Autoimmune pancreatitis (AIP) was described more than a

CLINICAL GASTROENTEROLOGY AND HEPATOLOGY 2007;5:1229 1234 The Use of Immunoglobulin G4 Immunostaining in Diagnosing Pancreatic and Extrapancreatic Involvement in Autoimmune Pancreatitis MAESHA G. DEHERAGODA,* NICHOLAS I. CHURCH, MANUEL RODRIGUEZ JUSTO,* PHILIPPA MUNSON, NEOMAL SANDANAYAKE, EDWARD W. SEWARD, KEITH MILLER, MARCO NOVELLI,* ADRIAN R. W. HATFIELD, STEPHEN P. PEREIRA,, and GEORGE J. M. WEBSTER, *Department of Histopathology, and Department of Gastroenterology, University College Hospital, London, United Kingdom; the The Institute of Hepatology, and UCL Advanced Diagnostics, Department of Pathology, University College Hospital, London, United Kingdom Background & Aims: Autoimmune pancreatitis (AIP) is recognized increasingly as a multisystem disorder. We evaluated the use of immunoglobulin (Ig)G4 immunostaining of pancreatic and extrapancreatic biopsy specimens to make a definitive diagnosis of AIP. Methods: Seventeen biopsy specimens and 3 gallbladder resections were assessed from 11 patients with clinical and radiologic features of AIP. Biopsy specimens from pancreas, liver, colon, stomach, duodenum, bone marrow, salivary gland, and kidney were analyzed morphologically, immunostained for IgG4- positive plasma cells, and compared with controls. Results: Positive IgG4 immunostaining enabled a definitive diagnosis in 10 of 11 (91%) AIP patients. In both pancreatic and extrapancreatic tissues, high levels of IgG4 immunostaining (>10 IgG4-positive plasma cells/highpower field) were found in 17 of 20 (85%) specimens from AIP patients compared with 1 of 175 (0.6%) specimens from controls (P <.05). Positive extrapancreatic IgG4 immunostaining was found in 8 of 11 (73%) patients, including all those with diagnostic features in the pancreas. Increased tissue IgG4 was found irrespective of serum IgG4 level. Conclusions: The finding of IgG4 immunostaining within a range of clinically involved tissues supports the hypothesis that AIP is a multisystem disease. Positive IgG4 immunostaining in extrapancreatic tissues may allow a definitive diagnosis of AIP to be made in those with evidence of pancreatic disease, without the necessity of pancreatic biopsy or surgical exploration. Immunostaining of involved tissue for IgG4 may be particularly useful when AIP is suspected clinically but the serum IgG4 level is normal. Autoimmune pancreatitis (AIP) was described more than a decade ago. 1 It often presents with a pancreatic mass and stricturing of the bile and pancreatic ducts, which may mimic pancreatobiliary cancer. Making a diagnosis is vital because steroid therapy may be effective, 2 4 allowing unnecessary surgery to be avoided. The disease largely has been described in Japan, but small patient series from Europe and the United States suggest that this rare condition occurs globally. 4 6 Diagnostic criteria for AIP have centered on clinical, radiologic, and pathologic features within the pancreas, and have relied on the demonstration of a lymphoplasmacytic pancreatic infiltrate to make a definitive diagnosis. 1,7,8 However, a percutaneous pancreatic biopsy procedure may be difficult technically, with a complication rate of up to 4.6%, 9 and so alternative diagnostic options would be preferable. In addition, the concept of AIP now is changing to one of a multisystem disorder, in which there may be clinical involvement of a range of organs. Both extrapancreatic gastrointestinal sites (eg, intrahepatic and hilar bile ducts, gallbladder, colon, duodenum, stomach, and duodenal papilla 4,5,10 12 ) and extragastrointestinal tissues (eg, retroperitoneal structures, lymph nodes, bone marrow, salivary gland, lung, kidney, mediastinum, thyroid, and prostate 13 18 ) may be involved. Cases also have been reported in which pancreatic disease was not a feature at the time of diagnosis. 