The Pathology of IgG4-Related Disease in the Bile Duct and Pancreas

Transcription

1 242 The Pathology of IgG4-Related Disease in the Bile Duct and Pancreas Yoh Zen, MD, PhD, FRCPath 1 1 Department of Diagnostic Pathology, Kobe University Graduate School of Medicine, Kobe, Japan Semin Liver Dis 2016;36: Address for correspondence Yoh Zen, MD, PhD, FRCPath, Department of Diagnostic Pathology, Kobe University Graduate School of Medicine, Kusunoki-Cho, Kobe , Japan ( yohzen@med.kobe-u.ac.jp). Abstract Keywords immunoglobulin G4 sclerosing cholangitis autoimmune pancreatitis Immunoglobulin G4-related disease (IgG4-RD) is widely accepted as a systemic condition that affects various organs. 1,2 Although presenting symptoms and clinical features vary among patients, histopathological findings are relatively uniform across the affected anatomical sites. 1,2 Therefore, most organ manifestations of IgG4-RD have been identified histologically. 3 7 Tissue examinations also play a central role in the diagnostic process in clinical practice. Although elevations in serum IgG4 concentrations and imaging features suggest this condition, tissue sampling is required to establish a conclusive diagnosis in most cases. An exception to this is the pancreatic manifestation, for which the combination of elevated serum IgG4 concentrations and imaging features leads to a definite diagnosis because of highly specific imaging abnormalities Although the diagnosis of IgG4-RD was previously achieved based on surgically resected specimens, there is increasing demand to diagnose this condition using biopsy samples. Here the pathological features of the pancreatobiliary manifestations of IgG4-RD are summarized. The clinical Immunoglobulin G4-related disease (IgG4-RD) in the pancreatobiliary system manifests as sclerosing cholangitis (SC), hepatic inflammatory pseudotumors, and type 1 autoimmune pancreatitis (AIP). The pathology of IgG4-RD involves an inflammatory process and fibrogenic pathway, the combination of which damages the affected organs. Fibroinflammatory injury is characterized by three microscopic findings: a diffuse lymphoplasmacytic infiltrate rich in IgG4-positive plasma cells, obliterative phlebitis, and storiform fibrosis. Although the diagnosis of IgG4-related pancreatocholangitis is relatively straightforward in surgical specimens, the current clinical requirement is to diagnose patients using biopsy samples, which remains challenging. Histological differential diagnoses include primary SC, follicular cholangitis/pancreatitis, SC with granulocytic epithelial lesions, and type 2 AIP. Although the massive infiltration of IgG4-positive plasma cells is a histological hallmark of IgG4-RD, many other immune cells (e.g., Th2 lymphocytes, regulatory T cells, and M2 macrophages) appear to be strongly involved in orchestral immune reactions. features and molecular aspects are also described briefly. Immunoglobulin G4-related chronic active hepatitis, another manifestation of IgG4-RD at this anatomical site, is beyond the scope of this review; it is described elsewhere in this issue. General Pathological Features of IgG4-RD The pathology of IgG4-RD consists of an inflammatory process and fibrogenic pathway, the combination of which damages the affected organs ( Fig. 1A, B). The severity of inflammation and fibrosis vary among lesions, but are relatively uniform in different areas of the same lesion. The fibroinflammatory process is characterized by three microscopic findings including diffuse lymphoplasmacytic infiltration, fibrosis arranged in a storiform pattern, and obliterative phlebitis. 11 Because lymphoplasmacytic infiltration is less specific and observed in virtually all chronic inflammatory conditions, storiform fibrosis and obliterative phlebitis have greater diagnostic value. Inflammatory infiltrates are Issue Theme IgG4-Associated Hepatobiliary and Pancreatic Disorders; Guest Editors, Kazuichi Okazaki, MD, PhD,andM.EricGershwin,MD,PhD Copyright 2016 by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA. Tel: +1(212) DOI /s ISSN

2 Pathology of IgG4-Related Disease in the Bile Duct and Pancreas Zen 243 Fig. 1 Histological features of immunoglobulin G4-related disease (IgG4-RD) in salivary and lacrimal glands. (A) The salivary gland is involved in the fibroinflammatory process with acinar atrophy. (B)Theinflammatory infiltrate mainly consists of lymphocytes and plasma cells. (C)Thereare many IgG-positive (þ) plasma cells. (D) IgG4þ plasma cells account for > 40% of IgGþ plasma cells. diffusely present with occasional lymphoid follicles, which are more commonly observed in sialo-dacryoadenitis than in pancreatobiliary manifestations. 12 Lymphocytes and plasma cells are polyclonal in nature, and are accompanied by lymphoid cells showing variable maturation (e.g., immunoblasts and plasmablasts). Tissue eosinophilia is another common indicator of this condition. 11 Macrophages are difficult to identify on hematoxylin and eosin- (H&E-) stained sections; however, immunostaining reveals the extensive infiltration of macrophages (particularly M2-type) in both inflamed and fibrotic areas. 