Antal Csepregi

APC promoter methylation and protein expression in hepatocellular carcinoma

PurposeWe investigated the impact of promoter methylation on APC protein expression in patients with hepatocellular carcinoma (HCC).Materials and methods50 patients [HCC (n=19), liver metastasis (n=19), cholangiocellular cancer (n=7), and benign liver tumors (n=5)] were studied for methylation using Methylight analysis. APC mutation was investigated by protein truncation test and direct sequencing of genomic DNA. The protein expression was evaluated by immunohistochemistry and Western blot analysis.ResultsThe APC promoter was hypermethylated in 81.8% of non-cancerous liver tissue samples. All HCC samples and ten patients with liver metastasis (52.6%) exhibited APC promoter methylation. The degree of methylation was significantly higher in samples from HCC compared to the non-cancerous liver tissue samples (63.1% vs. 24.98%; p=0.001). The level of APC protein expression was significantly reduced in HCC samples compared to that of the corresponding non-tumor liver tissue (p<0.05).ConclusionsPromoter methylation of the APC gene seems to be of significance in hepatocarcinogenesis and results in reduced protein expression in HCC. Interestingly, APC promoter methylation is also present in the vast majority of non-cancerous liver tissue whose (patho)physiological function remains unresolved.

Impact of the operator's experience on value of high-resolution transabdominal ultrasound in the diagnosis of choledocholithiasis : A prospective comparison using endoscopic retrograde cholangiography as the gold standard

Objective. Transabdominal ultrasound (US) is the most frequently used imaging method for the diagnosis of choledocholithiasis. The aim of this prospective study was to evaluate the diagnostic accuracy of high-resolution US in the diagnosis of common bile duct stones depending on the operators experience and in comparison with endoscopic retrograde cholangiography (ERC) as the gold standard. Material and methods. From April 2003 through November 2004, 126 patients referred because of clinically and biochemically suspected common bile duct stones were included in the study. Two patients were excluded because they refused to undergo ERC. Consequently, the study comprised 124 patients (86 F, 38 M, mean age 63.2 years, range 21–91 years). High-resolution US was performed (2–5 MHz sector scanner; Siemens Elegra, Erlangen, Germany) by operators who were unaware of the results of other imaging procedures. The definitive diagnosis was established by means of ERC. Results. Thirty-five out of 124 patients were investigated by experienced examiners. Twenty-seven of 35 patients (77%) were found to have stones at ERC. Bile duct stones were correctly found by US in 22 out of 27 patients (sensitivity 82%, 95% CI: 63–92). Of the 8 patients without stones at ERC, one false-positive diagnosis was made with US (specificity 88%, 95% CI: 53–98). Correct diagnoses were made in 29 out of 35 (accuracy 83%, 95% CI: 67–92) patients investigated by experienced examiners. Eighty-nine out of 124 patients were investigated by less-experienced examiners. Fifty-four of 89 patients (61%) were found to have stones at ERC. Choledocholithiasis was found correctly in only 25 out of 54 patients (sensitivity 46%, 95% CI: 34–59). Of the 35 patients without stones at ERC, three false-positive diagnoses were made with US (specificity 91%, 95% CI: 78–97). In conclusion, correct diagnoses were observed in 57 of 89 patients (accuracy 64%, 95% CI: 54–73) investigated by less-experienced examiners (p<0.05 in comparison with the results of experienced examiners). Conclusions. High-resolution US carried out by experienced examiners has a high diagnostic accuracy in the diagnosis of choledocholithiasis. Therefore, good training and continued experience are prerequisites for successful sonographic detection of bile duct stones using US. Under these conditions, further expensive and invasive methods such as ERC, magnetic resonance cholangiopancreatography and endoscopic ultrasonography may not be necessary in cases with a clear sonographic diagnosis.

