See “Autoantibodies in Hepatitis C: Red Flag or Bystander Effect?” by Tana and Heller on page 243.
The literature is replete with reports of de novo manifestations of autoimmune diseases occurring during the treatment of chronic hepatitis C (CHC) with interferon (IFN)-based therapies. The most common diseases reported are thyroid disease (1), type 1 diabetes mellitus (2), and autoimmune hepatitis (3). Rarer manifestations have included celiac disease (4), autoimmune thrombocytopenia (5), and autoimmune hemolytic anemia (6). These complications are serious and, in some cases, permanent after cessation of therapy. Thyroid disease can occur in patients with CHC before treatment (7) and in as many as 15% of adults during IFN treatment (1,7). A nearly 5% incidence has been reported for diabetes (2). These findings have implications for the risk and benefit of treating CHC with IFN-based therapies. To limit potential risk, some clinicians have suggested using antibodies to screen and monitor the onset of disease; however, it is unclear whether the presence of antibodies before treatment correlates with subsequent development of autoimmune disease (8).
Although combination therapy with pegylated IFN (PEG-IFN) and ribavirin (RV) has become an established and effective therapy for CHC in children (9), little is known about the development of autoantibodies and their predictive utility in children. The PEDS-C trial provided a unique opportunity to study a well-characterized cohort of North American children regarding the effect of IFN on the development of autoantibodies, the predictive value of antibody presence on the development of symptomatic complications, and the effect of baseline antibody on response to treatment (10). In the present study, we applied a standardized approach to screening our population to minimize risk to our treatment population. All children entering the treatment portion of the clinical trial were excluded if they had increased titers of anti-nuclear antibody (ANA), anti-smooth muscle antibody (ASMA), anti-liver-kidney-microsomal antibodies (LKM) or anti-thyroid peroxidase (TPO) antibodies. In the 114 remaining children, ages 5 to 17 years, a population typical of those a clinician would treat in practice, we assessed autoantibodies, using stored frozen sera obtained before, during, and after treatment in the PEDS-C trial of PEG-IFN α-2a with and without RV. We evaluated the effect of autoantibodies on response to therapy, tolerance of therapy, and development of de novo autoimmune disease.
Eligible children included treatment-naïve children from 5 to 17 years old with CHC infection documented by hepatitis C virus (HCV) RNA in serum on 2 occasions at least 6 months apart and a liver biopsy consistent with CHC. Details of the trial design and therapeutic outcomes were published (10,11). Subjects were enrolled by investigators at 11 US medical centers from December 2004 until May 2006. The present study was approved by institutional review boards at each site and was conducted in compliance with the Declaration of Helsinki, Good Clinical Practice Guidelines, and local regulatory requirements. Children were excluded if there was evidence of decompensated liver disease or serological tests suggesting hepatitis A, hepatitis B, human immunodeficiency virus infection, or autoimmune hepatitis (ANA >1:160, ASMA >1:80, LKM >60 IU/mL, Covance Laboratory, Princeton, NJ) (10,11). A total of 9 children were excluded from the study at screening: 5 with ANA, 1 with ASMA, and 3 with LKM.
Subjects were randomly assigned 1:1 to receive either PEG-IFN α-2a and RV or PEG-IFN α-2a and placebo (10). PEG-IFN 2a was administered at a dose of 180 μg/1.73 m2 body surface area (maximum 180 μg) subcutaneously once per week. RV was administered at a dose of 15 mg · kg−1 · day−1 orally in 2 doses (maximum 1200 mg/day if >75 kg and 1000 mg if <75 kg). Placebo was supplied in the same dosing regimen as RV. Patients without detectable HCV RNA at 24 weeks were continued on treatment for another 24 weeks, whereas those who had detectable HCV RNA at 24 weeks were considered treatment failures. Treatment was stopped in those subjects who had been given PEG-IFN α-2a with RV and who did not achieve viral clearance at 24 weeks. Patients who failed treatment with PEG-IFN α-2a plus placebo were offered open-label therapy with PEG-IFN α-2a plus RV for another 48 weeks (stopping after 24 weeks if HCV RNA remained positive). Further details of the study design and outcome results of the trial have been reported (10,11). Drug dose reductions were instituted for toxicities according to the protocol.
All samples from weeks 0, 24, and 72 in the PEDS-C trial were stored at −80°C until analysis and were tested in a single laboratory simultaneously at the completion of the clinical trial. Figure 1 illustrates the times at which sera were obtained. Baseline and week-24 sera were tested from all subjects. Only those who were HCV negative at 24 weeks and continued on medication to complete a 48-week course of therapy were tested at the 72-week time point (24 weeks off therapy).
