Hepatitis C virus infection associated with liver-kidney microsomal antibody type 1 (LKM1) autoantibodies in children

HEPATITIS C VIRUS INFECTION ASSOCIATED WITH LIVER-KIDNEY MICROSOMAL ANTIBODY TYPE 1 (LKM1) AUTOANTIBODIES IN CHILDREN FLAVIA BORTOLOTTI, MD, LUIGI MURATORI, PHD, PALOMA JARA, MD, LORETO HIERRO, MD, GABRIELLA VERUCCHI, MD, RAFFAELLA GIACCHINO, MD, CRISTIANA BARBERA, MD, LUCIA ZANCAN, MD, MARIA GUIDO, MD, MASSIMO RESTI, MD, SABRINA PEDDITZI, MD, FRANCESCO BIANCHI, MD, AND ANGELO GATTA, MD

Objective To evaluate the clinical pattern and evolution of chronic hepatitis C in children with liver/kidney microsomal antibody type 1 autoantibodies (LKM1). Study design A multicenter, retrospective study, including the following groups of children with hepatitis C virus infection: (1) 21 consecutive LKM1-positive patients, (2) 42 age- and sex- matched LKM1-negative patients, and (3) 4 interferon-induced LKM1-positive cases. LKM1 reactivity to human microsomes and recombinant cytochrome P450IID6 (CYP2D6) was assayed by immunoblotting. Results Clinical and biochemical features overlapped in LKM1-positive and LKM1-negative children, but a fibrosis score >3 (range 0-6) was significantly more frequent (P = .04) in the former. Reactivity to microsomal protein and CYP2D6 was significantly (P = .02) associated with LKM1 titers ≥1:320 and was found in 39% of patients, including severe cases and both children (of 4 treated) who achieved a sustained alanine aminotransferase (ALT) normalization after steroid treatment. Five of 7 LKM1positive children treated with interferon had an ALT exacerbation. Conclusions LKM1-positive hepatitis C in children is characterized by a wide spectrum of biochemical, serologic, and histologic features. Whether autoimmunity may contribute to liver damage in a subgroup of patients with more severe liver disease, high LKM1 titers, and reactivity to CYP2D6 is a question deserving further investigation. (J Pediatr 2003;142:185-90)

nfection with hepatitis C virus (HCV) is often associated with autoimmune phenomena, possibly induced by molecular or epitope mimicry between self-antigens and viral proteins and/or by infection of B-lymphocytes.1-5 In children with chronic hepatitis C, the most peculiar autoimmune feature is the presence of liver-kidney microsomal antibody type 1 (LKM1), which can be detected in serum in about 10% of cases.6,7 LKM1 has been recognized as the marker of type 2 autoimmune hepatitis, an inflammatory liver disease that affects mainly children and benefits of long-term immunosuppressive treatment.8-10 It was first hypothesized that HCV could be a triggering factor for “true” autoimmune hepatitis. However, anti-HCV has been rarely detected in children with type 2 autoimmune hepatitis,10 suggesting that LKM1-positive hepatitis C is a different clinical entity.1,11 Further research attempted to characterize the target antigen of LKM1 in HCV-infected patients,12-19 to evaluate the contribution of autoimmunity in worsening virus-induced liver disease,20-23 and to analyze the effect of conventional immunosuppressive and antiviral therapy.24-27 One major limitation to this research in pediatric patients has been the low prevalence of HCV infection in childhood, preventing the enrollment of an adequate sample. The purpose of this multicenter, retrospective study was to describe the features and the short- to medium-term evolution of liver disease in a relatively large group of LKM1-positive, anti–HCV-positive children and in a comparable group of LKM1-negative, anti–HCV-positive cases.

