Development and validation of a nomogram based on clinical factors and standard laboratory tests for prediction of clinically significant liver fibrosis in chronic hepatitis C virus infection.

OBJECTIVES: Staging liver fibrosis in chronic viral hepatitis C (HCV) patients is essential for prompting surveillance and treatment. The aim of this study was to develop a nomogram, on the basis of simple clinical and laboratory variables, to predict three clinically significant stages of fibrosis (nil-mild, moderate, advanced/cirrhosis), using histology as reference, and to compare its performance with that of FibroTest, a widely used noninvasive fibrosis score. MATERIALS AND METHODS: Nomograms are graphical representations of a mathematical formula, used as predictive tools. The study retrospectively recruited 406 HCV patients undergoing liver biopsy. Nomogram was developed in a training set of 252 patients and tested in a validation set of 154 patients. Histology was staged according to the Metavir system. Fibrosis stages were subgrouped as follows: advanced fibrosis/cirrhosis (F3/F4, 24%), nil-mild (F0/F1, 36%), and moderate (F2, 40%). Age at biopsy, aspartate aminotransferase, γ-glutamyl transpeptidase, albumin, platelet count, and prothrombin activity formed the basis for the so-called Fibro-Nomogram, which, in one graphical representation, estimates probability for different stages of fibrosis. RESULTS: Areas under the receiver-operating characteristic curves for advanced fibrosis/cirrhosis were similar for training (0.86) and validation sets (0.87). For nil-mild fibrosis, area under the receiver-operating characteristics were 0.81 and 0.79. Compared with FibroTest, Fibro-Nomogram performed slightly better at predicting severe fibrosis (F3/F4) with positive likelihood ratio (LR+) 5.07 (95% confidence interval 3.08-8.37) versus LR+ 3.82 (95% confidence interval 2.56-5.71) for FibroTest. For nil-mild fibrosis, the two tests showed limited but comparable performances. CONCLUSION: In HCV patients, Fibro-Nomogram, an inexpensive and readily available predictive tool, could enable clinicians to interpret patients' profile, concurrently stratifying patients into three clinically relevant probability categories with good overall performance.

Immunological and molecular correlates of disease recurrence after liver resection for hepatocellular carcinoma.

The definition of the risk of hepatocellular carcinoma (HCC) recurrence after resection represents a central issue to improve the clinical management of patients. In this study we examined the prognostic relevance of infiltrating immune cell subsets in the tumor (TIL) and in nontumorous (NT) liver (LIL), and the expression of immune-related and lineage-specific mRNAs in HCC and NT liver derived from 42 patients. The phenotype of infiltrating cells was analyzed by flow cytometry, and mRNA expression in liver tissue was examined by real-time reverse transcription (RT)-PCR. The tumor immune microenvironment was enriched in inhibitory and dysfunctional cell subsets. Enrichment in CD4+ T-cells and in particular CD4 and CD8+ memory subsets within TIL was predictive of better overall survival (OS) and time to recurrence (TTR). Increased programmed death ligand 1 (PDL1) mRNA content and higher prevalence of invariant NKT (iNKT) cells were associated with shorter OS and TTR, respectively. By combined evaluation of infiltrating cell subsets along with mRNA profiling of immune and tumor related genes, we identified the intratumoral frequency of memory T-cells and iNKT-cells as well as PDL1 expression as the best predictors of clinical outcome. HCC infiltrate is characterized by the expression of molecules with negative regulatory function that may favor tumor recurrence and poor survival.

Radiofrequency thermal ablation for hepatocellular carcinoma stimulates autologous NK-cell response.

BACKGROUND & AIMS: Radiofrequency thermal ablation (RFA) is a minimally invasive technique used as standard local therapy of hepatocellular carcinoma and second-line treatment for metastatic liver tumors. Studies in preclinical models and in patients have shown that thermal destruction of tumor tissue can enhance anti-tumor cellular responses, but our knowledge of its impact on natural killer (NK) cells is still very limited. METHODS: Thirty-seven patients undergoing RFA for hepatocellular carcinoma were studied for peripheral blood lymphocytes counts followed by phenotypic and functional characterization of NK-cell population. RESULTS: Peripheral blood lymphocytes kinetics revealed an increased frequency and absolute number of NK cells expressing higher levels of activatory along with reduced levels of inhibitory NK receptors, and increased functional NK-cell activity. A prevalent expansion of the CD3(-)CD56(dim) NK subset was observed compared to the CD3(-)CD56(bright) counterpart. Interferon-gamma production, anti-K562 cell cytotoxicity, and antibody-dependent cell cytotoxicity, appeared consistently increased in terms of both absolute activity and killing efficiency at 4 weeks after RFA, as compared to baseline. Interestingly, when recurrence-free survival was assessed in 2 groups of patients separated according to higher vs lower enhancement of cytotoxicity and/or interferon-gamma production, a significant difference was observed, thus suggesting a potential predictive role of NK functional assays on efficacy of RFA. CONCLUSIONS: RFA can lead to stimulation of NK cells with a more differentiated and proactivatory phenotypic profile with general increase of functional activities. This observation may be relevant for development of adjuvant immunotherapeutic strategies aimed at enhancing NK-cell responses against primary and metastatic liver tumors.

Increased immunostimulatory activity conferred to antigen-presenting cells by exposure to antigen extract from hepatocellular carcinoma after radiofrequency thermal ablation.

