Hepatitis C virus (HCV) infection is a major. Cryoglobulinemia Is Associated With Steatosis and Fibrosis in Chronic Hepatitis C

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1 Cryoglobulinemia Is Associated With Steatosis and Fibrosis in Chronic Hepatitis C David Saadoun, 1 Tarik Asselah, 2 Mathieu Resche-Rigon, 3 Frédéric Charlotte, 4 Pierre Bedossa, 5 Dominique Valla, 2 Jean-Charles Piette, 1 Patrick Marcellin, 2 and Patrice Cacoub 1 The relationship between cryoglobulin and severity of liver lesions is debated. No study has focused on the relationship between cryoglobulin, liver steatosis, and fibrosis. The aim of this study was to determine the relationship between cryoglobulins and liver lesions (necroinflammation, fibrosis, and steatosis) in patients with hepatitis C virus (HCV) infection. Four hundred and thirty-seven consecutive patients with untreated chronic hepatitis C who had been admitted for liver biopsy were included in the study. Risk factors for fibrosis and steatosis were assessed. The mean age was years, and 49% were male. Cryoglobulin was present in 286 patients, 103 of whom had vasculitis. One hundred and eighty-six patients (43%) had steatosis greater than 10%, and 110 (25%) had advanced fibrosis (Metavir score F3-F4). On multivariate analysis, cryoglobulin increased by nearly threefold the risk of having advanced fibrosis and steatosis greater than 10%. Steatosis greater than 10% was associated with a higher body mass index (P <.001), HCV genotype 3 (P <.001), cryoglobulin (P.002), and advanced liver fibrosis (P.009). Advanced fibrosis (F3-F4) was associated with a higher level of -glutamyltransferase (P.04), cryoglobulin (P <.001), a high grade of necroinflammation (Metavir score A2-A3) (P <.001), and steatosis higher than 10% (P.04). In conclusion, our study shows an independent association between cryoglobulin and steatosis as well as advanced fibrosis. (HEPATOLOGY 2006;43: ) Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide; approximately 170 million people are infected. 1 The severity of the disease varies widely, ranging from asymptomatic chronic infection to cirrhosis and hepatocellular carcinoma. Liver lesions are thought to be primarily related to immunomediated mechanisms. Factors influencing the outcome of chronic hepatitis C including age, sex, and alcohol consumption are poorly understood. 2 It is clear that these factors account only Abbreviations: HCV, hepatitis C virus; BMI, body mass index. From 1 Service de Médecine Interne and CNRS UMR 7087, Hôpital Pitié- Salpétrière, Paris, France; 2 Service d Hépatologie and INSERM CRB 3, Hôpital Beaujon, Clichy, France; 3 Département de Biostatistique et Informatique Médicale, Hôpital Saint-Louis, Paris, France; 4 Service d Anatomo-Pathologie, Hôpital Pitié- Salpétrière, Paris, France; and 5 Service d Anatomo-Pathologie, Hôpital Beaujon, Clichy, France. Received November 14, 2005; accepted March 15, D. Saadoun and T. Asselah contributed equally to this study. Address reprint requests to: Patrice Cacoub, M.D., Department of Internal Medicine, Hôpital La Pitié-Salpêtrière, 83, Boulevard de l Hôpital, Cedex 13 Paris, France. patrice.cacoub@psl.aphp.fr; fax: (33) Copyright 2006 by the American Association for the Study of Liver Diseases. Published online in Wiley InterScience ( DOI /hep Potential conflict of interest: Nothing to report. partially for the various outcomes of the disease. Better understanding of the natural history of chronic hepatitis C and the identifying factors associated with progression of fibrosis are of major importance. HCV is associated with a spectrum of extrahepatic manifestations, mainly mixed cryoglobulin. Cryoglobulins may be asymptomatic or they may lead to clinical manifestations ranging from a mixed cryoglobulinemia syndrome (e.g., purpura, arthralgia, asthenia) to a more serious systemic vasculitis with neurological and/or renal involvement. 