I

ALT ANA CYP2D6 HAI HCV

Alanineaminotransferase Antinuclear antibodies Human cytochrome P450 II D6 Histologic activity index Hepatitis C virus

IFN LKM1 PCR RIBA SMA

Interferon Liver-kidney microsomal antibody type 1 Polymerase chain reaction Recombinant immunoblot assay Anti–smooth muscle antibodies

From Clinica Medica 5 and The Department of Pediatrics, University of Padua, Department of Internal Medicine, Cardioangiology, Hepatology Alma Mater Studiorum, University of Bologna, Policlinico S. Orsola, Department of Infectious Diseases, University of Bologna, 3° Clinica Pediatrica, Florence, Department of Infectious Diseases, Istituto Gaslini, Genoa, Clinica Pediatrica, University, Turin, Anatomia Patologica, Padua, Italy; and Hospital Infantil La Paz, Madrid, Spain. Submitted for publication Nov 9, 2001; revisions received Mar 20, 2002, and July 8, 2002; accepted Nov 4, 2002. Reprint requests: Flavia Bortolotti, MD, and reprint request Clinica Medica 5, Policlinico,Via Giustiniani 2, 35100 Padova, Italy. Copyright © 2003, Mosby, Inc. All rights reserved. 0022-3476/2003/$30.00 + 0

10.1067/mpd.2003.45

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Table I. Epidemiologic features in LKM1-positive (group A) and LKM1-negative (group B) patients LKM1+ Children (n = 21) Male/female Mean ± SD age (years) Type of EXPOSURE transfusions of blood or derivatives Anti-HCV + mother Other or unknown Mean ± SD duration of infection (y) Cause of first observation Symptoms (asthenia, anorexia, abdominal pain) Maternal infection Previous transfusion Intercurrent diseases* -Other causes

P value

LKM1- Children (n = 42)

9/12 5.91 ± 3.93

NSS NSS

18/24 5.63 ± 4.14

10 (48%) 8 (38%) 3 (14%) 2.64 ± 2.82

NS NS NSS NS

17 (40%) 17 (40%) 8 (20%) 1.57 ± 1.97

2 (9.5%) 6 (28.5%) 2 (10%) 11 (52%)

NS NSS NSS NSS

7 (17%) 14 (33%) 8 (19%) 13 (31%)

NSS, Not statistically significant. *Transient and benign.

METHODS Patients Three groups of patients were included. Group A included 21 white children with chronic HCV and LKM1 autoantibodies in serum. They represented all children with such features observed in 6 pediatric and hepatologic centers in Madrid (Spain), Bologna, Genoa, Padua, Turin, and Florence (Italy) from 1985 to 1999. Group B included 42 LKM1-negative children with chronic hepatitis C (control group). They were selected from a series of 202 consecutive anti–HCV-positive patients seen in 5 of the above-mentioned centers in the same period28 and pair-matched with LKM1-positive children for age, sex, country of origin and, when possible, for duration of illness. Group C included 4 previously negative children in whom LKM1 developed during interferon (IFN) treatment for chronic HCV. They represented 7% of 14 Italian and 7% of 42 Spanish children who received IFN treatment during a controlled29 and an uncontrolled30 trial, respectively, and were evaluated for LKM1 before and during treatment. None of the patients in the 3 groups had underlying systemic diseases such as previous or ongoing malignancy, hematologic disorders, or chronic uremia or was coinfected with human immunodeficiency virus or hepatitis B virus. None had metabolic disorders. None had a history of autoimmunity among first-degree relatives. The diagnosis of HCV infection was based on the presence of HCV RNA in serum or of anti-HCV, confirmed by recombinant immunoblot assay (RIBA), when HCV RNA testing was negative or not available. In chronically infected children, HCV RNA positivity supported the diagnosis of hepatitis even in the absence of alanineaminotransferase (ALT) elevations,31 whereas persistent or intermittent ALT elevations during a 6-12-month period were required for children who could not be tested for HCV RNA or were HCV RNA-negative. Liver biopsy was proposed to the parents of all 186 Bortolotti et al

LKM1-positive children to assess disease activity. It was performed in all patients who entered follow-up and whose parents gave their consent. Among control patients, liver biopsy was performed according to the policies of the different centers: Some of them proposed a diagnostic liver biopsy for all patients who entered follow-up; others proposed biopsy only in view of treatment. Children were usually seen in the outpatient clinic every 6 to 12 months for physical examination and biochemical and serologic testing. At each visit, a serum sample had been collected, when possible, and stored at –20°C.

