Elie-Serge Zafrani

Extrahepatic Immunologic Manifestations in Chronic Hepatitis C and Hepatitis C Virus Serotypes

Extrahepatic immunologic abnormalities have been shown to occur frequently in patients with chronic hepatitis C virus (HCV) infection. Hepatitis C virus now appears to cause those cases of mixed cryoglobulinemia that were previously considered essential [1-5]. Indeed, HCV RNA has been detected in the serum specimens of about 90% of patients with essential mixed cryoglobulinemia [1, 2, 4]. In addition, cryoglobulin is found, usually at low levels, in the serum specimens of one third to one half of patients with chronic hepatitis C [6, 7]; rheumatoid factor, which may play a role in cryoglobulinemia, is present in the serum specimens of about 70% of patients [6]. Various autoantibodies have been seen in the serum of 40% to 50% of patients with chronic HCV infection [6, 8], and HCV has been associated with cases of autoimmune thyroiditis [9]. Salivary gland lesions, characterized by lymphocytic capillaritis, are seen in about half of patients and are sometimes associated with lymphocytic sialadenitis resembling that of the Sjogren syndrome [6]. Finally, HCV may cause the chronic liver disease frequently associated with lichen planus [10]. Recently, sequences of different HCV variants were classified into different genotypes on the basis of overall sequence similarity [11-22]. A consensus nomenclature for HCV genotypes has been proposed [23], in which the six HCV genotypes identified so far are numbered in the order of their discovery. Within each genotype, subtypes have been identified by lower case letters, which are also given in order of discovery [23]. Correspondence among the classifications reported so far is presented in Table 1. Different techniques for determining HCV genotype have been developed in recent months. Currently, in addition to sequencing the genome, investigators can use three techniques based on the polymerase chain reaction (PCR). The technique described by Okamoto and colleagues [24, 25] is based on a nested PCR amplification of the HCV genome and uses primers located in the core region: The first round of PCR uses a pair of universal (non-type-specific) primers and the second uses a pair of type-specific primers. The method of McOmish and colleagues [17, 18] is based on PCR amplification of the 5 noncoding region of the genome done with a pair of universal primers, followed by enzymatic digestion of the amplified products and analysis of their restriction fragment length polymorphism. Stuyver and colleagues described a line probe assay for the determination of HCV genotypes [22], in which a PCR amplification is done using universal primers located in the 5 noncoding region of the genome. This is followed by hybridization of the amplified products to oligonucleotide probes attached as parallel bands on nitrocellulose strips. On the other hand, a serotyping immunoenzymatic assay to detect genotype-specific antibodies directed to epitopes encoded by the NS4 region of the HCV genome has been developed [26]. This technique, in its present form, allows the differentiation of HCV serotypes 1, 2, and 3, which correspond to HCV genotypes 1, 2, and 3 in the consensus nomenclature [23]. Table 1. Correspondence between the Major Published Classification Systems for Hepatitis C Virus Genotypes* Several studies indicate that particular HCV genotypes are associated with more severe liver disease and poorer response to interferon- therapy [27-30]. The factors determining immunologic abnormalities in patients with chronic hepatitis C are largely unknown. We used a serotyping assay to study whether the occurrence of extrahepatic immunologic abnormalities in patients with chronic hepatitis C is serotype dependent. Methods Patients Fifty-nine consecutive patients with chronic hepatitis C were prospectively studied. Thirty-four were men and 25 were women; their mean age was 52 years (range, 18 to 77 years). In all cases, the diagnosis of chronic hepatitis C was based on long-term elevation of serum alanine aminotransferase levels in the blood, positive serologic markers of HCV infection (found using second-generation enzyme-linked immunosorbent assay and recombinant immunoblot assay, Ortho Diagnostic Systems, Raritan, New Jersey), and the absence of any other cause of chronic liver disease. Specimens obtained by percutaneous liver biopsy showed chronic active hepatitis in all 56 patients tested and associated cirrhosis in 15 of the 56 (27%). Before any treatment was given, serum specimens were tested for cryoglobulin, rheumatoid factor, and many antitissue antibodies, and biopsy of labial salivary glands was done. Hepatitis C virus serotype was determined in all patients by immunoenzymatic assay. Study Methods Detection of Cryoglobulinemia Venous blood (20 mL) was taken from fasting patients in a room at 37 C, allowed to clot at this temperature, and then separated by centrifugation. After centrifugation, the supernatant was removed from the