Promoter methylation of CDKN2A and lack of p16 expression characterize patients with hepatocellular carcinoma

BackgroundThe product of CDKN2A, p16 is an essential regulator of the cell cycle controlling the entry into the S-phase. Herein, we evaluated CDKN2A promoter methylation and p16 protein expression for the differentiation of hepatocellular carcinoma (HCC) from other liver tumors.MethodsTumor and corresponding non-tumor liver tissue samples were obtained from 85 patients with liver tumors. CDKN2A promoter methylation was studied using MethyLight technique and methylation-specific PCR (MSP). In the MethyLight analysis, samples with ≥ 4% of PMR (percentage of methylated reference) were regarded as hypermethylated. p16 expression was evaluated by immunohistochemistry in tissue sections (n = 148) obtained from 81 patients using an immunoreactivity score (IRS) ranging from 0 (no expression) to 6 (strong expression).ResultsHypermethylation of the CDKN2A promoter was found in 23 HCCs (69.7%; mean PMR = 42.34 ± 27.8%), six (20.7%; mean PMR = 31.85 ± 18%) liver metastases and in the extralesional tissue of only one patient. Using MSP, 32% of the non-tumor (n = 85), 70% of the HCCs, 40% of the CCCs and 24% of the liver metastases were hypermethylated. Correspondingly, nuclear p16 expression was found immunohistochemically in five (10.9%, mean IRS = 0.5) HCCs, 23 (92%; mean IRS = 4.9) metastases and only occasionally in hepatocytes of non-lesional liver tissues (mean IRS = 1.2). The difference of CDKN2A-methylation and p16 protein expression between HCCs and liver metastases was statistically significant (p < 0.01, respectively).ConclusionPromoter methylation of CDKN2A gene and lack of p16 expression characterize patients with HCC.

LKM3 autoantibodies in hepatitis C cirrhosis: A further phenomenon of the HCV-induced autoimmunity

Chronic hepatitis C is frequently associated with laboratory markers—including LKM1 autoantibodies—of autoimmunity. A 62-yr-old woman with hepatitis C cirrhosis presented autoantibodies against liver and kidney microsomal proteins. By further evaluation of autoantibodies using ELISA and immunoblotting LKM1 and LKM3 autoantibodies could be revealed. The target antigen of LKM3 autoantibodies proved to be UGT-1.1 isoenzyme. In the absence of chronic hepatitis D infection or autoimmune hepatitis type 2, this is the first case that reports the occurrence of LKM3 autoantibodies in HCV-induced chronic liver disease.

The pathophysiology of portal hypertension.

Portal hypertension is defined by an elevation in blood pressure in the portal system. Different causes are known and include a pre-, intra-, or posthepatic block. Portal hypertension is also classified according to the sinusoidal system. Portal pressure becomes elevated by either an increase in blood flow (Q), an increase in resistance (R), or both. Regulation of the vascular tone in the splanchnic system includes intrinsic and extrinsic aspects. A variety of metabolic end-products (e.g. adenosine), endothelium-derived substances (e.g. nitric oxide), and certain neurotransmitters (e.g. acetylcholine) are known to relax the tone and thus produce vasodilation. Important vasoconstrictor influences on splanchnic arterioles include circulating agents (e.g. angiotensin), endothelium-derived substances (e.g. endothelin), and again neurotransmitters (norepinephrine). Besides vascular tone, structural changes (thrombosis, fibrosis, shear stress, and cell regeneration) add to overall hepatic resistance. Further consequences of portal hypertension include an increase in blood flow which leads to a hyperdynamic state with fluid retention, leading to secondary involvement of other organs, such as cirrhotic cardiomyopathy, hepatopulmonary syndrome and hepatorenal syndrome. Finally, portal hypertension will end up in the formation of collateral vessels. Varices can involve the whole gastrointestinal tract and are a frequent source of bleeding.

Sorafenib Therapy in Patients with Advanced Hepatocellular Carcinoma in Advanced Liver Cirrhosis

Background and Aim: Sorafenib has become the treatment standard for patients with advanced hepatocellular carcinoma (HCC). It is not clear whether patients with advanced liver function impairment (Child B) and patients undergoing additional locoregional therapy may tolerate treatment with sorafenib and benefit. We aimed to evaluate the tolerability and efficiency of sorafenib in patients with advanced HCC and different stages of liver cirrhosis, and in combination with locoregional therapy. Methods: In 50 patients with advanced HCC treated with sorafenib tolerability and efficiency of the therapy with respect to stage of liver cirrhosis, existence of extrahepatic tumor spread, and additional locoregional therapy were evaluated. Results: Fifty patients with advanced HCC were treated with sorafenib, and 13 received additional locoregional therapy. Tolerability of the systemic treatment was moderate in all patients, with no significant differences between the subgroups, while the median survival was better in patients with Child A than Child B cirrhosis. Conclusion: Tolerability and toxicity of a systemic treatment with sorafenib are moderate in patients with liver cirrhosis in Child A or B. Prospective randomized studies are required to evaluate the efficacy and tolerability of combined systemic and locoregional treatment approaches in patients with advanced HCC.