13 Xanthogranulomatous inflammation with foamy macrophages is not a feature of IgG4-RD. Neutrophils are also rare, even in the acute phase. Storiform fibrosis is characterized by collagen fibers arranged in an irregularly whorled pattern, somewhat resembling the spokes of a cartwheel or the net of a straw mat. 11 A caveat is that the definition of storiform fibrosis in IgG4-RD has not yet been standardized. Obliterative phlebitis is a more objective finding characterized by the partial or complete obliteration of small veins involved in the process of sclerosing inflammation. Venous obliteration may occur in other inflammatory conditions, but is typically devoid of an inflammatory infiltrate. A small inflammatory nodule adjacent to an artery is a histological sign for identifying obliterated veins, which become more obvious on elastic staining. In contrast, histological findings that are unlikely to occur in this condition include extensive neutrophilic infiltration, abscess formation, necrosis, discrete granuloma, and necrotizing vasculitis. 11 If at least one of these findings is detected, another diagnosis should be suggested. Immunostaining for IgG and IgG4 contributes to the characterization of infiltrating plasma cells ( Fig. 1C, D). Immunoglobulin G4-positive (þ) plasmacellinfiltration is a well-known histological hallmark of IgG4-RD. Many IgG4þ plasma cells are present in tissues irrespective of whether serum IgG4 concentrations are elevated. However, IgG4þ plasma cell infiltration appears to be less specificfor this condition than previously considered because it has been reported in other inflammatory conditions and malignant neoplasms. 14 Immunostaining for IgG4 needs to be carefully interpreted to avoid an overdiagnosis of IgG4-RD. Immunoglobulin G4þ plasma cells are diffusely present in IgG4-RD, but are usually patchy in other conditions. The absolute number of IgG4þ plasma cells needs to be assessed based on the proposed site-specific cutoff value (e.g., 100 cells/high power field [HPF] for the salivary gland). 11 The ratios of IgG4/IgGþ plasma cells also need to be calculated based on immunostained sections. Immunoglobulin G immunostaining often leads to strong background staining, which restricts the counting of positive

3 244 Pathology of IgG4-Related Disease in the Bile Duct and Pancreas Zen cells. In that case, the total number of plasma cells counted on H&E-stained sections is used as an alternative. In IgG4-RD, the ratio of IgG4/IgGþ plasma cells exceeds 40% ( Fig. 1C, D). This is the minimum criterion because the ratio is typically > 70% in IgG4-RD. 11,12 The disease concept of IgG4-RD has widely been accepted as a new systemic disease worldwide; as a consequence, the histological finding of IgG4þ plasma cell infiltration has been overemphasized. It is important to note that IgG4-RD cannot be diagnosed based on IgG4þ cell infiltration alone. Immunoglobulin G4 immunostaining requires careful interpretation in conjunction with other morphological findings. A close clinicopathological correlation is also necessary prior to establishing a diagnosis of IgG4-RD. Overview of IgG4-RD in the Pancreatobiliary System Fig. 2 depicts the spectrum of IgG4-related pancreatobiliary manifestations. 3 The pancreatic lesion is called type 1 autoimmune pancreatitis (type 1 AIP), 15 IgG4-related pancreatitis, 16 or lymphoplasmacytic sclerosing pancreatitis. 17 Biliary manifestations include sclerosing cholangitis (SC), inflammatory pseudotumors, and cholecystitis. 18 Cholangiopathy occurs in close association with pancreatitis. The intrapancreatic bile duct is affected in most patients with type 1 AIP. Immunoglobulin G4-related pancreatitis and cholangitis develop simultaneously in many patients. Cholangiopathy may become dominant at the time of relapseinpatientswithtype1aip.althoughpancreatitis may precede an episode of cholangitis in some patients, it is extremely uncommon for hepatobiliary diseases to occur before type 1 AIP. 19 We recently examined 235 patients with IgG4-RD, which is the largest cohort available in the literature. 20 We identified all patients with IgG4-RD who were diagnosed at eight general hospitals belonging to the same medical district in the past 9 years. This consecutive cohort has provided an overview of this multifaceted systemic condition. There were 486 manifestations identified in 235 patients at the time of diagnosis. Pancreatitis appeared to be the most common manifestation in this systemic condition; it was observed in 60% of patients. The second most common manifestation was sialadenitis (34%), followed by tubulointerstitial nephritis (23%), dacryoadenitis (23%), and periaortitis (20%). 20 Of the 235 patients examined, 95% had at least one of the top five manifestations. 20 Hepatobiliary manifestations (excluding intrapancreatic bile duct involvement) were ranked sixth (13%). Although pancreatobiliary manifestations were not described in detail in the original study, among the 173 patients with pancreatobiliary abnormalities, 98% had pancreatitis with or without cholangiopathy, whereas only 2% presented with isolated SC. In addition, all patients with isolated SC had IgG4- RD in other organs outside the pancreatobiliary system, suggesting that true isolated cholangiopathy is uncommon in IgG4-RD. Another large study conducted by the Mayo Clinic analyzed 53 patients with IgG4-related sclerosing cholangitis (IgG4-SC). 21 They found that 92% of patients with IgG4-related cholangiopathy had concomitant type 1 AIP, whereas 8% presented with isolated cholangitis. The proportion of isolated biliary abnormalities was larger than that in our cohort, but may have been due to selection bias. Our cohort was based on general hospitals, whereas the Mayo Clinic is a tertiary referral center. Another histology-proven cohort (n ¼ 52) also suggested that isolated SC accounts for 8% of IgG4-related pancreatobiliary abnormalities. 18 Fig. 2 Spectrum of immunoglobulin G4-related pancreatocholangitis. The majority of patients have pancreatitis with or without cholangitis, while only 2% of patients present with isolated biliary disease.

