Autoimmune pancreatitis (AIP) is a type of pancreatitis with presumed autoimmune etiology and is a relatively newly characterized disease entity, with much of our knowledge of it gained in only the last decade. However, it is increasingly being recognized as a bona fide disease. Autoimmune pancreatitis occurs most often in elderly males who experience obstructive jaundice as the most common initial symptom. Pancreatic exocrine and endocrine functions are sometimes impaired. Occasionally, there is an association with various extrapancreatic lesions. Radiologically, AIP is characterized by enlargement of the pancreas and irregular narrowing of the main pancreatic duct. Serologically, AIP is characterized by elevation of serum IgG4 levels. Histopathologically, dense infiltrations of CD4- or CD8-positive T lymphocytes and IgG4-positive plasma cells occur with fibrosis and obliterative phlebitis in the pancreas. Autoimmune pancreatitis responds well to steroid therapy, and both structural and functional changes in AIP recover promptly after steroid therapy. In contrast to ordinary chronic pancreatitis, AIP appears to be reversible.1-3 Although the precise long-term outcome for AIP remains unclear, the prognosis of AIP is generally better than that of chronic pancreatitis.2-4 However, several reports of AIP associated with pancreatic cancer occurring simultaneously or during follow-up have recently been described.5-8
Mutations in codon 12 of the K-ras gene have been found in more than 90% of pancreatic adenocarcinomas.9,10 K-ras mutation is also detected in mucous cell hyperplasia in chronic pancreatitis.11,12 K-ras mutation may occur during a relatively early stage in the multistep carcinogenesis process in the pancreas.9,11,12 Chronic pancreatitis is characterized by irreversible morphologic changes and is a generally accepted risk factor for pancreatic cancer.13 To assess the relationship between AIP and pancreatic cancer, we analyzed K-ras mutation in the pancreatobiliary tissues of 11 patients with AIP.
MATERIALS AND METHODS
Study Patients and Materials
Between 1989 and 2008, AIP was diagnosed in a total of 55 patients based on the Asian diagnostic criteria for AIP.14 No patients developed pancreatobiliary cancer during the mean ± SD follow-up period of 42.8 ± 25.4 months.
K-ras analysis and/or an immunohistochemical study were performed on tissue from 11 patients with AIP (9 men and 2 women; age range, 61-79 years; mean age, 68.9 ± 6.3 years). Histories of heavy drinking and smoking were observed in 0 and 2 patients, respectively. The initial symptom was obstructive jaundice due to stenosis of the common bile duct in 10 patients and abdominal pain in 1 patient (Table 1).
Study materials consisted of 6 pancreatoduodenectomied pancreases, common bile ducts, and gallbladders; surgical biopsy specimen of 1 pancreas; 2 surgically resected common bile ducts and gallbladders; 1 autopsied pancreas, common bile duct, and gallbladder; and 10 surgically resected pancreases from patients with chronic alcoholic pancreatitis. K-ras mutation was analyzed in samples of pure pancreatic juice collected from the pancreatic duct during endoscopic retrograde cholangiopancreatography with injection of secretin in 3 patients with AIP and in 20 patients with chronic alcoholic pancreatitis other than the 10 resected cases.
The surgically resected tissues, the tissue biopsy specimens, and the autopsied tissues were fixed in 10% neutral buffered formalin. Each 1-cm wide tissue section was examined. Tissue blocks were routinely processed and embedded in paraffin. Serial sections were cut at a thickness of 3 μm. All sections were stained with hematoxylin and eosin (H&E) and were examined immunohistochemically.
All subjects provided written informed consent. This study was approved by the relevant institutional review boards.
Immunohistochemistry was performed, on average, on 2 representative sections from each case using antibodies against IgG4 (The Binding Site, Birmingham, United Kingdom), Ki-67 (clone MIB-1; Immunotech SA, Marseille, France), p53 (CM1; Novocastra Laboratories, Newcastle, United Kingdom), and Foxp3 (clone 22509; Abcam, Oxford, United Kingdom). All sections were stained with the avidin-biotin horseradish peroxidase method (Vectastain elite ABC kit; Vector, Burlingame, Calif). Additional staining procedures used have all been previously reported.15,16 The degree of infiltrating IgG4-positive plasma cells was classified as 3+ (more than 30/high-power field [HPF]), 2+ (10-30/HPF), 1+ (5-10/HPF), +/− (1-4/HPF), and − (0/HPF).17 The Ki-67 labeling index was determined by counting a minimum of 500 cells in the area representing the most homogenous region of positive cells.
K-ras Mutation Analysis
Paraffin blocks were prepared for DNA extraction. Two lesions from 2 slides of the pancreatic duct, the common bile duct, and the gallbladder mucosal epithelia were used for DNA extraction from tissue from each patient. The target lesions, including the abnormal epithelium, were microdissected using a 20-gauge needle, comparing the slide with H&E staining in the same position. The extracted DNA was diluted with 5 mL of Takara DEXPAT (for DNA extraction from paraffin-embedded tissue; Takara Biomedical Inc, Otsu, Japan). DNA was extracted from the pancreatic juice by the standard phenol/chloroform method.