3,4 Although cryoglobulin is detectable in 40% to 60% of patients with chronic hepatitis C, only 10% to 15% of them will develop symptomatic cryoglobulinemia. Patients with chronic hepatitis C and cryoglobulin have been reported to have a higher incidence of cirrhosis 5,6 and increased fibrosis score 4 compared with patients without cryoglobulin; however, this matter is still controversial. Additionally, there are no large comparison studies to assess severity of liver disease according to the presence of symptomatic or asymptomatic cryoglobulin. There is some controversy with regard to the influence of steatosis on the progression of fibrosis in patients with chronic hepatitis C No study has focused on the relationship between cryoglobulin and liver steatosis. Prelim- 1337

2 1338 SAADOUN ET AL. HEPATOLOGY, June 2006 inary data may support a particular link between lipid metabolism and cryoglobulin in HCV infection. Agnello 15 has suggested that very low-density lipoprotein was selectively associated with HCV in mixed cryoglobulins and that the persistence in the circulation of HCV very low-density lipoprotein complexes that contain apolipoprotein E2 may be one mechanism for developing cryoglobulins. To address the relationship between presence of detectable cryoglobulin and liver lesions, we analyzed the liver biopsies of 437 consecutive untreated patients with chronic hepatitis C. Cryoglobulin was present in 286 patients, 103 of whom had vasculitis. For all these 437 patients, risk factors for fibrosis and steatosis were reported, and a liver biopsy was available and scored for fibrosis, necroinflammation, steatosis, and liver iron deposition. Multivariate analysis was performed, taking into consideration risk factors for the progression of fibrosis and steatosis. Patients and Methods Patients. Four hundred thirty-seven consecutive untreated patients with chronic hepatitis C from 2 medical centers (Internal Medicine Department of Pitié-Salpétrière Hospital, Paris and Liver Disease Department of Beaujon Hospital, Clichy, France) with available liver biopsy were included in the study. Chronic hepatitis C was defined as presence of HCV antibodies and detectable serum HCV RNA via PCR. Exclusion criteria for the study were HIV infection, detectable hepatitis B surface antigen, evidence of other liver disease (e.g., autoimmune hepatitis, primary biliary cirrhosis), previous treatment for HCV infection, or previous therapy with immunosuppressive drugs. Patients with cryoglobulin were defined as having a serum cryoglobulin level greater than 0.05 g/l. 16 Patients with symptomatic cryoglobulin (i.e., vasculitis) had serum cryoglobulin associated with the triad purpura-arthralgia-asthenia, and/or renal or neurologic involvement. 17 The following standardized criteria were used to define cryoglobulin-associated manifestations: (1) purpura, palpable purpura on the lower limb; (2) joint, inflammatory polyarthralgias with or without arthritis; (3) peripheral neurologic involvement, sensory and/or motor peripheral nerve disturbances confirmed via electrophysiological study and biopsy-proven cryoglobulinemia neuropathy (31/75, 41%); (4) renal involvement, proteinuria, hematuria and/or increased serum creatinine ( 140 mol/l), and membranoproliferative glomerulonephritis on renal biopsy (17/18, 94%). Patients with a serum cryoglobulin level greater than 0.05 g/l and without the above clinical symptoms were defined as having asymptomatic mixed cryoglobulin. Data Collection. We conducted a prospective community-based study to evaluate