Characteristics and Long-term Outcome of 535 Patients with Histologically Conrmed Autoimmune Hepatitis (AIH) - A Single Center Experience of 18 Years

Introduction: Autoimmune hepatitis (AIH) is a rare liver disease with a favorable prognosis following immunosuppression. In this work-up, we present our long-term experience with this patient collective. Patients and Methods: Overall, we were able to retrospectively evaluate data from 535 patients with histologically conrmed AIH over a period of 18 years between 2002 and 2020. Results: As expected, almost ¾ of the patients were women (74.5 %) of middle age (47.0 ± 14.9 years). Type I AIH was diagnosed in almost all patients (97.5 %). However, 88 patients (16.4 %) revealed an overlap to primary biliary cholangitis (PBC). In contrast, overlap to primary sclerosing cholangitis (PSC) was detected in only 22 patients (3.7 %). Regarding auto-antibody prole, positivity for anti-nuclear antibodies (ANA) was found in 388 patients (72.5 %); smooth-muscle actin (SMA)-titer was positive in 90 patients (16.8 %), anti-mitochondrial antibodies (AMA) were detected in 43 individuals (8.0 %), and we found positive p-anti-neutrophil cytoplasmic antibodies (p-ANCA) in 12 patients (2.2 %). More than ¾ of the patients (n = 417 ≈ 77.9 %) were initially treated with corticosteroids (1 mg/kg/day) and this therapy was continued in almost all subjects (97.4 %) in low dose (5 - 7.5 mg/day) as maintenance therapy. Steroid-saving and remission-maintaining therapy with azathioprine was carried out in 380 patients (71.0 %). Mycophenolate mofetil (MMF) or calcineurin-inhibitors (CNI) were used as second- or third-line immunosuppression. Acute liver failure (ALF) - as the rst manifestation of AIH - was diagnosed in 101 patients (18.8 %). Hepatocellular carcinoma (HCC) was detected in at least 6 patients (1.1 %). Liver transplantation (LT) was performed in 51 patients (9.5 %) who progressed to cirrhosis despite immunosuppression. Unfortunately, a total of 45 patients (8.4 %) died of cirrhosis-related complications (infected ascites, bleeding, encephalopathy) without chance for adequate organ offer. Conclusion: We here present our long-term experience with a signicant number of patients diagnosed with AIH over a long observation period of 18 years. In general, patients with AIH can adequately be managed with good clinical outcome at a liver center requiring immunosuppressive therapy. However, HCC-screening, acute-on-chronic (AOC) liver decompensation, or liver transplantation have to be taken into consideration carefully.


Introduction
Autoimmune hepatitis (AIH) is the result of disturbed immunologic homeostasis characterized by immune-mediated destruction of hepatic parenchyma, female gender bias, presence of auto-antibodies, hypergammaglobulinemia, association with other autoimmune conditions, and excellent response to corticosteroid or immunosuppressive treatment [1]. Genetic and environmental factors seem to play a crucial role. Despite being a rare liver disease with an estimated prevalence of 2-17 cases per 100'000 in Europe, we observe a notably increase in disease incidence within the last years [2]. In some cases, patients present an overlap to other hepato-biliary diseases such as primary biliary cholangitis (PBC) or primary sclerosing cholangitis (PSC).
Despite being a chronic in ammatory liver disease, approximately 20 % of the patients reveal an acute presentation which may be induced by a triggering agent such as previous viral infections or treatment with immune-modifying drugs (DILI-AIH) [3]. Infectious triggers are commonly indicated as being involved in the induction of autoimmune diseases, with Epstein-Barr (EBV) or Cytomegalovirus (CMV) being implicated in several autoimmune liver disorders, such as type I AIH or PBC [4,5]. A high proportion of patients with acute manifestation can even develop acute liver failure (ALF) as previously reported by our group, particularly in cases of delayed diagnosis or treatment [6,7].
In the majority of patients clinical and biochemical remission can be induced following oral corticosteroid therapy with 1 mg/kg/day and a tapering-down scheme to 5-7.5 mg steroids per day within 3 months. Usually, steroid-sparing immunosuppression is performed with the use of azathioprine. In cases of azathioprine intolerance, drug toxicity or low-/non-response, other immunosuppressive agents, such as mycophenolate mofetil (MMF) or calcineurin-inhibitors (CNI) like cyclosporine A (CsA) or tacrolimus (FK 506) are used [8].
In addition to on-set and regular monitoring of their drug therapy, patients must also be monitored for liver-related complications. Fortunately, patients with AIH, among all other chronic liver diseases, are those who develop the least number of liver cancers (HCC) or who require liver transplantation (LT) due to progression to liver cirrhosis or fulminant course of the disease [9].
The aim of our current retrospective single center study was to investigate demographic characteristics, laboratory pro les, response to immunosuppressive therapy, clinical course, and outcome of especially these patients in order to share our experience as an excellence center for liver diseases with a total number of 535 patients with histologically-proven AIH over a long time period of 18 years.

