Distinct pretreatment innate immune landscape and posttreatment T cell responses underlie immunotherapy-induced colitis.

Immune-related adverse events represent a major hurdle to the success of immunotherapy. The immunological mechanisms underlying their development and relation to antitumor responses are poorly understood. By examining both systemic and tissue-specific immune changes induced by combination anti-CTLA-4 and anti-PD-1 immunotherapy, we found distinct repertoire changes in patients who developed moderate-severe colitis, irrespective of their antitumor response to therapy. The proportion of circulating monocytes were significantly increased at baseline in patients who subsequently developed colitis compared with patients who did not develop colitis, and biopsies from patients with colitis showed monocytic infiltration of both endoscopically and histopathologically normal and inflamed regions of colon. The magnitude of systemic expansion of T cells following commencement of immunotherapy was also greater in patients who developed colitis. Importantly, we show expansion of specific T cell subsets within inflamed regions of the colon, including tissue-resident memory CD8+ T cells and Th1 CD4+ T cells in patients who developed colitis. Our data also suggest that CD8+ T cell expansion was locally induced, while Th1 cell expansion was systemic. Together, our data show that exaggerated innate and T cell responses to combination immunotherapy synergize to propel colitis in susceptible patients.

Life-threatening diarrhea in neuroendocrine tumors: two case reports.

BACKGROUND: Neuroendocrine tumors are rare, heterogeneous neoplasms that produce a wide variety of clinical symptoms. Diarrhea in neuroendocrine tumors is incredibly common and is usually benign in nature. We report two extreme cases of diarrhea in metastatic neuroendocrine tumors that threatened fatality and provide evidence for steroids as a novel agent in the management of vasoactive intestinal peptide tumors. CASE PRESENTATION: A 63-year-old Caucasian male with a grade 2 (Ki-67 17%) metastatic small bowel neuroendocrine tumor, and a 43-year-old female with a grade 2 (Ki-67 5%) metastatic pancreatic vasoactive intestinal peptide tumor. Both patients suffered life-threatening diarrhea despite extensive treatment modalities, including new systemic agents. This case explains how a lack of compliance and patient under-reporting of symptoms contributed to their challenging clinical course. Only steroids had a significant sustained effect on the diarrhea of the patient with vasoactive intestinal peptide tumor. CONCLUSIONS: This report discusses two rare cases of life-threatening diarrhea in neuroendocrine tumors and stresses the importance of accurate clinical history taking, patient education, and compliance for symptom control. The report suggests steroids as a potential novel pharmaceutical option in the management of vasoactive intestinal peptide tumors; this is of great significance as it may provide a new approach to their management and potentially act as a life-saving agent in other oncology patients.

Management, outcomes and survival of an Australian IgG4-SC cohort: The MOSAIC study.

BACKGROUND AND AIMS: IgG4 sclerosing cholangitis (IgG4-SC) is the biliary component of the multisystem IgG4-related disease. We aimed to investigate the clinical features, demographics, treatment response and outcomes of IgG4-SC in a large Australian cohort. METHODS: We conducted nationwide retrospective cohort via the Australian Liver Association Clinical Trials Network (ALA-CRN). 39 sites were invited to participate. IgG4-SC was defined by the clinical diagnostic criteria established by the Japanese Biliary Association in 2012. Data were collected on patient demographic, clinical and laboratory information, presenting features, response to therapy and clinical outcomes. RESULTS: 67 patients meet inclusion criteria from 22 sites. 76% were male with mean age of 63.3 ± 14.5 years and a median IgG4 level of 3.6 g/L [0.09-67.1]. The most frequent presenting symptom was jaundice (62%) and abdominal pain (42%) and Type 1 biliary stricturing (52%) at the distal common bile duct was the most frequent biliary tract finding. Prednisolone was used as a primary treatment in 61 (91%) and partial or complete response occurred in 95% of subjects. Relapse was common (42%) in those who ceased medical therapy. After a median follow up of 3.9 years there was one hepatocellular carcinoma and no cholangiocarcinomas. CONCLUSIONS: Our study confirms the preponderance of IgG4-SC in males and highlights the steroid response nature of this condition although relapse is common after steroid cessation. Progression to malignancy was uncommon.

