Impairment of CD4+ cytotoxic T cells predicts poor survival and high recurrence rates in patients with hepatocellular carcinoma.

UNLABELLED: The role of CD4(+) cytotoxic T cells (CTLs) in hepatocellular carcinoma (HCC) remains obscure. This study characterized CD4(+) CTLs in HCC patients and further elucidated the associations between CD4(+) CTLs and HCC disease progression. In all, 547 HCC patients, 44 chronic hepatitis B (CHB) patients, 86 liver cirrhosis (LC) patients, and 88 healthy individuals were enrolled in the study. CD4(+) CTLs were defined by flow cytometry, immunohistochemistry, and lytic granule exocytosis assays. A multivariate analysis of prognostic factors for overall survival was performed using the Cox proportional hazards model. Circulating and liver-infiltrating CD4(+) CTLs were found to be significantly increased in HCC patients during early stage disease, but decreased in progressive stages of HCC. This loss of CD4(+) CTLs was significantly correlated with high mortality rates and reduced survival time of HCC patients. In addition, the proliferation, degranulation, and production of granzyme A, granzyme B, and perforin of CD4(+) CTLs were inhibited by the increased forkhead/winged helix transcription factor (FoxP3(+) ) regulatory T cells in these HCC patients. Further analysis showed that both circulating and tumor-infiltrating CD4(+) CTLs were independent predictors of disease-free survival and overall survival after the resection of the HCC. CONCLUSION: The progressive deficit in CD4(+) CTLs induced by increased FoxP3(+) regulatory T cells was correlated with poor survival and high recurrence rates in HCC patients. These data suggest that CD4(+) CTLs may represent both a potential prognostic marker and a therapeutic target for the treatment of HCC.

Cross-sectional study of the relationship of peripheral blood cell profiles with severity of infection by adenovirus type 55.

BACKGROUND: The immunologic profiles of patients with human adenovirus serotype 55 (HAdV-55) infections were characterized in subjects diagnosed with silent infections (n = 30), minor infections (n = 27), severe infections (n = 34), and healthy controls (n = 30) during a recent outbreak among Chinese military trainees. METHODS: Blood was sampled at the disease peak and four weeks later, and samples were analyzed to measure changes in leukocyte and platelet profiles in patients with different severities of disease. Differential lymphocyte subsets and cytokine profiles were measured by flow cytometry and Luminex xMAP®, and serum antibodies were analyzed by ELISA and immunofluorescence staining. RESULTS: Patients with severe HAdV infections had higher proportions of neutrophils and reduced levels of lymphocytes (p < 0.005 for both). Patients with minor and severe infections had significantly lower platelet counts (p < 0.005 for both) than those with silent infections. The silent and minor infection groups had higher levels of dendritic cells than the severe infection group. Relative to patients with silent infections, patients with severe infections had significantly higher levels of IL-17+CD4+ cells, decreased levels of IL-17+CD8+ cells, and higher levels of IFN-γ, IL-4, IL-10, and IFN-α2 (p < 0.001 for all comparisons). CONCLUSIONS: Patients with different severities of disease due to HAdV-55 infection had significantly different immune responses. These data provide an initial step toward the identification of patients at risk for more severe disease and the development of treatments against HAdV-55 infection.

Human umbilical cord mesenchymal stem cells improve liver function and ascites in decompensated liver cirrhosis patients.

Decompensated liver cirrhosis (LC), a life-threatening complication of chronic liver disease, is one of the major indications for liver transplantation. Recently, mesenchymal stem cell (MSC) transfusion has been shown to lead to the regression of liver fibrosis in mice and humans. This study examined the safety and efficacy of umbilical cord-derived MSC (UC-MSC) in patients with decompensated LC. A total of 45 chronic hepatitis B patients with decompensated LC, including 30 patients receiving UC-MSC transfusion, and 15 patients receiving saline as the control, were recruited; clinical parameters were detected during a 1-year follow-up period. No significant side-effects and complications were observed in either group. There was a significant reduction in the volume of ascites in patients treated with UC-MSC transfusion compared with controls (P < 0.05). UC-MSC therapy also significantly improved liver function, as indicated by the increase of serum albumin levels, decrease in total serum bilirubin levels, and decrease in the sodium model for end-stage liver disease scores. UC-MSC transfusion is clinically safe and could improve liver function and reduce ascites in patients with decompensated LC. UC-MSC transfusion, therefore, might present a novel therapeutic approach for patients with decompensated LC.

Targeting the raft-associated Akt signaling in hepatocellular carcinoma.

