Integrated metabolomic profiling of hepatocellular carcinoma in hepatitis C cirrhosis through GC/MS and UPLC/MS-MS.

BACKGROUND & AIMS: The metabolic pathway disturbances associated with hepatocellular carcinoma (HCC) remain unsatisfactorily characterized. Determination of the metabolic alterations associated with the presence of HCC can improve our understanding of the pathophysiology of this cancer and may provide opportunities for improved disease monitoring of patients at risk for HCC development. To characterize the global metabolic alterations associated with HCC arising from hepatitis C (HCV)-associated cirrhosis using an integrated non-targeted metabolomics methodology employing both gas chromatography/mass spectrometry (GC/MS) and ultrahigh-performance liquid chromatography/electrospray ionization tandem mass spectrometry (UPLC/MS-MS). METHODS: The global serum metabolomes of 30 HCC patients, 27 hepatitis C cirrhosis disease controls and 30 healthy volunteers were characterized using a metabolomics approach that combined two metabolomics platforms, GC/MS and UPLC/MS-MS. Random forest, multivariate statistics and receiver operator characteristic analysis were performed to identify the most significantly altered metabolites in HCC patients vs. HCV-cirrhosis controls and which therefore exhibited a close association with the presence of HCC. RESULTS: Elevated 12-hydroxyeicosatetraenoic acid (12-HETE), 15-HETE, sphingosine, γ-glutamyl oxidative stress-associated metabolites, xanthine, amino acids serine, glycine and aspartate, and acylcarnitines were strongly associated with the presence of HCC. Elevations in bile acids and dicarboxylic acids were highly correlated with cirrhosis. CONCLUSIONS: Integrated metabolomic profiling through GC/MS and UPLC/MS-MS identified global metabolic disturbances in HCC and HCV-cirrhosis. Aberrant amino acid biosynthesis, cell turnover regulation, reactive oxygen species neutralization and eicosanoid pathways may be hallmarks of HCC. Aberrant dicarboxylic acid metabolism, enhanced bile acid metabolism and elevations in fibrinogen cleavage peptides may be signatures of cirrhosis.

Immune modulation of effector CD4+ and regulatory T cell function by sorafenib in patients with hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a difficult to treat cancer characterized by poor tumor immunity with only one approved systemic drug, sorafenib. If novel combination treatments are to be developed with immunological agents, the effects of sorafenib on tumor immunity are important to understand. In this study, we investigate the impact of sorafenib on the CD4+CD25- effector T cells (Teff) and CD4+CD25+ regulatory T cells (Tregs) from patients with HCC. We isolated Teff and Treg from peripheral mononuclear cells of HCC patients to determine immune reactivity by thymidine incorporation, ELISA and flow cytometry. Teff cultured alone or with Treg were supplemented with different concentrations of sorafenib. The effects of sorafenib on Teff responses were dose-dependent. Pharmacologic doses of sorafenib decreased Teff activation by down regulating CD25 surface expression. In contrast, sub-pharmacologic concentrations of sorafenib resulted in Teff activation. These low doses of sorafenib in the Teff cultures led to a significant increase in Teff proliferation, IL2 secretion and up-regulation of CD25 expression on the cell surface. In addition, low doses of sorafenib in the suppression Teff/Treg cocultures restored Teff responses by eliminating Treg suppression. The loss of Treg suppressive function correlated with an increase in IL2 and IL6 secretion. Our findings show that sub-pharmacologic doses of sorafenib impact subsets of T cells differently, selectively increasing Teff activation while blocking Treg function. In conclusion, this study describes novel immune activating properties of low doses of sorafenib by promoting immune responsiveness in patients with HCC.

Serum levels of soluble CD25 as a marker for hepatocellular carcinoma.

In a previous study, we showed that the level of soluble CD25 (sCD25) was elevated in a small series of patients with hepatocellular carcinoma (HCC). In the present study, we determined the capacity of serum levels of sCD25 to detect the presence and the early stage of HCC using a larger cohort of HCC patients and evaluated the correlation between sCD25 level and tumor burden. Serum levels of sCD25 were quantified using ELISA in patients with HCC (n=145), controls with advanced fibrosis (n=61) and healthy control subjects (n=30). The levels of sCD25 in patients with HCC (median, 6,955 pg/ml) were significantly higher than those in cirrhosis-only patients (4,310 pg/ml; P<0.0001). At a cut-off value of 2,180 pg/ml, sCD25 had a sensitivity of 92.3% and a specificity of 37.7% in detecting HCC presence [area under the curve (AUC) of 0.685; P<0.0001]. By comparison, α-fetoprotein (AFP) had a sensitivity of 53.8% and a specificity of 86.8% at a cut-off value of 32.8 ng/ml (AUC=0.755; P<0.0001) for HCC presence detection. For early HCC, the sensitivity of sCD25 was 89.6% and its specificity was 39.3% (AUC=0.630; P<0.0001) at a cut-off value of 2,859 pg/ml, while AFP had a sensitivity of 41.7% and a specificity of 82.6% at a cut-off value of 20.6 ng/ml (AUC=0.630; P=0.0257). We also found a significant positive correlation between serum levels of sCD25 and tumor stage. In the present study study, sCD25 was more effective than AFP at detecting the presence and early stages of HCC. This immune factor may hold promise as a novel predictive marker of HCC presence and may be useful in distinguishing early HCC from advanced cirrhosis, currently areas of global unmet need.