Radiofrequency thermal ablation represents an effective treatment for hepatocellular carcinoma (HCC) and it can also exert an "adjuvant" effect on spontaneous antitumor T-cell responses, as suggested by human and animal studies. The adjuvant effect is thought to depend on the huge amount of necrotic tumor antigen made available to the immune system by HCC thermal ablation. In addition, radiofrequency thermal ablation may result in the release of local stimuli responsible for activation and maturation of antigen-presenting cells (APCs). To test this hypothesis, we studied APC maturation and function in 19 patients undergoing thermal ablation for HCC. Patients' monocytes induced to differentiate with granulocyte macrophage colony-stimulating factor (GM-CSF), or GM-CSF plus IL-4, were cocultured in vitro with tumor debris generated by radiofrequency thermal ablation. Expression of costimulatory molecules, lymphnode homing chemokine receptor, antigen presentation, and cytokine secretion were enhanced by incubation with HCC treated tissue as compared with untreated HCC and nontumor liver tissue. Moreover, HCC-specific T-cell responses could be induced by monocytes activated with GM-CSF and incubated with thermally ablated HCC tissue. HCC thermal ablation can create an antigenic source along with stimuli appropriate for maturation of APCs to induce HCC-specific T-cell responses. These results contribute to explain at least in part the adjuvant effect of HCC thermal ablation and suggest a novel strategy to induce maturation of APCs and their loading with HCC antigens for active immunotherapy protocols aimed at reducing HCC recurrence after thermal ablation.

Combination of radiofrequency ablation and immunotherapy.

The enhancement of immune response against tumor antigens has shown some efficacy when used as a single mode of systemic treatment in patients with late stage disease. Novel strategies of active immunotherapy could be more effective in patients with less advanced disease who receive standard therapies supporting concomitant stimulation of the immune system. Radio-Frequency Ablation (RFA) is a minimally invasive technique which is used as standard local therapy of primary and metastatic liver tumors. Tumor ablation by RFA induces effects important for boosting anti-tumor immune responses. Tumor cell necrosis generates a permanent immunogenic source of tumor antigens. These antigens can be uptaken, processed and presented by dendritic cells for effective immunization without requirement for ex vivo antigen loading. Further immune activation can be originated by RFA through induction of heat shock proteins on tumor cells, acute phase response which causes the release of pro-inflammatory cytokines, and mobilization of antigen presenting cells and effector lymphocytes. Thus, RFA can facilitate immune responses to tumor antigens driven by active immunotherapy. On the other hand, immunotherapy is expected to eradicate residual disease after RFA and prevent tumor recurrences. The combination of RFA and active immunotherapy may well have synergistic effects for cancer treatment.

Radiofrequency thermal ablation of hepatocellular carcinoma liver nodules can activate and enhance tumor-specific T-cell responses.

Radiofrequency thermal ablation (RFA) destroys tumoral tissue generating a local necrosis followed by marked inflammatory response with a dense T-cell infiltrate. In this study, we tested whether hepatocellular carcinoma thermal ablation can induce or enhance T-cell responses specific for hepatocellular carcinoma-associated antigens. Peripheral blood mononuclear cells derived from 20 patients with hepatocellular carcinoma were stimulated before and a month after RFA treatment with autologous hepatocellular carcinoma-derived protein lysates obtained before and immediately after RFA treatment. The effect of thermal ablation on memory T-cell responses to recall antigens [tetanus toxoid, protein purified derivative (PPD), Escherichia coli] was also assessed. T-cell reactivity was analyzed in an IFN-gamma enzyme-linked immunospot assay and by intracellular IFN-gamma staining. Treatment was followed by a significant increase of patients responsive either to tumor antigens derived from both the untreated hepatocellular carcinoma tissue (P < 0.05) and the necrotic tumor (P < 0.01) and by a higher frequency of circulating tumor-specific T cells. T-cell responses to recall antigens were also significantly augmented. Phenotypic analysis of circulating T and natural killer cells showed an increased expression of activation and cytotoxic surface markers. However, tumor-specific T-cell responses were not associated with protection from hepatocellular carcinoma relapse. Evidence of tumor immune escape was provided in one patient by the evidence that a new nodule of hepatocellular carcinoma recurrence was not recognized by T cells obtained at the time of RFA. In conclusion, RFA treatment generates the local conditions for activating the tumor-specific T-cell response. Although this effect is not sufficient for controlling hepatocellular carcinoma, it may represent the basis for the development of an adjuvant immunotherapy in patients undergoing RFA for primary and secondary liver tumors.

Ex vivo characterization of tumor-derived melanoma antigen encoding gene-specific CD8+cells in patients with hepatocellular carcinoma.

BACKGROUND/AIMS: Members of the melanoma antigen encoding gene family are expressed in tumors of different histological types but not in normal tissue. For this reason, they are attractive targets for cancer immunotherapy. METHODS: In the present study, we analyzed the expression of MAGE-1 and -3 genes in the hepatocellular carcinoma (HCC) tissue as well as frequency, phenotype and function of circulating and tumor infiltrating CD8+ cells specific for HLA-A1 and -A2 restricted epitopes of MAGE-1 and -3. RESULTS: Our study shows for the first time the presence of MAGE/tetramer+ CD8 cells in the tumor tissue of patients with HCC. These cells are able to recognize the MAGE-1 sequence 161-169 and the MAGE-3 sequence 271-279. In a patient with a particularly high frequency of MAGE-1 sequence 161-169-specific T cells, phenotypic and functional analysis was performed showing a phenotype of recently-primed CD8 cells (CD28+CD27+CD45RA-CCR7). CONCLUSIONS: The observation of a spontaneous in vivo priming of a MAGE-specific T cell response in patients with HCC and the high frequency of MAGE antigens expression in this tumor, makes this antigen a potential candidate for a MAGE-specific immunotherapy in hepatocellular carcinoma.