4 Pathology of IgG4-Related Disease in the Bile Duct and Pancreas Zen 245 This may have been because many patients with AIP were not included; their diagnoses were established without tissue examinations. 8 Another aspect is that patients with isolated SC more frequently underwent surgical resection for suspected cholangiocarcinoma because the lack of concomitant pancreatitismadeadiagnosismoredifficult. 22 These two factors may have contributed to the higher incidence of isolated biliary abnormalities in the pathology cohort. Pathological Features of IgG4-Related Sclerosing Cholangitis Large Duct Cholangiopathy Extrahepatic, perihilar, and intrahepatic large ducts are typically involved in IgG4-RD. The gross appearance of the affected ducts resembles a pipestem with diffuse and circumferential wall thickening ( Fig. 3A). 8 In contrast to cholangiocarcinoma, the mucosal surface is relatively smooth. 3 These morphological changes are detectable using imaging modalities. Some patients with IgG4-SC occasionally have separate strictures on imaging; however, IgG4-related pathology is generally continuously present not only in stenotic areas, but also in the dilated part of the biliary tree between these areas. Immunoglobulin G4-related sclerosing cholangitis is characterized histologically by a transmural fibroinflammatory process ( Fig. 3B). 3 The severities of inflammation and fibrosis at mucosal and subserosal sides are relatively similar. Inflammation consists of massive lymphoplasmacytic infiltration and occasional eosinophils. Neutrophilic infiltration is exceptional unless there is mechanical mucosal injury by biliary stents. Cellular infiltrates are intermingled with fibrosis. A storiform pattern is observed, at least focally. Obliterative phlebitis is identified in most cases ( Fig. 3C). Perineural inflammatory extension is noted, particularly in the outer layer of the bile duct wall. The epithelial lining of the bile duct lumen is well preserved. Although peribiliary glands are commonly involved in the sclerosing process with periglandular concentric fibrosis, the glandular epithelium is also typically intact ( Fig. 3D). Epithelial damage is generally inconspicuous, which may explain why IgG4-RD does not increase the risk of epithelial malignancy. Dysplastic and metaplastic changes (e.g., intestinal metaplasia) are also uncommon. On immunostaining, there are many IgG4þ plasma cells, which are diffusely distributed. Cutoff values for IgG4þ plasma cells at this anatomical site are 50 cells/hpf for surgical specimens and > 10 cells/hpf for biopsy samples. 11 Fig. 3 Immunoglobulin G4-related sclerosing cholangitis (large duct disease). (A) The appearance of the affected bile duct resembles a pipestem with extensive wall thickening. (B)Theinflammatory process is evenly present from the mucosa to the subserosal layer. (C) Although obliteration is not obvious, a small vein is infiltrated by lymphocytes, plasma cells, and eosinophils. (D) Peribiliary glands are involved in the fibroinflammatory process, but the glandular epithelium is preserved.

5 246 Pathology of IgG4-Related Disease in the Bile Duct and Pancreas Zen Fig. 4 Immunoglobulin G4-related sclerosing cholangitis (small duct disease). (A) A whitish peculiar lesion is present along the peripheral biliary tree. (B) Histologyconfirms a dense inflammatory process in the small portal tract. As described above, the ratios of IgG4/IgGþ cell counts increase to more than 40%. Immunostaining also contributes to the better characterization of inflammatory cells. CD20þ B cells are more likely to be aggregated, while CD3þ Tcellsare more diffusely observed. Both CD4þ and CD8þ lymphocytes are present, whereas cells expressing cytotoxic markers such as granzyme B are scarce. CD168 immunostaining assists in identifying many of the M2 macrophages infiltrating the duct wall. Small Duct Cholangiopathy This category includes IgG4-RD affecting septal and interlobular bile ducts. In IgG4-RD, small duct disease is always associated with large duct cholangiopathy, suggesting a direct inflammatory extension from large bile ducts to intrahepatic small portal tracts. Unlike primary sclerosing cholangitis (PSC; small-duct PSC), IgG4-related pathology restricted to small bile ducts has not yet been documented. Immunoglobulin G4-related small duct cholangiopathy may be identified as a whitish periductal lesion in resected specimens ( Fig. 4A); however, the conformation of small duct involvement requires tissue examination. 18,23 Portal tracts are enlarged with marked inflammatory infiltrates containing many plasma cells ( Fig. 4B). The inflammatory components around small bile ducts are basically similar to those in large bile ducts. Because neutrophils may also be present around reactive bile ductules, the presence of neutrophils does not exclude the possibility of IgG4-RD at this particular anatomical site. Similar to large ducts, the epithelial lining is well preserved in small bile ducts. Small duct cholangiopathy rarely creates microscopic nodules consisting of inflammatory cell infiltration and storiform fibrosis in or around small portal tracts. This finding, originally described as a portal inflammatory nodule, is not commonly observed in liver biopsy specimens, but is highly specific for this condition. 