Gaurav Roy

Tackling hepatitis B virus-associated hepatocellular carcinoma—the future is now

Hepatocellular carcinoma (HCC) is one of the most lethal and prevalent cancers in many developing countries including India. Among the various etiological factors being implicated in the cause of HCC, the most important cause, however, is hepatitis B virus (HBV) infection. Among all HBV genes, HBx is the most critical carcinogenic component, the molecular mechanisms of which have not been completely elucidated. Despite its clinical significance, there exists a very elemental understanding of the molecular, cellular, and environmental mechanisms that drive disease pathogenesis in HCC infected with HBV. Furthermore, there are only limited therapeutic options, the clinical benefits of which are insignificant. Therefore, the quest for novel and effective therapeutic regimen against HBV-related HCC is of paramount importance. This review attempts to epitomize the current state of knowledge of this most common and dreaded liver neoplasm, highlighting the putative treatment avenues and therapeutic research strategies that need to be implemented with immediate effect for tackling HBV-related HCC that has plagued the medical and scientific fraternity for decades. Additionally, this review proposes a novel “five-point” management algorithm for HBV-related HCC apart from portraying the unmet needs, principal challenges, and scientific perspectives that are relevant to controlling this accelerating global health crisis.

Novel point and combo-mutations in the genome of hepatitis B virus-genotype D: characterization and impact on liver disease progression to hepatocellular carcinoma.

Background The contribution of chronic hepatitis B virus (HBV) infection in the pathogenesis of hepatocellular carcinoma (HCC) through progressive stages of liver fibrosis is exacerbated by the acquisition of naturally occurring mutations in its genome. This study has investigated the prevalence of single and combo mutations in the genome of HBV-genotype D from treatment naïve Indian patients of progressive liver disease stages and assessed their impact on the disease progression to HCC. Methods The mutation profile was determined from the sequence analysis of the full-length HBV genome and compared with the reference HBV sequences. SPSS 16.0 and R software were used to delineate their statistical significance in predicting HCC occurrence. Results Age was identified as associated risk factor for HCC development in chronic hepatitis B (CHB) patients (p≤0.01). Beyond the classical mutations in basal core promoter (BCP) (A1762T/G1764A) and precore (G1862T), persistence of progressively accumulated mutations in enhancer-I, surface, HBx and core were showed significant association to liver disease progression. BCP_T1753C, core_T147C, surface_L213I had contributed significantly in the disease progression to HCC (p<0.05) in HBeAg positive patients whereas precore_T1858C, core_I116L, core_P130Q and preS1_S98T in HBeAg negative patients. Furthermore, the effect of individual mutation was magnified by the combination with A1762T/G1764A in HCC pathogenesis. Multivariate risk analysis had confirmed that core_P130Q [OR 20.71, 95% CI (1.64–261.77), p = 0.019] in B cell epitope and core_T147C [OR 14.58, 95% CI (1.17–181.76), p = 0.037] in CTL epitope were two independent predictors of HCC in HBeAg positive and negative patients respectively. Conclusions Thus distinct pattern of mutations distributed across the entire HBV genome may be useful in predicting HCC in high-risk CHB patients and pattern of mutational combinations may exert greater impact on HCC risk prediction more accurately than point mutations and hence these predictors may support the existing surveillance strategies in proper management of the patients.

MicroRNAs in hepatocellular carcinoma – therapeutics and beyond: A systematic review

Hepatocellular carcinoma (HCC) causes major cancer-related deaths worldwide. The current therapeutic strategies pitted against early HCC are woefully inadequate with surgical interventions, radiation therapy, and chemotherapy to suffice for. Therefore, the quest for novel, effective, and decisive screening tools is paramount. In context, microRNAs (miRNAs) have emerged as useful biomarkers in HCC. Accordingly, PubMed, Medline, Embase, and Cochrane databases were explored for relevant literature in English with combination of keywords “microRNA and hepatocellular carcinoma,” “microRNA and diagnosis and hepatocellular carcinoma,” “microRNA and prognosis and hepatocellular carcinoma,” “microRNA and survival and hepatocellular carcinoma,” and “microRNA and therapy and hepatocellular carcinoma” that were extracted till January 2017. Manuscripts relating to long noncoding RNAs and other concomitant small molecules involved in HCC were excluded from the review. Studies revealed a plethora of miRNAs and their altered expression profiles being significantly implicated in the diagnosis, prognosis, recurrence, and overall survival in HCC. Several miRNAs are currently being tested in different phases of clinical trials. Efforts should aim at a better validation and establishment of miRNAs as powerful diagnostic and prognostic investigating aid for HCC. However, despite extensive research, a consensus on the universal set of miRNAs to be used as diagnostic, prognostic, or recurrence markers for HCC is yet to be achieved. In addition, various targeted approaches should focus to reduce the possibility of deleterious off-target effects of miRNAs. On this background, this systematic review discusses latest developments on miRNAs as a marker of diagnosis, prognosis, recurrence, overall survival as well as a therapeutic target in HCC (REVIEW registry216).

