A multistep cytological approach for patients with jaundice and biliary strictures of indeterminate origin.

AIMS: Fluorescence in situ hybridisation (FISH) increases the sensitivity for detecting pancreatobiliary tract cancer over routine cytology. In this study, diagnostic accuracy and costs of cytology and FISH in detecting cancer in patients with jaundice with biliary strictures were assessed. METHODS: Brushing specimens from 109 patients with jaundice were obtained during endoscopic retrograde cholangiopancreatography and examined by cytology and FISH. The specimens were considered FISH-positive for malignancy if at least five polysomic cells or 10 cells with homozygous or heterozygous 9p21/p16 deletion were detected. Definitive diagnosis of the stricture as benign or malignant relied on surgical pathology (45 cases) or clinical-radiological follow-up >18 months (64 cases). We calculated costs of cytology and FISH based on the reimbursement from the Piedmont region, Italy (respectively, €33 and €750). RESULTS: Ninety of 109 patients had evidence of malignancy (44 pancreatic carcinomas, 36 cholangiocarcinomas, 5 gallbladder carcinomas, 5 other cancers), while 19 had benign strictures. Routine cytology showed 42% sensitivity, but 100% specificity for the diagnosis of malignancy, while FISH-polysomy showed 70% sensitivity with 100% specificity and FISH-polysomy plus homozygous or heterozygous 9p21/p16 deletion showed 76% sensitivity with 100% specificity. The cost per additional correct diagnosis of cancer obtained by FISH, in comparison with cytology, was €1775 using a sequential cytological approach (ie, performing FISH only in patients with negative or indeterminate cytology). CONCLUSIONS: FISH should be recommended as the second step in detecting cancer in patients with jaundice with pancreatobiliary tract strictures and cytology negative or indeterminate for malignancy.

A case-control histological study on the effects of phlebotomy in patients with chronic hepatitis C.

OBJECTIVE: The aim of this study was to assess the actual effectiveness of long-term phlebotomy by comparing histological improvement (HI) in 69 Caucasian HCV-RNA-positive CHC patients undergoing phlebotomy or receiving an interferon-based therapy without virological response [nonresponders to interferon therapy(IBT-NR)]. METHODS: HI was defined by at least one point reduction of the staging score or, in the case of unchanged stage, by at least two points reduction of the grading score (Knodel's Activity Index) and was retrospectively evaluated by comparing two consecutive (56 ± 28 months apart) liver biopsies from 30 phlebotomized and 39 IBT-NR patients. RESULTS: HI was observed in 15 of 30 (50%) patients treated with phlebotomy and in six of 39 (15%) IBT-NR subjects (P=0.002). Furthermore, AST, ALT, and GGT serum levels were significantly reduced only in phlebotomized patients (P ≤ 0.003) at the time of the second biopsy. Univariate and multivariate analysis showed that histological grading score before therapy (P=0.001) and phlebotomy (P=0.002) were independently predictors of HI. CONCLUSION: HI induced by long-term phlebotomy effectively exceeds that spontaneously occurring in patients IBT-NR confirming the efficacy of iron depletion in attenuating CHC progression when other therapies have failed.

Fluorescence in situ hybridisation in the cytological diagnosis of pancreatobiliary tumours.

AIMS: To assess the sensitivity, specificity, positive and negative predictive values of fluorescence in situ hybridisation (FISH) and conventional cytology in identifying bile duct stricture malignancies. METHODS: Brushing samples were collected from 64 patients by means of endoscopic retrograde cholangiopancreatography, and assessed cytologically and by means of a multi-probe FISH set. The cytological diagnoses were: positive, negative and suspicious, whereas criteria for FISH positivity were: more than five polysomic cells or more than 10 trisomic cells for chromosomes 3 or 7. RESULTS: Forty-eight of the 64 patients showed histological or clinical signs of malignancy. The sensitivity of cytology was high (77%) if suspicious cases were considered positive, but was significantly lower than that of FISH if suspicious cases were considered negative (58% versus 90%; p < 0.05). The specificity of cytology was 81% (positive and suspicious) or 100% (negative and suspicious), and the specificity of FISH was 94% (p = 1). FISH yielded one false negative result (isolated chromosome 7 trisomy). FISH allowed a definite diagnosis of 9/12 cytologically inconclusive cases. CONCLUSIONS: Our findings suggest using FISH in the case of bile duct strictures cytologically negative or inconclusive; a FISH diagnosis of malignancy should only be made in the presence of polysomic pattern.

