Dominique Wendum

Genotype–phenotype correlation in hepatocellular adenoma: New classification and relationship with HCC

Hepatocellular adenomas are benign tumors that can be difficult to diagnose. To refine their classification, we performed a comprehensive analysis of their genetic, pathological, and clinical features. A multicentric series of 96 liver tumors with a firm or possible diagnosis of hepatocellular adenoma was reviewed by liver pathologists. In all cases, the genes coding for hepatocyte nuclear factor 1α (HNF1α) and β‐catenin were sequenced. No tumors were mutated in both HNF1α and β‐catenin enabling tumors to be classified into 3 groups, according to genotype. Tumors with HNF1α mutations formed the most important group of adenomas (44 cases). They were phenotypically characterized by marked steatosis (P < 10−4), lack of cytological abnormalities (P < 10−6), and no inflammatory infiltrates (P < 10−4). In contrast, the group of tumors defined by β‐catenin activation included 13 lesions with frequent cytological abnormalities and pseudo‐glandular formation (P < 10−5). The third group of tumors without mutation was divided into two subgroups based on the presence of inflammatory infiltrates. The subgroup of tumors consisting of 17 inflammatory lesions, resembled telangiectatic focal nodular hyperplasias, with frequent cytological abnormalities (P = 10−3), ductular reaction (P < 10−2), and dystrophic vessels (P = .02). In this classification, hepatocellular carcinoma associated with adenoma or borderline lesions between carcinoma and adenoma is found in 46% of the β‐catenin–mutated tumors whereas they are never observed in inflammatory lesions and are rarely found in HNF1α mutated tumors (P = .004). In conclusion, the molecular and pathological classification of hepatocellular adenomas permits the identification of strong genotype–phenotype correlations and suggests that adenomas with β‐catenin activation have a higher risk of malignant transformation. (HEPATOLOGY 2006;43:515–524.)

Assessment of biliary fibrosis by transient elastography in patients with PBC and PSC.

Noninvasive measurement of liver stiffness with transient elastography has been recently validated for the evaluation of hepatic fibrosis in chronic hepatitis C. The current study assessed the diagnostic performance of liver stiffness measurement (LSM) for the determination of fibrosis stage in chronic cholestatic diseases. One hundred one patients with primary biliary cirrhosis (PBC, n = 73) or primary sclerosing cholangitis (PSC, n = 28) were prospectively enrolled in a multicenter study. All patients underwent liver biopsy (LB) and LSM. Histological and fibrosis stages were assessed on LB by two pathologists. LSM was performed by transient elastography. Efficiency of LSM for the determination of histological and fibrosis stages were determined by a receiver operating characteristics (ROC) curve analysis. Analysis failed in six patients (5.9%) because of unsuitable LB (n = 4) or LSM (n = 2). Stiffness values ranged from 2.8 to 69.1 kPa (median, 7.8 kPa). LSM was correlated to both fibrosis (Spearmans ρ = 0.84, P < .0001) and histological (0.79, P < .0001) stages. These correlations were still found when PBC and PSC patients were analyzed separately. Areas under ROC curves were 0.92 for fibrosis stage (F) ≥2, 0.95 for F ≥ 3 and 0.96 for F = 4. Optimal stiffness cutoff values of 7.3, 9.8, and 17.3 kPa showed F ≥ 2, F ≥ 3 and F = 4, respectively. LSM and serum hyaluronic acid level were independent parameters associated with extensive fibrosis on LB. In conclusion, transient elastography is a simple and reliable noninvasive means for assessing biliary fibrosis. It should be a promising tool to assess antifibrotic therapies in PBC or PSC. (HEPATOLOGY 2006;43:1118–1124.)