Hepatocellular carcinoma immunopathogenesis: clinical evidence for global T cell defects and an immunomodulatory role for soluble CD25 (sCD25).

BACKGROUND: The mechanisms involved in hepatocellular carcinoma (HCC) establishing an immunologically tolerogenic tumor environment remain poorly characterized. AIMS: This study evaluates effector T cell responses and soluble IL-2 receptor alpha chains (sCD25) in relation to HCC stage/survival and characterizes the impact of sCD25 on effectors. METHODS: Effector cell responses with serum from HCC patients and in serum free conditions were assessed by IFN-gamma ELISpot, proliferation and ATP production assays at baseline, after depletion of sCD25, and after supplementation with recombinant sCD25. Sera sCD25 were measured by ELISA and any relationship with stage/survival was determined. RESULTS: Hepatocellular carcinoma patients had marked global impairment in T cell responses at baseline which correlate with tumor burden and poor outcome. The impairment in immune responses is characterized by low IFN-gamma production, cell proliferation, and ATP production. Effector responses are impaired by serum from HCC patients in a dose-dependent manner, implicating soluble factors in the observed immunosuppression. Significant elevations in serum levels of sCD25 are found in patients with HCC, which correlate with tumor burden and a worse survival. T cell reactivity is inversely proportional to serum level of sCD25. Impaired T cell responses improve with sCD25 depletion from HCC serum or IL-2 supplementation suggesting impairment in IL-2 signaling. In contrast, adding increasing doses of sCD25 suppresses effector T cells, which partly involves induction of apoptosis. CONCLUSIONS: These findings show that HCC patients have blunted T cell immunity that is partly related to elevated levels of sCD25, supporting a novel immuno-inhibitory role for this soluble receptor.

Using an immune functional assay to differentiate acute cellular rejection from recurrent hepatitis C in liver transplant patients.

In transplant recipients transplanted for hepatitis C, presentation of abnormal transaminases can herald the presentation of recurrent hepatitis C, cellular rejection, or both. Given the sometimes ambiguous histology with these 2 entities, the ability to distinguish them is of great importance because misinterpretation can potentially affect graft survival. We used an immune functional assay to help assess the etiology of abnormal liver function test results in liver transplant recipients. Blood samples for the immune functional assay were taken from 42 recipients prospectively at various times post-transplant and compared with clinical and histologic findings. In patients whose liver biopsy showed evidence of cellular rejection, the immune response was noted to be very high, whereas in those with active recurrence of hepatitis C, the immune response was found to be very low. This finding was found to be statistically significant (P < 0.0001). In those patients in whom there was no predominant histologic features suggesting 1 entity over the other, the immune response was higher than in those with aggressive hepatitis C but lower than in those with cellular rejection. In conclusion, these data show the potential utility of the ImmuKnow assay as a means of distinguishing hepatitis C from cellular rejection and its potential usefulness as a marker for outlining the progression of hepatitis C.

Preferential transformation of human neuronal cells by human adenoviruses and the origin of HEK 293 cells.

The 293 cell line was derived by transformation of primary cultures of human embryonic kidney (HEK) cells with sheared adenovirus (Ad)5 DNA. A combination of immunostaining, immunoblot, and microarray analysis showed that 293 cells express the neurofilament (NF) subunits NF-L, NF-M, NF-H, and a-internexin as well as many other proteins typically found in neurons. Three other independently derived HEK lines, two transformed by Ad5 and one by Ad12, also expressed NFs, as did one human embryonic retinal cell line transformed with Ad5. Two rodent kidney lines transformed with Ad12 were also found to express NF proteins, although several rodent kidney cell lines transformed by Ad5 DNA and three HEK cell lines transformed by the SV40 early region did not express NFs. These results suggest that human Ads preferentially transform human neuronal lineage cells. We also demonstrate that the widely used HEK293 cells have an unexpected relationship to neurons, a finding that may require reinterpretation of many previous studies in which it was assumed that HEK293 cells resembled more typical kidney epithelial cells.