14 Extrapancreatic disease can be treated effectively with steroid therapy, 10 making recognition of this component an important aspect of disease management. Chari et al 5 recently proposed a new diagnostic scheme based on the use of Histology, Imaging, Serology, Other organ involvement, and Response to steroid therapy the (HISORt) criteria. In contrast to previous criteria, this classification takes greater account of extrapancreatic involvement in making a diagnosis of AIP. However, their criteria still propose that a definitive diagnosis of AIP can be made only on morphologic and immunohistochemical appearances in pancreatic tissue. Finkelberg et al 19 also proposed diagnostic criteria that include histologic evaluation of a restricted range of extrapancreatic tissues. However, there are little published data on the reliability of extrapancreatic biopsies from a wide range of clinically involved organs in making a diagnosis of AIP. 20 Furthermore, most published studies on the involvement of extrapancreatic tissues in AIP have relied on the histologic assessment of surgical resections, not core biopsy specimens. The histologic diagnosis of AIP may be difficult, in part because of the small volume of tissue often available from core biopsy specimens, and patchy disease distribution. 21 Specific features of AIP have been documented for the pancreas, 21 23 but in extrapancreatic organs there is a paucity of consistently reported features for AIP, beyond a lymphoplasmacytic infiltrate, fibrosis, and obliterative phlebitis, 10 which may be seen in chronic inflammation as a result of a range of causes. This limits the use of morphology alone in making a definitive histologic diagnosis in extrapancreatic organs. Increased serum immunoglobulin (Ig)G4 levels has been reported to be more than 95% specific and sensitive for AIP. 24 However, recent data from the United States 5 and the United Abbreviations used in this paper: AIP, autoimmune pancreatitis; HPF, high-power field; Ig, immunoglobulin. 2007 by the AGA Institute 1542-3565/07/$32.00 doi:10.1016/j.cgh.2007.04.023

1230 DEHERAGODA ET AL CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 5, No. 10 Table 1. Demographic, Clinical, and Initial Histologic Findings in 11 Patients Included in the Study Patient no. Patient age and race Serum IgG4 (N 1.64 g/l) Tissue from clinically involved organs Original diagnosis on H&E sections 1 53-year-old male Caucasian Increased Liver Large duct obstruction 2 51-year-old male Afro-Caribbean Increased Pancreas Liver Kidney Chronic pancreatitis Nonspecific features a Chronic tubulointerstitial nephritis a 3 64-year-old male South Asian Increased Bone marrow Duodenum Stomach Salivary gland 4 52-year-old male South Asian Normal Pancreas Gallbladder Liver Nonspecific features a Eosinophilic duodenitis a Chronic gastritis a Chronic sialadenitis a Chronic pancreatitis Chronic cholecystitis a Large duct obstruction 5 54-year-old male Caucasian Normal Pancreas Chronic pancreatitis 6 47-year-old male Caucasian Normal Liver Large duct obstruction 7 77-year-old male Caucasian Increased Gallbladder Chronic cholecystitis a 8 28-year-old male Caucasian Normal Liver AIP-associated biliary disease 9 49-year-old male Japanese Increased Liver AIP-associated biliary disease 10 60-year-old male South Asian Normal Liver Pancreas Large duct obstruction a Chronic pancreatitis a 11 77-year-old male Caucasian Normal Gallbladder Colon a Indicates diagnosis before referral. Chronic cholecystitis a Nonspecific features Kingdom 6 have shown lower sensitivities of 71% and 68%, respectively, for increased serum IgG4 levels on testing at a single time point. The aim of this study was to assess the diagnostic use of IgG4 immunostaining of pancreatic and extrapancreatic tissues in AIP and assess the sensitivity of tissue IgG4 immunostaining compared with measurement of serum IgG4 in making a diagnosis of AIP. Materials and Methods Patients Eleven consecutive patients (all male) seen in our center between 2004 and 2006 in whom a diagnosis of AIP was made and in whom archival tissue and biopsy specimens obtained at admission to our unit were available for assessment, were included in the study. The diagnosis of AIP was suspected on the basis of clinical and radiologic features 4 using published diagnostic criteria for the disease. 