Objective: α-fetoprotein (AFP) is utilized as a biomarker in patients with hepatocellular carcinoma (HCC) to predict disease recurrence after curative resection. It is not clear whether a peri-operative drop of AFP suggests any prognostic correlation. This study aims to review the specific peri-operative kinetics of serum AFP in patients with hepatitis B-associated hepatocellular carcinoma (HBV-HCC) and their outcomes. Methods: Data was analyzed from 175 HBV-HCC patients who underwent liver resection with curative intent at a single institution. Patients had pre-operative serum AFP measurement and paired post-operative AFP measurement within 8-24 weeks of surgery. Quintiles of AFP were used to categorize AFP levels, and the impact on outcome was analyzed by univariate and multivariate analysis. Results: Sixty percent of all patients (quintiles 1–3) had post-operative AFP lower than 5.8 ng/ml within 8–24 weeks of surgery. These patients did not differ significantly in overall survival or disease-free survival when stratified based on pre-operative AFP levels. 35 patients (quintile 5) had post-operative AFP greater than 30.4 ng/ml, and these patients had significantly lower 5 year overall survival (31%, p = 0.008) compared to patients with post-operative AFP <30.4 ng/ml (OS = 82%). 83% of patients in Q5 had HCC recurrence, all of which occurred within 24 months of surgery. Multi-variate analysis showed that post-operative AFP greater than 30.4 ng/ml was significantly associated with poor overall survival independent of other variables (HR 5.1, p < 0.001). Conclusion: Post-operative AFP within 8–24 month of surgery identifies a subset of patients with HBV-HCC at high risk of recurrence, regardless of pre-operative AFP levels.

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.

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. 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). 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. Viral replicative activity in non-neoplastic liver is associated with HCC recurrence through mechanisms that are distinct from and independent of Ishak fibrosis stage.

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-β 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-β suggests that tumor regression by DC vaccination may be associated with an altered immunosuppressive tumor microenvironment.

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.

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.

e14023 Background: The immune status of the tumor microenvironment influences tumor progression in HCC. Regulatory T cells (Tregs) inhibit effector cell responses, and play a pivotal role in effective cancer surveillance. This study examines the role on overall survival of intratumoral Tregs in patients with HCC, and evaluates the impact of autologous dendritic cell (DC) immunotherapy on Treg depletion, tumor progression, and overall survival in an orthotopic murine HCC model. Methods: The expression of Foxp3 and CD4 in 63 paired tumor and non-neoplastic liver resection tissue specimens of HCC patients was evaluated using RT-PCR. Orthotopic murine HCC was established by implanting Hepa1-6 cells in the liver. The effect of DC immunotherapy loaded with Hepa1-6 cell lysate on tumor progression, survival, tumor infiltrating lymphocytes and cytokines was examined. Results: High intratumoral Foxp3 expression in HCC patients treated with resection was associated with poor overall survival. DC immunotherapy in HCC bearing mice resulted in reduced Foxp3+CD4+ regulatory T cell accumulation and TGF-b production within the tumor microenvironment. Moreover, DC immunotherapy reversed murine HCC progression, as assessed by plasma a-fetoprotein levels and in vivo bioluminescence imaging, and increased survival to 90% by day 60, as compared to < 5% in untreated tumor-bearing mice. This anti-tumor response was accompanied by inhibition of STAT3 phosphorylation within tumor tissue Conclusions: High levels of intratumoral Tregs in patients undergoing hepatectomy is with associated poor overall survival. Tumor cell lysate-loaded DC immunotherapy results in tumor regression and increased survival in a clinically relevant orthotopic murine HCC model. The inhibitory effect of DC immunotherapy on the accumulation of Foxp3+CD4+ regulatory T cells within the tumor microenvironment and on the production of TGF-b suggests that effective DC immunotherapy can alter the immunosuppressive tumor microenvironment, thus resulting in tumor regression.

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.

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.