24 Obliterative phlebitis is also rare in interlobular or septal portal tracts. Fibro-obliterative changes such as periductal concentric fibrosis and ductopenia are not a feature in IgG4-RD. 25 On immunostaining, IgG4þ plasma cells (> 10 cells/hpf) are present in the affected portal tracts. The IgG4/IgGþ cell ratios increase to more than 40%. Similar to other chronic cholangiopathies, orcein staining reveals copper-associated protein deposits in periportal hepatocytes in approximately 30% of cases, a histological finding representative of chronic cholate stasis. 23 Controversy remains as to whether and how fast IgG4-related cholangitis progresses to liver cirrhosis. A previous study reported that 4 out of 53 patients developed portal hypertension and cirrhosis within 5 years of the onset of initial symptoms. 21 In our cohort, only one patient with nontreated chronic cholangitis exhibited early cirrhotic transformation, whereas fibrosis was less conspicuous in the remaining cases (bridging fibrosis in the worst cases). 18 Pathological Features of IgG4-Related Inflammatory Pseudotumors in the Liver Immunoglobulin G4-related sclerosing cholangitis occasionally manifests as periductal mass lesions typically involving perihilar ducts, which have been called inflammatory pseudotumors. 3,22 Immunoglobulin G4-related inflammatory pseudotumors are best regarded as a pseudotumorous exaggeration of SC. Before the concept of IgG4-RD was established, several case reports had described inflammatory pseudotumors associated with PSC; these may have been IgG4-related inflammatory pseudotumors. 26 Because radiological features mimic hilar cholangiocarcinoma ( Fig. 5A), patients often undergo unnecessary surgical resections. Another common clinical scenario is that IgG4-related inflammatory pseudotumors develop in the hepatic hilum at the time of relapse in patients with type 1 AIP. Grossly, the lesion is characterized by a whitish solid mass, within which penetrating bile ducts are identified ( Fig. 5B). The inflammatory process is generally restricted to hilar connective tissue with no direct involvement in the adjacent liver parenchyma ( Fig. 5C). Therefore, an imaging finding of intraparenchymal infiltration may suggest that hilar cholangiocarcinomas are more likely. In rare examples, large portal veins are partly obliterated due to obliterative phlebitis. The histological features of IgG4-related inflammatory

6 Pathology of IgG4-Related Disease in the Bile Duct and Pancreas Zen 247 Fig. 5 An Immunoglobulin G4-related hepatic inflammatory pseudotumor. (A) Imaging shows a periductal mass lesion at the hepatic hilum (arrow), the appearance of which is reminiscent of hilar cholangiocarcinoma. (B) Grossly, the lesion is a solid whitish mass affecting the perihilar large bile ducts. (C) Theinflammatory process is confined to the portal connective tissue. (D) Storiform fibrosis is noted. pseudotumors are similar to those of IgG4-SC ( Fig. 5D). Bile ducts and peribiliary glands are closely involved in the inflammatory process, but the epithelial lining is well preserved. Pathological Features of IgG4-Related Pancreatitis (Type 1 AIP) Type 1 AIP is a prototypic manifestation of IgG4-RD. This unique form of pancreatitis was previously called lymphoplasmacytic sclerosing pancreatitis based on the original description by Kawaguchi et al. 17 A pancreas affected by IgG4-RD typically shows diffuse enlargement with the surface nodular architecture being less clear. Rare examples may form single or multiple small nodules of up to 3 cm at the greatest diameter. In such cases, a differential diagnosis from pancreatic cancer becomes more challenging. The nodular lesions are grossly well circumscribed and surrounded by an unremarkable pancreatic parenchyma. 2 Histologically, sclerosis is more pronounced between lobules, while inflammatory infiltration is more obvious inside the acini. Due to interlobular fibrosis, the lobular architecture of the pancreatic parenchyma remains as type 1 AIP. The histological triad (lymphoplasmacytic infiltration, storiform fibrosis, and obliterative phlebitis) is easy to identify in most cases ( Fig. 6). 19 The fibroinflammatory process extends into peripancreatic adipose tissue, a histological finding corresponding to an imaging feature called a capsule-like rim. 9,10,19 Storiform fibrosis is commonly observed in the peripancreatic area. Although periductal inflammation is pronounced, the epithelial lining is well preserved. In contrast, the inflammatory process leads to acinar extinction, which is one reason why the pancreas becomes atrophic after steroid therapy. Islets are preserved or slightly enlarged in size, similar to the other forms of chronic pancreatitis. Some cases associated with extensive eosinophilic infiltration may have been mislabeled as eosinophilic pancreatitis. The IgG4-related inflammatory process may extend to the ampulla of Vater along the pancreatic duct system. The affected ampulla becomes enlarged, and may be reminiscent of ampullary neoplasms. Features on immunostaining are similar to those of IgG4-SC. The required numbers of IgG4þ plasma cells are also the same (> 50 cells/hpf for surgical specimens, > 10 cells/hpf for biopsy samples). 11 Biopsy Diagnosis If large specimens (e.g., Whipple s specimens) are submitted for pathological examinations, the diagnosis of IgG4-related