Calcineurin inhibitors in steroid and anti-TNF-alpha refractory immune checkpoint inhibitor colitis.

BACKGROUND AND AIM: Immune checkpoint inhibitor (ICI) colitis is an increasingly common problem encountered as the use of checkpoint inhibitors (CPIs) grows in the management of cancers. Corticosteroids and tumour necrosis factor (TNF)-alpha inhibitors are widely recommended in the management of ICI colitis; however, the experience is limited when patients are refractory. Different authors have reported success with vedolizumab, mycophenolate, and cyclosporine. This case series describes our experience with calcineurin inhibitors in the management of corticosteroid and anti-TNF-alpha refractory ICI colitis. METHODS: Data from electronic medical records were identified and reviewed retrospectively from a cohort of patients treated at a single oncology center. All patients who were identified between March 2018 and May 2020 with ICI colitis refractory to treatment with infliximab and corticosteroids were included. RESULTS: There were 11 patients who developed ICI colitis after receiving CPIs for advanced melanoma and required rescue therapy with either cyclosporine or tacrolimus after treatment failure of infliximab. Median age was 53 (±8.48) years, with nine patients (81%) receiving combination Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) / programmed cell death protein 1 (PD-1) immunotherapy. Median time after first CPI infusion to ICI colitis was 4.43 (±19.53) weeks. The median time from onset of symptoms to commencement of rescue therapy with calcineurin inhibitors was 70 days (±66.06). Eight of the 11 patients (72.7%) responded to calcineurin inhibition. In patients who responded, calcineurin inhibitors were continued for a median of 54 (±28.96) days. CONCLUSION: The calcineurin inhibitors cyclosporine and tacrolimus appear to be a safe and effective option for the management of patients with infliximab-refractory ICI colitis. The therapeutic benefit is observed rapidly, and adverse effects appear to be limited with close monitoring.

Clinicopathological characteristics and management of colitis with anti-PD1 immunotherapy alone or in combination with ipilimumab.

BACKGROUND: Colitis is one of the common immune-related adverse events that leads to morbidity and treatment discontinuation of immunotherapy. The clinical presentation, endoscopic and histopathological features and best management of this toxicity are not well defined. PATIENTS AND METHODS: Patients with metastatic melanoma who received immunotherapy (programmed cell death protein 1 (PD1) antibodies, alone or in combination with a cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) antibody (PD1 +CTLA-4)) and who developed clinically significant colitis (requiring systemic corticosteroids) were identified retrospectively from two academic centers. Clinical data were collected for all patients; endoscopic and histopathological data were examined in a subset. RESULTS: From May 2013 to May 2019, 118/1507 (7.8%) patients developed significant colitis; 80/553 (14.5%) after PD1+CTLA-4, 35/1000 (3.5%) PD1 alone, and three patients after Ipilimumab (IPI) alone. Combination therapy-induced colitis was more frequent (14.5% vs 3.5% in PD1 alone, p=<0.0001), had an earlier onset (6.3 weeks vs 25.7 weeks, p=<0.001), was more severe (grade 3/4 69% vs 31%, p=<0.001), and are more likely to require higher doses of steroids (91% vs 74%, p=0.01) than PD1 colitis. Among all patients treated with steroids (N=114), 54 (47%) responded and required no further therapy (steroid sensitive), 47 patients (41%) responded to infliximab (infliximab sensitive), and 13 (11%) were infliximab refractory and needed further immunosuppressive drugs. Infliximab-refractory patients all had onset within 4 weeks of immunotherapy commencement and were more likely to have an underlying autoimmune disease, have higher grade colitis, and require longer immunosuppression, yet had similar response and survival than other patients with colitis. Of 43 (37%) patients re-resumed treatment with PD1 monotherapy after colitis resolution, 16 (37%) of whom developed recurrent colitis. Endoscopic and histopathologic data were available for 64 patients. Most had left-sided colitis, with an increase in chronic inflammatory cells and neutrophils within the lamina propria, an increase in neutrophils in the surface epithelium, without increased lymphocytes or increased eosinophils. Infliximab-refractory colitis had a trend towards more confluent pancolitis with edema, erythema, ulceration, and absent vascularity with neutrophilic infiltration and erosion. CONCLUSION: Clinically significant colitis varies in presentation, response to immunosuppression, and endoscopic/histologic features depending on the immunotherapy type. Infliximab-refractory colitis occurs early, is often high grade, and has adverse endoscopic and histopathologic features.