Gefitinib, an EGFR inhibitor, prevents hepatocellular carcinoma development in the rat liver with cirrhosis†

Epidermal growth factor receptor (EGFR) binds transforming growth factor α (TGF‐α) which is mitogenic for hepatocytes. Diverse lines of evidence suggest that activation of the TGF‐α /EGFR pathway contributes to hepatocellular carcinoma (HCC) formation. Herein, we developed an experimental model of cirrhosis giving rise to HCC and tested the antitumoral effect of gefitinib, a selective EGFR tyrosine kinase inhibitor, in this model. Rats received weekly intraperitoneal injections of diethylnitrosamine (DEN) followed by a 2‐week wash‐out period that caused cirrhosis in 14 weeks and multifocal HCC in 18 weeks. Hepatocyte proliferation was increased in diseased tissue at 14 weeks compared with control liver and at even higher levels in HCC nodules compared with surrounding diseased tissues at 18 weeks. Increased proliferation was paralleled by upregulation of TGF‐α messenger RNA expression. A group of DEN‐treated rats received daily intraperitoneal injections of gefitinib between weeks 12 and 18. In rats treated with gefitinib, the number of HCC nodules was significantly lower than in untreated rats (18.1 ± 2.4 vs. 3.7 ± 0.45; P < .05), while EGFR was activated to a lesser extent in the diseased and tumoral tissues of these animals compared with untreated rats. HCC nodules from both untreated and gefitinib‐treated animals displayed insulin‐like growth factor 2 overexpression that contributed to tumor formation in treated animals. In conclusion, the blockade of EGFR activity by gefitinib has an antitumoral effect on the development of HCC in DEN‐exposed rats, suggesting that it may provide benefit for the chemoprevention of HCC. (HEPATOLOGY 2005,41:307–314.)

Diffusion-weighted magnetic resonance imaging for the assessment of fibrosis in chronic hepatitis C†

Liver biopsy is the gold standard for assessing fibrosis but has several limitations. We evaluated a noninvasive method, so‐called diffusion‐weighted magnetic resonance imaging (DWMRI), which measures the apparent diffusion coefficient (ADC) of water, for the diagnosis of liver fibrosis in patients with chronic hepatitis C virus (HCV). We analyzed 20 healthy volunteers and 54 patients with chronic HCV (METAVIR: F0, n = 1; F1, n = 30; F2, n = 8; F3, n = 5; and F4, n = 10) prospectively included. Patients with moderate‐to‐severe fibrosis (F2‐F3‐F4) had hepatic ADC values lower than those without or with mild fibrosis (F0‐F1; mean: 1.10 ± 0.11 versus 1.30 ± 0.12 × 10−3 mm2/s) and healthy volunteers (mean: 1.44 ± 0.02 × 10−3 mm2/s). In discriminating patients staged F3‐F4, the areas under the receiving operating characteristic curves (AUCs) were 0.92 (±0.04) for magnetic resonance imaging (MRI), 0.92 (±0.05) for elastography, 0.79 (±0.08) for FibroTest, 0.87 (±0.06) for the aspartate aminotransferase to platelets ratio index (APRI), 0.86 (±0.06) for the Forns index, and 0.87 (±0.06) for hyaluronate. In these patients, the sensitivity, specificity, positive predictive value, and negative predictive value were 87%, 87%, 72%, and 94%, respectively, with an ADC cutoff level of 1.21 × 10−3 mm2/s. In discriminating patients staged F2‐F3‐F4, the AUC values were 0.79 (±0.07) for MRI, 0.87 (±0.05) for elastography, 0.68 (±0.09) for FibroTest, 0.81 (±0.06) for APRI, 0.72 (±0.08) for the Forns index, and 0.77 (±0.06) for hyaluronate. Conclusion: This preliminary study suggests that DWMRI compares favorably with other noninvasive tests for the presence of significant liver fibrosis. (HEPATOLOGY 2007.)