7 248 Pathology of IgG4-Related Disease in the Bile Duct and Pancreas Zen Fig. 6 Immunoglobulin G4-related autoimmune pancreatitis. (A) Aseverefibroinflammatory process is associated with extensive parenchymal extinction, while ducts are affected less. (B) Collagen fibers are arranged in an irregularly whorled pattern, consistent with storiform fibrosis. (C) The small aggregate of inflammatory cells adjacent to the artery represents obliterative phlebitis. (D) The vascular structure becomes obvious on Elastica van Gieson staining. pancreatocholangitis may be established without difficulty. However, the current clinical requirement is to make diagnoses using biopsy samples, which remains challenging. One reason is that because obliterative phlebitis and storiform fibrosis are not commonly detected in small biopsy samples, a histological interpretation needs to be more dependent on IgG4 immunostaining. In addition, in taking a biopsy sample from patients with suspected IgG4-RD, we may be able to obtain additional diagnostic evidence for IgG4-RD; we also may be able to totally exclude IgG4-RD. It is important to note that difficulties are associated with the exclusion of inflammatory conditions using biopsy. One example is sarcoidosis, in which a diagnosis cannot be excluded, even if there is no evidence of granuloma in transbronchial biopsies. However, this may be possible for IgG4-RD because there are wellknown histological findings that counter the diagnosis of this condition. For example, the presence of necrosis, abscess, or discrete granuloma in biopsy specimens will argue against the possibility of IgG4-RD. Therefore, not only positive, but also negative histological findings for this condition need to be assessed in biopsy samples. Three commonly considered biopsy procedures for patients with suspected IgG4-related pancreatocholangitis are liver needle biopsy, bile duct biopsy, and pancreatic biopsy, which are discussed below. Liver Needle Biopsy This approach is commonly indicated for patients with the imaging features of SC Pathologists need to determine whether cholangiopathy is PSC or IgG4-SC. Portal inflammation, a bile ductular reaction, and copper-associated protein deposition in periportal hepatocytes may be observed in both conditions; therefore, these findings do not contribute to a differential diagnosis. 18 Histological findings that are highly specific for IgG4-SC are IgG4þ plasma cell infiltration, storiform fibrosis, and obliterative phlebitis; however, the latter two are rarely detected in biopsy samples ( Fig. 7). In contrast, fibro-obliterative changes such as periductal concentric fibrosis and bile duct loss suggest PSC over IgG4-RD. If the ductopenic process is confirmed in a liver biopsy, IgG4-SC will be excluded. In our experience, approximately 40% of patients show some histological findings suggestive of either PSC or IgG4-SC, whereas liver biopsy findings are not conclusive in the remaining cases. Bile Duct Biopsy Bile duct biopsy is useful for confirming or excluding the possibility of cholangiocarcinomas. 27 In contrast, its diagnostic value is limited for IgG4-RD. A histological examination with IgG4 immunostaining may only lead to a conclusive

8 Pathology of IgG4-Related Disease in the Bile Duct and Pancreas Zen 249 Fig. 7 Liver biopsy findings of immunoglobulin G4- (IgG4-) related sclerosing cholangitis. (A) Asmallportaltractisdenselyinfiltrated by lymphocytes, plasma cells, and occasional eosinophils. (B) IgG4 immunostaining reveals many IgG4-positive plasma cells. diagnosis of IgG4-SC when a large piece of tissue is obtained. Obliterative phlebitis and storiform fibrosis are not observed in superficial biopsies. 27 Pancreatic Biopsy This may be the most commonly performed biopsy procedure for patients with suspected IgG4-related pancreatocholangitis. 28 Although pancreatic tissue was previously obtained by percutaneous biopsy, endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) has been increasingly used for this purpose. Even if obliterative phlebitis is not suspected on H&E-stained sections, elastic staining is highly recommended because it often unexpectedly discloses completely obliterated veins embedded in the fibrotic stroma ( Fig. 8). The presence of > 10 IgG4þ plasma cells also suggests type 1 AIP. However, strong background staining in immunostained sections for IgG4 or IgG is a common issue in biopsy samples, and often restricts histological interpretations. In our experience, pancreatic biopsies are useful for IgG4-RD and pancreatic cancer. More than 70% of patients with type 1 AIP show histological features suggestive of IgG4-RD, while malignancy is confirmed in > 80% of patients with pancreatic cancer. Follow-up biopsies are not necessary in patients with IgG4-RD, but may provide useful information. Patients with IgG4-RD respond rapidly to corticosteroids with enlarged organs shrinking in the first few weeks posttreatment. However, imaging does not provide information regarding whether residual inflammation is present in tissue. Biopsies from patients with treated IgG4-RD exhibit variable histological changes. Some patients still have some inflammatory activities, even at clinical remission, whereas others show dense fibrosis with weak inflammation ( Fig. 9). On-treatment biopsies may assist in determining whether the complete withdrawal of corticosteroids is possible on a case-by-case basis, and this needs to be tested in a large, prospective cohort. Histological Differential Diagnosis From a clinical aspect, the leading differential diagnosis of IgG4-related pancreatocholangitis is malignancy. Many earlier cases of type 1 AIP or IgG4-SC underwent surgery for suspected cancer. From a pathological point of view, other forms of fibroinflammatory conditions are listed for Fig. 8 Endoscopic ultrasound-guided pancreatic biopsy of IgG4-related autoimmune pancreatitis. (A) Afibroinflammatory process affecting the pancreatic parenchyma is noted. (B) Elastica van Gieson staining reveals obliterative phlebitis (arrow).