the relationship of cryoglobulins with liver lesions (necroinflammation, fibrosis, and steatosis) in patients with HCV infection. The study started at the time of the first liver biopsy before antiviral therapy in 2 French medical centers (Internal Medicine Department of Pitié-Salpétrière Hospital, Paris and Liver Disease Department of Beaujon Hospital, Clichy, France). A questionnaire was completed on the day of the liver biopsy for every patient that included sex, age ( 50 yr or 50 yr), source of infection, estimated duration of infection, alcohol consumption (grams per day), height, and weight. The duration of infection was calculated for 174 patients based on the onset of drug use or date of blood transfusion. The body mass index (BMI) was calculated as weight (in kilograms) divided by height (in meters) squared, and the definitions for overweight (BMI 25 kg/m 2 ) and obesity (BMI 30 kg/m 2 ) were used. The symptoms related to cryoglobulinemia were recorded at the time of the first evaluation. A fasting blood sample was taken on the day of liver biopsy for analysis of the following parameters: serum alanine aminotransferase, -glutamyltransferase, glucose, cholesterol, triglycerides, and cryoglobulins. Cryoglobulins were measured as previously described, 16 wherein they were isolated from the patient sera, purified, and then characterized by immunoblotting at 37 C. Following the technique of Brouet et al., 18 all positive patients had either type II or type III mixed cryoglobulins, characterized, respectively, by the presence of a monoclonal or polyclonal rheumatoid factor component. HCV genotyping was performed in the 5 -noncoding region of the HCV genome using reverse hybridation with the line probe assay (LiPA; InGenn, Rungis, France.). 19 Laboratory evaluation included C4 fraction of complement and rheumatoid factor activity in symptomatic cryoglobulinemia patients. Serum HCV RNA quantification was performed with bdna version 2.0 assay according the manufacturer s instructions (Quantiplex HCV RNA version 2.0; Bayer, Puteaux, France). 20 Quantitative serum HCV RNA was available in 242 patients. Histological Evaluation. Liver histology, which was performed before treatment, was available for all 437 patients. All liver biopsies were graded (necroinflammation) and staged (fibrosis score) according to the Metavir scoring system. 21 The Metavir score incorporates 5 progressive stages of fibrosis (F0, normal; F1, portal fibrosis; F2, few fibrous septae; F3, numerous septae; and F4, cirrhosis) and 4 grades of necroinflammatory activity (A0, no activity; A1, mild activity; A2, moderate activity; A3, se-

3 HEPATOLOGY, Vol. 43, No. 6, 2006 SAADOUN ET AL vere activity), taking into account the severity of portal and lobular necroinflammatory lesions. Steatosis was calculated according to the percentage of hepatocytes containing cytoplasmic vacuoles. Then, steatosis was graded as absent, mild ( 10% of hepatocytes), moderate (10%- 30%), or marked ( 30%). Liver sections were stained with Perl s Prussian stain for iron grading. Stainable iron in the liver was graded as absent or present. Statistical Analysis. Data are expressed as the mean SD. Three end points were considered: (1) presence of high stage of fibrosis (F3-F4), (2) presence of more than 10% hepatocytes containing cytoplasmic vacuoles, and (3) presence of serum cryoglobulin. For each end point, association with sex, age ( 50 years), duration of infection ( 20 years), alcohol consumption ( 50 g/d), BMI ( 25 kg/m 2 ), cholesterol ( 6.5 mmol/l), triglyceride level ( 1.6 mmol/l), glycemia ( 7 mmol/l), -glutamyltransferase level ( 60 mmol/l), alanine aminotransferase level (more than a twofold increase in upper limit of normal value [40 