Section 2 -

Pathology of Autoimmune Overlap Syndromes Stefan G Hübscher Department of Pathology University of Birmingham Birmingham B15 2TT, U.K.

Introduction – Basic Concepts and Diseases Involved During the past 20 years there has been increasing interest in the concept of "overlap syndromes" involving three liver diseases that are thought to have an immune-mediated basis – autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC). Although all three diseases are often referred to as being autoimmune, only AIH fulfils the criteria for a classical autoimmune disease. This article will focus on histological aspects of overlap syndromes involving AIH, PBC and PSC and the role of liver biopsy in the diagnosis and management of this rather poorly-defined group of conditions. The term overlap syndrome is applied to describe cases that are difficult to classify within a single diagnostic category of AIH, PBC or PSC and instead appear to have biochemical, immunological or histological features of more than one disease process [1, 2, 3] . Three main types have been recognised: (i) The "true" overlap syndrome is the commonest form and mostly involves simultaneous presentation with combined features of AIH and PBC or AIH and PSC. Overlap syndromes involving combinations of PBC and PSC have also been described, but are much less common [4, 5, 6] . There have been infrequent reports of cases that appear to have features of all 3 diseases [6] . (ii) The "cross-over" or "outlier" syndrome refers to cases where there are some features suggesting one disease process and others favouring an alternative diagnosis. The best example is so-called "autoimmune cholangitis" (AIC), which is characterised by biochemical and histological features favouring PBC and an autoantibody profile more in keeping with AIH. Most people now regard this as a variant of PBC (AMA-negative PBC), although the precise nature of AIC remains controversial (see later). (iii) The "sequential" syndrome is an uncommon variant of overlap syndrome in which patients present initially with classical features of one autoimmune liver disease and subsequently develop features of another autoimmune liver disease, sometimes after a lag interval of many years. The commonest examples involve AIH and PSC, with AIH usually presenting first [7, 8, 9, 10] . Less frequently there is progression from PSC to AIH [10, 11, 12] . A few cases of PBC and AIH presenting with temporal dissociation have also been reported [13, 14, 15, 16] . The significance of overlap syndromes in terms of classifying autoimmune liver diseases and understanding the relationship between them is uncertain. A number of possible explanations have been put forward [2, 3] . The most likely is that AIH, PBC and PSC are three distinct disease processes, which happen to share overlapping biochemical, serological or histological features. Alternatively they may represent co-existence of different autoimmune diseases, possibly related to shared genetic susceptibility. Least likely is the suggestion that autoimmune liver disease forms a continuous disease spectrum, within which AIH, PBC and PSC are different manifestations. Although the "outlier syndrome" autoimmune cholangitis is generally regarded to be a variant of PBC, various other possibilities have been suggested. These include a variant of AIH with biliary features [17, 18] , a small duct variant of PSC [19] or a distinctive syndrome that has features different from all three of the other diseases [20, 21] . The recent use of more sensitive and specific tests to measure serum anti-mitochondrial antibodies (AMA) has shown that these may be present in some cases previously classified as AIC [22] . Fluctuating levels of AMA have also been recognised to occur during different phases in the evolution of PBC, in some cases becoming undetectable [23, 24] , which also suggests that AIC and PBC may be different manifestations of a common disease process. It has been suggested that the term "autoimmune cholangitis" could be applied to all cases with clinical, biochemical and histological features compatible with a diagnosis of PBC , irrespective of their AMA status [25, 26] but this has not been widely applied. Overlap syndromes involving AIH with hepatitis C, alcoholic liver disease, non-alcoholic fatty liver disease and other chronic liver diseases have also been described [1, 3, 27] . These will not be considered in further detail here. However, it is worth noting that autoantibodies are commonly present in low titre in many chronic liver diseases, where they are considered to be a non-specific response to hepatocellular injury [28, 29]. Incidence and Diagnostic Criteria The reported incidence of autoimmune overlap syndromes varies widely, most likely due to the use of different diagnostic criteria. Overall, approximately 20% of people with autoimmune liver disease may be considered to have features compatible with an overlap syndrome [30, 31, 32]. Although the diagnostic features of the three main disease involved are well described (see Table 1), the criteria required to diagnose overlap syndromes have not been clearly defined or standardized. Table 1. Diagnostic Features of AIH, PBC and PSC Autoimmune Hepatitis Primary Biliary Cirrhosis Primary Sclerosing Cholangitis Autoantibodies ANA, SMA (type 1) LKM (type2) SLA/LP (type 3) AMA Anti-M2 pANCA Biochemistry Hepatitic (raised AST/ALT) Cholestatic (raised Alk Phos) Cholestatic (raised Alk Phos) Immunoglobulins Raised IgG Raised IgM Normal levels or polyclonal elevation Radiology (ERCP) Normal