Phlebotomy improves histology in chronic hepatitis C males with mild iron overload.

AIM: To investigate the usefulness of mild iron depletion and the factors predictive for histological improvement following phlebotomy in Caucasians with chronic hepatitis C (CHC). METHODS: We investigated 28 CHC Caucasians with persistently elevated serum aminotransferase levels and non responders to, or unsuitable for, antiviral therapy who underwent mild iron depletion (ferritin < or = 70 ng/mL) by long-term phlebotomy. Histological improvement, as defined by at least one point reduction in the staging score or, in case of unchanged stage, as at least two points reduction in the grading score (Knodell), was evaluated in two subsequent liver biopsies (before and at the end of phlebotomy, 48 +/- 16 mo apart). RESULTS: Phlebotomy showed an excellent safety profile. Histological improvement occurred in 12/28 phlebotomized patients. Only males responded to phlebotomy. At univariate logistic analysis alcohol intake (P = 0.034), high histological grading (P = 0.01) and high hepatic iron concentration (HIC) (P = 0.04) before treatment were associated with histological improvement. Multivariate logistic analysis showed that in males high HIC was the only predictor of histological improvement following phlebotomy (OR = 1.41, 95% CI: 1.03-1.94, P = 0.031). Accordingly, 12 out of 17 (70%) patients with HIC > or = 20 micromol/g showed histological improvements at the second biopsy. CONCLUSION: Male CHC Caucasian non-responders to antiviral therapy with low-grade iron overload can benefit from mild iron depletion by long-term phlebotomy.

Combination of oxidative stress and steatosis is a risk factor for fibrosis in alcohol-drinking patients with chronic hepatitis C.

BACKGROUND AND AIMS: Alcohol and HCV have been shown to interact in stimulating hepatic oxidative damage. Thus, we investigated the contribution of oxidative mechanisms in the progression of chronic hepatitis C (CHC) in alcohol consumers. METHODS: An increased IgG reactivity against lipid peroxidation-derived antigens was used as the marker for alcohol-induced oxidative damage in 125 CHC patients. RESULTS: Alcohol intake significantly increased the frequency of the subjects with elevated lipid peroxidation-related IgG. However, no association was evident between oxidative stress markers and the severity of steatosis, necroinflammation, or fibrosis. Multivariate analysis revealed that age (P= 0.014) and hepatic iron content (P= 0.034) were the only independent predictors of fibrosis in these patients. However, the risk of fibrosis in the subjects with both steatosis and oxidative stress-induced immune responses was 6- (OR 6.2, 95% CI 1.2-31.0) and 14-fold (OR 14.6, 95% CI 3.1-68.1) higher than in the subjects with steatosis alone or without steatosis, respectively. Multivariate analysis confirmed that the combination of steatosis and oxidative stress (P= 0.045) was, together with age (P= 0.021) and hepatic iron content (P= 0.027), an independent risk factor for fibrosis in CHC patients with alcohol intake. CONCLUSIONS: These results demonstrate that oxidative stress interacts with steatosis to promote the progression of CHC in alcohol-consuming patients.

Heterozygous beta-globin gene mutations as a risk factor for iron accumulation and liver fibrosis in chronic hepatitis C.