IU/L]), necroinflammatory activity (A2 or A3), and presence of liver iron was tested using Fisher s exact test. HCV genotype (type 1, type 2, type 3, and others) were tested using a Wald test in a logistic model. The OR and 95% CI was also computed for each parameter. Adjusted analyses were performed using multiple logistic models in which all factors with a P value lower than.05 in the univariate analysis were considered statistically significant. Associations between cryoglobulin type and advanced fibrosis or steatosis greater than 10% were tested using a Fisher s exact test. Associations between cryoglobulin level and advanced fibrosis or steatosis greater than 10% were tested using a Wilcoxon test. Comparisons of liver fibrosis and steatosis according to the presence of symptomatic or asymptomatic cryoglobulin were tested using Fischer s exact test, as were associations between viral load and advanced fibrosis (F3-F4) or steatosis greater than 10%. All tests were 2-sided at the.05 significance level. Analyses were performed using the R statistical package (R Development Core Team; R Foundation for Statistical Computing, Vienna, Austria [ Results Characteristics of the Patients. The characteristics of all 437 patients are presented in Table 1. Forty-nine percent were male, and the mean age was years. Sources of infection included blood transfusion (n 131 [30%]), intravenous drug use (n 66 [15%]), and unknown causes (n 240 [55%]). The mean BMI was kg/m 2. The distribution of HCV genotypes was: genotype 1 (n 253 [58%]), genotype 2 (n 62 Table 1. Characteristics of the 437 Patients With Chronic Hepatitis C Infection Characteristics Values Male sex, n (%) 213 (49) Age, years (mean SD) Duration of infection, years (mean SD) Alcohol consumption 50 g/d, n (%) 21 (5) BMI, kg/m 2 (mean SD) Cholesterol, mmol/l (mean SD) Triglyceride, mmol/l (mean SD) Glycemia, mmol/l (mean SD) GGT, mmol/l (mean SD) ALT 2 ULN, n (%) 208 (48) Genotype, n (%) (58) 2 62 (14) 3 74 (17) Other 48 (11) Liver steatosis, n (%) Mild ( 10%) 251 (57) Moderate (10%-30%) 91 (21) Marked ( 30%) 95 (22) Liver necroinflammation, n (%) A0 33 (8) A1 259 (59) A2 130 (30) A3 15 (3) Liver fibrosis, n (%) F0 17 (4) F1 204 (47) F2 106 (24) F3 46 (10) F4 64 (15) Presence of liver iron, n (%) 114 (26) Presence of cryoglobulin, n (%) 286 (65) Presence of symptomatic cryoglobulin, n (%) 103 (24) NOTE. Liver necroinflammation and fibrosis were evaluated according to the Metavir score. 42 Abbreviations: BMI, body mass index; GGT, -glutamyltransferase; ALT, alanine aminotransferase; ULN, upper limit of normal. [14%]), genotype 3 (n 74 [17%]), and other genotypes (n 48 [11%]). Among the 242 patients with available HCV viral load, the value was 2 Meq/mL in 130 patients and 2 Meq/mL in 112 patients. Factors Associated With Presence of Cryoglobulin. Cryoglobulin was detectable in 286 (65%) of the 437 patients. Symptomatic cryoglobulin was present in 103 patients. Type II and type III mixed cryoglobulin were found in 166 (58%) and 120 (42%) HCV-infected patients, respectively. The mean cryoglobulin level was g/l. The clinical manifestations of cryoglobulinemic-vasculitis were: arthralgia (n 58 [56%]), purpura (n 63 [61%]), nerve involvement (n 75 [73%]), and renal involvement (n 18 [17%]) (Table 2). Factors associated with cryoglobulin were age 50 years, steatosis 10%, and advanced fibrosis (Table 3). In multivariate analysis, age 50 years (OR 1.58, 95% CI ), steatosis 10% (OR 1.61, 95% CI ),