Autoantibodies were measured by Food and Drug Administration–approved enzyme-linked immunosorbent assay (ELISA) in a Clinical Laboratories Improvement Amendments (CLIA)–approved laboratory and reported in World Health Organization (WHO) international units. All of the tests were performed in the fully accredited Immune Disorders Laboratory at Johns Hopkins University (CLIA #21D0709511). Positive cutoff values were all based on WHO standards established for each test. Thyroid-associated autoantibodies included antibodies to thyroglobulin (TG) (QUANTA Lite ELISA) and TPO (QUANTA Lite TPO ELISA) and were measured by commercially manufactured ELISA kits (Inova Diagnostics, San Diego, CA). Positive values were defined as levels exceeding the normal reference range of 100 U. Autoantibodies LKM and ANA were measured using Inova Diagnostics QUANTA Lite ELISA assays. Positive results for LKM-1 autoantibodies included values exceeding 25 U. A moderate positive result for ANA autoantibodies fell within the range of 20 to 60 U; however, for the purpose of the present study, only results exceeding 60 U (strong positive) were considered to be positive. Detection of F-actin antibodies was done via commercial QUANTA Lite Actin IgG ELISA (Inova Diagnostics, San Diego, CA). Values exceeding 30 U (“moderate to strong positive”) were considered positive. Glutamic acid decarboxylase (GAD) and insulin islet antigen-2 autoantibodies, associated with the presence of type 1 diabetes mellitus, were measured using a GAD/IA-2 Autoantibody Screen ELISA Kit (KRONUS Inc, Star, ID). GAD-positive values were defined as values exceeding 5 IU/mL. IA-2–positive values were defined as values exceeding 20 U.
Routine laboratory assessments were performed at Covance and included free T4, thyroid-stimulating hormone (TSH), complete blood cell count, and alanine aminotransferase (ALT) among other tests that are fully described by Schwarz et al (10,11).
The generalized estimating equation was used to analyze the study outcome by determining the correlation between autoantibody presence and treatment outcome. The presence of positive autoantibodies at 24 and 72 weeks, treatment type, and visit time point functioned as covariates. All category variables were summarized using frequency and percentage. The comparison of adverse events between patients with and without autoantibodies was performed using Fisher exact test. All continuous factors were reported as mean and standard deviation at each group. A 2-sided t test or a Wilcoxon rank-sum test was used to compare the significance of autoantibody presence and absence in relation to continuous variables (eg, Knodell score). A P value ≤0.05 was considered significant.
Antibody Presence at Baseline, 24 Weeks, and 72 Weeks
A total of 114 treatment-naïve children with HCV entered the randomized trial. Mean age was 10.7 years, 55% were male, 75% were white, and the majority of children acquired their infection in the perinatal period (10). Genotype 1 accounted for 81%. Autoantibodies common to autoimmune hepatitis (ANA, LKM, anti-F-actin), thyroid disease (TPO, TG), and diabetes mellitus (IA-2, GAD) were assessed (Table 1). At baseline 19% of children had autoantibodies. The most common autoantibody was ANA (8%) followed by LKM (4%) and GAD (4%). Children with hepatitis-associated antibodies at baseline were younger, 7.8 vs 11.1 years (P = 0.0007), and had slightly lower body mass index z scores, −0.11 vs 0.70 (P = 0.01) but did not differ from those without antibodies in terms of sex, genotype, viral load, Knodell inflammation, or fibrosis scores (P = 1.00, 0.46, 0.13, 0.84, and 0.41, respectively).
After 24 weeks of PEG-IFN therapy, the overall prevalence of antibodies was 23% with ANA remaining the most common. At the 72-week time point, 26% of subjects had autoantibodies. The antibody titers were not significantly different between time points before, during, or 24 weeks after completion of therapy (P = 0.58 for TPO, 0.53 for TG, 0.86 for F-actin, 0.22 for ANA, 0.68 for IA-2). Mean antibody titers did not increase at 24 or at 72 weeks, whether assessed in all subjects or only in those with initial positive titers. The rate of antibodies becoming positive in those who were negative at baseline was up to 3% for liver-specific antibodies; among those who were positive for hepatitis-related autoantibodies at baseline, antibody subsequently became negative in 1 of 9 children with ANA, 2 of 5 children with LKM, and the 1 child with F-actin.
