin-Yan Chung, Kevin Yuk-Lap Yip, Ken Kai-Yuen Tso, Sau-Dan Lee, Siu-Tim Cheung, Sai-Wah Tsao, Pierre Busson, Ka-Fai To, Kwok-Wai Lo. Whole-transcriptome analyses of EBV-associated nasopharyngeal carcinoma using next-generation transcriptome sequencing. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3425. doi:10.1158/1538-7445.AM2014-3425