The Myofibroblastic Conversion of Peribiliary Fibrogenic Cells Distinct from Hepatic Stellate Cells Is Stimulated by Platelet-Derived Growth Factor During Liver Fibrogenesis

The origin of myofibroblasts and the factors promoting their differentiation during liver fibrogenesis remain uncertain. During biliary-type fibrogenesis, the proliferation and chemoattraction of hepatic stellate cells (HSC) toward bile ducts is mediated by platelet-derived growth factor (PDGF), while myofibroblastic conversion of peribiliary cells distinct from HSC also occurs. We herein examined the phenotype of these peribiliary myofibroblasts as compared with myofibroblastic HSC and tested whether their differentiation was affected by PDGF. Biliary-type liver fibrogenesis was induced by common bile duct ligation in rats. After 48 hours, periductular fibrosis in portal tracts colocalized with smooth muscle α-actin–immunoreactive myofibroblasts, the majority of which were desmin negative. Simultaneously, in sinusoids, desmin immunoreactivity was induced in a large number of HSC, which were smooth muscle α-actin negative. Cultures of peribiliary myofibroblasts were expanded from isolated bile duct segments and compared with myofibroblastic HSC. Peribiliary myofibroblasts outgrowing from bile duct segments expressed smooth muscle α-actin, α1 (I) collagen mRNA, and PDGF receptor-β subunit. Desmin immunoreactivity gradually decreased in cultured peribiliary myofibroblasts, contrasting with constant labeling of all myofibroblastic HSC. In addition, IL-6 expression in peribiliary myofibroblasts was up to 100-fold lower than in myofibroblastic HSC, whereas the expression of the complement-activating protease P100 in both cell types showed little difference and that of the extracellular matrix component fibulin 2 was similar. The expression of smooth muscle α-actin protein in cultured peribiliary myofibroblasts was stimulated by PDGF-BB and inhibited by STI571, a PDGF receptor tyrosine kinase inhibitor, whereas in bile duct–ligated rats, the administration of STI571 caused a significant decrease in peribiliary smooth muscle α-actin immunoreactivity, and to a lesser extent, a decrease in peribiliary fibrosis. These results indicate that peribiliary cells distinct from HSC undergo a PDGF-mediated conversion into myofibroblasts expressing IL-6 at lower levels than myofibroblastic HSC and contribute to the initial formation of biliary-type liver fibrosis.

Hepatocellular Hypoxia-Induced Vascular Endothelial Growth Factor Expression and Angiogenesis in Experimental Biliary Cirrhosis

We tested the potential role of vascular endothelial growth factor (VEGF) and of fibroblast growth factor-2 (FGF-2) in the angiogenesis associated with experimental liver fibrogenesis induced by common bile duct ligation in Sprague-Dawley rats. In normal rats, VEGF and FGF-2 immunoreactivities were restricted to less than 3% of hepatocytes. One week after bile duct ligation, hypoxia was demonstrated by the immunodetection of pimonidazole adducts unevenly distributed throughout the lobule. After 2 weeks, hypoxia and VEGF expression were detected in >95% of hepatocytes and coexisted with an increase in periportal vascular endothelial cell proliferation, as ascertained by Ki67 immunolabeling. Subsequently, at 3 weeks the density of von Willebrand-labeled vascular section in fibrotic areas significantly increased. Semiquantitative reverse transcription polymerase chain reaction showed that VEGF(120) and VEGF(164) transcripts, that correspond to secreted isoforms, increased within 2 weeks, while VEGF(188) transcripts remained unchanged. FGF-2 mainly consisting of a 22-kd isoform, according to Western blot, was identified by immunohistochemistry in 49% and 100% of hepatocytes at 3 and 7 weeks, respectively. Our data provide evidence that in biliary-type liver fibrogenesis, angiogenesis is stimulated primarily by VEGF in response to hepatocellular hypoxia while FGF-2 likely contributes to the maintenance of angiogenesis at later stages.