Patient information, data collection, and ethical considerations
In this retrospective study between 07/2002 and 06/2020, a total of 535 consecutive patients with histologically con rmed AIH were analyzed. AIH was diagnosed according to the "Simpli ed Diagnostic Scoring System of the International Autoimmune Hepatitis Group" including the analysis of autoantibodies, immunoglobulins, histological ndings, and viral markers [10,11]. Liver histology was available for all of our patients and was obtained either by percutaneous-or laparoscopy-guided biopsy ( Figs. 1 and 2). The University Clinic of Essen ethics committee approved the retrospective, anonymous analysis of the data, and the study was conducted according to the principles expressed in the Declaration of Helsinki. All patients gave their written informed consent prior to study inclusion.

Immunosuppressive therapy
After diagnosis of AIH, most of the patients received corticosteroid therapy (1 mg/kg body weight) with a consecutive down-tapering to 5-7.5 mg steroids daily as maintenance therapy. Further immunosuppressive therapy was conducted in dependence to steroid response and patient's clinical pro le using predominantly azathioprine (1- However, a small number of patients (n = 6; 1.1 %) developed hepatocellular carcinoma (HCC). Despite immunosuppressive therapy, a remarkable proportion of patients with AIH progressed to end-stage liver cirrhosis with the necessity of liver transplantation (LT). Finally, this procedure was performed in 51 patients (9.5 %) out of our cohort. Favorable course was documented in these transplanted patients during their out-clinic visits. Patients' demographics and further laboratory data are summarized in Table 1.
An overview of the crucial blood values at initial presentation is depicted in Table 2. Notably, patients with acute liver failure (ALF) as their rst presentation of autoimmune hepatitis showed extremely high liver enzymes, partly in combination with severe jaundice and deterioration of the coagulation.

Overlap to autoimmune diseases
In the next step, we examined which extra-hepatic autoimmune diseases were present in our collective. It should be emphasized that a total of 64 patients (11.9 %) suffered from autoimmune thyroiditis type Hashimoto -followed by patients with in ammatory bowel disease (IBD). There was a total of 26 patients (1.6 %) with autoimmune hemolytic anemia (AIHA) (Fig. 1).

Viral hepatitis serology
Of course, virological status of our patients is also of clinical importance. Patients with active replicative viral disease were excluded from the study.

Liver histology
Liver biopsy (either percutaneous-or laparoscopy-guided) was performed in all of our patients (Fig. 2) and exemplarily demonstrated severe in ammation with typical interface-hepatitis. Furthermore, many plasma cells extending from the portal tract into the adjacent liver parenchyma were also detected in numerous AIH samples (Fig. 3).

Immunosuppressive therapy and survival analysis
Most of the patients (n = 417) were initially treated with corticosteroids and steroid therapy was continued to maintain remission in 97.4 % (n = 406 patients). Immunosuppression with azathioprine was preferentially used as rst-line in 380 patients (71.0 %). However, due to drug intolerance or toxic hepatitis, therapy with azathioprine had to be stopped in 75 patients (14.0 %). Patients were switched to second or third-line immunosuppressive therapy with cyclosporine A (CsA), tacrolimus (FK 506) or mycophenolate mofetil (MMF), respectively. Remission of underlying AIH within the rst year of diagnosis could be achieved in 394 patients (73.6 %). Moreover, 20-year cumulative survival was signi cantly improved in patients with AIH maintaining remission (91.6 vs. 56.0 %; p < 0.0001) (Fig. 4). In addition, liver-related complications and 20-year mortality was increased in patients without biochemical remission as compared to AIH patients with normal liver enzymes and remission (97.8 vs. 73.5 %; p < 0.0001) (Fig. 5).
Liver transplantation was indicated and performed in 51 patients (9.5 %) who presented acute liver failure or progressed to cirrhosis despite immunosuppression. Regrettably, a total of 45 patients (8.4 %) died of cirrhosis-related complications (infected ascites, bleeding, encephalopathy) without chance for adequate organ offer.