Serum CA19-9 is significantly upregulated up to 2 years before diagnosis with pancreatic cancer: implications for early disease detection.

PURPOSE: Biomarkers for the early detection of pancreatic cancer are urgently needed. The primary objective of this study was to evaluate whether increased levels of serum CA19-9, CA125, CEACAM1, and REG3A are present before clinical presentation of pancreatic cancer and to assess the performance of combined markers for early detection and prognosis. EXPERIMENTAL DESIGN: This nested case-control study within the UKCTOCS included 118 single and 143 serial serum samples from 154 postmenopausal women who were subsequently diagnosed with pancreatic cancer and 304 matched noncancer controls. Samples were split randomly into independent training and test sets. CA19-9, CA125, CEACAM1, and REG3A were measured using ELISA and/or CLIA. Performance of markers to detect cancers at different times before diagnosis and for prognosis was evaluated. RESULTS: At 95% specificity, CA19-9 (>37 U/mL) had a sensitivity of 68% up to 1 year, and 53% up to 2 years before diagnosis. Combining CA19-9 and CA125 improved sensitivity as CA125 was elevated (>30 U/mL) in approximately 20% of CA19-9-negative cases. CEACAM1 and REG3A were late markers adding little in combined models. Average lead times of 20 to 23 months were estimated for test-positive cases. Prediagnostic levels of CA19-9 and CA125 were associated with poor overall survival (HR, 2.69 and 3.15, respectively). CONCLUSIONS: CA19-9 and CA125 have encouraging sensitivity for detecting preclinical pancreatic cancer, and both markers can be used as prognostic tools. This work challenges the prevailing view that CA19-9 is upregulated late in the course of pancreatic cancer development.

Identification of potential serum peptide biomarkers of biliary tract cancer using MALDI MS profiling.

BACKGROUND: The aim of this discovery study was the identification of peptide serum biomarkers for detecting biliary tract cancer (BTC) using samples from healthy volunteers and benign cases of biliary disease as control groups. This work was based on the hypothesis that cancer-specific exopeptidases exist and that their activities in serum can generate cancer-predictive peptide fragments from circulating proteins during coagulation. METHODS: This case control study used a semi-automated platform incorporating polypeptide extraction linked to matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) to profile 92 patient serum samples. Predictive models were generated to test a validation serum set from BTC cases and healthy volunteers. RESULTS: Several peptide peaks were found that could significantly differentiate BTC patients from healthy controls and benign biliary disease. A predictive model resulted in a sensitivity of 100% and a specificity of 93.8% in detecting BTC in the validation set, whilst another model gave a sensitivity of 79.5% and a specificity of 83.9% in discriminating BTC from benign biliary disease samples in the training set. Discriminatory peaks were identified by tandem MS as fragments of abundant clotting proteins. CONCLUSIONS: Serum MALDI MS peptide signatures can accurately discriminate patients with BTC from healthy volunteers.

Pyruvate kinase M2 is a novel diagnostic marker and predicts tumor progression in human biliary tract cancer.