8 Patient details, the histologic material available for analysis, and the initial histologic diagnoses at referral are shown in Table 1. Serum IgG4 levels were measured in all patients at presentation and during follow-up evaluation. Patients diagnosed with AIP were commenced on steroid therapy and their response to treatment was monitored. Materials All tissue was formalin fixed and paraffin embedded. Twenty-two tissue specimens from AIP patients were available for analysis, of which 19 were biopsy specimens (4 pancreatic, 7 liver, 1 renal, 1 colonic, 1 duodenal, 2 gastric, 2 bone marrow, and 1 salivary gland) and 3 were gallbladder resections. For controls, both normal biopsy specimens and inflamed tissue samples were selected. The latter were chosen to include diseases with a similar degree of inflammatory cell infiltration (number of specimens): pancreas: chronic calcific pancreatitis (10), normal (10), pancreatic ductal adenocarcinoma (20); liver: chronic hepatitis C (10), normal (10); gallbladder: chronic cholecystitis (10), normal (10); salivary gland: chronic sialadenitis (10), normal (8); duodenum and colon: active chronic duodenitis/colitis (10/10), normal duodenum (10), normal colon (10); bone marrow: myeloma (10), normal (10); kidney: chronic tubulointerstitial nephritis (10), normal (7). Methods All pancreatic and extrapancreatic biopsy specimens were reviewed by 2 pathologists (M.G.D. and M.R.-J.) familiar with the morphologic appearances of AIP. The original diagnosis from the corresponding pathology report was recorded. H&E-stained tissue sections from AIP patients were assessed for the presence of morphologic features reported to be specific for AIP. These included fibrosis and a lymphoplasmacytic infiltrate (with periductal involvement, preservation of ductal epithelium, and loss of exocrine tissue in the pancreas) and obliterative phlebitis with preservation of arterial anatomy. 21 23 Successive serial sections were immunostained with CD138, 1/100 dilution (Dako, Ely, UK) to detect plasma cells. Mouse monoclonal CD38, 1/200 dilution (Novocastra Laboratories, Newcastle, UK), was used to detect plasma cells in the liver. Plasma cells identified in this way were assessed for their expression of IgG4 by immunostaining with mouse anti-human monoclonal IgG4 antibody, 1/200 dilution (The Binding Site, Birmingham, UK). All immunostained sections had dewaxing and antigen retrieval performed on Bond MaX machines (Vision Biosystems, Newcastle, UK). Slides for CD138 were retrieved in ER1 for 20 minutes and those for CD38 and IgG4 for 30 minutes. The primary antibody was developed using Bond Polymer Refine Detection (DS9800, Vision Biosystems) and visualized with DAB (3,3 diaminobenzidine). Numbers of IgG4-positive plasma cells per high-power field (HPF) (magnification, 400 ) were counted in 10 HPFs containing the densest lymphoplasmacytic

October 2007 IgG4 IMMUNOSTAINING IN AIP 1231 infiltrate. Where tissue volume was small, a minimum of 5 HPFs was assessed. The mean number of IgG4-positive plasma cells in a single HPF was calculated and the result was expressed as IgG4-positive plasma cells/hpf. Specimens were considered as positive for a diagnosis of AIP if there was a lymphoplasmacytic infiltrate and there were greater than 10 IgG4-positive plasma cells/hpf. 15 The Mann Whitney U test was chosen to assess for any statistically significant differences between the mean numbers of IgG4-positive plasma cells in patient tissue vs normal and inflamed controls for pancreatic, liver, and gallbladder specimens. Results Morphologic Appearances of Biopsy Tissue on H and E Sections Two tissue samples (1 bone marrow trephine from patient 2, and a gastric biopsy specimen from patient 4) were normal morphologically and therefore were not analyzed further. In 18 of 20 specimens (9 of 11 patients) the original diagnosis made on H&E sections alone was that of an organ-specific chronic inflammatory condition, or nonspecific features (Table 1). On review of the pancreatic biopsy specimens, characteristic morphologic features of AIP were seen in 3 of 4 (75%). In addition, the investigators observed nuclear streaming artifact, which refers to the elongated and distorted nuclei of fibroblasts and inflammatory cells, in all pancreatic biopsy specimens (Figure 1A). Review of extrapancreatic tissues