4 1340 SAADOUN ET AL. HEPATOLOGY, June 2006 Table 2. Characteristics of the 103 Patients With Symptomatic Cryoglobulin Characteristics Values ALT 2 ULN, n (%) 53 (51) HCV genotype 1, n (%) 47 (46) Clinical features, n (%) Peripheral neuropathy 75 (73) Purpura 63 (61) Arthralgia 58 (56) Renal involvement 18 (17) Cryoglobulin level, g/l (mean SD) Cryoglobulin type, n (%) II 85 (82) III 18 (18) Low C4 complement factor level, n (%) 60 (58) Rheumatoid factor, n (%) 71 (69) Abbreviations: ALT, alanine aminotransferase; ULN, upper limit of normal. and advanced fibrosis (F3-F4) (OR 3.27, 95% CI ) were independently and significantly associated with cryoglobulin (Table 3). Among patients with detectable cryoglobulin, no difference was found in degree of fibrosis or steatosis between symptomatic (i.e., with vasculitis) versus asymptomatic patients. Mild fibrosis (F0-F1-F2) was present in 67 (66%) patients with symptomatic cryoglobulin compared with 127 (68%) of those with asymptomatic cryoglobulin. Advanced fibrosis was present in 34 (34%) patients with symptomatic cryoglobulin compared with 58 (32%) of those with asymptomatic cryoglobulin (P.69). Steatosis lower than 10% was observed in 46 (46%) patients with symptomatic cryoglobulin compared with 103 (56%) of those with asymptomatic cryoglobulin. Steatosis greater than 10% was observed in 55 (54%) patients with symptomatic cryoglobulin compared with 82 (44%) of those with asymptomatic cryoglobulin (P.11). No association was found with steatosis 10% nor with advanced fibrosis (F3-F4) according to the type and level of cryoglobulin (data not shown). Factors Associated With Presence of Steatosis >10%. Two hundred and fifty-three patients (58%) had steatosis. Among these patients, steatosis was graded as mild for 67 (26%) patients, moderate for 91 (36%), and marked for 95 (38%). Patients with steatosis 10% had more frequently a higher BMI ( 25 kg/m 2 ), HCV genotype 3, advanced fibrosis, and cryoglobulin (Table 4). In multivariate analysis, higher BMI ( 25 kg/m 2 ) (OR 2.73, 95% CI ), HCV genotype 3 (OR 3.83, 95% CI ), advanced fibrosis (OR 2.06, 95% CI ), and cryoglobulin (OR 2.40, 95% CI ) were independently and significantly associated with the presence of steatosis 10% (Table 4). Factors Associated With High Stage of Fibrosis (F3-F4). Three hundred twenty-seven (75%) patients had a mild or moderate stage of fibrosis (F0-F1-F2), whereas 110 (25%) had a high stage of fibrosis (F3-F4). Patients with advanced fibrosis were more frequently older than 50 years, had a higher level of -glutamyltransferase ( 60 mmol/l), had a higher grade of necroinflammation (A2-A3), had steatosis 10%, and had detectable cryoglobulin (Table 5). In multivariate analysis, a higher level of -glutamyltransferase (OR 1.75, 95% CI ), steatosis 10% (OR 1.76, 95% CI ), high grade of necroinflammation (A2-A3) (OR 3.09, 95% CI ), and cryoglobulin (OR 3.40, 95% CI ) were independently and significantly associated with advanced fibrosis (F3-F4) (Table 5). Furthermore, in patients with advanced fibrosis, a significant interaction was found between steatosis 10% and cryoglobulin (P.02). The association between advanced fibrosis (F3-F4) and steatosis 10% was stronger in the cryoglobulin-positive group (OR 2.61, 95% CI ) than in the cryoglobulin-negative group (OR 0.51, 95% CI ). Among the 242 patients with available HCV RNA, no association was found between viral load and steatosis 10%, nor with advanced fibrosis (F3-F4) (data not shown). Discussion Among 437 consecutive untreated chronic hepatitis C patients, 286 had cryoglobulin, 103 of whom were symptomatic (i.e., vasculitis). The high prevalence of cryoglobulin and vasculitis observed in our population is likely explained by the biased referrals from centers specializing in extrahepatic complication of HCV. In multivariate analysis, age 50 years, steatosis 10%, and high grade of fibrosis (F3-F4) were independently associated with cryoglobulin. Older age is commonly associated with the