Glatiramer acetate induced acute exacerbation of autoimmune hepatitis in a patient with multiple sclerosis.

Sirs: Glatiramer acetate (Copaxone ) is a synthetic random copolymer of four amino acids and is antigenically similar to myelin basic protein [1]. It represents an established second-line therapy for relapsing-remitting type of multiple sclerosis (MS). Recently, glatiramer acetate was suggested to induce autoimmune diseases [2, 3]. We report here a case of a 71year-old man who had suffered from MS since 1992, and was treated with interferon beta-1b between 1994 and 2004. In 1999, a weak positive test result for antinuclear antibodies (ANA) with a titre of 80 (normal <80) was detected in his serum. Liver blood tests were normal. In January 2004, the interferon therapy was interrupted because of elevated (<3· ULN) serum liver enzyme activities. EDSS was performed giving a result of 3.5. Two months later, glatiramer acetate in a dose of 20 mg once a day was initiated because of increasing disability of the patient (EDSS 4.5). In May 2004, he presented first in our clinic with malaise and jaundice which was associated with elevated serum liver enzyme activities (Table 1). There was no evidence of any metabolic, viral (hepatitis A, B C and E, HSV, VZV, EBV, CMV) or alcoholic diseases or bile duct obstruction. The patient had no medical history that might have predisposed him for developing AIH (e.g. nitrofurantoin and minocycline). His physical examination showed only a slightly enlarged liver without stigmata of chronic liver disease. Blood tests showed elevated serum alanine aminotransferase and bilirubin levels (Table). Immunological studies revealed a significantly elevated titre of 1,280 of ANA, while serum IgG and total immunoglobulin levels remained normal. Further serological markers of AIH including autoantibodies against F-Actin, SLA, LC, LKM and c-ANCA, p-ANCA, SMA, and AMA were all negative. Liver histology suggested a drug-induced liver-injury without fibrotic changes of the liver. The treatment with Copaxone was stopped. The liver tests returned to normal in four weeks. Any medication was stopped over the next 15 months after first episode of acute hepatitis. The liver serum tests were controlled monthly and remained within the normal range. The MS showed, however, an unfavourable course giving an EDSS score of 6.5. In November 2005, our patient re-presented with fever, uncharacteristic abdominal symptoms, and elevated serum liver enzyme activities (Table 1). There was no evidence of viral hepatitis or obstruction of bile ducts. At this stage, the diagnosis of autoimmune hepatitis (AIH) was made. Our patient was commenced on budesonide (Budenofalk Caps) 3 mg tid. The therapy led to a significant improvement of liver function tests. Subsequently, he was put on mycophenolate mofetil (CellCept ). Glatiramer acetate is reported to be well tolerated. Most commonly reported adverse events include local reactions on the injection site and transient postinjection systemic reactions. In the context with the treatment with glatiramer acetate, autoimmune diseases such as myasthenia gravis [2] and autoimmune thyreoiditis [3] have been reported. Significant liver complications including the development of AIH have not been previously published. In our case, the optimal response to immunosuppressive treatment, the presence of high-titre autoantibodies, the second exacerbation of hepatitis, and the association of AIH with MS, especially in males, [4, 5] argues against a drug-induced liver disease. Glatiramer acetate can induce T helper type 2 cells that cross-react with myelin basic protein [6]. These cells release cytokines like IL-4, IL-6, and IL-10 which therefore may enhance the production of autoantibodies [7] and lead to induction of autoimmune diseases in genetically predisposed patients. The commercial formulation of Copaxone contains mannitol that was recently identified as an immunoactive hapten capable of provoking anaphylaxis [8]. Mannitol may play a distinct role in the H. Neumann (&) Æ A. Csepregi P. Malfertheiner Dept. of Gastroenterology, Hepatology, and Infectious Diseases Otto-von-Guericke University Leipziger St. 44 39120 Magdeburg, Germany E-Mail: Helmut.Neumann@Medizin.UniMagdeburg.de

Acquired angioedema associated with chronic hepatitis C

C1-esterase inhibitor (C1-INH) deficiency is known to result in generalized angioedema.1 In addition to its hereditary form, an increasing number of diseases, including tumors and autoimmune disorders, were described in association with C1-INH deficiency (acquired angioedema [AAE]).1 Hepatitis C virus (HCV) has been considered among the most important causes of chronic liver disease, as well as an inducer of autoimmunity.2 The cases of 2 women with chronic hepatitis C are reported herein; both had frequent, severe attacks of angioedema caused by deficiency of C1-INH.