Expression signature of lysosomal-associated transmembrane protein 4B in hepatitis C virus-induced hepatocellular carcinoma:

Introduction: Hepatocellular carcinoma is a lethal disease worldwide and therefore the establishment of novel diagnostic biomarkers is imperative. In this study, it was hypothesized that an abnormal expression of the lysosomal-associated protein transmembrane 4 beta (LAPTM4B) gene is crucial in the pathogenesis of hepatitis C virus-mediated hepatocellular carcinoma; hence we investigated the expression profile of LAPTM4B in hepatitis C virus-induced hepatocellular carcinoma. Methods: A group of 189 consecutive patients (hepatitis C virus-related hepatocellular carcinoma as tumor cases; n=93, hepatitis C virus-related cirrhotics as disease controls; n=96) opting for living donor liver transplantation as a therapeutic surgical regimen were recruited with informed consent. Additionally, paired adjacent non-tumorous tissues (n=93) obtained from cases were also included. Serum LAPTM4B protein concentrations were assessed by third-generation enzyme-linked immunosorbent assay and LAPTM4B mRNA, and protein expressions at tissue level were determined by quantitative real time reverse transcription-polymerase chain reaction (qRT-PCR) and immunohistochemistry techniques, respectively. Results: LAPTM4B protein concentrations in sera of patients were higher (p<0.001) in tumor cases (1.25±0.25 ng/ml) compared to disease controls (0.53±0.28 ng/ml). Our study also depicts positive clinicopathological correlations between alpha-fetoprotein titers (b=0.65; p<0.001), quantitative hepatitis C virus RNA copies (b=0.33; p<0.001), and LAPTM4B protein concentrations, all in sera of patients. In addition, qRT-PCR and immunohistochemistry analyses revealed a significantly higher (p<0.05) tissue LAPTM4B mRNA and protein expression, respectively, in tumor cases rather than in non-tumorous tissues and disease controls. Conclusions: Together, our results illustrate the LAPTM4B gene as a diagnostic biomarker in patients with hepatocellular carcinoma having documented evidence of chronic hepatitis C virus infection.

Abstract LB-71: Carcinogenic significance of integrated hepatitis b viral factors and its genotype in hepatocarcinogenesis.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Integration of Hepatitis B Virus (HBV) is one of the major causes underlying Hepatocellular carcinoma (HCC) development. Recent studies revealed that altered expression of multiple cellular genes due to multiple random integration of viral factors lead to development of HCC. The present study aims to identify the carcinogenic significance of integrated HBV genome and its genotype in HCC development. Host-Viral Junctions were determined in HCC (T) /adjacent tissues (NT) and cirrhotic (-HCC) tissues by sequencing of Alu PCR product followed by sequence blast. Host gene expression was checked by real time PCR. Competition between two coexisting genotypes was studied by replication assay using genotype specific PCR from media and viral core particle isolated from genotype specific plasmid transfected HepG2 cell line. Similarly, each viral genotype mediated DNA damage (γH2aX foci by confocal microscopy), ROS generation (DCFDA quantitation by Facs analysis and ER stress marker GRP78 luciferase assay), homologous recombination efficiency were also compared. HBV integration was observed in 66.7% of T (8/12), 33.3% (4/12) NT and 75% (6/8) cirrhotic tissue samples in 18, 4 and 8 different locations respectively. These viral integration analysis within or close to several host genes, such as genes involved in differentiation, signaling, stress response, cell cycle, telomere regulations (8/30, 26.7%) etc and most of them showed altered expression. Interestingly, C-terminal truncated HBX which lost its growth suppressive domain were observed more in T (5/8, 62.5%) than NT (2/4, 50%) tissue samples. Most importantly, upon genotype analysis of each five clones in serum and tissue showed genotype C preferentially integrated in coexistence of both C and D genotype as “free virus” in tissue but D genotype observed in circulation. Genotype specific PCR from HBV/C & D cotransfected media showed that in presence of HBV/C, D replicates better than C but HBV/C with high recombination frequency generates more DNA damage and facilitate viral integration. Thus random integration of HBV in host chromosome causes alterations in oncogenic gene expression and these alterations presumably lead to clonal selection of hepatocyte that acquires a growth advantage and proceeds towards HCC. Genotype of HBV is an important determining factor for HBV integration and liver inury. Citation Format: Soma Banerjee, Somenath Datta, Shrabasti Roychoudhury, Debanjali Dasgupta, Amit Ghosh, Gaurav Roy, Simanti Datta, Abhijit Chowdhury. Carcinogenic significance of integrated hepatitis b viral factors and its genotype in hepatocarcinogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-71. doi:10.1158/1538-7445.AM2013-LB-71