BACKGROUND:This study aims to evaluate the impact of liver fibrosis severity on prognosis following liver resection among HBV-HCC patients. METHODS:Data were extracted from a prospective database of 189 HBV-HCC patients treated at Mount Sinai between 1995 and 2008. Fibrosis staging of each surgical resection specimen using the modified Ishak method was performed by a single liver pathologist. RESULTS:A wide range of Ishak fibrosis stage was observed among this patient population, with 29% having established cirrhosis (Ishak stage 6). Ishak stage 6 was independently associated with poor overall and recurrence-free survival. In patients with Ishak stage 1-5, Ishak stage did not affect survival; rather, tumour size was associated with poor overall survival, and tumour size, histologic activity index and serum AFP>20 ng ml(-1) were associated with poor recurrence-free survival. In patients with Ishak stage 6, poorly differentiated histology and tumour size were associated with poor overall survival, and tumour size was associated with poor recurrence-free survival. CONCLUSION:HBV-HCC develops with varying degrees of underlying liver fibrosis; however, progressive liver fibrosis does not affect the outcomes following resection until cirrhosis is reached. Established cirrhosis, as defined histologically by Ishak stage 6, is an independent predictor of poor overall and recurrence-free survival among these patients.

Aims: The intent of this analysis is to assess clinico-pathologic and prognostic characteristics of HCC in patients with minimal liver fibrosis (Ishak stage 1-2) after primary surgical liver resection as compared to patients with moderate to severe fibrosis (Ishak stage 3-6) in order to improve screening and treatment of HCC.Methods: Data were obtained from 200 patients with HBV-related HCC who underwent primary surgical liver resection at a single North American medical institution between 1988 and 2012. A dedicated liver pathologist performed fibrosis staging for each resection specimen using the modified Ishak method. Univariate and multivariate analyses of clinico-pathologic variables were performed to determine those associated with prognosis.Results: Twenty-two percent of patients had minimal fibrosis defined as Ishak stage 1 or 2. Kaplan-Meier analysis of 5-year survival showed a non-significant trend toward better outcome among Ishak 1-2 patients compared to Ishak 3-6 (p = 0.09). Ishak 1-2 was associated with lower hazard of death compared to Ishak 3-6 (adjusted HR = 0.38, 95% CI = 0.15-0.99) Ishak 1-2 retained statistical significance after multivariate analysis for overall survival (p = 0.05), but not recurrence-free survival.Conclusions: A significant proportion of HBV-HCC cases arise in the minimally fibrotic liver. Patients with Ishak 1-2 fibrosis have better overall survival compared to those with Ishak 3-6, indicating that minimally fibrotic patients should be treated as a separate cohort. There is a need to better understand the mechanisms underlying hepatocarcinogenesis and to formulate unique diagnostic and therapeutic algorithms for minimally fibrotic HCC patients. (C) 2013 Elsevier Ltd. All rights reserved.

Background and Aims: Foxp3-expressing regulatory T cells impede effective immune surveillance against cancer and inhibit anti-tumor immune responses, thereby influencing the biological behavior of many, but not all tumors.This study aims to determine the microenvironment-specific influence of Foxp3 expression on tumor biology of hepatitis B-associated hepatocellular carcinoma (HBV-HCC).Methods: This prospective study includes 90 HBV-HCC surgical resection patients enrolled between 2008-2012 at the Mount Sinai Medical Center in New York.Foxp3 mRNA in four 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 for each case using real-time PCR.Resection specimens were assessed by a single dedicated liver pathologist to evaluate tumor characteristics as well as liver fibrosis using the Ishak method.Results: Foxp3 mRNA was detectable in all four regions of all the samples.While no differences were observed in median Foxp3 expression between tumor and non-neoplastic liver, Foxp3 expression in center of the tumor showed the greatest variance and skewed most towards the highest values (variance: 1603, 371, 66 and 110; and skewness: 6.8, 4.0, 3.4, 2.3, in center of the tumor, periphery of the tumor, non-neoplastic liver bordering tumor, nonneoplastic liver distant from tumor, respectively).This high variance in the tumor was due to the high variance in the highest quintile of Foxp3 expression, and was not observed in tumors that were associated with less fibrotic liver (Ishak stage 1-4), or in tumors that were well-differentiated.High Foxp3 expression in the tumor (highest quintile) was associated with mortality (6/17 (35%) vs. 5/64 (8%), p = 0.009).Likewise, mortality was associated with high Foxp3 expression (13.1 vs. 2.7, p = 0.015) and high variance (9325 vs. 524) in center of the tumor, but not in the other three compartments.Conclusions: A small population of HBV-HCC patients (20%) had high Foxp3 expression within the tumor microenvironment that is highly variable and associated with mortality following surgical resection.Therefore, HCC predominated by a Foxp3dependent immune-suppressive environment is associated with poor prognosis following surgical resection.

