MicroRNA Profiling of Laser-Microdissected Hepatocellular Carcinoma Reveals an Oncogenic Phenotype of the Tumor Capsule

Several microRNAs (miRNAs) are associated with the molecular pathogenesis of hepatocellular carcinoma (HCC). However, previous studies analyzing the dysregulation of miRNAs in HCC show heterogeneous results. We hypothesized that part of this heterogeneity might be attributable to variations of miRNA expression deriving from the HCC capsule or the fibrotic septa within the peritumoral tissue used as controls. Tissue from surgically resected hepatitis C–associated HCC from six well-matched patients was microdissected using laser microdissection and pressure catapulting technique. Four distinct histologic compartments were isolated: tumor parenchyma (TP), fibrous capsule of the tumor (TC), tumor-adjacent liver parenchyma (LP), and cirrhotic septa of the tumor-adjacent liver (LC). MiRNA expression profiling analysis of 1105 mature miRNAs and precursors was performed using miRNA microarray. Principal component analysis and consecutive pairwise supervised comparisons demonstrated distinct patterns of expressed miRNAs not only for TP versus LP (e.g., intratumoral down-regulation of miR-214, miR-199a, miR-146a, and miR-125a; P< .05) but also for TC versus LC (including down-regulation within TC of miR-126, miR-99a/100, miR-26a, and miR-125b; P< .05). The tumor capsule therefore demonstrates a tumor-like phenotype with down-regulation of well-known tumor-suppressive miRNAs. Variations of co-analyzed fibrotic tissue within the tumor or in controls may have profound influence on miRNA expression analyses in HCC. Several miRNAs, which are proposed to be HCC specific, may indeed be rather associated to the tumor capsule. As miRNAs evolve to be important biomarkers in liver tumors, the presented data have important translational implications on diagnostics and treatment in patients with HCC

Combination of DNA prime--adenovirus boost immunization with entecavir elicits sustained control of chronic hepatitis B in the woodchuck model.

A potent therapeutic T-cell vaccine may be an alternative treatment of chronic hepatitis B virus (HBV) infection. Previously, we developed a DNA prime-adenovirus (AdV) boost vaccination protocol that could elicit strong and specific CD8+ T-cell responses to woodchuck hepatitis virus (WHV) core antigen (WHcAg) in mice. In the present study, we first examined whether this new prime-boost immunization could induce WHcAg-specific T-cell responses and effectively control WHV replication in the WHV-transgenic mouse model. Secondly, we evaluated the therapeutic effect of this new vaccination strategy in chronically WHV-infected woodchucks in combination with a potent antiviral treatment. Immunization of WHV-transgenic mice by DNA prime-AdV boost regimen elicited potent and functional WHcAg-specific CD8+ T-cell response that consequently resulted in the reduction of the WHV load below the detection limit in more than 70% of animals. The combination therapy of entecavir (ETV) treatment and DNA prime-AdV boost immunization in chronic WHV carriers resulted in WHsAg- and WHcAg-specific CD4+ and CD8+ T-cell responses, which were not detectable in ETV-only treated controls. Woodchucks receiving the combination therapy showed a prolonged suppression of WHV replication and lower WHsAg levels compared to controls. Moreover, two of four immunized carriers remained WHV negative after the end of ETV treatment and developed anti-WHs antibodies. These results demonstrate that the combined antiviral and vaccination approach efficiently elicited sustained immunological control of chronic hepadnaviral infection in woodchucks and may be a new promising therapeutic strategy in patients

Enhancing Virus-Specific Immunity <i>In Vivo</i> by Combining Therapeutic Vaccination and PD-L1 Blockade in Chronic Hepadnaviral Infection

<div><p>Hepatitis B virus (HBV) persistence is facilitated by exhaustion of CD8 T cells that express the inhibitory receptor programmed cell death-1 (PD-1). Improvement of the HBV-specific T cell function has been obtained <i>in vitro</i> by inhibiting the PD-1/PD-ligand 1 (PD-L1) interaction. In this study, we examined whether <i>in vivo</i> blockade of the PD-1 pathway enhances virus-specific T cell immunity and leads to the resolution of chronic hepadnaviral infection in the woodchuck model. The woodchuck PD-1 was first cloned, characterized, and its expression patterns on T cells from woodchucks with acute or chronic woodchuck hepatitis virus (WHV) infection were investigated. Woodchucks chronically infected with WHV received a combination therapy with nucleoside analogue entecavir (ETV), therapeutic DNA vaccination and woodchuck PD-L1 antibody treatment. The gain of T cell function and the suppression of WHV replication by this therapy were evaluated. We could show that PD-1 expression on CD8 T cells was correlated with WHV viral loads during WHV infection. ETV treatment significantly decreased PD-1 expression on CD8 T cells in chronic carriers. <i>In vivo</i> blockade of PD-1/PD-L1 pathway on CD8 T cells, in combination with ETV treatment and DNA vaccination, potently enhanced the function of virus-specific T cells. Moreover, the combination therapy potently suppressed WHV replication, leading to sustained immunological control of viral infection, anti-WHs antibody development and complete viral clearance in some woodchucks. Our results provide a new approach to improve T cell function in chronic hepatitis B infection, which may be used to design new immunotherapeutic strategies in patients.</p></div

Kinetics of PD-1 expression on CD8 T cells during acute WHV infection.