Discussion
This is a long-term observational study over a time period of 18 years in a German cohort with a signi cant number of patients (n = 535) diagnosed with AIH [13][14][15]. Moreover, liver histology as a hallmark of the "International AIH-Score" was gained in each included patient.
In daily clinical practice, AIH commonly presents as a chronic in ammatory liver disease. The majority of patients are diagnosed due to repeatedly elevated transaminases in check-up examinations without having any complaints. From the immunological perspective, disruption of the immune tolerance to autologous liver antigens may be a trigger for the induction of this rare autoimmune liver disease [16-18].
As expected, patients of our cohort were middle-aged and predominantly of female gender. In addition, type I AIH with ANA-positivity, was almost thoroughly observed in our study population. Overlap to other autoimmune liver diseases like PBC and PSC or to further extra-hepatic disorders were frequently observed [19]. Compared to other common liver diseases in Germany as viral or (non-) alcoholic steatohepatitis (NASH), patients diagnosed with autoimmune liver disease have fortunately a benign course under initial corticosteroid therapy followed by a su cient immunosuppressive therapy using in general azathioprine with credible liver-related complications. As expected and demonstrated by us, clinical and biochemical remission in patients diagnosed with AIH is associated with a signi cantly improved overall-survival and reduced acute-on-chronic liver-related complications.
Compared to e.g. HCV-or alcohol-induced cirrhosis (ASH), development of HCC is not very common in AIH patients. Therefore, we did not focus on the different possibilities to treat HCC like the use of molecular-targeted therapies or selective internal radiation therapy (SIRT). Nevertheless, there is still a small group of AIH patients revealing low or even non-response to different immunosuppressive agents like mycophenolate mofetil, calcineurin inhibitors (tacrolimus/cyclosporine A), rituximab or plasmapheresis [20]. In these patients, liver in ammation with elevated transaminases progresses to brosis and in long-term course to end-stage liver cirrhosis with its sequela mentioned previously. In conclusion, liver transplantation is the only therapeutical option in these refractory patients -as we performed it in 51 patients.
In future, we should work on more potent immunosuppressive regimes and the goal should be to avoid the necessity of liver transplantation in patients with AIH because of its benign character. Risk factors for non-recovery among our patients in a sub-group analysis were increased age at AIH diagnosis, missing remission of liver enzymes following immunosuppression after 1 year, and a high MELD-score > 26 points [21].
Limitations of our study are of course the retrospective character and the case-by-case observational study at a single liver center. Nevertheless, we would like to emphasize that we were able to include a remarkable number of patients offering liver histology and a long-term follow-up with important data for the hepatologist. In summary, we do observe an increase in AIH incidence world-wide. Despite its benign character, some of the AIH patients need a close follow-up -on the one hand -to detect cirrhosis or HCC in early stages and -on the other hand -to escalate immunosuppressive therapy to induce and keep remission in disease activity.

Declarations
Ethics approval and consent to participate: The University Clinic of Essen ethics committee approved the retrospective, anonymous analysis of the data, and the study was conducted according to the principles expressed in the Declaration of Helsinki. All patients gave their written informed consent prior to study inclusion.

Consent for publication:
Not applicable.
Availability of data and materials: The datasets (raw data) used and analyzed during the current study are available from the corresponding author on reasonable request. Figure 1 Overlap to extra-hepatic autoimmune disorders are shown.

Figure 2
Exemplarily, mini laparoscopy of a patient with AIH demonstrating the right liver lobe with diffuse swelling, capsular brosis, and regenerative nodules. Left panel (magni cation x100) showing severe in ammation with interface-hepatitis. Right panel (magni cation x400) revealing many plasma cells extending from the portal tract into the adjacent liver parenchyma.

Figure 4
Cumulative survival is signi cantly improved in patients with AIH maintaining remission (black line).