Normal Abnormal (beading, stictures) Histology Bile duct lesions Bile duct loss Chronic cholestasis Hepatitic Mild No No Biliary Inflammatory Yes Yes Biliary Fibrosing Yes Yes Studies of adult patients with an initial diagnosis of AIH [33] , PBC [34] or PSC [10, 35] have suggested that approximately 5-10% of cases have additional features supporting a diagnosis of an overlap syndrome. A higher prevalence of overlap syndromes involving AIH and PSC has been documented in children, amongst whom 50% with an initial diagnosis of AIH were found to have cholangiographic findings typical of PSC [36] . The term "autoimmune sclerosing cholangitis" (ASC) has been proposed in this context. Histological Features Although the classical histological features of AIH, PBC and PSC are distinctive, areas of histological overlap exist, even amongst cases which are otherwise typical in terms of their clinical, biochemical and serological features [37] . Thus inflammatory bile duct lesions and other biliary features can be seen in otherwise typical cases of AIH, whilst portal inflammation and interface hepatitis can be considered as part of the normal histological spectrum of PBC and PSC. Autoimmune Hepatitis AIH is characterised by a predominantly mononuclear plasma cell rich infiltrate, mainly involving portal and periportal regions. Interface hepatitis is an important feature in establishing a diagnosis of AIH [28] and is also likely to be important in determining disease progression - it is often associated with ballooning, rosetting and fibrous entrapment of periportal hepatocytes and thus forms a potential pathway for the development of progressive periportal fibrosis. Parenchymal inflammatory changes are also seen in AIH and typically mainly involve centrilobular regions. More severe cases may be involved with bridging necrosis or panacinar necrosis, which are adverse prognostic features associated with disease progression and poor response to immunosuppressive therapy [38] . Inflammatory bile duct lesions are also recognised to occur in otherwise typical cases of AIH [39, 40, 41, 42] . The changes seen are usually mild, with focal lymphocytic infiltration of bile ducts that are otherwise well preserved. Less commonly there are more severe changes with biliary epithelial cell damage or disruption, in some instances associated with bile duct loss. Cases of AIH with destructive cholangitis or ductopenia tend to have higher alkaline phosphatase levels than those without biliary changes, but are otherwise similar in terms of disease behaviour and response to immunosuppression [39, 41] . Features favouring an alternative or additional diagnosis of autoimmune biliary disease include extensive bile duct loss and features of chronic cholestasis (marginal ductular reaction, cholate stasis and accumulation of copper-associated protein). However, ductular reaction can also be seen in cases of AIH with an acute presentation and/or prominent lobular inflammatory component and as part of the spectrum of progressive disease with the development of bridging fibrosis or cirrhosis. Primary biliary cirrhosis and primary sclerosing cholangitis The initial targets for damage in these diseases are bile ducts. PBC is characterised by inflammatory lesions involving small (interlobular) ducts, whereas PSC is associated with lesions affecting bile ducts of all sizes. Typical bile duct lesions are only seen in approximately 30% of liver biopsies from patients with PBC and 10% of those with PSC, so that a definite diagnosis is rarely possible on histological grounds [43] . Most cases of PBC and PSC are now diagnosed on the basis of serological and radiological findings respectively and liver biopsy is not used routinely in many centres. However, histological assessment may still carried out in cases with unusual features or an uncertain diagnosis, including those with a suspected overlap syndrome. The bile duct lesions in PBC comprise a histological spectrum including lymphocytic and granulomatous cholangitis. Although lymphocytic cholangitis can also be seen in AIH, the presence of florid duct lesions with duct disruption should still suggest a diagnosis of PBC. Likewise granulomatous duct lesions are not recognised to be a feature of AIH. The classical fibrosing bile duct lesions in PSC tend mainly to affect medium-sized ducts, which explains why these are rarely seen in needle biopsy specimens. Other biliary features occur in PBC and PSC as a consequence of bile duct loss. They include marginal ductular reaction, features of cholate stasis in periportal hepatocytes (feathery degeneration, ballooning and Mallory's hyaline formation) and the accumulation of copper and copper-associated protein. These changes are associated with the development of progressive periportal fibrosis and enable a diagnosis of chronic biliary disease to be made, even if typical bile duct lesions are not present. Inflammatory changes are also recognised to occur as part of the histological spectrum of autoimmune biliary disease. They are generally more prominent in PBC than PSC. In common with the changes seen in AIH, inflammation mainly involves portal and periportal regions. PBC is characterised by a mixed population of cells including lymphocytes, plasma cells, neutrophils and eosinophils. Interface hepatitis is also a common finding and appears to be important in the development of fibrosis in PBC [44] . Inflammatory changes also occur in the liver parenchyma and may include granulomatous foci. They are typically mild, but again appear to be involved in