H A El Aggan

MAGE-1 and MAGE-3 mRNA expressions as molecular biomarkers in patients with hepatitis C virus-related hepatocellular carcinoma

Introduction The melanoma antigen (MAGE) family members are tumour-specific antigens exclusively expressed in neoplastic cells. Therefore, the present work was designed to study the expression of MAGE-1 and MAGE-3 mRNAs in the peripheral blood and cancerous tissues of patients with hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC). Methods 30 patients with HCV-related cirrhosis (15 patients with HCC and 15 patients without HCC) and 15 healthy subjects were enrolled in the present study. Expression of MAGE-1 and MAGE-3 mRNAs in peripheral blood samples, HCC specimens and surrounding non-neoplastic liver tissues, were studied by a reverse-transcription PCR (RT-PCR) with the specific primers after RNA extraction. The sensitivity and specificity of MAGE-1 and MAGE-3 mRNAs as markers for diagnosis of HCC have been assessed by plotting a receiver-operating characteristic (ROC) curve. Results In HCC patients, the positive rate of MAGE-1 and MAGE-3 mRNA expression was 53.3% and 33.3% in peripheral blood samples respectively, while the positive rate was 53.3% and 40.0% in HCC tissue samples, respectively. By contrast, MAGE-1 and MAGE-3 mRNA were not detected in the adjacent non-neoplastic liver tissues or in the peripheral blood samples of cirrhotic patients without HCC and healthy subjects. No relationship was found between MAGE-1 and MAGE-3 mRNA expression and age, gender, Child-Pugh score, tumour size, clinical stage and histopathological grade (p>0.05). The sensitivity and specificity of MAGE-1 mRNA as a marker for the diagnosis of HCC was 53.3% and 100% respectively while MAGE-3 mRNA has a sensitivity of 40% and a specificity of 100%. Conclusion MAGE-1 and MAGE-3 mRNA are highly expressed in HCV-related HCCs and may play a role in hepatocarcinogenesis. These tumour-specific antigens can be used as molecular markers for early diagnosis of HCC and detection of disseminated tumour cells and may act as a potential target for immunotherapy in HCC patients.

PTU-109 Plasma Heat Shock Protein-32 Levels in Patients with Hepatitis C Virus-Related Cirrhosis: Relation to Renal Function and Hemodynamics

Introduction Heat shock protein-32 (HSP-32) is a microsomal enzyme that has hemodynamic effects and may play a role in the pathogenesis of renal diseases. Therefore, the present work was designed to study the plasma levels of HSP-32 and its product carbon monoxide (CO) in patients with hepatitis C virus (HCV)-related cirrhosis in relation to renal function and hemodynamics. Methods Thirty patients with HCV-related cirrhosis and 15 healthy subjects were included in the study. The severity of liver disease was assessed using Child-Pugh classification and The Model for End-Stage Liver Disease (MELD) score. Renal function was evaluated by serum creatinine (sCr) level, estimated glomerular filtration rate (eGFR) and urine sodium (UNa)concentration. Plasma HSP-32 levels were measured using commercially available enzyme-linked immunosorbant assay. Blood carboxyhemoglobin (COHB) concentration, an index of CO production, was assayed by spectrophotometry. Renal hemodynamics including renal artery peak systolic velocity (PSV), end-diastolic velocity (EDV), mean velocity (MnV), resistive index (RI) and pulsatility index (PI) and renal blood flow (RBF), were measured using Doppler ultrasonography. Results Patients with HCV-related cirrhosis showed significant increases in plasma HSP-32 levels, blood COHB concentration and renal artery RI and PI and significant decreases in RBF and renal artery EDV and MnV compared with healthy subjects (P < 0.001) The plasma HSP-32 levels and blood COHB concentration showed positive correlations with Child-Pugh and MELD scores, sCr and renal artery RI and PI and negative correlations with eGFR, UNa concentration, RBF and renal artery EDV and MnV (P < 0.05). No correlations were found between plasma HSP-32 levels and blood COHB concentration on one hand and age of the patient, apparent duration of HCV infection and serum HCV RNA levels on the other hand (P > 0.05). Conclusion The increased HSP-32 activity with enhanced endogenous CO generation may play an important role in renal dysfunction in HCV-related cirrhosis and could be a potential therapeutic target. Disclosure of Interest None Declared

PTU-110 Circulating Bone Marrow-Derived Stem Cells and Stem Cell Factor Serum Level in Chronic Hepatitis C: Relation to Hepatic Proliferation and Fibrosis