BACKGROUND: Iron accumulation is a well-known risk factor for the progression of chronic hepatitis C (CHC) to fibrosis. However, the profibrogenic role of the genes controlling iron homeostasis is still controversial. AIM: To evaluate the relative role of haemachromatosis (HFE), ferroportin and beta-globin gene mutations in promoting iron accumulation and fibrosis in patients with CHC. METHODS: Genetic analysis was performed together with the assessment of hepatic iron content and histology in 100 consecutive HIV-antibody and hepatitis B surface antigen-negative patients with biopsy-proven CHC. RESULTS: Among the patients investigated, 12 were heterozygous for various beta-globin gene mutations (39[C-->T], IVS1.1[G-->A], 22 7 bp deletion and IVS1.6[T-->C]) and 29 carried HFE (C282Y, H63D and S65C) gene mutations. One further patient was heterozygous for both HFE (H63D) and beta-globin (39[C-->T]) variants, whereas 58 had the wild-type alleles of both the genes. Hepatic iron concentration (HIC) and hepatic stainable iron were significantly higher (p<0.05) in patients with CHC carrying beta-globin mutations than in those with HFE mutations or the wild-type alleles. Multivariate analysis confirmed that the presence of beta-globin mutations was independently associated with both HIC (p = 0.008) and hepatic-stainable iron (odds ratio (OR) 6.11; 95% CI 1.56 to 23.92; p = 0.009). Moderate/severe fibrosis or cirrhosis (Ishak's score >2) was observed in 48 of 100 patients. Logistic regression demonstrated that age (OR 1.05; 95% CI 1.02 to 1.09; p<0.005) and beta-globin mutations (OR 4.99; 95% CI 1.22 to 20.3; p = 0.025) were independent predictors of the severity of fibrosis. CONCLUSIONS: Heterozygosis for beta-globin mutations is a novel risk factor for both hepatic iron accumulation and the progression to fibrosis in patients with CHC.

Anti-phospholipid antibodies associated with alcoholic liver disease target oxidized phosphatidylserine on apoptotic cell plasma membranes.

BACKGROUND/AIMS: Circulating anti-phospholipid antibodies (aPL) are often present in patients with alcoholic liver disease (ALD). The observations that defects in the disposal of apoptotic corpses leads to the development of aPL prompted us to investigate whether ALD-associated aPL might recognize antigens in apoptotic cells. METHODS: Apoptosis was induced in HuT-78 human T-lymphoma and HepG2 hepatoma cells by, respectively, FAS ligation with CH11 monoclonal antibodies or the incubation with ethanol (400 mmol/L). RESULTS: Flow cytometry revealed that IgG from ALD patients with high aPL titers selectively bind to the surface of apoptotic, but not to viable cells. No binding was instead evident using either control or aPL-negative ALD sera. ELISA assays using different oxidized phospholipids as antigens showed that anti-phospholipid reactivity of ALD sera was mainly directed towards oxidized cardiolipin and phosphatidylserine. The pre-adsorption of aPL-positive sera with oxidized phosphatidylserine, but not with oxidized cardiolipin, lowered aPL binding to apoptotic HuT-78 cells by about 50%. No effect was instead observed by pre-adsorption with oxidation-protected phospholipids or with human serum albumin adducted with different lipid peroxidation products. CONCLUSIONS: aPL associated with ALD target apoptotic cells by specifically recognizing oxidized phosphatidylserine, suggesting a possible link between hepatocyte apoptosis and anti-phospholipid auto-reactivity in ALD.

Evaluation of liver fibrosis in chronic hepatitis C with a computer-assisted morphometric method.

Objective methods are needed to quantitatively assess the burden of fibrous tissue in liver biopsy specimens and its changes after treatment. The aim of this study was to assess the validity of a computer-assisted morphometric method in the evaluation of liver fibrosis in patients with chronic hepatitis C. Sixty-nine liver biopsy specimens stained with Sirius red were evaluated by two independent observers with a computer-assisted morphometric method to measure the percentage of fibrous tissue in the optic fields examined (fibrosis ratio). Furthermore, 11 pairs of liver biopsy specimens obtained before and after treatment from patients with chronic hepatitis C were evaluated with morphometry by two independent observers in order to assess in which direction fibrosis changed. In the 69 patients, the correlation of the morphometry-measured fibrosis ratio pairs by the two observers was high (r = 0.781). However, the differences between paired values were large, reaching +/- 5% in 95% of instances. The fibrosis ratios observed with morphometry by the two examiners correlated poorly with the Ishak's staging score. The two examiners agreed in 10 out of 11 instances in judging in which direction fibrosis had changed. In conclusion, using our present technique of computer-assisted morphometry, the quantitative assessment of the percentage extension of fibrous tissue was not sufficiently accurate. However, computer-assisted morphometry proved to be useful when evaluating the direction of fibrous changes in pairs of liver biopsy specimens from patients with chronic hepatitis C before and after treatment.