<p>Four adult woodchucks were inoculated with 1×10⁷ or 1×10⁹ WHV genome equivalents. Sera and PBMCs of infected woodchucks were freshly isolated at indicated time points. WHV viral loads (black dots) were quantified by real-time PCR, and percentages of PD-1+ CD8 (CD3+ CD4−) T cells (gray squares) were analyzed by FACS. LOD: lower limit of detection.</p

<i>In vivo</i> PD-L1 blockade synergizes with therapeutic vaccination to control WHV replication.

<p>Serum WHV DNA (A) and WHsAg (B) concentrations of different treatment groups are presented at indicated time points. <b>C</b>: control group without any treatment; <b>E</b>: ETV treated only group; <b>ED</b>: ETV in combination with DNA vaccinations; <b>EDA</b>: ETV and DNA vaccination in combination with anti-PDL1 antibody treatment. LOD: lower limit of detection. (C) The kinetics of serum anti-WHs antibodies levels of woodchucks with triple combination treatment is presented. Anti-WHs antibodies level was determined using the following formula: S/N ratio = sample OD value/negative control OD value. Cut-off value of S/N ratio was 2.1. (D) Detection of WHV replication intermediates in liver tissues of differently treated woodchucks. Liver samples were taken 14 weeks after cessation of ETV treatment. Total DNA were extracted from the liver samples and subjected to Southern blotting (upper), PCR (middle), and realtime PCR (lower). RC: relaxed circular DNA, DL: double stranded linear DNA, SS: single stranded DNA. (E) Detection of WHV cccDNA in liver tissues of differently treated woodchucks. Extracted liver DNA samples were digested with Plasmid-Safe ATP-Dependent DNase and subjected cccDNA-specific PCR with primers amplifying the WHV gap-spanning region.</p

Expression of PD-1 on PBMCs and T cells during chronic WHV infection.

<p>(A) PBMCs were isolated from naïve (n = 4), WHV infection resolved (n = 4), and untreated chronically WHV infected woodchucks (n = 7). The absolute copy numbers of PD-1 mRNA of total PBMCs were analyzed by real-time PCR. The percentages of PD-1+ CD8 (CD3+ CD4−) T cells were analyzed by FACS. (B) The mean fluorescence intensity (MFI) of PD-1 expression on CD8 T cells was calculated and compared between different groups. Representative dot plot shows the population of PD-1^hi CD8 T cells of chronically WHV infected woodchucks. (C) PD-1 expression in chronically WHV infected woodchucks before and 6 weeks after ETV treatment (n = 5) were compared. Left: The absolute copy numbers of PD-1 mRNA of total PBMCs were analyzed by real-time PCR. Right: The percentages of PD-1+ CD8 T cells (black dots) are coordinated with the WHV viral loads (gray columns) in ETV treated woodchucks. (D) The changes of mean fluorescence intensity (MFI) of PD-1 expression on CD8 T cells before and 6 weeks after ETV treatment were compared. Representative dot plot shows the disappearance of PD-1^hi CD8 T cells in chronically WHV infected woodchucks after ETV treatment. (E) The percentages of PD-1+ CD4 T cells (CD3+ CD4+) were analyzed by FACS and compared to that of CD8 T cells (CD3+ CD4−). Representative dot plot shows the populations of PD-1+ CD4 T cells and PD-1+ CD8 T cells of chronically WHV infected woodchucks. (F) The percentages of PD-1+ CD4 T cells of chronically WHV infected woodchucks before and after ETV treatment (n = 5) were compared.</p

Liver transaminase GOT levels of woodchucks with combination treatment.

<p>The GOT values (gray areas) of the woodchuck sera were measured up to the end of the observation period. The duration of ETV treatment is indicated with a gray box, DNA vaccinations are indicated with black triangles, and αPD-L1 treatment is indicated with a black arrow. WHcAg-specific CD8 T cell responses of the woodchucks are indicated with black bars.</p

<i>In vivo</i> PD-L1 blockade synergizes with therapeutic vaccination to enhance WHV-specific T cell immunity.

<p>(A) WHcAg-specific T cell responses of differently treated woodchucks were analyzed by CD107a degranulation assay. The kinetics of WHcAg-specific CD8 T cell response of 4 differently treated groups of woodchucks is presented. <b>C</b>: control group without any treatment; <b>E</b>: ETV treated only group; <b>ED</b>: ETV in combination with DNA vaccinations; <b>EDA</b>: ETV and DNA vaccination in combination with anti-PDL1 antibody treatment. (B) Upper: Comparison of the strength of WHcAg-specific T cell response of woodchucks with different treatments at week 25 and week 38. Lower: Representative dot plots show the population of CD107a+ CD8 T cells of treated woodchuck PBMCs in response to WHcAg-specific peptide stimulation (week 25). (C) WHcAg-specific T cell responses of differently treated woodchucks were analyzed by proliferation assay. The kinetics of PBMCs proliferation in response to WHV core protein stimulation of woodchucks with different treatments is presented. (D) Comparison of the strength of PBMCs proliferation of woodchucks with different treatments at week 38. Stimulation index (SI) is calculated with the formula: (stimulated cpm - blank cpm)/(unstimulated cpm - blank cpm).</p