Introduction Bone marrow-derived stem cells (BMSCs) are pluripotent cells that can be mobilised into circulation and recruited to sites of inflammation where they promote local tissue repair. Therefore, the present work was designed to study circulating BM-derived hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) and serum levels of stem cell factor (SCF), a stem cell mobilising factor, in patients with chronic hepatitis C (CHC) in relation to hepatic proliferation and fibrosis. Methods Thirty treatment-naïve patients with CHC and 15 healthy subjects were included in the study. The BM-derived HSCs and MSCs cells in fresh blood samples were identified as CD34+CD45+CD117+ and CD34-CD45-CD106+ cells respectively using flow cytometric assay. Serum SCF levels were measured using an enzyme linked immunosorbant assaykit. Liver biopsies were examined to assess METAVIR histological activity grade and fibrosis stage and steatosis grade. Immunohistochemical staining of liver specimens was done using monoclonal antibodies against cytokeratin (CK)7 for detection of hepatic progenitor cells (HPCs), Ki-67 as proliferation marker and a-smooth muscle actin (a-SMA) for identification of activated hepatic stellate cells (HpSCs). Results Patients with CHC showed significant increases in the percentages of HSCs and MSCs in peripheral blood and serum SCF levels compared with healthy subjects (P < 0.05). Numerous CK7+ HPCs were detected mostly lining primitive bile ducts or as individual positive cells in the portal tracts. Hepatic proliferative activity evidenced as nuclear positivity for Ki-67, was observed in proliferated ductal profiles in portal tracts and within hepatocytes and directly correlated with HPC expansion. Based on the percentages of BMSCs in peripheral blood, patients with CHC were distinguished into two types of patients: “mobilizers” and “non-mobilizers”. BMSC mobilizers showed a significant increase in serum SCF levels and significant decreases in HPC expansion, hepatic proliferative activity, serum levels of aminotransferases, histological activity grade, fibrosis stage and a-SMA expression compared with BMSC non-mobilizers (P < 0.01). Conclusion Chronic HCV infection is associated with mobilisation of BMSCs from the BM into the circulation in parallel with an increased production of SCF, particularly when HPC activation and hepatic proliferative activity are impaired. Although the mobilised BMSCs are not sufficient to bring about hepatic repopulation, they may play an important role in limiting hepatic necroinflammation and fibrosis in HCV-induced liver damage. Disclosure of Interest None Declared

P52 Macrophage inflammatory protein-1 α/CC chemokine ligand 3 and tumour-associated macrophages in hepatitis C virus-related hepatocellular carcinoma: relation to tumour progression and angiogenesis

Introduction Hepatitis C virus (HCV) is a major risk factor for development of hepatocellular carcinoma (HCC), however, the mechanism of hepatocarcinogenesis in HCV infection is still undefined. Chemokines, which can induce the migration of leucocytes and activate inflammatory/immune responses, have recently been implicated in the regulation of tumour growth. Aim Therefore, the aim of the present work was to study the role of macrophage inflammatory protein-1 α/CC chemokine ligand 3 (MIP-1α/CCL3), a potent macrophage chemoattractant, in the pathogenesis of HCV-related HCC in relation to tumour progression and angiogenesis. Method 30 patients with HCV-related cirrhosis (15 patients with histologically-proven HCC and 15 patients without HCC) and 15 healthy subjects were enrolled in the study. The severity of liver disease was assessed according to Child-Pugh classification and the Model for End Stage Liver Disease (MELD) score. The tumour stage was classified using the Cancer of the Liver Italian Program (CLIP) scoring system. Histological tumour grading was performed according to the Edmondson and Steiners criteria and the surrounding liver tissue was examined for assessing the modified histological activity index (HAI), presence of cirrhosis and the grade of steatosis. Expressions of MIP-1α/CCL3, CD68 [a marker for tumour-associated macrophages (TAM)] and CD105 (Endoglin) [for tumour angiogenesis and determination of microvessel density (MVD)] were studied in HCC and adjacent non-neoplastic liver tissues by immunohistochemistry. Serum MIP-1α/CCL3 levels were measured using solid phase sandwich enzyme linked immunosorbant assay kit. The sensitivity and specificity of serum levels of MIP-1α/CCL3 as markers for diagnosis of HCC have been assessed by plotting a receiver-operating characteristic (ROC) curve. Results Patients with HCV-related HCCs showed significant increases in MIP-1α/CCL3 expression, CD68+ TAM count and CD105+ MVD in tumour tissues compared with adjacent non-neoplastic liver tissues (p=0.0004, p<0.001 and p<0.001 respectively). Serum MIP-1α/CCL3 levels were significantly higher in patients with and without HCC than in healthy subjects and in HCC patients than in patients without HCC (p<0.001). By plotting a ROC curve, the sensitivity and specificity of serum MIP-1α/CCL3 in discriminating cirrhotic patients with and without HCC were found to be 100% and 93.3% respectively at a cut-off value of 17.5 pg/ml. The MIP-1α/CCL3 expression in HCC tissues showed positive correlations with serum MIP-1α/CCL3 levels; tumour size, stage, histopathological grade; serum α-fetoprotein levels and CD68+ TAM count and CD105+ MVD in HCCs (p<0.05). Also, CD68+ TAM count and CD105+ MVD in HCC tissues were positively correlated (p<0.001). On the other hand, no correlations were found between MIP-1α/CCL3 expression, CD68+ TAM count and CD105+ MVD in HCCs on one hand and serum levels of aminotransferases, Child-Pugh score, MELD score and HAI and steatosis grade in the surrounding liver tissue (p>0.05). Conclusion The CC chemokine, MIP-1α/CCL3, may play an important role in the pathogenesis and progression of HCC in HCV-related liver disease, possibly, through migration of macrophages to tumour microenvironment and enhancement of angiogenesis. MIP-1α/CCL3 may also serve as a potential serum biological marker and a useful therapeutic target for HCC.

