Title: Clinical and histopathological features of immunoglobulin G4-associated autoimmune hepatitis in children Short title: Immunoglobulin G4-associated autoimmune hepatitis in children Yusuf Aydemir¹, MD, Assistant Professor Zuhal Akcoren 2, MD, Associate Professor Hulya Demir 3, MD, Professor Inci Nur Saltik Temizel 3, MD, Professor Hasan Ozen 3, MD, Professor Aysel Yuce 3, MD, Professor ¹Department of Pediatric Gastroenterology and Hepatology, Eskisehir Osmangazi University School of Medicine, Eskisehir, Turkey. 2 Department of Pediatric Pathology, Hacettepe University School of Medicine, Ankara, Turkey. 3 Department of Pediatric Gastroenterology and Hepatology, Hacettepe University School of Medicine, Ankara, Turkey. Corresponding Author: Yusuf Aydemir, MD Address for Correspondence: Pediatric Gastroenterology and Hepatology, Eskisehir Osmangazi University School of Medicine, 26480 Eskisehir, TURKEY. Phone Number: +90 (532) 3841477 Fax: +90 (222) 2290064 E-mail: dryusufaydemir@yahoo.com Disclosure statement: The authors declare no conflicts of interest. Authors Contributions: Y.A. and A.Y. conceived the research, Y.A., H.D. and I.N.S.T conducted the analysis, Z.A. evaluated the histopathological preparations and H.O. and A.Y. interpreted the findings from a clinical perspective. All authors participated in interpreting the results, drafting the This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/jgh.14525
manuscript, and revising it for intellectual content. All authors have read and approved the submitted version. ABSTRACT Background and Aim We aimed to examine the frequency and the characteristics of IgG4-associated autoimmune hepatitis among pediatric patients with autoimmune hepatitis. Methods Immunostaining for IgG and IgG4 were performed in liver biopsies of 40 pediatric patients with autoimmune hepatitis. The patients with more than 10 IgG4-positive plasma cells/hpf were defined as IgG4-associated autoimmune hepatitis. Clinic, laboratory and histopathological results were compared between groups. Results Among the 40 pediatric patients, 34 patients were type 1 and six patients were type 2 autoimmune hepatitis. Six patients (15%), four of the type 1 and 2 of the type 2 autoimmune hepatitis patients, were diagnosed as IgG4-associated autoimmune hepatitis. Clinical, laboratory and histopathological data were initially similar in both forms. There was a positive correlation between IgG4-positive plasma cell count and degree of portal (r:0.406, p:0.009) and lobular inflammation (r:0.37, p:0.019), grade of interface hepatitis (r:0.33, p:0.03) and fibrosis (r:0.318, p:0.046). Time required for normalization of liver transaminases and serum IgG level were significantly shorter in IgG4- associated autoimmune hepatitis [3.3±0.5 vs 6.6±3.5 for ALT, 3.7±0.8 vs 6.7±1.2 for AST, 4.3±1.2 vs 7.1±2.7 for GGT, 7.2±3.1 vs 12.8±4.5 for IgG]. Conclusions IgG4-associated autoimmune hepatitis can be found in pediatric age group and also in type 2 autoimmune hepatitis patients. As steroid response may be better in IgG4-associated autoimmune hepatitis, biopsy specimens should be evaluated for this entity at diagnosis. Key words: Autoimmune hepatitis, IgG4-associated AIH, IgG4-related diseases
INTRODUCTION Autoimmune hepatitis (AIH) is an immune-mediated disorder characterized by hyperimmunoglobulinemia, elevated transaminases, presence of autoantibodies in serum and interface hepatitis on biopsy, in the absence of a known etiology [1]. Diagnostic criteria of AIH were established at the meeting of the International Autoimmune Hepatitis Group (IAIHG) [2]. There are two types of AIH; type 1 characterized by the presence of smooth muscle and/or antinuclear antibodies and type 2 which is mainly a pediatric condition, characterized by presence of liver kidney microsome type 1 and/or anti-liver cytosol type 1 antibodies [3]. The annual incidence of AIH is 0.8 1.9 cases per 100,000 people in Europe and less frequent with an estimated incidence between 0.08 and 0.15 cases per 100,000 people in Asia. The incidence of childhood AIH is unknown. It is three times more frequent in females than males and AIH-1 accounts for two third of cases. Autoimmune hepatitis presents acutely and has a more aggressive course in pediatric patients. Positive family history of autoimmune diseases is found in 