DNA degrades during storage in formalin-fixed and paraffin-embedded tissue blocks.

Formalin-fixed paraffin-embedded (FFPE) tissue blocks are widely used to identify clinically actionable molecular alterations or perform retrospective molecular studies. Our goal was to quantify degradation of DNA occurring during mid to long-term storage of samples in usual conditions. We selected 46 FFPE samples of surgically resected carcinomas of lung, colon, and urothelial tract, of which DNA had been previously extracted. We performed a second DNA extraction on the same blocks under identical conditions after a median period of storage of 5.5 years. Quantitation of DNA by fluorimetry showed a 53% decrease in DNA quantity after storage. Quantitative PCR (qPCR) targeting KRAS exon 2 showed delayed amplification of DNA extracted after storage in all samples but one. The qPCR/fluorimetry quantification ratio decreased from 56 to 15% after storage (p < 0.001). Overall, remaining proportion of DNA analyzable by qPCR represented only 11% of the amount obtained at first extraction. Maximal length of amplifiable DNA fragments assessed with a multiplex PCR was reduced in DNA extracted from stored tissue, indicating that DNA fragmentation had increased in the paraffin blocks during storage. Next-generation sequencing was performed on 12 samples and showed a mean 3.3-fold decrease in library yield and a mean 4.5-fold increase in the number of single-nucleotide variants detected after storage. In conclusion, we observed significant degradation of DNA extracted from the same FFPE block after 4 to 6 years of storage. Better preservation strategies should be considered for storage of FFPE biopsy specimens.

Germline and somatic DICER1 mutations in familial and sporadic liver tumors.

BACKGROUND & AIMS: Growing evidence suggests that genetic predisposition significantly increases the risk of hepatocellular carcinoma (HCC), independently from the presence of other risk factors. Here, we report a novel germline DICER1 mutation associated with familial recurrent liver tumors. We then aimed to investigate the contribution of constitutional and somatic DICER1 mutations on HCC occurrence. METHODS: We investigated two individuals of a single family that developed recurrent well-differentiated hepatocellular tumors over the years. Histological slides from surgically resected tumors were reviewed. Exome sequencing was performed on constitutional DNA from circulating lymphocytes in both patients. The presence of somatic DICER1 mutations was analyzed in 243 liver tumors. MicroRNA (miRNA) sequencing was performed in 50 liver tumors to identify groups of tumors with similar profiles and differentially expressed miRNAs (DEMs). RESULTS: A pathological study identified hepatocellular adenomas and well-differentiated carcinomas in both patients. Tumors exhibited Wnt/ß-catenin pathway activation, with strong and diffuse glutamine synthetase expression. Interestingly, non-tumor liver tissues showed abnormal liver zonation as previously reported in Dicer1 knockout mouse livers. Screening for DICER1 mutations in 243 sporadic liver tumors identified six tumors with somatic DICER1 mutations. In HCCs, DICER1 mutations were significantly associated with CTNNB1 mutations (p=0.03). miRNA profiling identified a specific expression profile in DICER1-mutated tumors with a decreased expression of mature miRNAs compared to the other samples. Among the DEMs, downregulation of let-7a and miR-365b was closely related to DICER1 mutations. CONCLUSIONS: Our results highlight the role of DICER1 mutations in liver carcinogenesis in a specific subtype of familial and sporadic hepatocellular carcinomas associated with ß-catenin activation. LAY SUMMARY: DICER1 germline mutations are known to predispose individuals to the development of malignant tumors, mainly pleuropulmonary blastoma and ovarian Sertoli-Leydig cell tumor. Here, we described familial HCC associated with a novel DICER1 germline mutation and altered liver zonation. Familial and sporadic HCCs carrying DICER1 mutations are associated with CTNNB1 mutation and characterized by a reduced expression of specific mature miRNAs.

Impact of hepatobiliary phase liver MRI versus Contrast-Enhanced Ultrasound after an inconclusive extracellular gadolinium-based contrast-enhanced MRI for the diagnosis of benign hepatocellular tumors.