9 250 Pathology of IgG4-Related Disease in the Bile Duct and Pancreas Zen Fig. 9 Pancreatic biopsy after steroid therapy for immunoglobulin G4-related autoimmune pancreatitis. Due to the treatment, inflammation has become less conspicuous on hematoxylin and eosin-stained sections (A), whereas CD4 immunostaining shows residual CD4-positive lymphocytes in the pancreas (B). In another patient, the pancreas is replaced by densely fibrotic scarring (C) with almost no CD4-positive lymphocytes (D). These two patients are clinically in remission with low-dose steroids. differential diagnoses. These are histologically characterized by plasma cell-rich inflammation. Because these conditions share some clinicopathological features with IgG4-RD, careful tissue examinations and clinicopathological corrections are needed. The pathological features of individual conditions in differential diagnoses are described below. Primary Sclerosing Cholangitis Age and a history of inflammatory bowel disease (IBD) assist in the discrimination of PSC from IgG4-SC. A younger age (< 40 years) and history of IBD suggest that PSC is more likely. Imaging analyses are also useful, as explained in detail in another article in this issue. Histologically, if a large bile duct specimen is available for histological assessments, this discrimination is relatively straightforward. Patterns of inflammation markedly differ from each other. Unlike IgG4-SC, which has transmural inflammation, PSC is characterized by inflammation that is more pronounced on the luminal side with mucosal ulceration and xanthogranulomatous inflammation. Intestinal metaplasia and dysplastic changes are also sometimes observed. 3 Obliterative phlebitis and storiform fibrosis are not features of PSC. Small veins are often obliterated in PSC, but are not associated with an inflammatory infiltrate. 29 Tissue eosinophilia may be present in PSC and IgG4-SC. The fibrous obliteration of the bile duct is nearly diagnostic for PSC ( Fig. 10). Caution is needed when interpreting IgG4 immunostaining because the large bile ducts of PSC may be infiltrated by moderate to large numbers of IgG4þ plasma cells. In a series of explanted livers with PSC, 23 out of 98 livers (23%) had more than 10 IgG4þ cells per HPF. 30 Another study also demonstrated that 2 out of 41 (5%) explanted livers with PSC showed more than 100 IgG4þ cells per HPF. 29 However, IgG4þ cells are only focally aggregated, and the IgG4/IgGþ cell ratio is typically less than 40% in PSC. Non-IgG4-Related Inflammatory Pseudotumors Hepatic inflammatory pseudotumors are not always IgG4- related. Of the two major subtypes (lymphoplasmacytic and fibrohistiocytic) of hepatic inflammatory pseudotumors, the former corresponds to IgG4-related inflammatory pseudotumors, while the latter is not related to IgG4. 22 These two subtypes have distinct clinicopathological features. Immunoglobulin G4-related pseudotumors more commonly affect males, while the non-igg4-related type occurs in males and females with no significant gender difference. As described above, IgG4-related inflammatory pseudotumors typically affect perihilar bile ducts, whereas non-igg4-related pseudotumors more commonly develop in the liver parenchyma.

10 Pathology of IgG4-Related Disease in the Bile Duct and Pancreas Zen 251 Fig. 10 Liver biopsy findings of primary sclerosing cholangitis. (A) There is periductal concentric fibrosis. (B) The attenuated bile duct (arrow) appearstobemarkedlysmallerthantheadjacentartery(usuallysimilarinsize), a feature that likely represents the ductopenic process. Histologically, the non-igg4-related fibrohistiocytic type is characterized by an extensive xanthogranulomatous reaction. In addition to lymphocytes and plasma cells, many foamy macrophages and neutrophils are noted ( Fig. 11). Multinucleated giant cells may also be present. This discrimination may be achieved based on H&E-stained sections in most cases. Immunoglobulin G4þ plasma cells may be present to variable degrees in the fibrohistiocytic type, while the IgG4/IgGþ cell ratio is typically less than 40%. Non-IgG4- related pseudotumors sometimes show spontaneous regression. The etiology of this subtype remains unclear; however, infection is suspected in at least a subset of patients. Follicular Cholangitis and Follicular Pancreatitis Follicular cholangitis is a rare form of cholangiopathy that typically affects perihilar large bile ducts. 31 This condition is characterized by many lymphoid follicles around the bile ducts, the appearance of which is somewhat reminiscent of follicular cholecystitis. Patients are generally adults and present with hilar duct stricture with a periductal plaquelike mass. Serological autoimmune abnormalities and extrabiliary diseases such as IBDs are typically absent. Because no serological markers are available, tissue examinations are needed to reach a diagnosis. Follicular cholangitis differs from IgG4-SC in that lymphoid follicle formation is more extensive ( Fig. 12). 31 Obliterative phlebitis and storiform fibrosis are not observed. Similar to IgG4-SC, the biliary epithelial lining is commonly preserved. Immunoglobulin G4þ plasma cells may be present, but with a less diffuse distribution and smaller number. The IgG4/IgGþ cell ratios are always less than 40%. Responsiveness to steroid therapy is unknown because all reported cases underwent surgical resection. 31,32 A similar inflammatory process rich in lymphoid follicles may develop in the pancreas. 31 This condition, which is currently called follicular pancreatitis, may correspond to cases reported as reactive lymphoid hyperplasia or pseudolymphoma of the pancreas. 33 Type 2 Autoimmune Pancreatitis Two subtypes of AIP with distinct clinical and histological characteristics have increasingly been recognized. 8 Unlike type 1 AIP, which is a pancreatic manifestation of IgG4-RD, type 2 AIP is not related to IgG4. Type 1 is 10 times more common than type Type 1 AIP typically affects adults Fig. 11 A non-immunoglobulin G4- (IgG4-) related hepatic inflammatory pseudotumor. (A) Theinflammatoryprocessisassociatedwith xanthogranulomatous inflammation and many foamy macrophages. (B) Only a small number of IgG4-positive plasma cells are present.