40% of cases. Accompanying autoimmune diseases including thyroiditis, insulin-dependent diabetes, vitiligo, hypoparathyroidism, inflammatory bowel disease, nephrotic syndrome, and Addison disease are present in about 20% of the patients [4,5]. Immunoglobulin G4-related diseases (IgG4-RD) are systemic fibro-inflammatory diseases characterized by tissue infiltration with IgG4-positive plasma cells, development of fibrosis, and often elevated serum levels of IgG4 [6]. The IgG4-RD predominantly affects males and the average age at onset of symptoms is 50 60 years [7]. There is only one article that reviews the literature for IgG4- RD in pediatric age group. In children, IgG4-RD can affect mostly the orbit, the salivary gland, the pancreas and the lymph nodes [8]. The diagnosis of IgG4-RD can be established by histopathological examination. Clinical symptoms, serological tests and radiological findings support the diagnosis [9]. The IgG4-RD can cause irreversible organ damage and timely treatment is important [10]. Glucocorticoids are the first choice of the treatment. Azathioprine, mycophenolate mofetil and methotrexate are other treatment options. Some cases may need surgical intervention [11]. Recently, isolated liver involvement with IgG4-positive plasma cell infiltrate has been reported in adult patients with AIH and named as IgG4-associated AIH. We aimed to examine the frequency and the characteristics of IgG4-associated AIH among pediatric patients with AIH.
METHODS Subjects and Settings This retrospective study was conducted in Hacettepe University School of Medicine Pediatric Gastroenterology and Hepatology Department between January 2011 and December 2014. The ethics committee of Hacettepe University approved the study. Liver biopsies of pediatric AIH patients obtained at diagnosis were examined. Patients were scored by the IAIHG scoring system before the treatment and all patients satisfied the criteria for a definite diagnosis of AIH. Patients with positive serology for smooth muscle and/or antinuclear antibodies taken as type 1 while liver kidney microsome type 1 and/or anti-liver cytosol type 1 taken as type 2 AIH. All the patients were treated with prednisolone at an initial dose of 2 mg/kg/day (maximum 60 mg/day) for one month, then azathioprine therapy 2 mg/kg/day (maximum 100 mg/day) was started and steroid treatment was tapered and discontinued in three months. All the patients underwent magnetic resonance cholangiopancreatography. The patients with autoimmune sclerosing cholangitis or other accompanying autoimmune diseases and those who lost to follow-up were excluded. Demographic features, laboratory investigations [complete blood count (CBC), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamaglutamile transpeptidase (GGT), direct and indirect bilirubin, total protein and albumin, IgG and international normalized ratio (INR)], medications and time required for normalization of liver enzymes under treatment were recorded. Patients were divided into two groups according to the presence of IgG4-positive plasma cells in the liver biopsy. The patients with more than 10 IgG4-positive plasma cells/hpf according to the criteria defined by Umemura et al [12] were defined as IgG4-associated AIH while remaining AIH patients were defined as classical AIH. Clinic, laboratory and histopathological results were compared between groups. Histopathology and immunohistochemistry Liver tissues embedded in paraffin taken before treatment were used. An experienced pediatric pathologist blind to clinical and serological characteristics of the patients evaluated the samples. Fibrosis was graded as; 0 (No fibrosis), 1 (Fibrous expansion of some portal areas, with or without short fibrous septa), 2 (Fibrous expansion of most portal areas, with or without short fibrous septa), 3 (Fibrous expansion of most portal areas with occasional portal to portal bridging), 4 (Fibrous expansion of portal areas with marked bridging), 5 (Marked bridging with occasional nodules) and 6 (Cirrhosis, probable or definite). Portal inflammation was graded as; 0 (No inflammation), 1 (Mild, some or all portal areas), 2 (Moderate, some or all portal areas), 3 (Moderate/marked, all portal areas)
