The Woodchuck Hepatitis Virus Model of Hepatocellular Carcinoma for Interventional Radiology Research.

Overexpression of a p-glycoprotein in hepatocellular carcinomas from woodchuck hepatitis virus-infected woodchucks (Marmota monax)

The leading cause of human hepatocellular carcinomas (HCCs) is hepatitis B virus (HBV) infection. Woodchucks infected with a closely related hepadnavirus, woodchuck hepatitis virus (WHV), serve as a model for HBV because woodchucks chronically infected with WHV also develop hepatocellular carcinomas. Increased expression of p-glycoprotein (pgp) in human HCCs is a common obstacle in successful cancer chemotherapy. Pgps are encoded by a family of multidrug-resistance (MDR) genes. Livers from uninfected and WHV-infected woodchucks were examined to determine if pgp was expressed in HCCs and if there was a difference in expression between HCCs and nonneoplastic liver. A 170-kd protein was identified by Western blot in HCCs, whereas, constitutive pgp was not detected in normal liver taken from the same animals in 3 of 3 cases. Immunolocalization of the pgp with a panel of monoclonal antibodies revealed intensification of staining in 7 of 20 foci and 12 of 22 HCCs from six animals. Using primers for the human MDR1 gene, a single product was detected by reverse-transcribed polymerase chain reaction (RT-PCR) from HCCs. We have shown an increase in pgp in HCCs compared with normal liver from WHV-infected woodchucks. This is the first example of the induction of a pgp in a naturally hepadnavirus infected rodent system. It suggests the woodchuck can be a useful model for the study of the acquisition of resistance to chemotherapeutic agents in virally induced HCCs.

Woodchuck hepatitis virus-induced carcinoma as a relevant natural model for therapy of human hepatoma

Woodchucks (Marmota monax) have a high incidence of hepatocellular carcinoma (HCC) associated with chronic infection with woodchuck hepatitis virus (WHV) and serve as a model of hepatitis B virus-associated HCC in humans. Helicobacter hepaticus, an enterohepatic helicobacter in mice, is known to cause hepatocellular adenomas and carcinomas in susceptible mouse strains. In long-term chemical bioassays conducted with B6C3F(1) mice, H. hepaticus has been regarded as a confounding factor because of its tumor-promoting activity. In order to determine if woodchucks harbor a Helicobacter sp. that might play a role in potentiating hepatic inflammation or neoplasia, a study was undertaken to determine whether woodchucks' livers were infected with a Helicobacter sp. Frozen liver samples from 20 (17 WHV-infected and 3 noninfected) woodchucks, 10 with WHV-associated hepatic tumors and 10 without tumors, were cultured by microaerobic techniques and analyzed by using genus- and species-specific helicobacter PCR primers. A 1,200-bp Helicobacter sp.-specific sequence was amplified from 14 liver samples. Southern hybridization confirmed the specific identity of the PCR products. Nine of the 10 livers with tumors had positive Helicobacter sp. identified by PCR, whereas 5 of the 10 livers without tumors were positive. By use of 16S rRNA species-specific primers for H. marmotae, two additional liver samples from the nontumor group had positive PCR amplicons confirmed by Southern hybridization. A urease-, catalase-, and oxidase-positive bacterium was isolated from one liver sample from a liver tumor-positive woodchuck. By 16S rRNA analysis and biochemical and phenotypic characteristics, the organism was classified as a novel Helicobacter sp. Subsequently, four additional bacterial strains isolated from feces of cats and characterized by biochemical, phenotypic, and 16S rRNA analysis were determined to be identical to the woodchuck isolate. We propose the name Helicobacter marmotae sp. nov. for these organisms. Further studies are required to ascertain if this novel Helicobacter sp. plays a tumor promotion role in hepadnavirus-associated tumors in woodchucks or causes enterohepatic disease in cats.