PURPOSE: To compare the added values of hepatobiliary phase (HBP) MRI and contrast-enhanced ultrasound (CEUS) in addition to inconclusive extracellular gadolinium-based contrast-enhanced MRI (CE-MRI) to characterize benign hepatocellular tumors (BHT). METHODS: Eighty-three BHT-46 focal nodular hyperplasia (FNH) and 37 hepatocellular adenomas (HCA)-with inconclusive CE-MRI in 54 patients (43 women and 11 men, mean age 42 years old ± 14.8) were retrospectively analyzed. All patients underwent both HBP-MRI and CEUS. Two radiologists independently reviewed 2 sets of images, SET-1: CE-MRI and HBP-MRI; SET-2: CE-MRI and CEUS, and classified lesions as "definite FNH," "possible FNH," or "definitely not FNH." Sensitivity (Se) and specificity (Spe) were compared between the two sets; subgroup analyses according to the lesion's size were performed. RESULTS: Regardless of lesion size, the respective Se and Spe of both datasets were not statistically different (95.7 and 100% vs. 76.1 and 94.6% for set-1 and -2 respectively; p = 0.18). For lesions larger than 35 mm, although both sets had similar specificity (100%), sensitivity was higher for SET-1 (100% vs. 40%); p = 0.04. Tumor classifications using SET-1 and SET-2 could have changed patient management in 35/54 (64.8%) and 33/54 (61.1%) of all patients, respectively. CONCLUSIONS: HBP-MRI or CEUS should be performed after an inconclusive CE-MRI. Both can change patient management by avoiding unnecessary biopsy or surveillance. The use of HBP-MRI should be advocated over CEUS in larger (>35 mm) lesions.

Programmed death ligand 1 expression in hepatocellular carcinoma: Relationship With clinical and pathological features.

The prognosis of hepatocellular carcinoma (HCC) remains poor, with only one third of patients eligible for curative treatments and very limited survival benefits with the use of sorafenib, the current standard of care for advanced disease. Recently, agents targeting the programmed death ligand 1 (PD-L1)/programmed death receptor 1 (PD-1) immune checkpoint were shown to display impressive antitumor activity in various solid or hematological malignancies, including HCC. PD-L1 immunohistochemical expression is thought to represent a biomarker predictive of drug sensitivity. Here, we investigated PD-L1 expression in a series of 217 HCCs and correlated our results with clinical and histological features and immunohistochemical markers (PD-1, cytokeratin 19, glutamine synthetase, and ß-catenin expression). PD-L1 expression by neoplastic cells was significantly associated with common markers of tumor aggressiveness (high serum alpha-fetoprotein levels, P = 0.038; satellite nodules, P < 0.001; macrovascular invasion, P < 0.001; microvascular invasion, P < 0.001; poor differentiation, P < 0.001) and with the progenitor subtype of HCC (cytokeratin 19 expression, P = 0.031). High PD-L1 expression by inflammatory cells from the tumor microenvironment also correlated with high serum alpha-fetoprotein levels (P < 0.001), macrovascular invasion (P = 0.001), poor differentiation (P = 0.001), high PD-1 expression (P < 0.001), and the so-called lymphoepithelioma-like histological subtype of HCC (P = 0.003). CONCLUSION: PD-L1 expression by either neoplastic or intratumoral inflammatory cells is related to tumor aggressiveness and suggests that the response to treatments targeting the PD-L1/PD-1 immune checkpoint could be restricted to particular HCC variants; thus, enrichment of these tumor subtypes in future clinical trials should be considered. (Hepatology 2016;64:2038-2046).

Inflammatory hepatocellular adenomas developed in the setting of chronic liver disease and cirrhosis.