e14583 Background: Little is known about the prognostic significance of total intrahepatic HBV DNA (ihHBV DNA) and cccDNA, a stable episome that accumulates in the nuclei and serves as the template for viral replication. This study aims to quantitate ihHBV DNA and cccDNA in the non-neoplastic liver and to assess their impact on prognosis in HBV-HCC patients. Methods: 111 patients, many on HBV antivirals, who underwent liver resection for HBV-HCC from 1991 to 2008 were assessed by real time PCR for ihHBV DNA, cccDNA, and albumin. Liver fibrosis and necroinflammation was assessed using the modified Ishak method. Independent variables associated with survival were analyzed using multivariate Cox regression model, with a median follow up for survivors of 52 months. Results: Serum HBV DNA was detectable in only 42% of patients, but 106 patients (95%) had detectable ihHBV DNA (median: 0.018copy/hepatocyte); and 89 patients (80%) had detectable cccDNA (0.00058 copy/hepatocyte). Median cccDNA/ihHBV DNA ratio was 0.019. ihHBV DNA correlated with histologic activity index (p = 0.04) and serum ALT (p = 0.004). Patients in the lowest quartile of cccDNA/ihHBV DNA ratio (<0.0032) trended towards poor overall 5-year survival by univariate analysis (p = 0.09), with higher mortality at 2 years (89% vs. 45%, p = 0.03). In multivariate analysis, AFP > 20, Ishak fibrosis stage 6 (established cirrhosis), cccDNA/ihHBV DNA < 0.0032, and large tumor diameter were independently associated with poor overall survival (Table). Conclusions: ihHBV DNA levels were associated with severity of liver necroinflammation and injury. ihHBV DNA and cccDNA levels were not associated with survival; rather, low proportion of total HBV DNA in the form of cccDNA was independently associated with poor overall survival. Thus, viral behavior at the time of liver resection influences clinical outcome for HBV-HCC patients. [Table: see text]

BACKGROUND:To determine clinical-pathologic variables in patients with a new diagnosis of hepatocellular carcinoma (HCC) and underlying hepatitis B vs. C infection. METHODS:Patients presenting to a single urban hospital with a new diagnosis of HCC were entered into a clinical database. Variables including number and size of tumors, presence of metastases, serum alpha-Fetoprotein, hepatitis serologies, severity of hepatic dysfunction, and presence of cirrhosis were evaluated in 127 patients. RESULTS:Patients with hepatitis B (HBV) were more likely to develop HCC at a younger age than patients with hepatitis C (HCV) (HBV-26% under age 40, HCV-0% under age 40; p < 0.001), with greater serum alpha-Fetoprotein production (median level: HBV-1000 ng/ml vs. HCV-37 ng/ml; p = 0.002), with larger tumors (HBV-78% >5 cm, HCV-28% >5 cm; p < 0.001), in the absence of cirrhosis (HBV-40%, HCV-0%; p < 0.001), and a decreased eligibility for curative treatment (HBV-14%, HCV-34%; p < 0.05). Conversely, patients with HCV were more likely to develop HCC in association with multiple co-morbidities, cirrhosis, and older age. CONCLUSIONS:Significant clinical-pathologic differences exist among HCC patients with underlying HBV vs. HCV. These differences impact eligibility for potentially-curative therapy and prognosis.

OBJECTIVE: The aim of this study was to determine the impact of hepatocelluar carcinoma (HCC) screening in chronic hepatitis B patients who did not meet the current screening recommendations. METHODS: Patients who were admitted to Bellevue Hospital Center with HCC were assessed for risk factors, cirrhosis and tumor-specific factors. Eligibility for liver transplantation or resection with favorable outcome was determined by applying Milan criteria. RESULTS: In all 93 patients were diagnosed with hepatitis B virus (HBV)-associated HCC, 18 of whom were under 40 years. Cirrhosis was infrequently associated with HCC in this group, with most cancers occurring in non-cirrhotic patients (12/ 18, 66.7%). No patient developed HCC outside the American Association for the Study of Liver Diseases (AASLD) cancer screening recommendations (young age, noncirrhotic) were eligible for liver transplantation or resection with favorable outcomes (within Milan criteria). However, HCC patients who were diagnosed within AASLD screening recommendations did meet Milan criteria in 17.3% (14/ 81) patients. CONCLUSIONS: Current guidelines for HCC screening in patients with HBV may lead to a delay in diagnosis in non-cirrhotic patients under 40 years. Consideration should be given to modifying current recommendations to advocate entering HBV patients into a cancer-screening program at young age.