showed all cases to contain a lymphoplasmacytic infiltrate with a mild to moderate increase in eosinophils. With the exception of 1 gallbladder and the bone marrow sample, all extrapancreatic tissues showed variable degrees of fibrosis with nuclear streaming artifact (Figure 1E, H, and K). Beyond this, no other specific features were noted. Nuclear streaming artifact was not seen in any of our control tissues. Liver biopsy specimens from 2 patients (patients 8 and 9) showed the histologic features of large duct obstruction. However, concurrent IgG4 immunostaining in these cases (in conjunction with suggestive clinical and radiologic features) led to an initial diagnosis of AIP-related biliary disease. Immunoglobulin G4 Immunostaining Increased numbers of IgG4-positive plasma cells ( 10/ HPF) were seen in 18 of 20 tissue specimens (Figures 1 and 2). In contrast, 174 of 175 control tissues showed less than 3 IgG4-positive plasma cells/hpf (Figure 2). The Mann Whitney U test showed a statistically significant difference in the mean numbers of IgG4-positive plasma cells between patient tissues and normal and inflamed controls (pancreas: range of IgG4- positive plasma cells/hpf in patient tissue, 8 22; median, 20; P.0021; liver: range of IgG4-positive plasma cells/hpf in patient tissue, 8 28; median, 17; P.0001; gallbladder: range of IgG4-positive plasma cells/hpf in patient tissue, 28 61; median, 28; P.007). The small number of specimens available from other extrapancreatic tissues precluded statistical analysis. Nevertheless, an increased level of IgG4-positive plasma cells was seen in samples of salivary gland, stomach, duodenum, colon, kidney, and bone marrow in AIP patients, which was not found in comparative controls (Figure 2). IgG4 immunostaining of extrapancreatic tissues was positive in 8 of 11 (73%) patients. Positive IgG4 immunostaining of extrapancreatic tissue was found in all 3 patients in whom pancreatic histology was diagnostic of AIP. In 6 of 11 patients (55%), serum IgG4 was normal at presentation to our center. Nevertheless, IgG4 immunostaining was positive in 5 of 6 (83%) of these patients. False Positives One of 20 (5%) pancreatic ductal adenocarcinoma biopsy specimens used as controls showed 10 IgG4-positive plasma cells/hpf. The morphologic appearances in this biopsy specimen were unequivocally those of adenocarcinoma and no features of AIP were present. The IgG4-positive plasma cells were associated closely with adenocarcinoma cells. False Negatives Biopsy specimens from 2 AIP patients showed fewer than 10 IgG4-positive plasma cells/hpf. The pancreatic biopsy specimen from patient 5 showed extensive fibrosis and fewer than 4 plasma cells/hpf were present for assessment. Patient 4 had a liver biopsy specimen in which there was very little portal tract tissue for the assessment of plasma cells (only 4 HPFs could be assessed). Patient Response to Steroid Therapy All patients with AIP were commenced on oral steroid therapy. All had a clinical, radiologic, and biochemical response, further supporting the diagnosis of AIP, as we have reported in 9 of these patients recently. 4 Discussion AIP is now accepted to be a global disease, 6,25 and not confined to Japan, from where most reports have arisen. As more cases and small series have been published, the involvement of extrapancreatic tissues has been appreciated, and it may be that the pancreas represents just one target of a multisystem disease. 5 Although new diagnostic criteria for AIP have placed greater importance on extrapancreatic disease 5,19 than did previous criteria, 1 all require pancreatic histology to make a definitive diagnosis. An alternative to a pancreatic biopsy examination would be attractive, particularly in those with extrapancreatic involvement. A diagnosis of AIP made solely on morphologic grounds requires the presence of a constellation of histologic features, and although the lymphoplasmacytic infiltrate, fibrosis, and obliterative phlebitis that is seen in the pancreas has been reported in extrapancreatic tissue, 10,15 consistent and specific morphologic features have, to date, only been described for the pancreas. In this study IgG4 immunostaining added to the diagnostic accuracy that was possible by morphologic examination alone. The value of nuclear streaming artifact as a specific diagnostic feature for AIP awaits further evaluation on larger numbers of tissue specimens. Beyond this feature, H&E analysis of extrapancreatic tissue showed characteristic features of AIP in less than 20% of patients, but the addition of IgG4 immunostaining increased this yield to 73%. With respect to pancreatic tissue analysis, it is of note that although a histologic diagnosis of AIP could be made on pancreatic biopsy specimens in 3 of 4 patients, all 3 of these