BACKGROUND: The early diagnosis of biliary tract cancer (BTC) remains challenging, and there are few effective therapies. This study investigated whether the M2 isotype of pyruvate kinase (M2-PK), which serves as the key regulator of cellular energy metabolism in proliferating cells, could play a role in the diagnosis and therapy of BTC. METHODS: Plasma and bile M2-PK concentrations were measured by enzyme-linked immunosorbent assay in 88 patients with BTC, 79 with benign biliary diseases, and 17 healthy controls. M2-PK expression was assayed in a BTC tissue array by immunohistochemistry. The role of M2-PK in tumor growth, invasion, and angiogenesis was evaluated in BTC cell lines by retrovirus-mediated M2-PK transfection and short hairpin RNA silencing techniques. RESULTS: Sensitivity (90.3%) and specificity (84.3%) of bile M2-PK for malignancy were significantly higher than those for plasma M2-PK and serum carbohydrate antigen 19-9. M2-PK expression was specific for cancer cells and correlated with microvessel density. M2-PK positivity was a significant independent prognostic factor by multivariable analysis. Transfection of M2-PK in a negatively expressed cell line (HuCCT-1 cells) increased cell invasion, whereas silencing in an M2-PK-positive cell line (TFK cells) decreased tumor nodule formation and cellular invasion. A significant increase in endothelial tube formation was noted when supernatants from M2-PK-transfected cells were added to an in vitro angiogenesis assay, whereas supernatants from silenced cells negated endothelial tube formation. CONCLUSIONS: Bile M2-PK is a novel tumor marker for BTC and correlates with tumor aggressiveness and poor outcome. Short hairpin RNA-mediated inhibition of M2-PK indicates the potential of M2-PK as a therapeutic target.

Whole genome RNA expression profiling of endoscopic biliary brushings provides data suitable for biomarker discovery in cholangiocarcinoma.

BACKGROUND & AIMS: Molecular analyses of biliary brushings using microarray and qPCR have the potential to provide valuable information on the biology of biliary diseases. Microarray analysis of biliary strictures has rarely been applied to endoscopic biliary brushings. METHODS: Biliary brushings were obtained from patients with benign and malignant biliary disease at the time of ERCP. Microarray analysis of mRNA isolated using brushings from 10 patients was validated for a selection of genes by qPCR using the same source mRNA and a second fresh set of nine biliary brushings as well as surgical resection tissue. Cultured cholangiocytes were used to assess the impact of bile or X-ray contrast solution on RNA quality. RESULTS: RNA was of variable quantity (100-1500 ng) and poor quality (Agilent RNA Integrity Number (RIN)<5, estimated to be fragments 100 to 600 base pairs long). Reliable qPCR results required primer pairs designed to produce amplicons <130 bp. Differential gene expression by microarray analysis identified 1140 up-regulated genes and 1001 down-regulated genes between benign and malignant biliary strictures. The trends in a selection of 45 up-regulated genes, including various HOX genes, collagens, PVT1, MUC4, MUC5AC, and LEF1, were validated by qPCR using RNA from biliary strictures with a moderate to strong correlation coefficient between microarray and qPCR (r=0.41 to r=0.57). Immunohistochemistry of surgical resection tissues (n=23) showed elevated CD9, SERPINA3, and PNMA2 protein expression in cancer samples. CONCLUSIONS: RNA isolated from biliary brushings is suitable for molecular analysis of biliary diseases using qPCR and microarray.

Endoscopic retrograde cholangiography does not reliably distinguish IgG4-associated cholangitis from primary sclerosing cholangitis or cholangiocarcinoma.

BACKGROUND & AIMS: Distinction of immunoglobulin G4-associated cholangitis (IAC) from primary sclerosing cholangitis (PSC) or cholangiocarcinoma is challenging. We aimed to assess the performance characteristics of endoscopic retrograde cholangiography (ERC) for the diagnosis of IAC. METHODS: Seventeen physicians from centers in the United States, Japan, and the United Kingdom, unaware of clinical data, reviewed 40 preselected ERCs of patients with IAC (n = 20), PSC (n = 10), and cholangiocarcinoma (n = 10). The performance characteristics of ERC for IAC diagnosis as well as the κ statistic for intraobserver and interobserver agreement were calculated. RESULTS: The overall specificity, sensitivity, and interobserver agreement for the diagnosis of IAC were 88%, 45%, and 0.18, respectively. Reviewer origin, specialty, or years of experience had no statistically significant effect on reporting success. The overall intraobserver agreement was fair (0.74). The operating characteristics of different ERC features for the diagnosis of IAC were poor. CONCLUSIONS: Despite high specificity of ERC for diagnosing IAC, sensitivity is poor, suggesting that many patients with IAC may be misdiagnosed with PSC or cholangiocarcinoma. Additional diagnostic strategies are likely to be vital in distinguishing these diseases.

R0 but not R1/R2 resection is associated with better survival than palliative photodynamic therapy in biliary tract cancer.