Hepatocellular adenoma is considered to occur exclusively in non-fibrotic livers. It is a heterogeneous entity and a molecular classification is now widely accepted. The most frequent hepatocellular adenoma subtype, namely inflammatory adenoma, harbor somatic activating mutations of genes involved in the interleukin-6 pathway that lead to high C-reactive protein and serum amyloid A expression. The aim of our study was to investigate a series of benign hepatocellular neoplasms developed on cirrhotic livers and characterized by an unequivocal histological diagnosis. We performed a clinical, pathological, and molecular study of 10 benign hepatocellular neoplasms developed in three patients with cirrhosis. Markers allowing hepatocellular adenoma classification were assessed by quantitative real-time PCR and immunohistochemistry. Samples were sequenced for CTNNB1, HNF1A, IL6ST, GNAS, STAT3, and TERT (promoter) mutations. A control series of 32 classical macronodules developed in cirrhosis related to various etiologies was screened by immunohistochemistry and gene sequencing. The three patients had cirrhosis related to metabolic syndrome and/or alcohol intake; two had a single tumor, while the third developed more than 30 lesions. Microscopic examination showed well-differentiated neoplasms sharing features with inflammatory adenoma including inflammatory infiltrates, sinusoidal dilatation, and dystrophic vessels. Sequencing revealed classical hotspot somatic mutations (IL6ST, n=8; STAT3, n=1; and GNAS, n=1) known to be responsible for IL-6/JAK/STAT pathway activation. Two classical high-grade macronodules demonstrated high serum amyloid A and/or C-reactive protein expression, without gene mutations. Altogether, our findings support the existence of rare inflammatory adenoma developed in cirrhosis.

Differentiation of focal nodular hyperplasia from hepatocellular adenoma: Role of the quantitative analysis of gadobenate dimeglumine-enhanced hepatobiliary phase MRI.

PURPOSE: To determine the value of quantitative analysis of the hepatobiliary phase (HBP) in gadobenate dimeglumine (Gd-BOPTA)-enhanced magnetic resonance imaging (MRI) to differentiate focal nodular hyperplasia (FNH) from hepatocellular adenoma (HCA). MATERIALS AND METHODS: Thirty-eight patients bearing 67 lesions (40 FNH; 27 HCA) were retrospectively included in this Institutional Review Board-approved study. The same volumetric interpolated breath-hold examination (VIBE) T1 -weighted sequences were performed before and after contrast injection on a 1.5T MRI, with HBP images acquired with a mean delay of 80 minutes (range 60-120 min). After a visual assessment of lesions enhancement (qualitative HBP analysis), the HBP signal intensity ratio (SIR) and the lesion-to-liver contrast enhancement ratio (LLCER) were calculated for each lesion by two observers (Mann-Whitney test). The sensitivities, specificities (receiver operating characteristic [ROC] curve analysis) and interobserver correlation (intraclass coefficient, ICC) of quantitative HBP analysis were determined. RESULTS: All FNH and 44.4% of HCA appeared hyper- or isointense relative to the adjacent liver on qualitative HBP analysis. The mean SIR (P < 0.01) and LLCER (P < 0.0001) of FNH were significantly higher than that of HCA. The area under the ROC curve for the differentiation of FNH from HCA with LLCER was 0.98 for both observers. With a cutoff value of -0.3%-observer 1 with highest experience- LLCER assessment provided respective sensitivity and specificity values of 100% and 96.2% for the differentiation of FNH from HCA. The ICC was 0.7 for SIR measurements and 0.8 for LLCER measurements. CONCLUSION: Quantitative LLCER assessment allows an accurate differentiation of FNH from HCA, even in hyper- or isointense HCA on HBP images.

BRAF V600E mutational status in bile duct adenomas and hamartomas.

AIMS: Bile duct adenomas (BDA) and bile duct hamartomas (BDH) are benign bile duct lesions considered neoplastic or secondary to ductal plate malformation, respectively. We have reported previously a high prevalence of BRAF V600E mutations detected by allele-specific polymerase chain reaction assay in BDA, and suggested that BDA may be precursors to a subset of intrahepatic cholangiocarcinomas harbouring V600E mutations. The aim of the present study was to assess the existence of BRAF V600E mutations, using immunohistochemical methods, in additional BDA as well as in BDH. METHODS AND RESULTS: Fifteen BDA and 35 BDH were retrieved from the archives of the pathology departments of two French university hospitals. All cases were reviewed by two pathologists specialized in liver diseases. BRAF V600E mutational status was investigated by immunohistochemistry. Mutated BRAF mutant protein was detected in 53% of the BDA and in none of the cases of BDH. CONCLUSION: Our findings suggest that BDA and BDH are different processes, and that BDA represent true benign neoplasms. They also support the hypothesis that mutated BDA might precede the development of the subset of intrahepatic cholangiocarcinomas harbouring BRAF V600E mutations.

Primary biliary cirrhosis: proposal for a new simple histological scoring system.