OC-029 Expression of heat shock protein 27 and caspase-3 in hepatitis C virus-related hepatocellular carcinoma: relation to tumour progression

Introduction Hepatitis C virus (HCV) is a major risk factor for development of hepatocellular carcinoma (HCC), however, the mechanism of hepatocarcinogenesis in HCV infection is still undefined. Heat shock protein (HSP) 27 is a ubiquitous chaperone molecule induced in cells exposed to different stress conditions, including carcinogenesis. It also has potent anti-apoptotic properties through inhibition of caspase-3 activation. Therefore, the aim of the present work was to study the expression of HSP27 and caspase-3 in HCV-related HCCs in relation to tumour progression. Methods Twenty cirrhotic patients with HCV-related HCC were enrolled in the study. The severity of liver disease was assessed according to the Model for End Stage Liver Disease (MELD) score. Serum levels of α-fetoprotein (AFP) were measured by enzyme immunoassay kit. The tumour stage was classified using the scoring system proposed by the Cancer of the Liver Italian Program (CLIP). Histological grading of tumour s was performed according to the Edmondson and Steiners criteria and the surrounding liver tissue was examined for the presence of cirrhosis and steatosis. Expression of HSP27 and caspase-3 was studied in HCC and adjacent non-neoplastic liver tissues by immunohistochemistry and the staining intensity of the tumour was designated as “negative/low expression” or “high expression” if <25% and >25% of cells were positively-stained, respectively. Results HCV-related HCCs showed a significant increase in HSP27 expression and a significant decrease in caspase-3 expression as compared with adjacent non-neoplastic liver tissues (p=0.029 and p=0.040, respectively). The expression of HSP27 showed an inverse correlation with caspase-3 expression in HCC tissues (r=−0.691, p=0.001). High HSP27 expression and negative/low caspase-3 expression in HCCs were associated with significant increases in serum levels of aminotransferases, HCV RNA and AFP, MELD score, tumour size, tumour stage, histological tumour grade, body mass index and the presence of steatosis in the surrounding liver tissue (P<0.05). No relationship was found between expression of HSP27 and caspase-3 in HCCs and age, gender, apparent duration of HCV infection, and tumour encapsulation, multiplicity, location and lymphocyte infiltration (P>0.05). Conclusion HSP27 plays an important role in the pathogenesis and progression of HCC in HCV infection through inhibition of caspase-3 mediated cell apoptosis and may serve as a potentially useful therapeutic target.

PTU-054 Peripheral blood and intrahepatic natural killer cells in chronic hepatitis C: relation to disease activity and hepatic fibrosis

Introduction Cellular immune responses are thought to play a key role in the pathogenesis of hepatitis C virus (HCV)-related liver damage. Recently, increasing attention has been drawn towards components of the innate immune system to HCV, including natural killer (NK) cells. The present work was designed to study peripheral blood and intrahepatic NK cells in patients with chronic hepatitis C in relation to disease activity and severity of hepatic fibrosis. Methods Fifteen patients with untreated chronic hepatitis C (CHC) and 12 healthy subjects were included in the current study. The NK and NKT cells in fresh whole blood samples were identified using two-colour flow cytometry as CD3-CD56+ and CD3+CD56 cells, respectively, and the results were expressed as percentages of the total lymphocyte count. Liver biopsies were taken from all patients and the specimens were evaluated as regards the histological activity grade and fibrosis stage according to METAVIR scoring system and for the presence and grade of steatosis. Immunohistochemical staining was done using antibodies against CD56 and smooth muscle actin (SMA) for detection of intrahepatic NK cells and activated hepatic stellate cells (HSCs), respectively. A semi-quantitative method was used to score the intensity of immunostaining. Results The percentages of CD3-CD56+ NK cells and CD3+CD56+ NKT cells in peripheral blood showed significant decreases in patients with CHC compared with healthy subjects (p<0.01) and was positively correlated with the intensity of intrahepatic NK cells (p=0.001). The CD56+ NK cell infiltrate was found to be absent or minimal in about 70% of the liver biopsies of patients with CHC. Patients presented with chronic fatigue showed significantly lower percentages of circulating NK and NKT cells and intensity of intrahepatic NK cells than patients who were asymptomatic (p<0.05). The percentages of peripheral blood NK cells and NKT cells and the intensity of intrahepatic NK cells showed significant inverse correlations with serum HCV RNA levels, steatosis grade, METAVIR fibrosis stage and intensity of activated HSCs (p<0.05) and statistically insignificant correlations with serum levels of aminotransferases and the histological activity grade (p>0.05). Conclusion The deficiency of peripheral blood and intrahepatic NK cells in patients with chronic hepatitis C may provide a mechanism for immune suppression resulting in viral persistence, disease chronicity and progression of hepatic fibrosis. Restoration of the NK cell population may be one of the potential manipulations for resolution of HCV infection and for therapeutic modulation of hepatofibrogenesis.