BACKGROUND: There is a need for better management strategies to improve the survival and quality of life in patients with biliary tract cancer (BTC). AIM: To assess prognostic factors for survival in a large, non-selective cohort of patients with BTC. METHOD: We compared outcomes in 321 patients with a final diagnosis of BTC (cholangiocarcinoma n = 237, gallbladder cancer n = 84) seen in a tertiary referral cancer centre between 1998 and 2007. Survival according to disease stage and treatment category was compared using log-rank testing. Cox's regression analysis was used to determine independent prognostic factors. RESULTS: Eighty-nine (28%) patients underwent a surgical intervention with curative intent, of whom 38% had R0 resections. Among the 321 patients, 34% were given chemo- and/or radiotherapy, 14% were palliated with photodynamic therapy (PDT) and 37% with biliary drainage procedures alone. The overall median survival was 9 months (3-year survival, 14%). R0-resective surgery conferred the most favourable outcome (3-year survival, 57%). Although patients palliated with PDT had more advanced clinical T-stages, their survival was similar to those treated with attempted curative surgery but who had positive resection margins. On multivariable analysis, treatment modality, serum carbohydrate-associated antigen 19-9, distant metastases and vascular involvement were independent prognostic indicators of survival. CONCLUSION: In this large UK series of BTC, palliative PDT resulted in survival similar to those with curatively intended R1/R2 resections. Surgery conferred a survival advantage only in patients with R0 resection margins, emphasising the need for accurate pre-operative staging.

Endoscopic retrograde pancreatography criteria to diagnose autoimmune pancreatitis: an international multicentre study.

BACKGROUND: Characteristic pancreatic duct changes on endoscopic retrograde pancreatography (ERP) have been described in autoimmune pancreatitis (AIP). The performance characteristics of ERP to diagnose AIP were determined. METHODS: The study was done in two phases. In phase I, 21 physicians from four centres in Asia, Europe and the USA, unaware of the clinical data or diagnoses, reviewed 40 preselected ERPs of patients with AIP (n=20), chronic pancreatitis (n=10) and pancreatic cancer (n=10). Physicians noted the presence or absence of key pancreatographic features and ranked the diagnostic possibilities. For phase II, a teaching module was created based on features found most useful in the diagnosis of AIP by the four best performing physicians in phase I. After a washout period of 3 months, all physicians reviewed the teaching module and reanalysed the same set of ERPs, unaware of their performance in phase I. RESULTS: In phase I the sensitivity, specificity and interobserver agreement of ERP alone to diagnose AIP were 44, 92 and 0.23, respectively. The four key features of AIP identified in phase I were (i) long (>1/3 the length of the pancreatic duct) stricture; (ii) lack of upstream dilatation from the stricture (<5 mm); (iii) multiple strictures; and (iv) side branches arising from a strictured segment. In phase II the sensitivity (71%) of ERP significantly improved (p<0.05) without a significant decline in specificity (83%) (p>0.05); the interobserver agreement was fair (0.40). CONCLUSIONS: The ability to diagnose AIP based on ERP features alone is limited but can be improved with knowledge of some key features.

Circulating CYFRA 21-1 is a Specific Diagnostic and Prognostic Biomarker in Biliary Tract Cancer.

BACKGROUND: Biliary tract cancer (BTC) has a poor prognosis, in part related to difficulties in diagnosis. Cytokeratin 19 (CK19) is a constituent of the intermediate filament proteins of epithelial cells. CK19 fragments (CYFRA 21-1) are rarely identified in the blood of healthy individuals. We assessed the utility of CYFRA 21-1 as a diagnostic and prognostic marker of BTC. METHODS: Blood was prospectively collected from patients with benign biliary disease (n = 39), primary sclerosing cholangitis (n = 19), PSC-related cholangiocarcinoma (n = 6) and sporadic BTC (n = 60). CYFRA 21-1 levels were measured in duplicate by ELISA. RESULTS: CYFRA 21-1 (≥ 1.5 ng/mL) had a sensitivity of 56% and specificity of 88%, compared with figures of 79% and 78% for CA 19-9 (≥ 37U/mL). Using a higher cut-off of 3 ng/mL, CYFRA 21-1 had a sensitivity of 30% and specificity of 97%. Combination of CYFRA 21-1 (≥ 1.5 ng/mL) and CA 19-9 (≥ 37 U/mL) resulted in sensitivity and specificity of 45% and 96%. In contrast to CA 19-9, CYFRA 21-1 (≥ 3.0 ng/mL) alone was a strong predictor of prognosis (median survival 2 months vs 10 months, p = 0.001). CONCLUSION: Elevated circulating CYFRA 21-1 is a specific, but less sensitive diagnostic marker than CA 19-9, predicts a poor outcome and may act as a surrogate marker of circulating tumor cells in BTC. Further prospective studies of its utility in assessing operability and response to chemotherapy are needed.