presence of cryoglobulin in patients with chronic hepatitis C. 4 We report an independent association between cryoglobulin and the presence of steatosis in chronic hepatitis C. Steatosis 10% was found in 48% of patients with cryoglobulin versus 32% of those without cryoglobulin. The development of steatosis in chronic HCV patients occurs in 2 distinct ways: metabolically or virally induced. Preliminary data may support a particular link between lipid metabolism and cryoglobulin in HCV infection. Very low-density lipoprotein may be selectively associated with HCV in mixed cryoglobulins. 15 One mechanism for developing cryoglobulins may be the persistence in the circulation of HCV very low-density lipoprotein complexes that contain apolipoprotien E2. 15 Furthermore, it has been demonstrated that low-density lipoprotein re-

5 HEPATOLOGY, Vol. 43, No. 6, 2006 SAADOUN ET AL Table 3. Factors Associated With Presence of Cryoglobulin Univariate Analysis Multivariate Analysis Parameter N n (%) OR (95%CI) P Value OR (95%CI) P Value Sex Male (61.0) 1 Female (69.6) 1.46 ( ).07 Age (yr) (54.1) (66.1) 1.5 ( ) ( ).04 Duration of infection (yr) (80.9) (72.9) 0.64 ( ).28 Alcohol consumption (g/d) (66.1) (61.9) 0.83 ( ).81 BMI (kg/m 2 ) (70.5) (61.7) 0.67 ( ).09 Cholesterol (mmol/l) (69.3) (81.8) 1.99 ( ).51 Triglyceride (mmol/l) (70.7) (72.9) 0.78 ( ).50 Glycemia (mmol/l) (73.9) (71.43) 1.11 ( ) 1 GGT (mmol/l) (66.0) (65.5) 0.98 ( ).91 ALT (ULN) (64.7) (67.8) 1.15 ( ).54 Genotype (64.6) (74.0) 1.56 ( ) (61.7) 0.88 ( ).68 Other (68.4) 1.19 ( ).64 Liver steatosis 10% (59.4) 1 10% (73.7) 1.91 ( ) ( ).04 Liver necroinflammation A0-A (63.0) 1 A2-A (70.3) 1.39 ( ).14 Liver fibrosis F0-F1-F (59.3) 1 F3-F (83.6) 3.49 ( ) ( ).0001 Liver iron Absent (63.2) 1 Present (71.9) 1.49 ( ).12 Abbreviations: BMI, body mass index; GGT, -glutamyltransferase; ALT, alanine aminotransferase; ULN, upper limit of normal. ceptors are upregulated on keratinocytes in cutaneous vasculitis lesions of HCV-infected patients compared with normal skin. 22 A higher proinflammatory cytokine production (i.e., tumor necrosis factor ) has been observed in the peripheral blood and liver of HCV-cryoglobulinemic patients compared with their cryoglobulin-negative couterparts. 23,24 Hence, upregulation of low-density lipoprotein receptors associated with inflammation may result in lipid accumulation in the liver of HCV-cryoglobulinemic patients. Among 437 patients with chronic hepatitis C, 55% had steatosis. This is consistent with previous studies that have found prevalence of steatosis in chronic hepatitis C ranging from 40% to 86% of adults in the United States and Europe. 10,14,25-28 The presence of steatosis 10% was independently associated with higher BMI ( 25 kg/m 2 ),

6 1342 SAADOUN ET AL. HEPATOLOGY, June 2006 Table 4. Factors Associated With Presence of Steatosis >10% Univariate Analysis Multivariate Analysis Parameter N n (%) OR (95%CI) P Value OR (95%CI) P Value Sex Male (46.0) 1 Female (39.3) 0.76 ( ).18 Age (yr) (42.6) (37.8) 0.82 ( ).35 Duration of infection (yr) (50.6) (41.2) 0.69 ( ).23 Alcohol consumption (g/d) (41.6) (61.9) 2.28 ( ).07 BMI (kg/m 2 ) (35.6) (59.4) 2.64 ( ) ( ).0001 Cholesterol (mmol/l) (43.2) (72.7) 3.5 ( ).07 Triglyceride (mmol/l) (44.5) (42.9) 0.94 ( ).88 Glycemia (mmol/l ) (43.1) (57.1) 1.76 ( ).26 GGT (mmol/l) (42.5) (43.2) 1.03 ( ).92 ALT (ULN) (38.8) (46.2) 1.35 ( ).14 Genotype (34.9) (48.0) 1.72 ( ) ( ) (63.3) 3.21 ( ) ( ).0001 Other (47.4) 1.68 ( ) ( ).46 Liver necroinflammation A0-A (41.1) 1 A2-A (45.52) 1.2 ( ).41 Liver fibrosis F0-F1-F (38.53) 1 F3-F (54.55) 1.91 ( ) ( ).009 Liver iron Absent (42.72) 1 Present (42.11) 1.03 ( ) 1 Cryoglobulin Absent (32.45) 1 Present (47.9) 1.91 ( ) ( ).002 Abbreviations: BMI, body mass index; GGT, -glutamyltransferase; ALT, alanine aminotransferase; ULN, upper limit of normal. HCV genotype 3 infection, presence of cryoglobulin, and advanced fibrosis (F3-F4). Similar to previous studies, we found a strong association between BMI 25 kg/m 2 and steatosis 10%. 