239 Background: Anti-angiogenic agents have become the backbone of advanced HCC treatment and provide clinical benefit despite infrequent radiographic response. Animal models that mimic human HCC and treatment response are greatly needed. The laboratory woodchuck (Maramota monax) is a unique animal model that replicates the complex human liver milieu of HCC in the context of chronic hepatitis B viral infection and was selected for this translational study. Methods: Humans: Patients with inoperable HCC were consented and prospectively treated with sunitinib 37.5mg PO qd for one week prior to liver directed therapy(chemoembolization). Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) was done pre-sunitinib and after one week as part of the trial. Woodchucks: Laboratory bred eastern woodchucks were inoculated at birth with known titers of sera from a chronic infectious pool of chronic woodchuck hepatitis virus (WHV), confirmed by ultrasound exam at 12-18 months to have developed HCC. Methods for assessment of woodchuck HCC blood flow using DCE-MRI were optimized. Woodchucks received sunitinib 12mg/kg/day poQD and identical image acquisition and analysis techniques were used for both species and identical parameter sets were obtained for cross comparison done by Dr. Ashton in a blinded fashion. Results: A total of seven patients and eight woodchucks with HCC were included (2 scans per patient/woodchuck, pre and post sunitinib). The “Exact Wilcoxon rank-sum test” was used for statistical analysis. Comparison between median Ktrans, AUCBN(90), necrotic and total volumes of the tumors in both groups revealed all parameters to be similar and only total tumor volume reached statistical significance (P=0.002). Conclusions: Our data indicate similarities between human and woodchucks in the tumor histology, blood flow and baseline and post antiangiogenic therapy functional imaging assessment by DCE-MRI. The current sample size of n=7+8 can detect large differences of roughly 1.5 standard deviation units between humans and woodchucks for our measures at alpha=0.05 and power=0.80. Further studies that explore the translational potential of this unique animal model are warranted.

Altered glycosylation of alpha-fetoprotein in hepadnavirus-induced hepatocellular carcinoma of the woodchuck

Tumor immune microenvironment (TIME) has become a new hotspot in cancer research over the past few years. Tumor immune microenvironment of hepatocellular carcinoma (HCC) is especially intriguing as HCC is reported to be highly heterogeneous by previous genomic and cytological studies. It is also closely related to patient prognosis and therapeutic outcome. The recently emerged single-cell RNA sequencing (scRNAseq) technique provides a new tool for TIME study, and current studies have made great advances in defining the roles of TIME in HCC pathogenesis and therapy. Current evidence suggests that heterogeneity is a key player influencing therapeutic response, drug resistance, and prognosis. However, our understanding is limited on the roles of TIME heterogeneity in HCC development, prognosis, and therapeutic response, especially in the era of immunotherapy. This review aims to unravel the heterogeneity of TIME in HCC by scRNAseq, with specific focuses on the cellular, transcriptional, and marker perspectives of TIME heterogeneity in HCC, as well as assessing prognostic and therapeutic response by heterogeneity markers. By summarizing current discoveries regarding TIME heterogeneity, we hope to provide clues on the crucial roles of various cellular components in the development and progression of HCC. We also hope to identify potential markers and therapeutic targets for prognosis assessment and personalized treatment to improve patient outcomes. Combined therapies from multiple dimensions regarding heterogeneity may provide new opportunities to treat HCC more effectively.

Peripheral blood mononuclear cells (PBMCs) from 10 woodchuck hepatitis virus (WHV)-infected woodchucks were examined for the presence of WHV surface (WHs) and core (WHc) antigens (WHsAg and WHcAg) by cytofluorometry using fluorescein isothiocyanate-conjugated anti-WHs and anti-HBc-purified immunoglobulins from woodchuck and human sera. The presence of viral DNA and RNA was detected in the serum and PBMCs from the same blood samples by polymerase chain reaction (PCR) with two primer sets located in the S and C genes of the WHV genome. Seven animals were found positive for both WHsAg and WHcAg on the surface of PBMCs: four WHV-chronic carriers, two WHsAg-positive animals with acute WHV infection, and one woodchuck which was bled during the incubation phase of WHV infection and which became WHsAg-positive only 1 month later. Sixteen to 71% of the studied leukocyte population expressed WHsAg with a low density of expression whereas 7 to 72% expressed WHcAg with a high density of expression. Only two cases were positive for WHsAg without WHcAg on PBMCs, one WHV chronic carrier and one anti-WHs-positive animal. All woodchucks positive for WHcAg and/or WHsAg by cytofluorometry were positive also for WHV DNA and RNA in PBMCs by PCR. The tenth animal was found negative for both viral antigens as well as for WHV DNA and RNA in PBMCs despite the presence of persistent viral DNA in the serum as detected by PCR. Five healthy woodchucks devoid of WHV serological markers served as negative controls. These results obtained with a novel approach further confirm, in the woodchuck model, that a significant proportion of PBMCs are probably permissive for WHV replication. The possible immunopathogenic implications of the phenomenon are discussed.