11 252 Pathology of IgG4-Related Disease in the Bile Duct and Pancreas Zen Fig. 12 Follicular cholangitis. (A, B) There is a dense inflammatory infiltrate with many lymphoid follicles in the bile duct wall. (often older than 50 years), while type 2 occurs at any age. 35 Type 2 AIP is not a systemic condition, with IBD being the single potential extrapancreatic disease (20%). Serum IgG4 concentrations are within the normal range or only slightly elevated (200 mg/dl) in patients with type 2 AIP. Similar to type 1 AIP, type 2 manifests as the diffuse enlargement of the pancreas. There are no known imaging features that reliably discriminate between the two types. Type 1 and type 2 AIP share some histological features including diffuse lymphoplasmacytic infiltration and fibrosis; however, these are distinguishable based on purely histological grounds. Type 2 AIP is characterized by neutrophilic infiltration into the pancreatic duct epithelium (granulocytic epithelial lesion [GEL]) ( Fig. 13). 36,37 Lobular neutrophilic infiltration is another, although less specific, histological finding. 38 Immunoglobulin G4þ plasma cells may occasionally be present, but at a smaller number in type 2 AIP. Because there is no serological marker for type 2 AIP, the histological confirmation of GEL or neutrophilic lobular inflammation is required to establish a diagnosis. Sclerosing Cholangitis with Granulocytic Epithelial Lesion We recently proposed an entity of SC with GEL. 39 We found neutrophilic bile duct injury in liver biopsy samples obtained from 5 of 254 patients (2%) with PSC. Many neutrophils were present in the epithelial layer of the small bile ducts, associated with an irregular configuration of the lining epithelium ( Fig. 14). Their appearance differs from suppurative cholangitis in that neutrophils are present in the epithelial layer, but not inside the duct lumen. All patients went into remission with prednisolone and/or ursodeoxycholic acid and their liver function tests remained entirely normal without relapses for years, leading to the hypothesis that SC with GEL may be a biliary counterpart of type 2 AIP. 39,40 Type 2 AIP is currently regarded as a pancreas-specific inflammatory condition with no known biliary involvement. However, a similar neutrophil-rich inflammatory process responsive to corticosteroids may also occur in the biliary tree. It is important to note that steroid-responsive SC is not always IgG4-RD. Fig. 13 Type 2 autoimmune pancreatitis. (A) Pancreatitis is characterized by duct-centered inflammation. (B) The pancreatic duct is severely damaged with massive intraepithelial neutrophilic infiltration (granulocytic epithelial lesion).

12 Pathology of IgG4-Related Disease in the Bile Duct and Pancreas Zen 253 infiltration of many Tregs in tissues. This is in clear contrast to classic autoimmune diseases, in which Tregs are expected to be fewer in number and have impaired function. This may also challenge the hypothesis that IgG4-RD is an autoimmune disorder. Interleukin-10, a regulatory cytokine that is overexpressed in tissue, is supposed to be produced by infiltrating Tregs. Given the fact that an IgG4 class switch is induced when both IL-4 and IL-10 react to B cells simultaneously, 46 the combined activation of Th2 and Tregs may lead to the selective induction of IgG4. Tregs are also supposed to overexpress transforming growth factor beta, which is a well-known fibrogenic cytokine. Therefore, Tregs appear to play a crucial role in both inflammatory and fibrosing processes. 43,44 Fig. 14 Sclerosing cholangitis with granulocytic epithelial lesions. The small bile duct obtained by a needle biopsy is irregular in shape with many intraepithelial neutrophils. Immunopathology of IgG4-RD The pathogenetic mechanisms underlying IgG4-related pancreatocholangitis have not yet been elucidated in detail. Previous studies identified the genetic predisposing factors and unique immunological features of AIP, raising the possibility that the process is multifactorial. 35 As with most immunomediated conditions, a likely pathogenetic mechanism is that the disease develops in genetically susceptible individuals exposed to environmental factors. Although most basic studies on IgG4-RD used the sera of patients to characterize the immunological aspects of this condition, some pathological studies attempted to address its immunological features by phenotyping infiltrating lymphocytes. These tissue studies are mainly reviewed herein to provide a better understanding of the immune reactions expected to underlie histopathological findings. T-Cell Response Earlier studies mainly investigated T-cell responses in IgG4-RD. Although two studies using the sera of patients suggested that Th1 cells were activated more than Th2 cells, 41,42 asubsequent pathological study indicated that the Th1/Th2 balance shifted in favor of Th2 in tissues. 43 The mrna expression values of interferon gamma (INF-γ) were similar between IgG4-related pancreatocholangitis and other inflammatory conditions, whereas the expression of interleukin- (IL-) 4, IL-5, and IL-10 was significantly higher in IgG4-RD. 43 In situ hybridization also revealed that, in addition to INF-γþ cells, many lymphocytes expressing IL-4 were present in the pancreas and bile duct. 43,44 In line with tissue studies, bile samples from patients with IgG4-related pancreatocholangitis were found to contain significantly increased levels of IL-4 and IL Although these findings indicate that the Th1 reaction is not completely suppressed, the Th2 immune response appears to be dominant in tissues of IgG4-RD. 43,44 Another important subset of immune cells is regulatory T cells (Tregs). Immunostaining for FOXP3 demonstrates the Chemotactic Factors Although a massive cellular infiltrate is a histological hallmark of this condition, the chemotactic process is poorly understood. Only approximately 10 chemotactic factors are known to be upregulated (e.g., CXCL13, CCL1, CCL17, CCR4, and CCR8). 