Methods Anti-HCV was investigated by second- or third-generation ELISA tests (Ortho Diagnostic Systems, Raritan, NJ, and Chiron Corporation, Emeryville, Calif ). RIBA was performed in selected cases by a commercial assay (Ortho Diagnostic Systems, Raritan NJ). HCV-RNA was assayed in fresh or frozen serum samples never thawed before, by homemade nested polymerase chain reaction (PCR) in Madrid for Spanish children, and in Florence for Italian children. Genotyping was performed, analyzing PCR products by a reverse-hybridization assay (InnoLipa HCV II, Innogenetics, Zwijndrecht, Belgium) in the same laboratories. Quantitative analysis of viremia, performed by Amplicor HCV Monitor (Roche Diagnostic Systems, Branchburg, NJ) was available only in a subgroup of patients. Assays for autoimmunity were performed by a single investigator under identical experimental conditions in a single laboratory in Bologna. Antinuclear antibodies (ANA), anti–smooth muscle antibodies (SMA), and LKM1 were assayed by indirect immunofluorescence at serum dilutions of 1:10 on cryostatic sections of rat liver, kidney, and stomach. LKM1 reactivity was characterized further by Western immunoblot, with liver microsomes and recombinant human cytochrome P450 II D6 (CYP2D6) used as antigenic substrates.14-19 Liver cytosol antibody type 1 was investigated by counterimmunoelecThe Journal of Pediatrics • February 2003

Table II. Features at baseline in LKM1-positive (group A) and LKM1-negative (group B) patients LKM1+ CHILDREN (21 cases) ALT times  normal mean ± SD range Gammaglobulin > (n°) HCV RNA + (n°) HCV genotype 1a 1b 2 3 Other or undetermined No. HCV RNA copies/mL* median range Liver histology† HAI score > 5 Fibrosis score > 3

P value

LKM1- CHILDREN (42 cases)

3.55 ± 2.7 1–10.8 1 20/20

ns ns

3.08 ± 2.23 1–10.5 0 39/41

4/19 (21%) 11/19 (58%) 3/19 (16%) 1/19 (5%) 0

ns ns ns ns ns

8/35 (23%) 20/35 (57%) 2/35 (6%) 3/35 (8%) 2/35 (6%)

6  105 3  104–4.9  106

ns

4  105 3  104–5  106

5/15 (33%) 4/15 (27%)

ns 0.04

5/22 (23%) 0/22

NS, Not statistically significant. *Test performed in 14 LKM1-positive children and in 24 control patients. †See text.

trophoresis and Western immunoblot, with a preparation of human liver cytosol used as previously described.32 Liver biopsy was performed by use of the Menghini technique after informed consent of parents or guardians. Specimens were reviewed by an experienced pathologist who was unaware of any clinical details. Grade and stage of hepatitis were evaluated by means of a semiquantitative numerical score, according to Ishak et al.33 The histologic activity index (HAI) ranged between 0 and 18 and the score for fibrosis between 0 and 6. Each biopsy was finally labeled with a conclusive diagnosis of minimal, mild, moderate, or severe hepatitis or of cirrhosis. In LKM1-positive patients who underwent liver biopsy, the score for autoimmune hepatitis was calculated according to a recently revised scoring system.34 For the statistical analysis of data, we used the χ2 test, the Fisher exact probability tests, and the Student t test as appropriate.

RESULTS Baseline Features in LKM1-Positive and LKM1Negative Children The diagnosis of chronic HCV infection was based on the detection of anti-HCV and HCV RNA in all but one case in LKM1-positive children (group A) and in all but 3 cases in LKM1-negative children (group B). In the remaining 4 patients, the diagnosis was based on anti-HCV positivity by ELISA and RIBA; 2 children, one with persistently abnormal ALT and the other with intermittent ALT elevations, were found to be HCV RNA positive later during follow-up. Hepatitis C Virus Infection Associated With Liver-Kidney Microsomal Antibody Type 1 (LKM1) Autoantibodies in Children