Caveolin-1 and flotillin-1 are considered as markers of lipid rafts which can be regarded as sorting platforms for targeted transport of transmembrane proteins and are involved in fundamental cellular events such as signal transduction, cell adhesion, lipid/protein sorting, and human cancer. We addressed caveolin-1 and flotillin-1 expression in 90 human hepatocellular carcinoma (HCC) and adjacent noncancerous tissues (ANT) samples by SDS-PAGE and immunoblotting with specific antibodies. Significant caveolin-1 and flotillin-1 overexpression was found in HCC tissues compared to ANT and was confirmed by immunohistochemistry. Raft-associated Akt signaling pathway components involved in the regulation of cell survival were altered by western blotting in HCC microdomain-enriched subcellular fractions purified from paired HCC and ANT samples. Our results demonstrated that the activity of raft-associated but not total membrane Akt determines its cellular functions. Lipid rafts differ in different types of tissues, which allows for the possibility of tissue-type-specific targeting for cell survival.

Prevalence, virology and antiviral drugs susceptibility of hepatitis B virus rtN238H polymerase mutation from 1865 Chinese patients with chronic hepatitis B.

Amino acid substitutions at positions rtN238T/D of the hepatitis B virus (HBV) polymerase have been reported as potential mutations associated with adefovir (ADV) resistance. In this study, we characterized the prevalence of the rtN238H mutation and determined the susceptibility to LAM and ADV using phenotypic analyzes in vitro. One thousand eight hundred and sixty-five HBsAg-positive patients with chronic HBV (CHB) infection were included in this study. HBV genotypes and reverse transcriptase (RT) mutations were determined by direct sequencing. Replication-competent HBV constructs containing the naturally occurring rtN238H mutation were generated and replication capacity and susceptibility to LAM and ADV in transiently transfected hepatoma cell lines were determined. Among 1865 enrolled HBV infected patients, 8.8% (165/1865) showed mutations in the rtN238 locus (143 males/22 females, 91 treatment-naive, 42 ADV-treated, 16 LAM-treated and 16 ADV+LAM-treated), namely 86% rtN238H (142/165), 5.5% rtN238S (9/165), 5.5% rtN238T (9/165) and 3% rtN238D (5/165). Among the rtN238H mutant strains, there were no significant differences between ADV- or/and LAM- treated patients and treated-naive patients (42% vs. 58%). Compared with wild-type HBV, this mutant displayed an equivalent susceptibility to LAM or ADV in phenotypic assays. Importantly, we found that the incidence rate of rtN238H was higher in HBV genotype B infected patients than HBV genotype C subsets (80.3% vs. 19.7%), even without exogenous selection pressures. As rtN238H did neither impair the viral replication efficiency nor susceptibility to LAM or ADV in vitro, rtN238H likely represents background polymorphisms rather than resistance mutations with clinical implications. The incidence of rtN238H may be associated with HBV genotype.

Transarterial chemoembolization with/without cryotherapy is associated with improved clinical outcomes of sorafenib for the treatment of advanced hepatocellular carcinoma.

Sorafenib may prolong survival in patients with advanced hepatocellular carcinoma (HCC), but with limited efficacy. The present study aimed to prospectively investigate the efficacy and analyze the prognostic factors for survival in sorafenib-treated patients with advanced HCC. The baseline characteristics and clinical outcomes of 110 patients with advanced hepatitis B virus-related HCC treated with sorafenib with/without local therapy (transarterial chemoembolization with/without cryoablation) at a single liver cancer center were recorded. Predictors of progression-free survival (PFS) and overall survival (OS) were determined by multivariate analysis. A total of 14 (12.7%) patients achieved complete response (CR), 16 (14.5%) achieved partial response (PR) and 40 (36.4%) achieved stable disease (SD) lasting longer than 8 weeks. The median OS and PFS for the whole cohort were 10.5 [95% confidence interval (CI), 8.7-12.3] and 5.0 months (95% CI, 3.7-6.3), respectively. Sorafenib in combination with local therapy was an independent predictor for longer PFS, whereas Eastern Cooperative Group (ECOG) performance status (PS) and Child-Pugh class were associated with reduced PFS. Local therapy was associated with longer OS while ECOG PS and α-fetoprotein were associated with reduced OS. In a subset of patients with radiological progressive disease, a significant difference was found in OS between patients who continued taking sorafenib and those who discontinued therapy (11 vs. 7.5 months, P<0.001). In conclusion, sorafenib in combination with local therapy (transarterial chemoembolization with/without cryoablation) was independently associated with longer OS and PFS in advanced HCC patients. Poor ECOG PS was associated with shorter OS and PFS and is thus a marker of poor outcomes in sorafenib-treated HCC patients.