serum, incubated at 4 C for 8 days, and examined daily for cryoprecipitation. Detection of Rheumatoid Factor Rheumatoid factor was measured using a nephelometer analyzer (BNA, Behring, Marburg, Germany); polystyrene particles coated with human globulin were agglutinated when mixed with samples containing rheumatoid factor. Normal values were those less than 18 IU/mL. Detection of Autoantibodies Antinuclear, anti-smooth muscle, type 1 anti-liver-kidney microsomal (anti-LKM1), and antimitochondrial antibodies were detected by indirect immunofluorescence using air-dried cryostat sections from rat or mouse livers and kidneys and HEp-2 cells (Kallestad, Chaska, Minnesota) as substrates. Antithyroid microsomal antibodies were detected by indirect immunofluorescence using surgical specimens of human thyrotoxic thyroid as substrate. In all cases, the classic Weller and Coon indirect immunofluorescence method was used with fluorescein-labeled goat immunoglobulin directed to IgG, IgA, and IgM (Pasteur Diagnostics, Marnes la Coquette, France) as a second layer [31]. The serum specimens were tested undiluted for anti-DNA antibodies, at a 1/5 dilution for antimicrosomal antibodies, and at a 1/10 dilution for other antibodies. The titers were established using increasing dilutions up to 1/2560. Antithyroglobulin antibodies were detected using an hemagglutination kit (Thymune-T, Wellcome Diagnostics, Dartford, United Kingdom). Labial Salivary Gland Examination All biopsies were done in macroscopically normal mucosa. The samples were fixed in Bouin fluid, embedded in paraffin, and stained with hematoxylin-eosin-safranin. All sections were examined blind by two pathologists and graded according to the Chisholm and Mason classification system [32]. Determination of Serotypes The 59 serum specimens in our study were tested for the presence of serotype-specific antibodies using the recently developed enzyme immunoassay [26]. A series of eight branched peptides, synthesized from two antigenic regions of HCV genotypes 1, 2, and 3, were used to coat polypropylene microtiter wells overnight at 4 C. After washing, the wells were blocked with 150 L of blocking solution (phosphate-buffered saline, 0.1% Tween 20, and 2% bovine serum albumin) for 1 hour at room temperature. Blocking assays were done using mixes of type-specific peptides at a final concentration of 1 mg/mL (for example, 100:1 excess over that used to coat the wells). Plasma specimens from the 59 patients were diluted in the blocking solution and 100 L were added to antigen-coated and blocked wells. The first incubation was done overnight at 4 C. Plates were washed four times in phosphate-buffered saline and 0.1% Tween 20 and then incubated with horseradish peroxidase-conjugated anti-human IgG (1/20 000 in phosphate-buffered saline and 0.1% Tween 20 for 1 hour at room temperature). The plates were finally washed four times in phosphate-buffered saline and 0.1% Tween 20 and incubated with substrate (50 g of O-phenylenediamine per milliliter and 0.1% H2O2 [30 volumes] for 30 minutes in the dark at room temperature). Optical densities were read at 490 nm; values ranged from 100 to 2000 mU. Results Prevalence of Immunologic Abnormalities Our results are presented in Table 2. Cryoglobulin was found in the serum specimens of 20 of the 56 patients tested (36%), and rheumatoid factor was present at abnormal levels in 42 of the 59 patients (71%). Table 2. Prevalence of the Different Immunologic Abnormalities according to Hepatitis C Virus Serotype At least one type of antitissue antibody was detected in the serum specimens of 24 of the 59 patients (41%). Thirteen patients (22%) had serum antinuclear antibodies and 13 (22%) had anti-smooth muscle antibodies at a significant titer (greater than 1/40). Anti-LKM1 antibodies were found in 3 patients (5%) and antithyroid antibodies were found in 5 (8%); 4 of these 5 had antithyroglobulin and 1 had antithyroid microsomes. No antimitochondrial antibodies were found. Labial salivary gland biopsies were done in those 49 of the 59 patients who had no contraindication and who gave informed consent; lesions were found in 24 of them (49%). In all patients, these lesions were characterized by lymphocytic capillaritis, as previously described [6]. In 7 patients (14%), they were associated with more severe lesions, grades 3 and 4 by the Chisholm and Mason classification (lymphocytic sialadenitis) [32], and resembled the lymphocytic sialadenitis of the Sjogren syndrome (14%). Only one of the patients with salivary gland lesions had a mild case of the ocular sicca syndrome shown by the Schirmer test. The prevalences of the different immunologic abnormalities did not vary significantly according to the presence of cirrhotic findings in liver specimens. Hepatitis C Virus Serotypes Thirty-five of the 59 patients (59%) were infected with HCV serotype 1, 6 (10%) were infected with serotype 2, and 7 (12%) were infected with serotype 3 (serotypes are here described using the proposed consensus nomenclature [23]). Two