and 4 (Marked, all portal areas). Interface hepatitis was graded as; 0 (None), 1 (Mild, focal, few portal areas), 2 (Mild/moderate focal, most portal areas), 3 (Moderate continuous around < 50% of tracts or septa) and 4 (Severe continuous around > 50% of tracts or septa). Confluent necrosis was graded as; 0 (None), 1 (Focal confluent necrosis) 2 (Zone 3 necrosis in some areas), 3 (Zone 3 necrosis in most areas), 4 (Zone 3 necrosis + occasional portal central bridging), 5 (Zone 3 necrosis + multiple portal central bridging) and 6 (Panacinar or multiacinar necrosis). Spotty necrosis was graded as; 0 (None), 1 (One focus or less per 10x objective), 2 (Two to four foci per 10x objective), 3 (Five to ten foci per 10x objective) and 4 (More than 10 foci per 10x objective) [13]. The following were defined depending on the count per high-power field (HPF): plasma cell infiltration (Negative: <9 cells/hpf, positive:>9 cells/hpf); steatosis (positive: >30% cells with fatty change/hpf); and eosinophil infiltration (positive:>4 cells/hpf) [13]. Canalicular cholestasis, ductular proliferation, rosette and giant cell formation were classed as positive or negative. Immunostaining for IgG (Abcam, Cambridge, UK) and IgG4 (Abcam, Cambridge, UK) were performed using an autostainer (Leica Bond Max) according to the manufacturer s guidelines. Immunostaining for IgG and IgG4 were performed in liver biopsies of 40 pediatric patients with AIH. Tonsil specimens were also stained with IgG and IgG4 as a positive control. Statistical Analyses Statistical analyses were performed using the SPSS software version 16.0 (SPSS Inc., Chicago, IL, USA). Data were expressed as numbers (n), frequencies (%), means with standard deviations, and median with minimum and maximum values. Categorical variables between groups were analyzed using the χ2 test. Continuous variables were compared by Mann-Whitney U tests. Correlation analyses were performed with the Spearman test. Differences were considered significant at p<0.05. RESULTS During the study period, 60 patients were diagnosed with AIH. Twenty patients (10 lost to follow-up, 6 had autoimmune sclerosing cholangitis, 4 had other auto-immune diseases) were excluded from the study. Among the remaining 40 pediatric patients (24 female and 16 male, age between 24-192 months, mean 116.4 40.6 months) with AIH included in our study, 34 patients (85%) were type 1 AIH and six patients (15%) were type 2 AIH. Six patients (15%), four of the type 1 and 2 of the type 2 AIH patients were diagnosed with IgG4-associated AIH. The IAIHG score was similar between groups. There was no statistically significant difference between groups for levels of ALT, AST, GGT, ALP, total protein, albumin, total and direct bilirubin, INR and IgG. The clinical and laboratory characteristics of the study groups were summarized in Table 1. IgG levels were decreased in both
IgG4-associated and classical AIH patients at time of normalization of liver transaminase (ALT) [1356 mg/dl (1102-1636) vs 1694 mg/dl (1327-1872), p>0.05]. Portal and periportal fibrosis were present in all patients, and nine (22.5%) of them showed bridging fibrosis (Figure 1e). Two patients with classical AIH showed incomplete cirrhosis (fibrosis stage 5). Portal inflammation and interface hepatitis were evident in all patients (Figure 1a). Lobular confluent necrosis was found in 27 (67.5%) patients. Spotty necrosis was found in 37 (92.5%) patients. Moderate to marked plasma cell infiltration (Figure 1b) and eosinophil infiltration (Figure 1d) in portal tracts were present in 18 (45%) and 13 (32.5%) patients, respectively. Giant cell change and rosette formation (Figure 1c) were detected in 12 (30%) and 21 (52.5%) patients, respectively. Bile duct damage was found in 19 (47.5%) patients, while chronic nonsuppurative destructive cholangitis or bile duct loss was not detected. Grade of portal and lobular inflammation, interface hepatitis, histological activity index, fibrosis and degree of plasma cell infiltration were similar between type 1 and 2 AIH groups. Immunohistochemistry showed IgG staining and IgG4-bearing plasma cells in the portal area (Figure 1f). IgG4/IgG bearing plasma cell ratio was 0.65 in our study. In our cohort, six patients had more than 10/HPF IgG4-positive cells. Grade of portal and lobular inflammation, plasma cell infiltration, interface hepatitis, histological activity index and fibrosis were higher in the IgG4-positive