7-11,13,14,25-27 Steatosis 10% was also associated with HCV genotype 3 infection. Among 60 patients with HCV genotype 3, 63% had steatosis 10% compared with 48% in patients with HCV genotype 1 infection. This suggests the existence of specific viral-induced steatosis with this genotype and is consistent with previous findings. 13,14,25,27 No association was found between steatosis 10% and high grade of necroinflammation (A2-A3). However, this issue is still a matter of debate, because several studies 10,11,13,14 have found a link between necroinflammation (or HCV replication) and steatosis, whereas others have not. 8,25 Steatosis was strongly associated with advanced fibrosis (F3-F4). Furthermore, in patients with advanced fibrosis, a significant interaction was found between steatosis

7 HEPATOLOGY, Vol. 43, No. 6, 2006 SAADOUN ET AL Table 5. Factors Associated With Presence of High-Grade Fibrosis (F3-F4) Univariate Analysis Multivariate Analysis Parameter N n (%) OR (95%CI) P Value OR (95%CI) P Value Sex Male (22.1) 1 Female (28.1) 1.38 ( ).15 Age (yr) (19.3) (29.4) 1.73 ( ) ( ).12 Duration of infection (yr) (22.5) (23.5) 1.06 ( ) 1 Alcohol consumption (g/d) (25.3) (14.3) 0.49 ( ).31 BMI (kg/m 2 ) (24.6) (26.3) 1.09 ( ).71 Cholesterol (mmol/l) (28.6) (9.1) 0.25 ( ).30 Triglyceride (mmol/l) (29.3) (20.4) 0.62 ( ).23 Glycemia (mmol/l) (25.9) (33.3) 1.43 ( ).45 GGT (mmol/l) (21.4) (33.8) 1.87 ( ) ( ).04 ALT (ULN) (25.9) (24.5) 0.93 ( ).82 Genotype (24.9) (30.0) 1.56 ( ) (15.0) 0.88 ( ).68 Other (39.5) 1.19 ( ).65 Liver steatosis 10% (19.9) 1 10% (32.3) 1.91 ( ) ( ).04 Liver necroinflammation A0-A (15.8) 1 A2-A (44.1) 4.21 ( ) ( ).0001 Liver iron Absent (24.6) 1 Present (25.4) 1.04 ( ).90 Cryoglobulin Absent (11.9) 1 Present (32.2) 3.49 ( ) ( ).0001 Abbreviations: BMI, body mass index; GGT, -glutamyltransferase; ALT, alanine aminotransferase; ULN, upper limit of normal. 10% and cryoglobulin. The association between advanced fibrosis and steatosis was stronger in the cryoglobulin-positive group than in the cryoglobulin-negative group. Although many studies have found an association between steatosis and the progression of fibrosis, this issue is still debated. Among 14 published studies, 7-12,14,25-27, found an association between the presence of steatosis and a high grade of fibrosis. 7-14,26,33 However, in most of these studies, this association had a low degree of statistical significance. 7,9,10,14 The link between steatosis and fibrosis is complex, and the exact sequence of events is unclear, though several hypotheses have been made. 34 A potential link between severity of the steatosis and fibrogenesis may come from the excess reactive oxygen species produced by steatotic hepatocytes. There were more patients with advanced fibrosis (F3- F4) among patients with cryoglobulin than those without cryoglobulin (32% vs. 12%). The positive association be-

8 1344 SAADOUN ET AL. HEPATOLOGY, June 2006 tween cryoglobulin and severity of liver disease in chronic hepatitis C remains a controversial issue. Geographic factors may influence different associations observed in HCV-infected patients with cryoglobulin. 35,36 Patients with cryoglobulin tend to have a longer estimated duration of disease, which is also an important risk factor for cirrhosis. 