the development of fibrosis [44] . The presence of unusually prominent interface hepatitis, with hepatocyte ballooning and rosetting, should raise the possible of an additional or alternative diagnosis of AIH. Confluent and bridging necrosis are also distinctly uncommon in PBC or PSC and again suggest another diagnosis. Autoimmune cholangitis Autoimmune cholangitis is associated with histological features closely resembling those occurring in PBC, including foci of granulomatous cholangitis and bile duct loss [45, 46, 47] . Subtle differences in the composition of portal inflammatory infiltrates have been described, including larger numbers of T lymphocytes [48] and plasma cells [49] in AIC compared with AMA-positive PBC. However, other aspects of inflammatory activity in cases of AIC are similar to those seen in PBC and do not suggest an additional diagnosis of AIH [20, 21, 45, 46] . Problems with the Diagnostic Criteria for Autoimmune Overlap Syndromes Given the intrinsic overlap that appears to exist between AIH, PBC and PSC, it is not surprising that problems have arisen with trying to establish criteria for the diagnosis of autoimmune overlap syndromes. A particular problem relates to the lack of a single diagnostic criterion for AIH. Instead the diagnosis of AIH is based on a constellation of clinical, biochemical, serological and histological findings. The International Autoimmune Hepatitis Group has devised a scoring system in which points are attributed to a range of individual diagnostic features – these are then combined to produce an aggregate score, which can be then interpreted as "definite" or "probable" AIH [28] . In addition to positive scores for typical features of AIH, negative scores are given for features considered to be atypical - these include a cholestatic biochemical profile, the presence of anti-mitochondrial antibodies and biliary features on liver biopsy, all of which are features one would expect to see in an overlap syndrome. It is not clear how the AIH scoring system should be applied or modified in cases where these other features are present [35, 50, 51, 52, 53, 54] . Another approach to the problem is to view it from the perspective of a patient with an initial diagnosis of PBC or PSC and then try to establish the minimum diagnostic criteria (biochemical, serological, histological or combinations of all three) required to diagnose an overlap syndrome. Again no clear agreement exists. Suggested criteria to be applied to patients with an initial diagnosis of PBC were 2 of 3 of the following : (1) ALT > 5x upper limit of normal (ULN), (2) Serum IgG > 2x ULN or smooth muscle antibody positivity (3) liver biopsy showing moderate or severe interface hepatitis [34, 55] . Although such an approach offers hope for a more standardised approach to future studies, there are still problems in applying the pathological criteria which are not clearly defined. Prognostic and Therapeutic Implications Given the lack of uniformity in the criteria used to diagnose overlap syndromes, it is not surprising that their prognostic significance and therapeutic implications are also poorly understood. There are no reports of controlled therapeutic trials in this setting [3] . A detailed discussion of therapeutic aspects is beyond the scope of this article, but a few general points will be considered. The inflammatory changes occurring in classical AIH respond to treatment with corticosteroids and other immunosuppressive agents [38, 56] . By contrast, ursodeoxycholic acid (UDCA) is increasingly emerging as the first line medical treatment for the cholestatic problems that occur in PBC and PSC [3, 57] . A similar approach has been advocated for the treatment of autoimmune cholangitis [2, 3] . For many overlap syndromes occurring in adults (PBC/AIH and PSC/AIH), the biliary disease is considered to be the main diagnosis with hepatitic features occurring as a secondary phenomenon. UDCA can still used as the first line of treatment in such cases [2, 58, 59] . However, other studies have suggested that some patients with an overlap syndrome involving PBC and AIH may benefit from combined therapy with UDCA and immunosuppression [34, 55, 60] . Similar observations have been made for cases with an AIH/PSC overlap syndrome [8, 10, 61] By contrast, the overlap syndrome involving AIH and PSC in children ("autoimmune sclerosing cholangitis") is considered to lie within the spectrum of AIH and immunosuppression is used as the first line of treatment, although UDCA may also be used as an adjunct [36] . Summary and Conclusions Although there are still problems with the diagnosis and classification of autoimmune overlap syndromes, there is increasing evidence to suggest that cases with overlapping features may behave differently from classical cases of "pure" AIH, PBC and PSC. Histological assessments are important in the investigation of patients with atypical presentations of autoimmune liver disease, and form an important component in the diagnosis of overlap syndromes. Mild hepatitic features are commonly present in PBC and PSC and biliary features can be seen in otherwise typical AIH. Diagnostic criteria for "overlap syndrome" are therefore difficult to define. Nevertheless, cases of PBC or PSC with unusually prominent histological inflammatory activity may benefit from immunosuppression, whereas in a person with an initial diagnosis of AIH, the presence of bile duct lesions or other biliary features may point to an underlying biliary disease. 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