AIH group, but differences were not statistically significant. However, there was a positive correlation between IgG4-positive plasma cell count and degree of portal (r:0.406, p:0.009) and lobular inflammation (r:0.37, p:0.019), grade of interface hepatitis (r:0.33, p:0.03) and fibrosis (r:0.318, p:0.046). There was also a positive correlation between IgG-positive plasma cell count and degree of portal (r:0.659, p:0.001) and lobular inflammation (r:0.472, p:0.002), grade of interface hepatitis (r:0.475, p:0.002) and fibrosis (r:0.363, p:0.021). Bile duct damage was seen in 50% of IgG4- associated AIH patients but chronic nonsuppurative destructive cholangitis was not detected. Rosette formation and giant cells were seen together in three of the patients two of whom were IgG4- associated type 2 AIH. Histopathological features of the both groups were presented in Table 2. Time required for normalization of liver transaminases under steroid therapy was significantly shorter in IgG4-associated AIH (Table 1). None of the patients had relapse on azathioprine maintenance therapy after discontinuation of steroids. Nine patients had follow-up liver biopsy before treatment cessation. Two of them were IgG4-associated AIH, histopathological examination of their liver biopsies showed minimal inflammatory activity and loss of IgG4 bearing plasma cell.
DISCUSSION After the Umemura et al had described a case with IgG4-associated AIH in 2007, three studies with adult population were reported up to date [12,14,15]. We report the first study of IgG4-associated AIH with pediatric patients. Chung et al [14] reported that 35% of their patients with AIH had IgG4- associated AIH. Umemura et al [12] assessed IgG4-associated AIH in a study conducted in 60 patients diagnosed with type 1 AIH and found two patients (3%) with IgG4-associated AIH. Canivet et al [15] evaluated 28 adult patients with AIH, seven (25%) had immunoglobulin G4-associated autoimmune hepatitis. We found six (15%) patients with IgG4-associated AIH among 40 pediatric AIH patients. Differences of frequencies between studies can be due to different criteria used to define IgG4-associated AIH and different ethnicity. We preferred Umemura s histological criteria to define IgG4-associated AIH because 10 IgG4 bearing plasma cells/hpf is often used to diagnosis for all other types of IgG4-RD. Chung et al [14] detected that degree of plasma cell infiltration, lobular and portal inflammation were significantly higher in the IgG4-positive group, while Canivet et al [15] found similar inflammatory activity and stage of fibrosis between groups. Although the grade of portal and lobular inflammation, interface hepatitis, histological activity index, fibrosis and degree of plasma cell infiltration were not significantly different between our groups, there was a moderate correlation between portal, lobular inflammation and IgG4-positive plasma cell count, and weak correlation between grade of interface hepatitis, fibrosis and IgG4-positive plasma cell count. While most adult patients with AIH-1 have a chronic disease course with nonspecific symptoms such as fatigue, nausea, abdominal pain, and arthralgia, in children and adolescents, AIH has a more aggressive phenotype and takes diagnosis earlier. Depending on the short disease course, cirrhosis is present in only about 10% of children with AIH at diagnosis [16]. In our study, the mean duration between the beginning of the symptoms and the diagnosis was 4.1 (1-12) weeks and 2 (5%) patients with classical AIH had cirrhosis. None of the patients with IgG4-associated AIH had cirrhosis. Chung et al [14] found that their patients with IgG4-associated AIH showed a marked response to steroid therapy, whereas Canivet et al [15] did not find difference in treatment response in their patients. They concluded that the difference in treatment response may be related to different ethnicity of patients and treatment modalities between two studies. In our cohort, patients with IgG4-associated AIH improved in a shorter period of time compared to classical AIH patients after initiation of steroid treatment. Steroid treatment seems to be effective in IgG4-associated AIH as it is in other IgG4- realated diseases. None of the patients had relapse on azathioprine maintenance therapy after discontinuation of steroids.