Mannose-binding lectin deficiency confers risk for bacterial infections in a large Hungarian cohort of patients with liver cirrhosis.

BACKGROUND & AIMS Mannose-binding lectin (MBL) is a serum lectin synthesized by the liver and involved in innate host defense. MBL deficiency increases the risk of various infectious diseases mostly in immune-deficient conditions. Bacterial infections are a significant cause of morbidity and mortality in liver cirrhosis due to the relative immuncompromised state. METHODS Sera of 929 patients with various chronic liver diseases [autoimmune liver diseases (ALD), 406; viral hepatitis C (HCV), 185; and liver cirrhosis (LC) with various etiologies, 338] and 296 healthy controls (HC) were assayed for MBL concentration. Furthermore, a follow-up, observational study was conducted to assess MBL level as a risk factor for clinically significant bacterial infections in cirrhotic patients. RESULTS MBL level and the prevalence of absolute MBL deficiency (<100 ng/ml) was not significantly different between patients and controls (ALD: 14.5%, HCV: 11.9%, LC: 10.7%, HC: 15.6%). In cirrhotic patients, the risk for infection was significantly higher among MBL deficient subjects as compared to non-deficient ones (50.0% vs. 31.8%, p=0.039). In a logistic regression analysis, MBL deficiency was an independent risk factor for infections (OR: 2.14 95% CI: 1.03-4.45, p=0.04) after adjusting for Child-Pugh score, co-morbidities, gender, and age. In a Kaplan-Meier analysis, MBL deficiency was associated with a shorter time to develop the first infectious complication (median days: 579 vs. 944, pBreslow=0.016, pLogRank=0.027) and was identified as an independent predictor in a multivariate Cox-regression analysis (p=0.003, OR: 2.33, 95% CI: 1.34-4.03). CONCLUSIONS MBL deficiency is associated with a higher probability and shorter time of developing infections in liver cirrhosis, further supporting the impact of the MBL molecule on the host defense.

Serum Anti-cholesterol Antibodies in Chronic Hepatitis-C Patients During IFN-α-2b Treatment

Previously we detected more than 3 times higher anti-cholesterol antibody (ACHA) levels in HIV positive patients compared to healthy individuals, however, this level significantly decreased during highly active anti-retroviral therapy (HAART). In our present study we examined whether these findings could also be detected in patients with chronic hepatitis C (CHC). We calculated the correlation between the ACHA levels and the C5b-9 complement activation product. 39 patients with CHC were treated with IFN-alpha-2b (Schering-Plough) 5 MU daily for 6 weeks, followed by 5 MU TIW. Serum levels of ACHA and complement activation products were measured with ELISA. Serum HCV RNA was measured by a highly sensitive branched DNA technique before and 3, 6 and 12 months after the beginning of IFN-alpha-2b therapy. 52 healthy persons served as controls. At the onset of treatment ACHA level was significantly (p = 0.0062) higher in patients (40 (24-69) AU/ml) (median (interquartile range)) than in control sera (26 (20-35) AU/ml). In the 26 responder patients ACHA levels decreased to the normal level during the therapy, but no change was observed in the 13 non-responders. In patients with a sustained response ACHA levels remained low till the end of the 12 months IFN treatment. ACHA levels were significantly (p = 0.0422) higher in the patients with low ( or = 4.0 mmol/l) cholesterol concentrations. The ACHA level before the therapy strongly correlated (r = 0.5499, p = 0.0014) with C5b-9 serum levels. ACHA levels are elevated in CHC, but this elevation is not as high as in HIV. Decrease of viral load by IFN-alpha-2b treatment in the responders results in normalization of ACHA concentration. High ACHA levels in patients with low serum cholesterol concentration suggest that high ACHA levels may contribute to the decrease in cholesterol levels. The correlation between the ACHA and C5b-9 levels indicate, that the ACHA may play a role in the complement activation in CHC.