Table I shows the baseline epidemiologic features of LKM1-positive and LKM1-negative children. The youngest LKM1-positive patient was a perinatally infected girl who was found to be LKM1-positive at first testing, when she was 23 months old. Table II shows the serologic, virologic, and histologic aspects of infection and liver disease in LKM1-positive patients and control patients. Liver histology showed that a high fibrosis score was significantly more frequent among autoantibody-positive children: 2 of these children had cirrhosis and 1 had severe hepatitis, whereas all 25 control patients who underwent liver biopsy had features of minimal to moderate chronic hepatitis. Cirrhosis was observed in a 14-year-old transfused boy with LKM1 titer of 1:320 and in a 2.5-year-old perinatally infected girl with LKM1 titer of 1:320, respectively. Severe HCV was seen in a girl 8.5 years of age, transfused at birth for prematurity, with autoantibody titer of 1:1280. Neither the HAI nor the score for fibrosis correlated with the levels of viremia or with genotype (data not shown). In the 15 LKM1-positive children who underwent liver biopsy, the score for autoimmune hepatitis ranged from –2 to 12 (median, 4); 4 (80%) of the 5 children with a score of 6 to 12 showed a reactivity to CYP2D6 and/or 50 kDa microsomal protein compared with 4 (40%) of 10 children with a lower score. A score of 12 was found in the girl with severe hepatitis. ANA, SMA, and antibodies to liver cytosol type 1 were never found in LKM1-positive cases, and low titers of ANA were detected in 4 control patients. 187

Table III. Features at the end of follow-up in untreated patients in groups A and B LKM1+ children (15 cases)

LKM1- children (26 cases)

4.4 ± 3.8 1*

5.1 ± 4.3 0

0 1 (7%)

0 5 (19%)

1 (7%) 13 (86%)

0 21 (81%)

Duration of follow-up (mean ± SD years) Symptoms ALT normalized with: HCV RNA– HCV RNA+† ALT persistently or intermittently abnormal with: HCV RNA– HCV RNA+†

*This boy had signs of liver failure and received transplantation at the age of 19 years. †Or intermittently positive.

Features During Follow-up in LKM1-Positive Children and Control Patients UNTREATED CASES. All LKM1-positive children maintained the autoantibody during follow-up without development of other features of autoimmunity (Table III). HCV RNA clearance was observed only in the youngest patient, mentioned above, who lost viremia at 20 months of age but remained LKM1-positive with normal or near-normal ALT. PATIENTS TREATED WITH STEROIDS. It was suggested that anti-HCV in autoantibody-positive hepatitis might represent a false-positive result 35 ; therefore, 3 LKM1-positive patients received conventional immunosuppressive treatment (prednisone or prednisone and azathioprine). Two girls with LKM1 titers of 1:320 and 1:1280, respectively, and ALT of 4 to 10 times the normal but minimal hepatitis on biopsy were treated for 6 and 18 months, respectively. They did transiently normalize ALT and loose LKM1, but autoantibody and abnormal ALT reappeared as soon as therapy was withdrawn. In one of these girls, a liver biopsy obtained 4 years after stopping treatment was still consistent with minimal hepatitis. The third patient, with 10-fold ALT elevation, mild hypergammaglobulinemia, LKM1 titer of 1:5120, and mild hepatitis on liver biopsy, had promptly normalized ALT after starting steroid treatment. Biochemical remission was sustained during maintenance therapy. HCV RNA could not be investigated. The girl left the referral center after 1 year of treatment and entered follow-up in another institution. PATIENTS TREATED WITH IFN. Three LKM1-positive children were included in therapeutic trials with recombinant IFN-α at a dose of 5 MU/m2 3 times weekly for 12 months. Treatment was withdrawn in 2 cases after 8 and 10 months, respectively, because of persistent viremia and ALT abnormalities. In the third case, described in a previous report,6 therapy was discontinued after 4 months after an ALT exacerbation of 25 times the upper normal value. Conventional immunosuppressive treatment was then started and is being maintained at 188 Bortolotti et al

low dose, with biochemical remission but persistence of HCV RNA. A baseline liver biopsy was consistent with mild hepatitis; histology had worsened (moderate hepatitis) after the ALT flare and improved (mild hepatitis) after 2 years of immunosuppressive therapy.