5 However, in our study this factor was not associated with cryoglobulin. In a meta-analysis, Kayali et al. 5 found a significant association between cirrhosis and cryoglobulin after adjusting for age, sex, and length of infection. However, data arise from small cohort populations (usually fewer than 100 patients) without precise characterization of cryoglobulin features (i.e., type, level, and associated symptoms). In this large study, cryoglobulin was a key independent factor associated with advanced fibrosis. The mechanisms responsible for the association between cryoglobulin and more severe liver fibrosis are still poorly understood. Some reports have indicated that cell-mediated host immunity is likely responsible for hepatic inflammation. 37 Inflammatory cells and hepatocytes will induce activation of hepatic stellate cells to initiate fibrogenesis. Emerging evidence suggests that patients with HCV-related mixed cryoglobulin display a particular immune response to HCV infection. 23,24,38 Tumor necrosis factor, which induces the recruitement of inflammatory cells and stimulates oxidative stress in hepatocytes, 39 is highly produced in the liver of HCV-cryoglobulinemic patients 24 and may account for a more severe course of liver disease. A recent study showed that B cells play an important role in the development of liver fibrosis. 40 In addition, overrepresentation of B cell clonal expansions that contribute to the formation of intraportal lymphoid nodules in the liver of HCVinfected patients are invariably associated with extrahepatic manifestations, including cryoglobulins. 41 This study allows important clarification in the severity of liver disease between symptomatic or asymptomatic cryoglobulin in chronic HCV infection. The majority of HCV-cryoglobulinemic patients are free of vasculitis and are either asymptomatic or have nonspecific findings (e.g., arthralgias, fatigue). Therefore, we selected stringent definition criteria of symptomatic cryoglobulin including purpura, renal involvement, or nerve involvement, which are known to be the most specific manifestations of mixed cryoglobulinemia vasculitis. 17 Patients with asthenia or arthralgia as the sole extrahepatic symptom were not considered as having symptomatic cryoglobulin. We did not observe significant difference in degree of fibrosis or steatosis between these 2 groups. This suggests that serum cryoglobulin may be the true covariate associated with severity of liver disease in symptomatic versus asymptomatic HCV-cryoglobulinemic patients. In conclusion, our study of a large cohort of patients with chronic hepatitis C shows an independent association between the presence of cryoglobulin and steatosis as well as advanced fibrosis. Cryoglobulins, with or without vasculitis, are associated with the severity of liver disease in patients with chronic hepatitis C. References 1. Wasley A, Alter MJ. Epidemiology of hepatitis C: geographic differences and temporal trends. Semin Liver Dis 2000;20: Marcellin P, Asselah T, Boyer N. Fibrosis and disease progression in hepatitis C. HEPATOLOGY 2002;36(5B):S47-S Gorevic PD, Kassab HJ, Levo Y, Kohn R, Meltzer M, Prose P, et al. Mixed cryoglobulinemia: clinical aspects and long-term follow-up of 40 patients. Am J Med 1980;69: Cacoub P, Poynard T, Ghillani P, Charlotte F, Olivi M, Piette JC, et al. Extrahepatic manifestations of chronic hepatitis C. MULTIVIRC Group. Multidepartment Virus C. Arthritis Rheum 1999;42: Kayali Z, Buckwold VE, Zimmerman B, Schmidt WN. Hepatitis C, cryoglobulinemia, and cirrhosis: a meta-analysis. HEPATOLOGY 2002;36: Lunel F, Musset L, Cacoub P, Frangeul L, Cresta P, Perrin M, et al. Cryoglobulinemia in chronic liver diseases: role of hepatitis C virus and liver damage. Gastroenterology 1994;106: Castera L, Hezode C, Roudot-Thoraval F, Bastie A, Zafrani ES, Pawlotsky JM, et al. 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