The immunobiology of hepatocellular carcinoma in humans and mice: Basic concepts and therapeutic implications

15107 Background: HCC is a common and rapidly fatal cancer. Current screening tools are inadequate for identification of potentially curable cases. Our aim was to determine whether H-NMR can identify HCC compared to controls in the woodchuck (WC) model of hepatitis related HCC. Methods: Eastern WCs were bred and inoculated at birth with dilute sera from WCs that are chronic carriers of Woodchuck Hepatitis B Virus (WHV). This resulted in chronic hepatitis in ∼60% animals and all carriers developed HCC by 24–36 months. Serum from 10 chronic WHV carriers with HCC (group 1), 5 WHV carriers with no HCC (group 2) and 15 matched non-infected controls (group 3) was obtained. 45uL serum was diluted with 5uL of D2O containing 27mM formic acid + 0.9% saline. Spectra were collected on a 600 MHz INOVA spectrometer using a CapNMR flow probe with 10uL flow cell at 298K without knowledge of group assignments. The resulting 1D spectra were processed using Nuts from AcornNMR. Results: Principle component analysis and supervised PLS-DA was performed using Simca P+ from Umetrics. Despite general separation of groups, the Q2 value of this model was relatively low (0.20). We trained a Support Vector Machine (SVM) algorithm, a supervised machine-learning algorithm, to learn to identify the groups. Evaluation of the performance of the algorithm using 10-fold validation on the data set achieved a Kappa value of 0.43. This algorithm learnt to identify HCC [0.765 ROC, 0.8 sensitivity, and 0.727 positive predictive value (PPV)] and controls (0.75 ROC, 0.69 sensitivity and 0.73 PPV) but not the WHV carrier group, likely due to the small numbers. In a second analysis of 10 HCC and 15 controls, PLS-DA showed clear separation using three components (Q2= 0.5). The corresponding SVM model showed a kappa value of 0.52 and ROC values of 0.767 for both classes. Conclusions: Our preliminary results indicate that H-NMR spectra alone can be used to distinguish HCC from healthy controls using the machine-learning algorithm for classification. Further validation in a larger cohort of woodchucks is ongoing and confirmation of these preliminary findings would support investigation of this technique as a screening tool in patients at risk for developing HCC. No significant financial relationships to disclose.

The woodchuck hepatitis virus (WHV)/woodchuck system is studied as animal model of human hepatocellular carcinoma (HCC) induced by chronic hepatitis B virus infection. The aim of the present study was the evaluation of ultrasound (US) liver examination in woodchuck as a routine method to detect HCC nodules and to follow their growth. Sixteen woodchucks were included in the study. US liver examination was carried out in all animals using a 5 MHz convex scanner. Macroscopic and microscopic examinations were performed to evaluate the US findings. The lower limit of nodule detection by US examination was a diameter of 5 mm. Macroscopic and microscopic examinations confirmed US findings in 14 of 16 animals (86.6%). No false negative results were obtained. Increase of nodule size was faster in the early phase of tumour growth. Small nodules (16 +/- 5 mm) appeared as hypoechoic lesions with well-defined margins and homogeneous structure. Large nodules (42 +/- 19 mm) appeared as hyperechoic lesions with irregular margins, heterogeneous or of mixed pattern; microscopical examination showed different degrees of necrosis, inflammation and fibrosis inside these latter neoplasms. The hepatitis reaction was conspicuously more severe around HCC nodules. No fibrosis and/or cirrhosis were found in normal liver parenchyma surrounding tumour nodules. On the whole, US appears to be helpful in the diagnosis of woodchuck HCC even at an early stage. Serial US evaluation can be used to study the growth rate of tumour nodules during natural history or experimental HCC treatments in woodchuck.

BackgroundWoodchucks chronically infected with woodchuck hepatitis virus (WHV), which resembles human hepatitis B virus, develop spontaneous hepatic tumors and may be an important biological and immunological model for human HCC. Nonetheless, this model requires further validation to fully realize its translational potential.MethodsWoodchucks infected at birth with WHV that had developed HCC (n=12) were studied. Computed tomography, ultrasound, and magnetic resonance imaging were performed under anesthesia. LI-RADS scoring and correlative histologic analysis of sectioned tissues were performed. For immune characterization of tumors, CD3 (T cells), CD4 (T helpers), NCAM (Natural killers), FOXP3 (T-regulatory), PDL-1 (inhibitory checkpoint protein), and the human hepatocellular carcinoma (HCC) biomarker alpha-fetoprotein (AFP) immunohistochemical stains were performed.ResultsForty tumors were identified on imaging of which 29 were confirmed to be HCC with 26 categorized as LR-4 or 5. The remainder of the tumors had benign histology including basophilic foci, adenoma, and lipidosis as well as pre-malignant dysplastic foci. LR-4 and LR-5 lesions showed high sensitivity (90%) and specificity (100%) for malignant and pre-malignant tumors. Natural killers count was found to be 2–5 times lower in tumors relative to normal parenchyma while other immune cells were located in the periphery of tumors. Tumors expressed AFP and did not express PD-L1.ConclusionWoodchucks chronically infected with WHV developed diverse hepatic tumor types with diagnostic imaging, pathology, and immune patterns comparable to that in humans. This unique animal model may provide a valuable tool for translation and validation of novel image-guided and immune-therapeutic investigations.