Outcomes of ultrasound-guided percutaneous argon-helium cryoablation of hepatocellular carcinoma.

PURPOSE: To evaluate the efficacy and safety of ultrasound (US)-guided percutaneous argon-helium cryoablation for hepatocellular carcinoma (HCC) and determine appropriate indications. METHODS: We reviewed outcomes of 300 HCC patients who underwent US-guided percutaneous cryoablation. RESULTS: Overall, 223 tumors (mean diameter 7.2 ± 2.8 cm) in 165 patients were incompletely ablated, while 185 tumors (mean diameter 5.6 ± 0.8 cm, P = 0.0001 vs. incomplete ablation) in 135 patients were completely ablated. Nineteen patients (6.3%) developed serious complications while in hospital, including cryoshock syndrome in six patients, hepatic bleeding in five, stress-induced gastric bleeding in four, liver abscess in one and intestinal fistulas in one. Two patients died because of liver failure. The median follow-up was 36.7 months (range 6-63 months). The local tumor recurrence rate was 31%, and was related to tumor size (P = 0.029) and tumor location (P = 0.037). The mean survival duration of patients with early, intermediate and advanced HCC (Barcelona Clinic Liver Cancer staging system) was 45.7 ± 3.8, 28.4 ± 1.2 and 17.7 ± 0.6 months, respectively. CONCLUSIONS: US-guided percutaneous cryoablation is a relatively safe and effective therapy for selected HCC patients.

Cryotherapy is associated with improved clinical outcomes of Sorafenib therapy for advanced hepatocellular carcinoma.

We assessed the safety and efficacy of sorafenib with cryotherapy (cryoRx) in advanced hepatocellular carcinoma (HCC). One hundred four HCC patients were enrolled, who met the following criteria: (i) Barcelona Clinic Liver Cancer stage C; (ii) HCC without distant metastasis; (iii) the presence of portal vein thrombosis (PVT); (iv) Child-Pugh class A or B; and (v) life expectancy of at least 12 weeks. The patients were randomly divided into sorafenib-cryoRx and sorafenib (control) groups. Primary endpoint was time to progression (TTP); secondary endpoints included overall survival (OS) and tolerability. Microvessel density (MVD) was assessed by CD34-immunostaining. After a median 10.5 (4-26) months follow-up, the data showed that median TTP was 9.5 (8.4-13.5) months in combinatorial therapy group vs. 5.3 (3.8-6.9) months in sorafenib group (P = 0.02). The median OS was 12.5 (95 % CI 10.6-16.4) months in combination therapy group vs. 8.6 (7.3-10.4) months in sorafenib group (P = 0.01). Low MVD patients in combination therapy exhibited significantly longer median TTP and OS than controls. High MVD was predictive of poor responses to sorafenib. CryoRx did not increase frequency/degree of sorafenib-related adverse events. Therefore, it was concluded that the addition of cryoRx significantly improved clinical outcomes of Sorafenib therapy in advanced HCC with acceptable tolerance and similar safety profiles as previously reported.

Cryotherapy is associated with improved clinical outcomes of sorafenib for the treatment of advanced hepatocellular carcinoma.

Sorafenib may prolong survival in patients with advanced hepatocellular carcinoma (HCC), but with limited efficacy. The present study aimed to assess the safety and efficacy of sorafenib combined with cryotherapy (cryoRx) for the treatment of advanced HCC. A total of 104 patients met the following criteria: advanced HCC without distant metastasis, presence of portal vein thrombosis, Child-Pugh class A or B and life expectancy of at least 12 weeks. All patients were randomly assigned to sorafenib and cryoRx (n=52) or sorafenib-alone (n=52) treatment groups. The primary end-point of the study was overall survival (OS). The secondary end-points included time to progression (TTP) and tolerability. Microvessel density (MVD) was assessed following immunostaining for CD34. In a median of 10.5 (4-26) months follow-up, the median OS was 12.5 months (95% CI 10.6-16.4) in the combination therapy vs. 8.6 months (7.3-10.4) in the sorafenib-alone (P=0.01) group. The median TTP was 9.5 months (8.4-13.5) in the combination therapy vs. 5.3 months (3.8-6.9) in the sorafenib alone (P=0.02) group. CryoRx was an independent factor associated with improved clinical outcomes of sorafenib for the treatment of advanced HCC. Patients with low intratumoral MVD receiving the combination therapy exhibited a significantly longer median TTP and OS compared to those receiving sorafenib. High intratumoral MVD was an independent predictor of poor responses to sorafenib for advanced HCC. Compared with previous reports of sorafenib-related adverse drug reactions (ADRs), cryoRx did not further increase the frequency and degree of sorafenib-related ADRs. In conclusion, compared to sorafenib alone, the addition of cryoRx to sorafenib significantly improves the clinical outcomes of sorafenib for the treatment of advanced HCC with acceptable tolerance and similar safety profiles as previously reported. High intratumoral MVD is predictive of poor responses to sorafenib in advanced HCC patients.

