Impact of non-neoplastic vs intratumoural hepatitis B viral DNA and replication on hepatocellular carcinoma recurrence.

BACKGROUND: This study aims to determine the impact of intracellular hepatitis B virus (HBV) DNA, covalently closed circular DNA (cccDNA) and viral replicative activity in both tumour and non-neoplastic liver on prognosis and to determine the relationship of viral replicative activity and Ishak fibrosis in predicting outcome following resection. METHODS: A total of 99 prospectively enrolled patients treated with primary liver resection for HBV-HCC are included. Intracellular HBV DNA and cccDNA were quantitated by real-time PCR. The RNA-sequencing (RNA-seq) was performed in a subset of 21 patients who had either minimal liver fibrosis (Ishak stages 0-2) or end-stage fibrosis (Ishak stage 6). RESULTS: Tumour tissue contained a lower cccDNA copy number compared with paired non-neoplastic liver, and larger tumours (>3 cm) had less cccDNA compared with small tumours (⩽3 cm). High viral replicative activity in non-neoplastic liver was associated with higher HCC recurrence rate independent of Ishak fibrosis stage. Genes correlated with viral replicative activity in non-neoplastic liver (620 genes) were distinct from those associated with end-stage fibrosis (1226 genes). Genes associated with viral replicative activity were preferentially distributed in regions on chr3, chr16 and chr19. CONCLUSIONS: Viral replicative activity in non-neoplastic liver is associated with HCC recurrence through mechanisms that are distinct from and independent of Ishak fibrosis stage.

Autologous Tumor Cell Lysate-Loaded Dendritic Cell Vaccine Inhibited Tumor Progression in an Orthotopic Murine Model for Hepatocellular Carcinoma.

The immune status of the tumor microenvironment influences tumor progression, and hepatocellular carcinoma (HCC) with an immunosuppressive signature often is associated with a poor prognosis. This study examined the impact of a bone marrow-derived dendritic cell (DC) vaccine loaded with autologous tumor cell lysate on tumor progression and the tumor microenvironment using an orthotopic murine HCC model. An orthotopic murine HCC was established by implantation of Hepa1-6 cells in the liver. The impact of DC vaccine loaded with Hepa1-6 cell lysate on tumor progression, survival, and tumor-infiltrating lymphocytes and cytokines was examined. Treating mice with DC vaccine loaded with Hepa1-6 cell lysate inhibited the progression of murine HCC generated through orthotopic implantation of Hepa1-6 cells and resulted in a 90 % survival rate by day 60 compared with a survival rate lower than 5 % for untreated mice. This anti-tumor response was associated with inhibition of STAT3 phosphorylation within the tumor. The DC vaccine reduced accumulation of Foxp3+CD4+ regulatory T cells within the tumor microenvironment and prevented TGF-ß production from the tumor tissue. Tumor cell lysate-loaded DC vaccine prevented HCC progression in a clinically relevant orthotopic murine HCC model. The effect of DC vaccine on the accumulation of Foxp3+CD4+ regulatory T cells within the tumor microenvironment and on the production of TGF-ß suggests that tumor regression by DC vaccination may be associated with an altered immunosuppressive tumor microenvironment.

Prognostic Role of Immune Cells in Hepatitis B-associated Hepatocellular Carcinoma Following Surgical Resection Depends on Their Localization and Tumor Size.

This study aims to evaluate localized expression of CD4, interleukin (IL)-17, Foxp3, and CD8 in hepatitis B-associated hepatocellular carcinoma (HBV-HCC) and to explore their potential effects on outcome following surgical resection. This prospective study includes 66 HBV-HCC surgical resection patients enrolled from 2008 to 2013. CD4, IL-17, Foxp3, and CD8 mRNA in 4 regions of the resection specimens (center of the tumor, periphery of the tumor, non-neoplastic liver bordering tumor, non-neoplastic liver distant from tumor) was quantitated using real-time polymerase chain reaction. The tumoral regions had lower CD4 and CD8 expression as compared with paired non-neoplastic regions, whereas the expression of IL-17 and Foxp3 did not differ. High Foxp3 in all regions except non-neoplastic liver distant from tumor was associated with poor overall survival, whereas low CD8 expression in distant non-neoplastic liver may be associated with high HCC recurrence rate. Although the expression of almost all molecules did not differ between small (≤3 cm) and large HCC (>3 cm), high IL-17 in periphery of tumor, high CD8 in center of tumor, or low CD8 in distant non-neoplastic liver was associated with high HCC recurrence rate in patients with small HCC, but not in those with large HCC. The effect of immune cells on HCC progression therefore depends on the expression level, localization, and tumor size, and an imbalance toward regulatory T cells is associated with poor outcome.