Relation Between Antibody Presence and EVR/SVR
Of the 114 subjects in the trial, 61% achieved viral suppression at week 24. Baseline hepatitis-associated antibody positivity did not significantly affect subsequent early viral response (EVR) and sustained viral response (SVR). No significant differences in response at 24 weeks were found when comparing those with any autoantibodies and those without (Table 2). EVR in those with antibodies at week 24 was 76% (19/25), and in those without antibodies at week 24 was 58% (48/83). SVR among this group of children who had undetectable HCV-RNA at 24 weeks was 69% (11/16) in those with antibodies and 65% (30/ 46) in those without (P = 0.46). Additionally, there was no difference in the rate of antibody positivity at week 72 in those who achieved SVR versus those who did not (27% vs 24%, P = 0.50). The children who relapsed at week 72 were compared with those who achieved SVR. Again, there was no statistically significant association of autoantibodies with SVR or relapse based on generalized estimating equation analysis. When comparing the hepatitis-associated antibody–positive and –negative groups that had achieved SVR, there was no significant difference in the frequency of nongenotype I or initial combination therapy, parameters that may have influenced SVR.
Autoantibodies and End-organ Disease
Three children developed significant autoimmune disease (Table 3). A 6-year-old girl developed type 1 diabetes mellitus after 32 weeks of study drug. She developed severe hyperglycemia at 32 weeks of therapy. Study drug was discontinued at week 32 and she continued to require insulin. Study testing on stored sera revealed negative IA-2 antibody at each time point and negative GAD at baseline and week 24; GAD was positive at week 72. Two girls, ages 11 and 13, developed clinical hypothyroidism requiring treatment; neither child had positive TG or TPO at baseline. In the 11-year-old at 24 weeks, TSH was elevated to 45.33 IU/mL at week 24 and her TG increased from 19 U at baseline to 264 U at week 24. Subsequent TSH normalized on replacement therapy and she continued thyroid medication after study completion. The 13-year-old girl developed elevated TSH 30 IU/mL and low free T4 0.6 ng/dL at 36 weeks of study treatment; thyroid-specific autoantibodies were not detected. She remained on thyroid hormone therapy with normal thyroid function tests after achieving SVR. Four children had elevated TPO and none of these children had abnormal T4 or TSH. Of the 7 with positive TG, only the one described above developed thyroid disease.
Symptoms, ALT, and Autoantibodies
ALT elevation was examined in those children with ANA, LKM, or F-actin. At baseline, week 24, and week 72, children with and without these antibodies had similar ALT values. Of those with autoimmune hepatitis–related antibodies, the highest ALT was 126 U/L (Table 4). In contrast, the highest ALT values, those exceeding 5 times the upper limit of normal (ULN), all occurred among those children without antibodies to ANA, LKM, or F-actin. The most common adverse effects of treatment were flu-like symptoms (67%), gastrointestinal symptoms (33%), and headaches (24%). Comparing 24-week data for flu-like symptoms, gastrointestinal symptoms, and headaches, side effects were not more frequent in those with or without antibodies.
This is the first study to prospectively and longitudinally assess the presence and development of autoantibodies in children with CHC. Despite use of conventional screening techniques that excluded 9 children who had antibodies to ANA, LKM, and SMA, we found that almost 20% of the enrolled subjects had baseline antibodies using an ELISA technique. We found thyroid-, diabetes-, and hepatitis-related antibodies to be common in these children. The frequency of autoantibodies, however, did not increase significantly with treatment in this cohort, nor did it change significantly 6 months after completion of treatment. The presence of autoantibodies did not affect response to therapy. Furthermore, despite screening, 3 children (3%), none of whom had organ-specific autoantibodies at baseline, developed de novo clinical autoimmune disease, 2 with hypothyroidism and 1 with type 1 diabetes mellitus.
Earlier studies have presented conflicting results about the importance of antibodies before and during treatment with IFN-based therapies. The present study has methodologic advantages. All samples were analyzed simultaneously on well-standardized Food and Drug Adminstration–approved and CLIA-certified (except for insulin) immunoassays carried out by ELISA. This technology allows large numbers of samples to be simultaneously and objectively measured in WHO units; the assays are quantitative and represent a continuous function, as opposed to immunofluorescence assays, which are subjective, and discontinuous measures not amenable to statistical analysis in an epidemiologic study. Because these tests are performed under the proficiency program of the College of American Pathologists, the results can be compared nationally from one clinical laboratory to another.