A vector based on Semliki Forest virus (SFV) expressing high levels of interleukin-12 (SFV-enhIL-12) has previously demonstrated potent antitumoral efficacy in small rodents with hepatocellular carcinoma (HCC) induced by transplantation of tumor cells. In the present study, the infectivity and antitumoral/antiviral effects of SFV vectors were evaluated in the clinically more relevant woodchuck model, in which primary HCC is induced by chronic infection with woodchuck hepatitis virus (WHV). Intratumoral injection of SFV vectors expressing luciferase or IL-12 resulted in high reporter gene activity within tumors and cytokine secretion into serum, respectively, demonstrating that SFV vectors infect woodchuck tumor cells. For evaluating antitumoral efficacy, woodchuck tumors were injected with increasing doses of SFV-enhIL-12, and tumor size was measured by ultrasonography following treatment. In five (83%) of six woodchucks, a dose-dependent, partial tumor remission was observed, with reductions in tumor volume of up to 80%, but tumor growth was restored thereafter. Intratumoral treatment further produced transient changes in WHV viremia and antigenemia, with >or=1.5-log(10) reductions in serum WHV DNA in half of the woodchucks. Antitumoral and antiviral effects were associated with T-cell responses to tumor and WHV antigens and with expression of CD4 and CD8 markers, gamma interferon, and tumor necrosis factor alpha in peripheral blood mononuclear cells, suggesting that immune responses against WHV and HCC had been induced. These experimental observations suggest that intratumoral administration of SFV-enhIL-12 may represent a strategy for treatment of chronic HBV infection and associated HCC in humans but indicate that this approach could benefit from further improvements.

New technological developments have frequently preceded major advances in biomedical research and medicine [1]. For example, the development of fluorescent DNA sequencing techniques made it possible to establish the large-scale high-throughput technology needed for human genome sequencing. Polymerase chain reaction (PCR), fluorescent DNA sequencing, and other techniques have enabled the discovery of about 1700 mendelian disease genes [2]. The advent of the DNA microarray based technologies has now made it possible to measure simultaneously the expression of tens of thousands of genes in different tissues under a variety of conditions. This high-throughput technology has afforded biomedical scientists a unique opportunity to integrate the descriptive characteristics (i.e. ‘phenotype’) of a biological system under study with the genomic readout (i.e. gene expression). The opportunity to contemplate the integrated view of biological systems has provoked a shift in biological sciences away from the classical reductionism to systems biology [1,3,4]. The systems approach to a disease is based on the hypothesis that disease processes perturb a regulatory network of genes and proteins in a way that differs from the respective normal counterpart. Consequently, by using multi-parametric measurements it may be possible to transform current diagnostic and therapeutic approaches and enable a predictive and preventive personalized medicine [4]. The application of microarray technologies to characterize tumors at the gene expression level has significantly impacted clinical oncology [5,6]. Global gene expression analysis of various human tumors has resulted in

The objective of this study was to evaluate, by developing one-step real-time PCR, the outcome of superinfection with hepatitis D virus (HDV) genotype I in woodchucks that were chronic carriers of woodchuck hepatitis virus (WHV) and did not show relevant signs of liver damage. Three woodchucks (Marmota monax) chronically infected with WHV were superinfected with a woodchuck HDV inoculum. The evolution of the WHV and HDV infections was monitored by quantifying HDV-RNA, WHV-DNA, and HDV-WHV antigens and antibodies. WHV and HDV sequencing was also performed and liver markers were evaluated. Liver damage was assessed using the Ishak method. All woodchucks showed a high HDV viral load, antigenemia and short survival after superinfection. Histopathological examination of autoptic liver samples showed massive liver necrosis compatible with an acute fatal course of hepatitis. The WHV sequencing showed that the virus population was not substituted by the WHV inoculum. The HDV sequencing performed during superinfection and at autopsy indicated amino acid changes in immune dominant regions of the HDV antigen. The strong correlation between acute infection with HDV genotype I and rapid and fatal liver failure indicates that HDV can be an important factor in the prognosis of HDV-WHV-superinfected woodchucks.