Serum hepatitis B surface antigen correlates with tissue covalently closed circular DNA in patients with hepatitis B-associated hepatocellular carcinoma.

Quantitation of hepatitis B surface antigen (HBsAg) in hepatitis B-associated hepatocellular carcinoma (HBV-HCC) remains to be clearly defined. This study aims to determine the association of HBsAg quantity with intrahepatic HBV viral load and activity in both tumor and non-neoplastic liver of HBV-HCC patients. Data were obtained from 89 prospectively enrolled patients treated with primary liver resection for HBV-HCC at a single Western institution (2008-2013). Circulating HBsAg was quantitated using ELISA. HBV DNA, covalently closed circular (cccDNA) and precore-pregenomic RNA (preC-pgRNA) in both tumor and non-neoplastic liver were quantitated by real-time PCR from fresh liver resection specimens. Circulating HBsAg was detectable in all 89 patients. HBsAg negatively correlated with age, and positively correlated with pre-operative serum AFP and ALT levels. HBsAg correlated with HBV cccDNA copy number in tumor or non-neoplastic liver tissue. It also correlated with preC-pgRNA copy number in non-neoplastic liver tissue. HBsAg did not correlate with serum HBV DNA, total intrahepatic HBV DNA, viral replicative activity or transcriptional activity. In HBV-HCC patients, HBsAg levels correlated with cccDNA copy number in tumor or non-neoplastic liver tissue, suggesting that a greater pool of cccDNA is associated with a higher rate of HBsAg production.

The Severity of Liver Fibrosis Influences the Prognostic Value of Inflammation-Based Scores in Hepatitis B-Associated Hepatocellular Carcinoma.

BACKGROUND: This study was designed to evaluate the prognostic value of three systemic inflammation markers, neutrophil to lymphocyte ratio (NLR), platelet to lymphocyte ratio (PLR), and prognostic nutritional index (PNI), for hepatocellular carcinoma (HCC) associated with hepatitis B (HBV). METHODS: This analysis included 234 HBV-HCC patients who underwent primary surgical resection at the Mount Sinai Medical Center between 1988 and 2013. Serum albumin and circulating neutrophil, lymphocyte, and platelet counts immediately before surgery were obtained to calculate NLR, PLR, and PNI. RESULTS: Patients with larger tumor size (>3 cm) had higher NLR, higher PLR, and lower PNI. Stratified analysis showed that the impact of three markers on outcome depends on the severity of liver fibrosis. High NLR, high PLR, or low PNI was associated with poor outcome only in patients without end-stage fibrosis (Ishak stage 0-5) and not in those with cirrhosis (Ishak stage 6). Multivariate analysis in Ishak stage 0-5 patients showed that only high NLR was associated with poor outcome independent of tumor size. Of the three markers, only NLR correlated with PD-L1 expression in center of tumor, but not in nonneoplastic liver. CONCLUSIONS: The prognostic value of these three markers following surgery was only significant for HBV-HCC patients without end-stage fibrosis, and among the three markers, only NLR remained a significant prognostic indicator independent of tumor size. The correlation of NLR with intratumoral PD-L1 expression raises a hypothesis for shared pathways leading to PD-L1-mediated local tolerance within tumor and systemic inflammatory responses represented by elevated NLR in HBV-HCC.

Assessing prognostic significance of preoperative alpha-fetoprotein in hepatitis B-associated hepatocellular carcinoma: normal is not the new normal.