Presentation and management of post-treatment relapse in autoimmune pancreatitis/immunoglobulin G4-associated cholangitis.

BACKGROUND & AIMS: Autoimmune pancreatitis (AIP) is a multisystem disorder that often has extrapancreatic manifestations such as immunoglobulin G4-associated cholangitis (IAC). Patients respond rapidly to steroids but can relapse after therapy. We assessed the clinical management of relapse in a group of patients with AIP/IAC. METHODS: We performed a prospective study of patients diagnosed with AIP from 2004-2007 who received steroids. Treatment outcome was defined clinically, radiologically, and biochemically as response to steroids, remission after steroids, failure to wean steroids, and relapse. Steroids +/- azathioprine (AZA) were used to treat patients who failed, relapsed, or could not be weaned from steroids. RESULTS: Twenty-eight patients with AIP were studied; 23 (82%) had IAC. All patients responded within 6 weeks to prednisolone therapy. Twenty-three patients achieved remission after a median of 5 months of treatment (range, 1.5-17 months), whereas 5 patients (18%) could not be weaned because of a disease flare. Of the patients who achieved remission, 8 of 23 (35%) subsequently relapsed. Overall, 13 of 23 patients (57%) with AIP/IAC relapsed, compared with 0 of the 5 with isolated AIP (P = .04, Fisher exact test). Steroids were increased/restarted in all patients who relapsed; 10 also received AZA. Remission was achieved and maintained in 7 patients; they remain on AZA monotherapy at a median of 14 months (range, 1-27 months). CONCLUSIONS: Relapse or failure to wean steroids occurred in 46% of patients with AIP. Patients with IAC are at particularly high risk of relapse. AZA appears to be effective in patients with post-treatment relapse or who cannot be weaned from steroids. To view this article's video abstract, go to the AGA's YouTube Channel.

Autoimmune pancreatitis: clinical and radiological features and objective response to steroid therapy in a UK series.

OBJECTIVE: Most cases of autoimmune pancreatitis (AIP) have been reported from Japan. We present data on a UK series, including clinical and radiological features at presentation, and longitudinal response to immunosuppression. METHODS: Over an 18-month period, all patients diagnosed in our center with AIP were studied. Endoscopic biliary stenting was performed as required, and patients were treated with prednisolone, with response assessed longitudinally. In cases of disease relapse following steroid reduction, azathioprine was instituted. RESULTS: Eleven patients met diagnostic criteria for AIP. Diffuse pancreatic enlargement was seen in eight patients (73%), and pancreatic duct strictures in all. Seven patients required biliary stents. Extrapancreatic involvement occurred in all, including intrahepatic stricturing and renal disease. Eight weeks after starting steroids, the median serum bilirubin level had fallen from 38 mumol/L to 11 mumol/L (P= 0.001), and ALT from 97 IU/L to 39 IU/L (P= 0.002). Stents were removed in all cases, with no recurrence of jaundice. Improvements in mass lesions and pancreaticobiliary stricturing occurred in all patients. During a median 18-month follow-up, six patients relapsed, four of whom responded to azathioprine. Two patients discontinued steroids and remained well. CONCLUSIONS: Extrapancreatic disease was an important feature of AIP in this UK series. Initial response to immunosuppressive therapy was excellent, but disease relapse was common. Optimal long-term management remains to be established.

The use of immunoglobulin g4 immunostaining in diagnosing pancreatic and extrapancreatic involvement in autoimmune pancreatitis.

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/high-power 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.