All three studies evaluated the type 1 AIH. For the first time, we evaluated the type 2 AIH which is mainly pediatric condition and report IgG4-associated AIH may be found in patient with type 2 AIH. Grade of portal and lobular inflammation, interface hepatitis, histological activity index, fibrosis and degree of plasma cell infiltration were similar between patients with type 1 and 2 AIH. Interestingly, rosette formation and giant cells were seen together in both of the type 2 AIH patients with IgG4- associated AIH. Systemic inflammatory diseases characterized by the infiltration of IgG4-positive plasma cells and elevated serum IgG4 levels were classified as IgG4-related disease. Portal inflammation and lobular hepatitis characterized by infiltration of IgG4-positive plasma cells is often seen in the livers of these patients. It is still unknown whether the IgG4-associated AIH is a subtype of AIH or a particular liver involvement of IgG4-RD [17]. As owing to the retrospective collection of the data, we could not assess the serum IgG4 levels. Elevated serum IgG4 levels may help the diagnosis of IgG4-RD. On the other hand, it has been reported normal in up to 40% of patients with IgG4-RD [17]. Furthermore, Chung et al [14] found normal IgG4 levels in their patients with IgG4-associated AIH and they concluded that tissue staining of IgG4 is more sensitive than serum IgG4 assays for the diagnosis of IgG4-associated AIH. The number of patients with IgG4-associated AIH detected in our study was small as were in other three studies, so a multicenter study is needed for better identification of characteristics of this new entity. In conclusion, we report that IgG4-associated AIH may be found in pediatric patients. Furthermore, for the first time we showed presence of IgG4-associated type 2 AIH which is mainly a pediatric condition. As response to steroid treatment can be better in IgG4-associated AIH, biopsy specimens should be evaluated for this entity at diagnosis.
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1a: Severe interface hepatitis and pan lobular hepatitis in hematoxylin-eosin stained liver biopsy 1b: Plenty of plasma cells 1c: Ballooning of hepatocytes, giant cells and rosette formation 1d: Eosinophils in the portal inflammation 1e: Bridging fibrosis and inflammation in Masson Trichrome stained liver biopsy 1f: IgG4 bearing cells in IgG4-immunostained liver biopsy
Table 1 The clinical and laboratory characteristics of the study groups IgG4-associated AIH (n:6) Classical AIH (n:34) P value Age (months) 120,1±63.1 115.8±36.6 >0.05 Gender (Female/Male) 3/3 21/13 NA AST (IU/L) 454.5(120-8357) 541.4(96-3448) >0.05 ALT (IU/L) 556.2(105-2369) 709(88-2157) >0.05 GGT (IU/L) 98.5(31-386) 78.2(13-310) >0.05 Albumin (gr/dl) 3.8(3.1-3.8) 4.1(2-5) >0.05 Total bilirubin (mg/dl) 2.35(0.6-16.2) 2.6(0.2-21.8) >0.05 INR 1.31±0.2 1.35±0.3 >0.05 IgG (mg/dl) 2636(1924-3822) 2872(2126-4102) >0.05 IAIHG score 17.8±1.6 17±1.8 >0.05 ALT normalization time (weeks) AST normalization time (weeks) GGT normalization time (weeks) IgG normalization time (weeks) 3.3±0.5 6.6±3.5 <0.05 3.7±0.8 6.7±1.2 <0.05 4.3±1.2 7.1±2.7 <0.05 7.2±3.1 12.8±4.5 <0.05 Abbreviations: AST; Aspartate aminotransferase, ALT; Alanine aminotransferase, GGT; Gama-glutamile transpeptidase, IAIHG; International Autoimmune Hepatitis Group, INR; International normalized ratio, NA; Not applicable, values are presented as means±sds or medians with min-max
Table 2 Histopathological features of the study groups IgG4-associated AIH (n:6) Classical AIH (n:34) P value Fibrosis stage 2.5 (2-4) 3 (1-5) >0.05 Portal inflammation grade 2 (1-3) 2 (1-3) >0.05 Interface hepatitis grade 2.5 (1-4) 2 (1-4) >0.05 Confluent necrosis grade 1.5 (1-6) 1 (0-6) >0.05 Spotty necrosis grade 1 (0-4) 1 (0-3) >0.05 Rosette formation (n) 5 16 >0.05 Giant cell formation (n) 3 9 >0.05 Eosinophilic infiltration (n) 3 10 >0.05 Plasma cell infiltration (n) 4 14 >0.05 Abbreviation: AIH; Autoimmune hepatitis, values are presented as medians (minimum-maximum) and numbers
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