Hepatitis C Virus-Induced Hepatocellular Steatosis

Two distinct forms of hepatocellular steatosis can be seen in patients with chronic hepatitis C virus (HCV) infection. Classical metabolic risk factors for hepatocellular steatosis account for the vast majority of cases of steatosis in patients infected by non-genotype 3 HCV strains. In contrast, in patients infected by HCV genotype 3, steatosis is generally induced by the virus itself through a direct cytopathic effect, the mechanisms of which remain debated. Mixed forms of steatosis can also be seen in HCV genotype 3-infected patients with metabolic risk factors. Hepatocellular steatosis appears to be associated with more rapid progression of hepatic fibrosis. However, it is unclear whether this association is due to steatosis itself, or rather to metabolic and host factors that promote steatosis and fibrosis concomitantly. This review discusses current knowledge of HCV-induced steatosis and its relation to chronic HCV-associated liver disease.

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 β‐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)

PD‐L1 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 β‐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)

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 β-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-1β, TNF-α, lymphotoxin-β, 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-β 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.

Is histological diagnosis of primary liver carcinomas with fibrous stroma reproducible among experts

Aims: In the era of targeted therapeutics, histological typing of hepatobiliary carcinomas has major clinical implications. Little is known about the reproducibility of the pathological diagnosis of primary liver carcinomas. Therefore, this study aimed to evaluate the worldwide variation in the pathological expert diagnoses of primary liver carcinomas with fibrous stroma in patients who did not have cirrhosis. Methods: A single set of slides was selected from 25 tumours, and this set was reviewed independently by 12 pathologists who have worldwide expertise in liver tumours. Reproducibility of the diagnoses was evaluated by Light’s κ, and diagnoses were clustered by multidimensional scaling. Immunohistochemistry was performed after histological review. Results: The interobserver reproducibility for diagnosis of hepatocellular carcinoma subtypes and cholangiocarcinomas was poor (κ 0.23–0.52), even when the experts considered that the diagnosis required no additional stains or clinical information. Interestingly, multidimensional scaling revealed three main clusters of tumours: hepatocellular carcinoma with no other specifications (n = 13), fibrolamellar hepatocellular carcinoma (n = 3) and cholangiocarcinoma (n = 9). Using immunohistochemistry, these histological clusters correlated with expression of anti-hepatocyte and anti-cytokeratin 19 (p<0.001). Conclusions: The results demonstrate the poor reproducibility among experts of the pathological diagnosis of primary liver carcinomas with fibrous stroma in patients who did not have cirrhosis, and highlight that the systematic use of immunohistochemistry may improve the diagnostic accuracy.

Carcinome hépatocellulaire en l’absence de cirrhose au cours d’une stéato-hépatite non alcoolique

Resume Tous les carcinomes hepatocellulaires jusqu’ici rapportes chez des malades atteints de steato-hepatite non alcoolique se sont developpes sur une cirrhose pre-existante. Nous rapportons ici l’observation d’un homme de 68 ans atteint d’une steato-hepatite non alcoolique et chez qui un carcinome hepatocellulaire est survenu en l’absence de cirrhose. Cette observation conduit a envisager une filiation entre la steato-hepatite non alcoolique et/ou l’obesite, et le carcinome hepatocellulaire independamment de la cirrhose et invite a la conduite d’etudes epidemiologiques pour preciser la reelle incidence de cette association.

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

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).