BACKGROUND & AIMS: A simple and reproducible evaluation of non diagnostic histological lesions related to prognosis remains crucial in primary biliary cirrhosis (PBC). Presently there is no satisfactory simple scoring system analysing them reliably. We elaborated a semi-quantitative scoring system that assesses fibrosis, lymphocytic interface hepatitis (LIH) and ductopenia, separately. This study was aimed to evaluate its intra/interobserver reproducibility and its correlation with the main biochemical data. METHODS: Liver biopsies from 33 consecutive newly diagnosed PBC patients were independently analysed by five liver pathologists. Fibrosis was classified into five stages (portal/periportal fibrosis/few septa/numerous septa/cirrhosis) and LIH into four grades. The bile duct ratio (BDR), i.e. ratio of the number of portal tracts with ducts to total number of portal tracts, Ludwig's and Scheuer's stages were evaluated. Intra and interobserver agreements were assessed. Histological results were correlated to the biochemical data. RESULTS: Most patients had an early disease on clinical and biological parameters. The biopsies measured 23 mm on average (range 12 - 40 mm). Intraobserver reproducibility was substantial for fibrosis (κ = 0.68), LIH (κ = 0.69) and BDR (ICC = 0.69). Interobserver agreement for fibrosis was fair with the 5-class system (κ = 0.36), moderate with a 4-class system (κ = 0.56). moderate for LIH (κ = 0.59) and BDR (ICC = 0.50). Ludwig's and Scheuer's staging showed a fair interobserver agreement (κ = 0.32, κ = 0.31 respectively). Our system showed better correlations with biochemistry than Ludwig's and Scheuer's systems did. CONCLUSIONS: This simple scoring system, assessing fibrosis, LIH and BDR separately, has a substantial intraobserver and a moderate interobserver reproducibility. Its prognostic relevance has to be evaluated.

Telomerase reverse transcriptase promoter mutation is an early somatic genetic alteration in the transformation of premalignant nodules in hepatocellular carcinoma on cirrhosis.

UNLABELLED: Genetic determinants of the early steps of carcinogenesis on cirrhosis are still poorly understood. We aimed to evaluate the occurrence of telomerase reverse transcriptase (TERT) promoter mutations in the transformation of cirrhotic nodules into hepatocellular carcinoma (HCC). We analyzed a series of 268 liver samples, including 96 nodules developed in 58 patients with cirrhosis and 114 additional cirrhosis. All samples were screened for TERT promoter mutations, and in 31 nodules, for 10 genes recurrently mutated in HCC. Immunohistochemistry (IHC) analyses were performed for glypican 3, glutamine synthase, and heat shock protein 70. Six liver pathologists reviewed all the samples. Among The 96 nodules, 88 were firmly diagnosed as low-grade dysplastic nodules (LGDNs; 32 cases), high-grade dysplastic nodules (HGDNs; 16 cases), early HCC (eHCC; 23 cases), or small and progressed HCC in 17 cases. The agreement between the initial diagnosis from pathological report and the final expert consensus report was moderate for the diagnosis of benign versus malignant nodules (weighted kappa = 0.530). TERT promoter mutations were highly related to the step-wise hepatocarcinogenesis because mutations were identified in 6% of LGDNs, 19% of HGDNs, 61% of eHCCs, and 42% of small and progressed HCC. TERT promoter mutation is the most frequent molecular alteration in eHCC given that the IHC criteria for diagnosis of malignancy were found in only 39% of the cases. TERT promoter mutation was also the earliest genetic alteration because mutations in 10 other genes were only identified in 28% of the small and progressed HCC. CONCLUSION: Frequency of TERT promoter mutations rapidly increases during the different steps of the transformation of premalignant lesions into HCC on cirrhosis. Consequently, somatic TERT promoter mutation is a new biomarker predictive of transformation of premalignant lesions into HCC.

Cannabinoid receptor 2 counteracts interleukin-17-induced immune and fibrogenic responses in mouse liver.