1232 DEHERAGODA ET AL CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 5, No. 10 Figure 1. (A) H&E-stained section (magnification, 100 ) of pancreatic biopsy specimen from patient 10 showing fibrosis, a lymphoplasmacytic infiltrate within pancreatic lobules, with nuclear streaming artifact surrounding a venule showing obliterative phlebitis (V). There is relative preservation of arterioles (A). (B) H&E-stained section of pancreatic biopsy from patient 10 (magnification, 200 ) with a lymphoplasmacytic infiltrate surrounding pancreatic ducts (D). There is atrophy of exocrine pancreatic tissue with preservation of islets (I). (C) IgG4 immunostaining of plasma cells within the pancreatic biopsy from patient 10 (magnification, 400 ). (D) H&E-stained section (magnification, 100 ) of liver biopsy from patient 6 showing the lymphoplasmacytic infiltrate within portal tracts. (E) H&E-stained section of a portal tract from the liver biopsy on patient 6 showing the features of large duct obstruction with a mild increase in eosinophils and nuclear streaming artifact (N) (magnification, 200 ). (F) IgG4 immunostaining of plasma cells within portal tracts from the liver biopsy on patient 6 (magnification, 400 ). (G) H&E-stained section (magnification, 100 ) of salivary gland from patient 3 showing a lymphoplasmacytic infiltrate with lymphoid follicle formation. (H) H&E-stained section (magnification, 200 ) of salivary gland from patient 3 showing a lymphoplasmacytic infiltrate and nuclear streaming artifact (N) within lobules (magnification, 200 ). (I) IgG4 immunostaining of plasma cells within salivary gland from patient 3 (magnification, 400 ). (J) H&E-stained section (magnification, 100 ) of duodenum from patient 3. (K) H&E-stained section of duodenal biopsy from patient 3 (magnification, 200 ) of duodenum showing fibrosis with nuclear streaming artifact (N) and a lymphoplasmacytic infiltrate with a prominence of eosinophils (E). (L) IgG4 immunostaining of duodenal biopsy from patient 3 (magnification, 400 ). Plasma cells expressing IgG4 were identified on the basis of CD138 staining of adjacent tissue sections and characteristic plasma cell morphology (an eccentrically placed nucleus that has a clock face appearance and abundant cytoplasm). patients had positive IgG4 immunostaining of extrapancreatic tissue. This suggests that pancreatic biopsy examination may not have been necessary diagnostically. Small studies have suggested that the diagnosis of AIP may be made on biopsy specimens from the stomach26 and duodenal papilla,11 both of which are easily and safely obtained endoscopically. In the case of AIP in which IgG4 immunostaining of a pancreatic biopsy specimen was negative (patient 5), histology showed severe fibrosis, and the numbers of plasma cells were too low for assessment. It is of note that the serum IgG4 was normal in this case. The appearances in this biopsy specimen may represent a late burned-out stage of disease and therefore high numbers of IgG4-expressing cells may not be a feature. A chronologic assessment of histologic features during the natural course of AIP has not been reported. It would be interesting to see if (as is our clinical impression) the intensity of the