Chronic liver injury during obstructive sleep apnea

Patients with obstructive sleep apnea (OSA) are at risk for the development of fatty liver as a result of being overweight. Several data suggest that OSA per se could be a risk factor of liver injury; ischemic hepatitis during OSA has been reported, and OSA is an independent risk factor for insulin resistance. Therefore, we investigated liver damage and potential mechanisms in 163 consecutive nondrinking patients with nocturnal polysomnographic recording for clinical suspicion of OSA. Serum levels of liver enzymes were measured in all patients. Liver biopsy was offered to patients with elevated liver enzymes. Intrahepatic hypoxia was assessed by the expression of vascular endothelial growth factor (VEGF) on liver biopsy specimens. Severe OSA (apnea‐hypopnea index [AHI] > 50/hr) was seen in 27% of patients; 52% had moderate OSA (AHI 10‐50/hr), and 21% had no OSA. Overall, 20% had elevated liver enzymes. Independent parameters associated with elevated liver enzymes were body mass index (BMI) (OR: 1.13; CI: 1.03‐1.2) and severe OSA (OR: 5.9; CI: 1.2‐29). Liver biopsy was performed in 18 of 32 patients with elevated liver enzymes and showed steatohepatitis in 12 cases, associated with fibrosis in 7 cases. Patients with severe OSA were more insulin‐resistant according to homeostasis model assessment, had higher percentage of steatosis as well as scores of necrosis and fibrosis, despite similar BMI. Hepatic immunostaining used as an indirect marker of hypoxia was not different between patients with or without severe OSA. In conclusion, severe OSA is a risk factor for elevated liver enzymes and steatohepatitis independent of body weight. Promotion of insulin resistance is probably involved. Further studies are needed to determine whether hypoxia contributes directly to liver injury. (HEPATOLOGY 2005;41:1290–1296.)

Impact of steatosis on progression of fibrosis in patients with mild hepatitis C.

In patients with mild hepatitis C, the usefulness of antiviral therapy is subject of debate, as a low risk for progression of fibrosis is assumed. Several studies have shown that steatosis is a strong and independent predictor of the severity as well as the progression of fibrosis in chronic hepatitis C. Therefore, this study assessed the impact of steatosis on the progression of fibrosis between paired liver biopsies in untreated patients with mild hepatitis on index biopsy. One hundred thirty‐five untreated patients (mean age, 38 years; M/F sex ratio, 1.43) with one known risk factor of infection (68 transfusions, 67 injecting drug use) had 2 liver biopsies after a median interval of 61 months (18‐158). All had METAVIR score of A1F1 or lower at first liver biopsy. Unequivocal progression of fibrosis was considered if patients had a fibrosis score of 3 or 4 at the second liver biopsy. The probability of progression of fibrosis was estimated by using the Kaplan‐Meier method. During follow‐up, progression of fibrosis occurred in 21 patients (16%) after a median delay of 65 months. Cumulative probabilities of the progression of fibrosis at 4 and 6 years were 5.2% and 19.8%, respectively. In multivariate analysis, steatosis was the only independent factor predictive of progression of fibrosis (RR, 4.8; CI, 1.3‐18.3). Probability of progression of fibrosis was significantly related to the percentage of hepatocytes with steatosis. In conclusion, steatosis is a major determinant of the progression of fibrosis in mild hepatitis C, regardless of the genotype. Our results argue for antiviral treatment in the subgroup of patients with mild hepatitis and steatosis. (HEPATOLOGY 2005;41:82–87.)

Gemcitabine and oxaliplatin with or without cetuximab in advanced biliary-tract cancer (BINGO): a randomised, open-label, non-comparative phase 2 trial