47,48 Because no antibodies that work on paraffinembedded tissue are commercially available for most of these molecules, the cellular origins of these chemotactic factors have not yet been examined in detail. We investigated the expression of CCL1 and CCR8 using in situ hybridization based on the assumption that they are involved in the recruitment of Th2 lymphocytes and Tregs because 50% of Th2 lymphocytes and 60% of FOXP3þ Tregs express CCR8. 49 CCL1 was found to be expressed in the ductal and glandular epithelia as well as in endothelial cells, including those involved in obliterative phlebitis. 47 CCR8þ lymphocytes were present around ducts, glands, and vessels that were positive for CCL1. The CCL1 CCR8 interaction may be involved in creating a unique microenvironment in which Th2 cells and Tregs are abundant. 49 Furthermore, this immunological reaction may be related to histopathological findings such as periductal and periglandular inflammation, and obliterative phlebitis in IgG4-related pancreatocholangitis. It currently remains unknown why intraepithelial lymphocytes are scarce even though the duct epithelium expresses CCL1. Th2 lymphocytes and Tregs may not be strong enough to infiltrate the basement membrane. A previous study demonstrated that the pancreatic duct epithelium was damaged at the molecular level despite its unremarkable morphological appearance. In the duct epithelium in untreated AIP, membranous proteins such as the cystic fibrosis transmembrane conductance regulator (CFTR) were mislocated in the cytoplasm. 50 A more recent study also suggested that the biliary epithelium in IgG4-SC has impaired barrier function because of abnormally expressed claudins that may be caused by an interaction between Th2 cytokines and their receptors expressed on cholangiocytes. 45 B Cells and Macrophages The effectiveness of B-cell depletion therapy with anti-cd20 antibodies for IgG4-RD has prompted us to speculate about the crucial roles of B cells in the pathogenesis of this condition. 51,52 We recently investigated protein expression profiles in the bile duct tissue of IgG4-RD using a robust proteomic

13 254 Pathology of IgG4-Related Disease in the Bile Duct and Pancreas Zen approach, which enabled us to identify 23,373 peptides and 4,870 proteins. 53 To the best of our knowledge, this is the first nonbiased global tissue examination of this particular condition. In the pathway analysis based on protein expression profiles, IgG4-SC was found to have 11 more activated signal cascades including three immunological pathways than PSC. The three immune pathways were all B cell- or immunoglobulin-related (Fc-gamma receptor-mediated phagocytosis, B-cell receptor signaling pathway, and Fc-epsilon receptor I signaling pathway). 53 Fc-gamma receptor-mediated phagocytosis occurs in macrophages, and is initiated by IgG molecules binding to Fc-gamma receptors. However, given that the capacity of IgG4 to bind to Fc receptors is poor, 54 this pathway may be activated by other IgG subclasses (e.g., IgG1). The B-cell receptor signaling pathway is initiated by the interaction between antigens and B-cell receptors on the cell membrane. The Fc-epsilon receptor I signaling pathway is a signaling cascade in mast cells, leading to Th2 responses and eosinophilic activation. Because no pathways directly related to T cells were significant, B-cell immune responses may better discriminate the immunological features of IgG4-RD and PSC. A proteomic study also identified individual proteins that were significantly overexpressed in IgG4-SC. Two immunological markers (FYN-binding protein and allograft inflammatory factor-1) that are upregulated in IgG4-RD were selected for a validation study using immunohistochemistry. They were mainly expressed in CD163þ M2 macrophages, 53 which is consistent with the findings of a recent study suggesting a possible role for M2 macrophages in the fibrosing process in IgG4-RD. 13 Because FYN-binding protein has mainly been examined in terms of its possible impact on the activation of T cells and myeloid cells, 55 the exact role of FYN-binding protein in macrophages remains unclear. Previous studies suggested the possible involvement of FYN-binding protein in the activation of a phagocytotic process in macrophages. 56 Allograft inflammatory factor-1 is also known to be involved in the activation process of macrophages. Its expression enhances the production of cytokines such as IL-6, IL-10, and IL-12, as well as the phagocytotic activity of macrophages. 57,58 These findings are consistent with the results of a pathway analysis showing that Fc-gamma receptor-mediated phagocytosis in macrophages was the most significantly activated immunological cascade. Therefore, macrophages, particularly the M2 type, may be activated by the IgG-Fc-gamma receptor interaction and bridge the inflammatory response to the fibrosing process. Conclusion In summary, IgG4-RD in the pancreatobiliary system was reviewed in terms of histopathological features, differential diagnoses, and immunopathological aspects. Although histological findings have been characterized in detail, more experience and knowledge are required to improve diagnoses based only on biopsy samples. Future immunopathological studies will assist in clarifying the underlying immunological features of IgG4-RD and may provide other diagnostic immunohistochemical markers. Abbreviations þ AIP CFTR EUS-FNA GEL H&E IBD IgG4-RD IgG4-SC IL INF-γ PSC Tregs positive autoimmune pancreatitis cystic fibrosis transmembrane conductance regulator endoscopic ultrasound-guided fine needle aspiration granulocytic epithelial lesion hematoxylin and eosin inflammatory bowel disease immunoglobulin G4-related disease IgG4-related sclerosing cholangitis interleukin interferon gamma primary sclerosing cholangitis regulatory T cells References 1 Stone JH, Zen Y, Deshpande V. IgG4-related disease. N Engl J Med 2012;366(6): Kamisawa T, Zen Y, Pillai S, Stone JH. IgG4-related disease. Lancet 2015;385(9976): Zen Y, Harada K, Sasaki M, et al. IgG4-related sclerosing cholangitis with and without hepatic inflammatory pseudotumor, and sclerosing pancreatitis-associated sclerosing cholangitis: do they belong to a spectrum of sclerosing pancreatitis? Am J Surg Pathol 2004;28(9): Kitagawa S, Zen Y, Harada K, et al. 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