Autoantibodies have been reported in both adults and children with CHC. Autoantibodies are seen in 70% of adults with CHC; 41% are ANA-positive, 9% are SMA-positive (12). Gregorio et al (13) screened for autoantibodies in 51 Italian children with CHC and found 65% to be positive (90% of treated patients, 65% of untreated, P = 0.12); SMA was the most common antibody (67%), followed by ANA (10%) and LKM (8%). Bortolotti reported positive SMA in 17.5%, LKM in 10%, and ANA in 7.5% of 40 children with CHC (14,15). Jara et al (16) found ANA in 13%, SMA in 3%, and LKM in 10% of 30 children, and 7 children developed ANA during treatment. In our prospectively studied North American cohort, the frequency is less and at least in part explained by our exclusion of children with high-titer autoimmune hepatitis–related antibodies. In the present study, the frequency of F-actin antibody (the cytoskeletal target of SMA) was <2%, LKM <5%, and ANA at most 13%. Other potential contributors to the difference in antibody frequency between this study population and that reported by Bortolotti include differences in the populations studied, differences in the laboratory techniques for measuring autoantibodies, as well as our use of a stringent standard of strong positivity as defined by the manufacturer for each assay, rather than designating moderate or weak results as positive. These methodologic and population differences may account for variability in frequencies reported among these different studies.
It is notable that no child in the present study, with or without antibodies, developed evidence of autoimmune hepatitis. Although we excluded 9 children with AIH-related antibodies, we still identified children who had moderate and strong positive antibody by ELISA in the archived samples. No child had an ALT flare during treatment that limited therapy, similar to the experience of Jara et al group, who did not exclude children with autoantibodies from treatment as long as they did not have features of autoimmune hepatitis (16). In contrast, Bertolotti et al reported in a study of only 40 subjects that 5 of 7 LKM-positive children had a hepatitis flare during treatment (14). Gregorio et al (13) reported the need to discontinue treatment in several autoantibody-positive children who developed worsening hepatitis while receiving interferon. New LKM antibodies during treatment and association with a hepatitis flare have been reported in 1 patient (17).
Like most adult studies, we did not detect a relation between autoantibody appearance and treatment outcome (8,18–20). In our study, there was no significant difference in 72-week treatment outcomes (only those who were virus-negative at 24 weeks were analyzed) between the autoantibody-positive and -negative groups, with SVR of 70% and 65%, respectively.
Of particular note is that neither our screening antibodies nor our archived samples assayed for thyroid and islet cell antibodies identified the subjects who developed the most serious complications. As discussed earlier, 1 child treated in the present study developed type 1 diabetes mellitus at 32 weeks of treatment. She was negative for GAD and IA-2 antibodies at the start of treatment and at 24 weeks. GAD antibody was positive in the study week 72 sample tested. Diabetes mellitus has been reported to occur rarely in patients with CHC who are treated with IFN (2); among 11,000 adults with viral hepatitis treated with interferon in a retrospective European report, 10 (0.08%) developed diabetes. Several adult patients with CHC with positive diabetes antibodies who did not develop diabetes with IFN treatment have been reported (21,22). Wirth et al (23) reported 1 girl who developed diabetes mellitus while being treated with PEG-IFN and RV for hepatitis C. In the present study, up to 9.3% of subjects had positive diabetes-related antibodies at some point, with the notable development of clinical diabetes in only 1 child, who was antibody negative at baseline and 24 weeks. For comparison, diabetes antibody positivity in the general pediatric population ranges from 0.5% to 2.9% % (24). Autoantibodies were thus not helpful in identifying the child who would develop diabetes in this study, although the literature suggests that these autoantibodies are strong predictors of subsequent development of diabetes in nonhepatitis C patients (25).
The development of thyroid disease is common in adults with CHC, both with and without treatment. Most studies report a rate of ≤10% (26,27) Autoantibody positivity before treatment has been associated with an increased risk (27) of clinical autoimmune disease and new autoantibodies develop during treatment in 5% to 45% (28). In the pediatric literature, about 20% of children treated with IFN and RV developed thyroid autoantibodies, and about 10% developed transient or permanent hypothyroidism requiring treatment (9,16,23,29). In the present report, the prevalence of thyroid autoantibodies was lower than in most series at around 3%, with the frequency of anti-TG trending up from 0% at baseline to 6.5% at week 72. It thus appears that thyroid autoantibodies as well as clinical thyroid disease occur uncommonly in children treated with IFN, and at-risk children cannot be identified prospectively.
We found that autoantibodies are commonly detected in children with CHC infection and de novo autoimmune disease still occurs despite screening at baseline. Even sensitive antibody measures did not predict subjects at risk. Three children developed clinical disease, 2 with hypothyroidism and 1 with type 1 diabetes mellitus. In all 3 cases, screening antibodies as well as baseline and on-treatment antibodies did not identify risk until the disease developed. These data affirm that a variety of autoantibodies are common in children with CHC before, during, and after treatment with PEG-IFN–based therapy. Our screening at baseline may have accounted for our lower frequency of complications compared with historic reports. Even with screening and subsequent autoantibody assays, we were not able to identify children who would develop autoimmune disease. We provide fresh insight into the limited clinical role for following these antibodies to predict complications when treating children with CHC.
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