UNLABELLED: Interleukin (IL)-17 is a proinflammatory and fibrogenic cytokine mainly produced by T-helper (Th)17 lymphocytes, together with the hepatoprotective and antifibrogenic cytokine, IL-22. Cannabinoid receptor 2 (CB2) is predominantly expressed in immune cells and displays anti-inflammatory and antifibrogenic effects. In the present study, we further investigated the mechanism underlying antifibrogenic properties of CB2 receptor and explored its effect on the profibrogenic properties of IL-17. After bile duct ligation (BDL), the hepatic expression of Th17 markers and IL-17 production were enhanced in CB2(-/-) mice, as compared to wild-type (WT) counterparts, and correlated with increased fibrosis in these animals. In contrast, IL-22-induced expression was similar in both animal groups. Inhibition of Th17 differentiation by digoxin lowered Th17 marker gene expression and IL-17 production and strongly reduced liver fibrosis in CB2(-/-) BDL mice. In vitro, differentiation of CD4(+) naïve T cells into Th17 lymphocytes was decreased by the CB2 agonist, JWH-133, and was associated with reduced Th17 marker messenger RNA expression and IL-17 production, without modification of IL-22 release. The inhibitory effect of JWH-133 on IL-17 production relied on signal transducer and activator of transcription (STAT)5 phosphorylation. Indeed, STAT5 phosphorylation and translocation into the nucleus was enhanced in JWH133-treated Th17 lymphocytes, and the addition of a STAT5 inhibitor reversed the inhibitory effect of the CB2 agonist on IL-17 production, without affecting IL-22 levels. Finally, in vitro studies also demonstrated that CB2 receptor activation in macrophages and hepatic myofibroblasts blunts IL-17-induced proinflammatory gene expression. CONCLUSION: These data demonstrate that CB2 receptor activation decreases liver fibrosis by selectively reducing IL-17 production by Th17 lymphocytes via a STAT5-dependent pathway, and by blunting the proinflammatory effects of IL-17 on its target cells, while preserving IL-22 production.

Contribution of the ELFG test in algorithms of non-invasive markers towards the diagnosis of significant fibrosis in chronic hepatitis C.

BACKGROUND AND AIMS: We aimed to determine the best algorithms for the diagnosis of significant fibrosis in chronic hepatitis C (CHC) patients using all available parameters and tests. PATIENTS AND METHODS: We used the database from our study of 507 patients with histologically proven CHC in which fibrosis was evaluated by liver biopsy (Metavir) and tests: Fibrometer®, Fibrotest®, Hepascore®, Apri, ELFG, MP3, Forn's, hyaluronic acid, tissue inhibitor of metalloproteinase-1 (TIMP1), MMP1, collagen IV and when possible Fibroscan™. For the first test we used 90% negative predictive value to exclude patients with F≤1, next an induction algorithm was applied giving the best tests with at least 80% positive predictive value for the diagnosis of F≥2. The algorithms were computed using the R Software C4.5 program to select the best tests and cut-offs. The algorithm was automatically induced without premises on the part of the investigators. We also examined the inter-observer variations after independent review of liver biopsies by two pathologists. A medico-economic analysis compared the screening strategies with liver biopsy. RESULTS: In "intention to diagnose" the best algorithms for F≥2 were Fibrometer ®, Fibrotest®, or Hepascore® in first intention with the ELFG score in second intention for indeterminate cases. The percentage of avoided biopsies varied between 50% (Fibrotest® or Fibrometer®+ELFG) and 51% (Hepascore®+ELFG). In "per-analysis" Fibroscan™+ELFG avoided liver biopsy in 55% of cases. The diagnostic performance of these screening strategies was statistically superior to the usual combinations (Fibrometer® or Fibrotest®+Fibroscan™) and was cost effective. We note that the consensual review of liver biopsies between the two pathologists was mainly in favor of F1 (64-69%). CONCLUSION: The ELFG test could replace Fibroscan in most currently used algorithms for the diagnosis of significant fibrosis including for those patients for whom Fibroscan™ is unusable.

Non-invasive assessment of liver graft fibrosis by transient elastography after liver transplantation.