BACKGROUND Gemcitabine plus a platinum-based agent (eg, cisplatin or oxaliplatin) is the standard of care for advanced biliary cancers. We investigated the addition of cetuximab to chemotherapy in patients with advanced biliary cancers. METHODS In this non-comparative, open-label, randomised phase 2 trial, we recruited patients with locally advanced (non-resectable) or metastatic cholangiocarcinoma, gallbladder carcinoma, or ampullary carcinoma and a WHO performance status of 0 or 1 from 18 hospitals across France and Germany. Eligible patients were randomly assigned (1:1) centrally with a minimisation procedure to first-line treatment with gemcitabine (1000 mg/m(2)) and oxaliplatin (100 mg/m(2)) with or without cetuximab (500 mg/m(2)), repeated every 2 weeks until disease progression or unacceptable toxicity. Randomisation was stratified by centre, primary site of disease, disease stage, and previous treatment with curative intent or adjuvant therapy. Investigators who assessed treatment response were not masked to group assignment. The primary endpoint was the proportion of patients who were progression-free at 4 months, analysed by intention to treat. This study is registered with ClinicalTrials.gov, number NCT00552149. FINDINGS Between Oct 10, 2007, and Dec 18, 2009, 76 patients were assigned to chemotherapy plus cetuximab and 74 to chemotherapy alone. 48 (63%; 95% CI 52-74) patients assigned to chemotherapy plus cetuximab and 40 (54%; 43-65) assigned to chemotherapy alone were progression-free at 4 months. Median progression-free survival was 6·1 months (95% CI 5·1-7·6) in the chemotherapy plus cetuximab group and 5·5 months (3·7-6·6) in the chemotherapy alone group. Median overall survival was 11·0 months (9·1-13·7) in the chemotherapy plus cetuximab group and 12·4 months (8·6-16·0) in the chemotherapy alone group. The most common grade 3-4 adverse events were peripheral neuropathy (in 18 [24%] of 76 patients who received chemotherapy plus cetuximab vs ten [15%] of 68 who received chemotherapy alone), neutropenia (17 [22%] vs 11 [16%]), and increased aminotransferase concentrations (17 [22%] vs ten [15%]). 70 serious adverse events were reported in 39 (51%) of 76 patients who received chemotherapy plus cetuximab (34 events in 19 [25%] patients were treatment-related), whereas 41 serious adverse events were reported in 25 (35%) of 71 patients who received chemotherapy alone (20 events in 12 [17%] patients were treatment-related). One patient died of atypical pneumonia related to treatment in the chemotherapy alone group. INTERPRETATION The addition of cetuximab to gemcitabine and oxaliplatin did not seem to enhance the activity of chemotherapy in patients with advanced biliary cancer, although it was well tolerated. Gemcitabine and platinum-based combination should remain the standard treatment option. FUNDING Institut National du Cancer, Merck Serono.

Noninvasive elastography‐based assessment of liver fibrosis progression and prognosis in primary biliary cirrhosis

The development of liver fibrosis markers in primary biliary cirrhosis (PBC) is needed to facilitate the assessment of its progression and the effectiveness of new therapies. Here, we investigated the potential usefulness of transient elastography (TE) in the noninvasive evaluation of liver fibrosis stage and disease progression in PBC. We performed, first, a prospective performance analysis of TE for the diagnosis of METAVIR fibrosis stages in a diagnostic cohort of 103 patients and, second, a retrospective longitudinal analysis of repeated examinations in a monitoring cohort of 150 patients followed‐up for up to 5 years. All patients were treated with ursodeoxycholic acid. Diagnostic thresholds of liver stiffness in discriminating fibrosis stages ≥F1, ≥F2, ≥F3, and =F4 were 7.1, 8.8, 10.7, and 16.9 kPa, respectively. TE showed high performance and was significantly superior to biochemical markers (e.g., aspartate aminotransferase [AST]/platelet ratio, FIB‐4, hyaluronic acid, AST/alanine aminotransferase ratio, and Mayo score) in diagnosing significant fibrosis, severe fibrosis, or cirrhosis. Analysis of the monitoring cohort data set using generalized linear models showed the following: (1) an overall progression rate of 0.48 ± 0.21 kPa/year (P = 0.02) and (2) no significant progression in patients with F0‐F1, F2, or F3 stages, but a significant increase (4.06 ± 0.72 kPa/year; P < 0.0001) in cirrhotic patients. A cut‐off value of 2.1 kPa/year was associated with an 8.4‐fold increased risk of liver decompensations, liver transplantations, or deaths (P < 0.0001, Cox regression analysis). Conclusion: TE is one of the best current surrogate markers of liver fibrosis in PBC. Over a 5‐year period, on‐treatment liver stiffness appears stable in most noncirrhotic PBC patients, whereas it significantly increases in patients with cirrhosis. Progression of liver stiffness in PBC is predictive of poor outcome. (HEPATOLOGY 2012;56:198–208)