IFN Therapy and Development of LKM1 in Previously Negative Patients During a controlled trial including 14 LKM1-negative, HCV RNA–positive Italian children with chronic hepatitis C29 who received recombinant IFN at a dose of 5 MU/m2 3 times weekly for 1 year, LKM1 developed in 1 (7%) patient. During an uncontrolled trial including 42 LKM1-negative, HCV RNA–positive Spanish children with chronic HCV30 treated with recombinant IFN at a dose of 3 MU/m2 3 times weekly for 1 year, LKM1 developed in 3 (7%) children. All 4 cases had a 3- to 4-fold increase of baseline ALT coincident with the development of autoimmunity. After therapy withdrawal, all remained HCV RNA–positive throughout observation; 3 patients remained LKM1-positive with abnormal ALT, whereas 1 child had normalized ALT 8 months after stopping treatment and lost LKM1 2.5 years after cessation of therapy.

LKM1 Reactivity in Serum LKM1 titers ranged between 1:10 and 1:5120 (median, 1:160). All patients with titers >1:1280 were female, and all patients with cirrhosis or severe hepatitis had LKM1 titers of ≥1:320. Anti-CYP2D6 and anti–human microsomal protein were investigated in 19 of 21 patients in group A and in the 4 patients in group C. A reactivity was found in 9 (39%) cases and was significantly more frequent in patients with LKM1 titres of 1:320 or greater (8 of 13 versus 1 of 10 with lower titer; P = .02). Reactivity to CYP2D6 was found in both patients on long-term steroid treatment and in the children with severe hepatitis or cirrhosis.

DISCUSSION The results of this retrospective study strengthen the hypothesis that LKM1-positive hepatitis C and type 2 autoimThe Journal of Pediatrics • February 2003

mune hepatitis may be different clinical entities, the latter being more severe. In the series of 20 children with autoimmune hepatitis type 2 observed by Gregorio et al10 during a 20-year period, 56% of cases had features of acute hepatitis, 44% had symptoms and/or complications, and 38% had cirrhosis on initial biopsy. Conversely, none of our LKM1-positive patients had a symptomatic acute hepatitis and only 9.5% had mild symptoms; increased levels of IgG were uncommon, and cirrhosis was detected in 13.3% of the cases who underwent liver biopsy. The clinical, serologic, and histologic patterns of LKM1-positive hepatitis C are heterogeneous in our series, including cases with mild liver lesions and very low autoantibody titers, with mild liver disease but high LKM1 titer, and cases with severe disease activity or cirrhosis, also with high LKM1 titer. Reactivity to CYP2D6 was detected in 39% of LKM1-positive children, particularly in those with high autoantibody titers. LKM1 from HCV-infected patients preferentially reacts with eukaryotically expressed conformational epitopes.16,17 It has been suggested that HCV, like other viruses, may trigger autoimmunity in genetically predisposed individuals.36 That LKM1 development may be a simple consequence of hepatocellular damage, without pathogenic significance, is supported by the following considerations: One patient in our series was LKM1-positive but had normal ALT. A more severe liver damage was associated with higher LKM1 titers and with the presence of antibodies to CYP2D6; LKM1 is considered to be an antigen-driven autoimmune response.5 On the other hand, the hypothesis that autoimmunity may have pathogenetic implications is supported by studies that show that CYP2D6, the main target of LKM1, is expressed on the plasma membrane of rat and human hepatocytes, where it is recognized by LKM1-positive sera of both patients with type 2 autoimmune hepatitis and patients with hepatitis C.19 In keeping with this hypothesis, our data show that patients with LKM1-positive hepatitis C are more prone to have fibrosis and severe liver disease than patients with LKM1-negative hepatitis C. These data must be interpreted cautiously because of the small number of biopsy specimens considered; nevertheless, recent reports in adult patients with chronic hepatitis C could also show a more severe biochemical and histologic activity in autoantibody-positive cases.22,23 These findings suggest that autoimmunity might contribute to liver damage in a subgroup of HCV-infected patients. To single out the subgroup parameters such as high LKM1 titers, reactivity to CYP2D6 antigens, severe liver histology, score for autoimmune hepatitis of ≥10, hypergammaglobulinemia, and female sex could be useful. Assessment of HLA genetic pattern may also be helpful.37 Treatment of LKM1-positive chronic hepatitis C remains a controversial issue.6,23-26 In this series, immunosuppressive therapy induced prompt ALT normalization and then sustained biochemical remission in 2 LKM1-positive children with high LKM1 titer and reactivity to CYP2D6. In 2 additional patients, ALT normalized during steroid treatment but increased again after therapy withdrawal; however, liver hisHepatitis C Virus Infection Associated With Liver-Kidney Microsomal Antibody Type 1 (LKM1) Autoantibodies in Children