BACKGROUND: Liver stiffness measurement (LSM) by transient elastography (TE) (FibroScan) is a validated method of quantifying liver fibrosis in non-transplanted patients with hepatitis C virus (HCV). It could be useful in follow-up after liver transplantation (LT). The aim of this study was to assess the diagnostic accuracy of LSM in evaluating liver fibrosis after LT in patients with and without recurrent HCV. PATIENTS AND METHODS: Forty-three patients (mean age 57.6 ± 9.9 years), 28 (65.1%) HCV-positive patients and 15 (34.9%) HCV-negative patients underwent gold standard liver biopsy and TE 55.8 ± 4.9 months after transplantation. Liver fibrosis was scored on biopsy specimens according to METAVIR (F0-F4). Accuracy of TE and optimal stiffness cut-off values for fibrosis staging were determined by a receiver-operating characteristics (ROC) curve analysis. RESULTS: Median stiffness values were significantly different for METAVIR score less than 2 (5.8 kPa) vs. METAVIR score greater to equal to 2 (9.6 kPa) (P<0.001). The area under the ROC curve was 0.83 for METAVIR score greater to equal to 2 (95%CI: 0.71-0.95). The optimal stiffness cut-off value was 7 kPa for METAVIR scores greater to equal to 2. The results were similar whether the patients had recurrent HCV infection or not. CONCLUSIONS: These results indicate that transient elastography accurately identifies LT recipients with significant fibrosis, irrespective of HCV status. It is a promising non-invasive tool to assess graft fibrosis progression after LT in patients with HCV recurrence, as well as for screening of late graft fibrosis of other etiologies. Transient elastography could reduce the use of invasive protocol biopsies.

Phase II study of sirolimus in treatment-naive patients with advanced hepatocellular carcinoma.

BACKGROUND: Rapalogs are emerging as promising targeted anticancer drugs. Activation of the PI3K/Akt/mTOR pathway has been observed in 15-50% of hepatocellular carcinomas. METHODS: In this phase II study, patients with advanced hepatocellular carcinoma and underlying cirrhosis received sirolimus (20 mg/week for 1 month then 30 mg/week). Tumour response was assessed every 8 weeks. The primary endpoint was the objective tumour response rate according to the Response Evaluation Criteria in Solid Tumours criteria. Secondary endpoints included the objective response according to the modified Response Evaluation Criteria in Solid Tumours criteria, safety, and pharmacokinetic parameters. RESULTS: Twenty-five patients received sirolimus for a median of 20.6 weeks. Two patients had an objective response (8%, 95CI: 0.98-26.03), including one complete response, and 8 patients had stable disease. There were 2 cases of grade 5 toxicity (infections) and 5 cases of grade 3 toxicity. The main grade 1/2 toxicity was mild transient fatigue (76%). Median time to radiological progression and overall survival were 15.3 weeks (range: 8.2-173.9) and 26.4 weeks (range: 8.2-173.9) respectively. Use of the modified Response Evaluation Criteria in Solid Tumours criteria did not identify any further responders. CONCLUSION: These data suggest that first-line sirolimus shows antitumoural efficacy in advanced hepatocellular carcinoma. Larger trials with Child A patients are needed.

Comparison of nine blood tests and transient elastography for liver fibrosis in chronic hepatitis C: the ANRS HCEP-23 study.

BACKGROUND & AIMS: Blood tests and transient elastography (Fibroscan™) have been developed as alternatives to liver biopsy. This ANRS HCEP-23 study compared the diagnostic accuracy of nine blood tests and transient elastography (Fibroscan™) to assess liver fibrosis, vs. liver biopsy, in untreated patients with chronic hepatitis C (CHC). METHODS: This was a multicentre prospective independent study in 19 French University hospitals of consecutive adult patients having simultaneous liver biopsy, biochemical blood tests (performed in a centralized laboratory) and Fibroscan™. Two experienced pathologists independently reviewed the liver biopsies (mean length=25±8.4 mm). Performance was assessed using ROC curves corrected by Obuchowski's method. RESULTS: Fibroscan™ was not interpretable in 113 (22%) patients. In the 382 patients having both blood tests and interpretable Fibroscan™, Fibroscan™ performed similarly to the best blood tests for the diagnosis of significant fibrosis and cirrhosis. Obuchowski's measure showed Fibrometer® (0.86), Fibrotest® (0.84), Hepascore® (0.84), and interpretable Fibroscan™ (0.84) to be the most accurate tests. The combination of Fibrotest®, Fibrometer®, or Hepascore® with Fibroscan™ or Apri increases the percentage of well classified patients from 70-73% to 80-83% for significant fibrosis, but for cirrhosis a combination offers no improvement. For the 436 patients having all the blood tests, AUROC's ranged from 0.82 (Fibrometer®) to 0.75 (Hyaluronate) for significant fibrosis, and from 0.89 (Fibrometer® and Hepascore®) to 0.83 (FIB-4) for cirrhosis. CONCLUSIONS: Contrarily to blood tests, performance of Fibroscan™ was reduced due to uninterpretable results. Fibrotest®, interpretable Fibroscan™, Fibrometer®, and Hepascore® perform best and similarly for diagnosis of significant fibrosis and cirrhosis.