Overexpression of metastasis-associated in colon cancer 1 predicts a poor outcome of hepatitis B virus-related hepatocellular carcinoma.

AIM: To investigate the intratumoral expression of metastasis-associated in colon cancer 1 (MACC1) and c-Met and determine their clinical values associated with hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). METHODS: A retrospective study admitted three hundred fifty-four patients with HBV-related HCC. The expression and distribution of MACC1 and c-Met were assessed by quantitative real-time polymerase chain reaction and immunohistochemistry staining. Prognostic factors influencing survival, metastasis and recurrence were assessed. RESULTS: Intratumoral MACC1 level was found to be associated with HCC disease progression. Both median tumor-free survival (TFS) and overall survival (OS) were significantly shorter in the postoperative HCC patients with high intratumoral MACC1 expression, as compared to those with low intratumoral MACC1 levels (TFS: 34 mo vs 48.0 mo, P < 0.001; OS: 40 mo vs 48 mo, P < 0.01). Multivariable analysis indicated that high MACC1 expression or co-expression with c-Met were independent predictors for HCC clinic outcome (P < 0.001). CONCLUSION: High intratumoral MACC1 expression can be associated with enhanced tumor progression and poor outcome of HBV-related HCC. MACC1 may serve as a prognostic biomarker for postoperative HCC.

[Increase in peripheral and liver infiltrating regulatory T cells favors development of primary hepatocellular carcinoma].

AIM: This study attempted to investigate the features of Treg cells in peripheral blood and liver of patients with primary hepatocellular carcinoma (HCC). METHODS: Peripheral blood samples were obtained from 30 HCC patients and 30 healthy control subjects, then were quantitatively analyzed for Treg cells by using flow cytometry. Liver infiltrating lymphocytes (LIL) isolated from resected tumor samples of 7 HCC patients were simultaneously analyzed. RESULTS: There was a significant increase in the frequency of peripheral Treg cells in HCC patients compared with healthy controls (P < 0.01). Furthermore, we also found that there was a higher frequency of infiltrated Treg within tumor samples than the counterpart in non-tumor region (P < 0.05). In addition, CD4(+) CD25(low) and CD4(+) CD25(-) T cells isolated from resected tumor samples were found to express higher level of FOXP3 molecules. CONCLUSION: Our findings showed that a dramatic increasing increase of Treg in peripheral blood and liver tissue of HCC patients may be associated with the significant increased development of Treg, which favors the disease progression through the suppressive effect of Treg on host immune response in these patients.

Upregulation of circulating PD-L1/PD-1 is associated with poor post-cryoablation prognosis in patients with HBV-related hepatocellular carcinoma.

BACKGROUND: The programmed cell death-1 receptor/programmed cell death-1 ligand (PD-1/PD-L1) pathway plays a crucial role in tumor evasion from host immunity. This study was designed to evaluate the association between circulating PD-L1/PD-1 and prognosis after cryoablation in patients with HBV-related hepatocellular carcinoma (HCC). METHODOLOGY/PRINCIPAL FINDINGS: In the present study, 141 HBV-related HCC patients were enrolled and of those 109 patients received cryoablation. Circulating PD-L1/PD-1 expression was tested by flow cytometry, and 23 patients were simultaneously evaluated for intratumoral PD-L1 expression by immunohistochemical staining. Circulating PD-1/PD-L1 expression was associated with severity of diseases in patients with HCC, and the circulating PD-L1 expression was closely correlated with intratumoral PD-L1 expression. Of the clinical parameters, PD-1/PD-L1 expression was associated with tumor size, blood vessel invasion and BCLC staging. Moreover, PD-1/PD-L1 expression dropped after cryoablation while being elevated at the time of tumor recurrence. Patients with higher expression of circulating PD-L1, as well as circulating PD-1, had a significantly shorter overall survival and tumor-free survival than those with lower expression. Multivariate analysis confirmed that circulating PD-L1 could serve as an independent predictor of overall survival and tumor-recurrence survival in HCC patients after cryoablation. CONCLUSIONS/SIGNIFICANCE: Upregulation of circulating PD-L1/PD-1 is associated with poor post-cryoablation prognosis in patients with HBV-related hepatocellular carcinoma.