October 2007 IgG4 IMMUNOSTAINING IN AIP 1233 Figure 2. Mean numbers of IgG4-positive plasma cells in AIP patients vs normal and inflamed controls. SG, salivary gland. lymphoplasmacytic infiltrate and the IgG4 positive staining is highest during the earlier phase of the disease, and whether the longer the disease persists the more that fibrosis predominates and the infiltrate reduces. Steroid treatment might be less effective in this late stage, emphasizing the need to make an early diagnosis. Inflammatory mass lesions in AIP are known to remit/improve spontaneously over time, 2 and increased IgG4 levels may normalize in response to treatment. 24 The finding of extrapancreatic involvement in many of our patients supports the argument that clinical features previously thought to represent other diseases occurring in association with AIP (eg, sclerosing cholangitis and renal involvement) may in fact all be manifestations of the same multisystem disease. 5 Our data also support the recent publication from the Mayo Clinic and their proposal of the HISORt criteria, which places greater emphasis on extrapancreatic disease than was the case for previous criteria for AIP. Restricting the nomenclature of this condition to the pancreas may be misleading and the suggested term IgG4-related systemic disease 27 may have merit over AIP, and is supported by our data on IgG4 immunostaining of extrapancreatic tissue. The majority of our patients had normal serum IgG4 levels on initial testing. Despite this we showed IgG4 immunostaining of pancreatic and extrapancreatic tissue in 10 of 11 (91%) patients, and in 5 of 6 patients (83%) with normal serum IgG4 levels at presentation. A caveat to these data is that serum IgG4 level often was not taken at the same time point as tissue samples, with some biopsy specimens taken when AIP was not clinically considered, and serum IgG4 levels therefore were not measured. With the knowledge that serum IgG4 levels may fluctuate over time, 4,24 levels may have settled by the time of serologic testing. We found that serum IgG4 became positive in 3 of 6 patients (50%) with normal levels on initial testing, and that through repeat testing increased levels were found in 8 of 11 patients (73%). Nevertheless, our findings are in accordance with data from Chari et al, 5 who showed normal serum IgG4 levels in 29% of their patients, but positive pancreatic IgG4 immunostaining in all of these cases. The presence of positive IgG4 immunostaining in 5% of our pancreatic adenocarcinoma controls illustrates the importance of combining the clinical, radiologic, and histologic appearances when interpreting the results of IgG4 immunostaining. The occasional presence of increased IgG4 in association with pancreatic cancer has been reported previously, 19,28 and is unexplained, but the morphologic appearances of adenocarcinoma and the lymphoplasmacytic infiltrates that characterize AIP are distinctly different. These observations emphasize the necessity not to interpret the results of IgG4 immunostaining in isolation. They perhaps further support the use of extrapancreatic biopsy specimens in making a diagnosis of AIP in patients presenting with pancreatic or hilar masses in whom the clinical and radiologic differential diagnosis between AIP and carcinoma is difficult. In summary, our study adds to the emerging data on the pathologic and clinical nature of AIP, providing further evidence of the multiorgan nature of this disease in many patients. Based on our experience we would argue that a definitive diagnosis of AIP should not have to rely on pancreatic biopsy specimens but also could be made on the basis of an IgG4-positive inflammatory infiltrate within clinically involved extrapancreatic tissue, if imaging also suggests pancreatic disease. In addition, our data have shown that IgG4 immunostaining of involved tissue may be particularly useful when AIP is suspected clinically, but the serum IgG4 level is normal. References 1. Yoshida K, Toki F, Takeuchi T, et al. Chronic pancreatitis caused by an autoimmune abnormality. Proposal of the concept of autoimmune pancreatitis. Dig Dis Sci 1995;40:1561 1568. 2. Wakabayashi T, Kawaura Y, Satomura Y, et al. Long term prognosis of duct narrowing chronic pancreatitis: strategy for steroid treatment. Pancreas 2005;30:31 39. 3. Kamisawa T, Yoshiike M, Egawa N, et al. Treating patients with autoimmune pancreatitis: results from a long term follow up study. Pancreatology 2005;5:234 238.

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