Gas6 deficiency prevents liver inflammation, steatohepatitis, and fibrosis in mice.

The Gas6/Axl pathway has been increasingly implicated in regeneration and tissue repair and, recently, in the control of innate immunity. In liver, we have demonstrated that Gas6 and its receptor Axl are expressed in macrophages, progenitor cells, and myofibroblasts and that Gas6 deficiency reduced inflammation and myofibroblast activation, causing delayed liver repair in response to acute injury. All these data suggest a role of Gas6/Axl signaling in pathogenesis of chronic liver diseases. In the present study, we address the role of Gas6 in steatohepatitis and progression to liver fibrosis using Gas6-deficient mice fed a choline-deficient ethionine-supplemented diet (CDE) or receiving a chronic carbon tetrachloride (CCl(4)) treatment. Gas6 deficiency attenuated hepatic steatosis by limiting CDE-induced downregulation of genes involved in ß-oxidation observed in wild-type animals. Moreover, Gas6-deficient mice displayed reduction of hepatic inflammation, revealed by limited F4/80-positive macrophage infiltration, decreased expression of IL-1ß, TNF-α, lymphotoxin-ß, and monocyte chemotactic protein-1, and attenuated hepatic progenitor cell response to CDE diet. Gas6 deficiency reduced CDE-induced fibrogenesis and hepatic myofibroblast activation and decreased expression of TGF-ß and collagen 1 mRNAs. After chronic CCl(4) injury, Gas6-deficient mice also exhibited reduced liver fibrosis as a consequence of defective macrophage recruitment compared with wild-type animals. We conclude that improvement of steatohepatitis and fibrosis in Gas6(-/-) mice is linked to an inhibition of the inflammatory response that controls lipid metabolism and myofibroblast activation. This study highlights the deleterious effect of Gas6 in the progression of steatosis to steatohepatitis and fibrosis.

Association of caffeine intake and histological features of chronic hepatitis C.

BACKGROUND & AIMS: The severity of chronic hepatitis C (CHC) is modulated by host and environmental factors. Several reports suggest that caffeine intake exerts hepatoprotective effects in patients with chronic liver disease. The aim of this study was to evaluate the impact of caffeine consumption on activity grade and fibrosis stage in patients with CHC. METHODS: A total of 238 treatment-naïve patients with histologically-proven CHC were included in the study. Demographic, epidemiological, environmental, virological, and metabolic data were collected, including daily consumption of alcohol, cannabis, tobacco, and caffeine during the six months preceding liver biopsy. Daily caffeine consumption was estimated as the sum of mean intakes of caffeinated coffee, tea, and caffeine-containing sodas. Histological activity grade and fibrosis stage were scored according to Metavir. Patients (154 men, 84 women, mean age: 45±11 years) were categorized according to caffeine consumption quartiles: group 1 (<225 mg/day, n=59), group 2 (225-407 mg/day, n=57), group 3 (408-678 mg/day, n=62), and group 4 (>678 mg/day, n=60). RESULTS: There was a significant inverse relationship between activity grade and daily caffeine consumption: activity grade>A2 was present in 78%, 61%, 52%, and 48% of patients in group 1, 2, 3, and 4, respectively (p<0.001). By multivariate analysis, daily caffeine consumption greater than 408 mg/day was associated with a lesser risk of activity grade>A2 (OR=0.32 (0.12-0.85). Caffeine intake showed no relation with fibrosis stage. CONCLUSIONS: Caffeine consumption greater than 408 mg/day (3 cups or more) is associated with reduced histological activity in patients with CHC. These findings support potential hepatoprotective properties of caffeine in chronic liver diseases.