BACKGROUND: Hepatitis B (HBV)-associated hepatocellular carcinoma (HCC) is often associated with alpha-fetoprotein (AFP) production. Although serum AFP has been demonstrated to be a prognostic factor for patient survival, optimal cutoff levels remain unclear. METHODS: Patients with HBV-associated HCC treated by primary liver resection were prospectively followed at a single institution between 1995 and 2008. AFP level was categorized into quintiles for Kaplan­Meier analysis and multivariable Cox proportional hazards regression models. RESULTS: Best 5-year survival after surgery was observed for patients with AFP in the first quintile (1.4-4.1 ng/mL), with progressively worse outcomes for patients in each increasing quintile. AFP was associated with overall survival (HR = 1.61; 95 % CI 1.30-1.98), disease-free survival (HR = 1.26; 95 % CI 1.10-1.44), and 2-year recurrence (HR = 1.30; 95 % CI 1.07-1.57) in multivariate analysis. Noncirrhotic patients (Ishak 1-5) with AFP in quintile 1 had 94 % 5-year survival, compared with 0 % survival for patients with AFP in quintile 5 (2,332.7-327,560.0 ng/mL) and Ishak stage 6 cirrhosis. CONCLUSIONS: Preoperative serum AFP is an independent predictor of prognosis among HBV-HCC patients following surgical resection. Categorizing AFP into quintiles creates the opportunity to observe differences in outcomes even at low serum levels within the normal range. Additionally, combining AFP quintiles and fibrosis staging provides a predictive model of prognosis for HCC. Thus, even small differences in AFP within the normal range may impact prognosis and disease progression for HBV-HCC.

Peritoneal carcinomatosis in patients with gastric cancer, and the role for surgical resection, cytoreductive surgery, and hyperthermic intraperitoneal chemotherapy.

BACKGROUND: The aims of this study were to create a model of peritoneal carcinomatosis in patients with gastric cancer and to evaluates outcomes in patients with gastric cancer treated using surgery and hyperthermic intraperitoneal chemotherapy (HIPEC). METHODS: A single-institution cohort of patients with gastric cancer was analyzed according to the development of gastric cancer with peritoneal carcinomatosis (GCPC). Variables were evaluated using regression analysis. Kaplan-Meier analysis was used to evaluate outcomes after surgical resection, cytoreductive surgery, and HIPEC. RESULTS: Age ≤60 years and local tumor stage (T3/T4) were significantly associated with GCPC (odds ratio, 3.95 and 3.94, respectively). Thirty-six-month survival was 57% for patients without peritoneal disease and 39% for patients with GCPC. There was no significant trend of improved survival after surgical management or HIPEC. CONCLUSIONS: Age ≤60 years and T3/T4 tumor stage are risk factors for GCPC. Intermediate-term survival of patients with GCPC treated with surgical resection or cytoreductive surgery and HIPEC was not improved, though future research should address the possible benefits of aggressive approaches to the treatment of GCRC.

Impact of intrahepatic hepatitis B DNA and covalently closed circular DNA on survival after hepatectomy in HBV-associated hepatocellular carcinoma patients.

BACKGROUND: Chronic hepatitis B virus (HBV) infection induces persistent but ineffective immune activation that contributes to necroinflammation, fibrosis, and risk of hepatocellular carcinoma (HCC). This study aims to determine the relationship of intrahepatic total HBV (ih HBV) DNA and covalently closed circular DNA (cccDNA) with necroinflammation and fibrosis, and their impact on prognosis after resection in HBV HCC patients. METHODS: Data are from 111 patients treated with primary liver resection for HBV HCC at Mount Sinai, New York (1991-2008). ih HBV DNA and cccDNA were quantitated by real-time PCR. Necroinflammation was graded according to histologic activity index (HAI), and liver fibrosis was staged by the modified Ishak method. RESULTS: A total of 106 (95%) and 89 patients (80%) had detectable ih HBV DNA and cccDNA, respectively, while 43% had detectable serum HBV DNA. cccDNA made up a small proportion of ih HBV DNA (median cccDNA/ih HBV DNA ratio = 0.022). Higher levels of ih HBV DNA were associated with higher HAI and serum alanine aminotransferase (ALT), while a lower ratio of cccDNA/ih HBV DNA was associated with higher HAI, ALT, and Ishak fibrosis stage. ih HBV and cccDNA were not associated with survival, but the lowest quintile of cccDNA/ih HBV DNA ratio (<0.0024) was independently associated with poor overall survival. CONCLUSIONS: A lower cccDNA/ih HBV DNA ratio was associated with greater necroinflammation and liver fibrosis, and was independently associated with poor overall survival. Thus, intracellular virus loads and relative proportions of virus DNA reflect histologic damage in the liver and influence the clinical outcome of HBV HCC patients.