tology was still consistent with minimal hepatitis in the girl who underwent liver biopsy 4 years later. Even if successful in terms of clinical and biochemical remission of liver disease in selected cases, immunosuppressive therapy cannot be considered satisfactory because it favors persistent HCV replication. On the other hand, IFN alone proved to have little benefit in the few LKM1-positive cases treated in this study; ALT exacerbations occurred in 5 of 7 treated cases and 7% of LKM1-negative children with hepatitis C had LKM1 after IFN therapy. More favorable results have been recently reported by Iorio et al38 in 4 LKM1-positive children, but the small sample size of these studies does not allow conclusions. In any case, a careful monitoring for LKM1 is indicated for HCV-infected children who receive IFN.

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26. Ijima Y, Kato T, Miyakawa H, Ogino M, Mizuno M, Sugihara K, et al. Effect of interferon therapy on Japanese chronic hepatitis C virus patients with antiliver/kidney microsome antibody type 1. J Gastroenterol Hepatol 2001;16:782-8. 27. Todros L, Saracco G, Durazzo M, Abate ML, Touscoz G, Scaglione L, et al. Efficacy and safety of interferon alpha therapy in chronic hepatitis C with autoantibodies to liver-kidney microsomes. Hepatology 1995;22:1374-8. 28. Jara P, Resti M, Zancan L, Giacchino, Hierro L, Sokal E, et al. Natural history of chronic HCV infection in children J Hepatol 1999;30(suppl 1):52. 29. Bortolotti F, Giacchino R, Vajro P, Barbera C, Crivellaro C, Alberti A, et al. Recombinant interferon-alpha therapy in children with chronic hepatitis C. Hepatology 1995;22:1623-6. 30. Jara P, Hierro E, Frauca MJ, Robledo C, Camarena C, De La Vega A, et al. Interferon alfa-2b in children with chronic HCV hepatitis. Proceedings of the 30th Annual Meeting of ESPGHAN, Tessaloniki 1997. p. 65. 31. Shiff ER, De Medina M, Kahn RS. New perspectives in the diagnosis of hepatitis C. Semin Liver Dis 1999;19(suppl 1):3-15. 32. Muratori L, Cataleta M, Muratori P, Manotti P, Lenzi M, Cassani F, et al. Detection of anti-liver cytosol antibody type 1 (anti-LC1) by immunodiffusion, counterimmunoelectrophoresis and immunoblotting: comparison of different techniques. J Immunol Methods 1995;187:259-64. 33. Ishak K, Baptista A, Bianchi L, Callea F, De Groote J, Goudat F, et al. Histological grading and staging of chronic hepatitis. J Hepatol 1995;22:696-9. 34. International Autoimmune Hepatitis Group Report: review of criteria for diagnosis of autoimmune hepatitis. J Hepatol 1999;31:929-38. 35. McFarlane IG, Smith HM, Johnson PJ, Bray GP, Vergani D, Williams R. Hepatitis C virus antibodies in chronic active hepatitis: pathogenetic factor or false-positive result? Lancet 1990;335:754-7. 36. Czaja AJ, Carpenter HA, Santrach PJ, Moore SB. Immunologic features and HLA associations in chronic viral hepatitis. Gastroenterology 1995;108:157-64. 37. Jurado A, Cardaba B, Jara P, Cuadrado P, Hierro L, de Andres B, et al. Autoimmune hepatitis type 2 and hepatitis C virus infection: study of HLA antigens. J Hepatol 1997;26:983-91. 38. Iorio R, Giannattasio A, Vespere G, Vegnente A. LKM1 antibody and interferon therapy in children with chronic hepatitis C. J Hepatol 2001; 35:685-7.

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