Mutation of K-ras codon 12 was analyzed and compared by enriched polymerase chain reaction-enzyme-linked mini-sequence assay (PCR-ELMA).18-20 In PCR-ELMA, the upstream primer for the first and second PCRs was 5′-TAAACTTGTGGTAGTTGGAACT-3′, the downstream primer for the first PCR was 5′-GTTGGATCATATTCGTCCAC-3′, and the downstream primer for the second PCR was 5′-CAAATGATCTGAATTAGCTG-3′. The first PCR reaction was performed using 1 μL of DNA lysate, 100 μmol/L of deoxyribonucleotide triphosphates, 1.5 mmol/L of MgCl2, 1 μmol/L of each primer, 0.625 U of Taq DNA polymerase (Perkin Elmer, Norwalk, Conn), and 1× PCR buffer (containing 10 mmol/L of Tris-HCl [pH 8.3 at 25°C], 50 mmol/L of KCl, and 0.001% [wt/vol] gelatin) in a thermal cycler (Perkin Elmer PJ-2000). Polymerase chain reaction amplifications proceeded at 95°C for 2 minutes, followed by 25 cycles at 95°C for 40 seconds, 60°C for 40 seconds, and 72°C for 40 seconds and a final extraction step of 7 minutes at 72°C. One microliter each of 10-fold dilutions of the first PCR-amplified product (93 base pairs) was taken for digestions with 0.5 μL (2.5 U) of BsrI (New England Biolabs, Inc, Ipswich, Mass), and the 3.5-μL reaction buffer was composed of 100 mmol/L of NaCl, 50 mmol/L of Tris-HCl, 10 mmol/L of MgCl2, and 1 mmol/L of dithiothreitol at 65°C for more than 15 hours in a total of 5 μL. For digestion, 45 μL of the second-stage reaction mixtures containing 1 μmol/L (each) of primers F1 and R2, and other components as those in the first-stage reaction mixtures were added to the previously mentioned restriction endonuclease reaction tubes. Subsequently, the second PCR amplifications were performed for 40 cycles with the same thermal cycle condition as the first PCR. Then, 10 μL of the denatured second PCR product was hybridized with probes to detect the K-ras codon 12 wild type (GGT) 6 mutants (GAT, GCT, GTT, AGT, CGT, and TGT). DNAs were immobilized at 55°C for 30 minutes, 100 μL of biotinalated A and 0.01 U of Tdq DNA polymerase were added, and incubation was continued at 55°C for 30 minutes.
To develop color, 100 μL of avidin-horseradish peroxidase conjugate was added, and the reaction was conducted at room temperature for 30 minutes. After a washing step, 100 μL of tetramethyl-benzidine substrate was added and the plates were left to develop in the dark at room temperature for 20 minutes. Finally, 100 μL of stop solution was decanted, and the light absorbance of each sample was measured by spectrophotometry (Multiskan Multisoft; Labsystems, Tokyo, Japan) with a 450-nm filter wavelength.
The amplified K-ras gene by PCR was captured to the probes that were designed to detect the K-ras codon 12 wild type (GGT) and 6 mutants (GAT, GTT, CGT, TGT, AGT, and GCT), which were finally measured using a microtiter plate reader for detection and quantification. The results of the semiquantitative analysis were scored as 3+, 2+, 1+, +/−, and − according to the percentage of mutant ras gene. Approximately, 3+, 2+, 1+, +/−, and − represented more than 20%, 2% to 20%, 0.2% to 2%, fewer than 0.2%, and none (not detected) of the mutants, respectively, according to the manufacturer.19,20
The degree of infiltration of IgG4-positive plasma cells and the frequencies of K-ras mutation were scored as 3, 2, 1, 0.5, and 0, and they were analyzed with a Mann-Whitney U test. All reported P values are 2 sided. A P < 0.05 was considered significant.
Histopathological and Immunohistochemical Findings
Histopathologically, marked lymphoplasmacytic infiltration, periductal and interlobular fibroses, obliterative phlebitis in the pancreas (Fig. 1A), and transmural fibrosis with marked lymphoplasmacytic infiltration in the common bile duct were detected in the tissues from all patients with AIP. Transmural fibrosis with lymphoplasmacytic infiltration was detected in the gallbladder wall of 5 patients (patients 1-4 and 7). Pancreatic intraepithelial neoplasia (PanIN)-1A21 (n = 6) and PanIN-1B (n = 2; patients 4 and 8) were focally detected. The pancreatic duct epithelium appeared almost normal in 2 patients (patients 3 and 6). There were no atypical changes in the epithelium of the common bile duct or the gallbladder in any patients. The mean (SD) Ki-67 labeling index was 2.0 ± 0.9% in the pancreatic duct, 1.5 ± 0.6% in the common bile duct, and 0.9 ± 0.5% in the gallbladder (Table 2). Dense infiltrations of IgG4-positive plasma cells (2+ or 3+) in the pancreas (Fig. 1B), the bile duct, and the gallbladder were detected in 100%, 89%, and 67% of patients, respectively (Table 3). Intensive fibroinflammation was detected in association with dense infiltration of IgG4-positive plasma cells. Foxp3-positive cells were distributed prominently in these fibroinflammatory lesions with many IgG4-positive plasma cells.
In chronic alcoholic pancreatitis, PanIN-1A and PanIN-1B were detected in all patients, and PanIN-2 was detected in 5 patients. The number of infiltrating IgG4-positive plasma cells was fewer than 5/HPF in the pancreas. The mean Ki-67 labeling index was 3.2 ± 0.9%. Overexpression of p53 was not observed in any specimen.
High-frequency K-ras mutation (2+ or 3+) was detected in the pancreas of all 8 patients and in the pancreatic juice of the other 2 patients. The mutant type of ras gene was GAT in all the 10 patients (Table 3; Fig. 2A). In patients with chronic alcoholic pancreatitis, high-frequency K-ras mutation was detected in 4 (GAT, n = 2; TGT, n = 1; and GAT/GCT, n = 1) of the 10 resected pancreases (40%) and in 2 (GAT, n = 1; GAT/GGT/GTT, n = 1) out of 20 (10%) of the samples of pancreatic juice.
High-frequency K-ras mutation was detected in the common bile duct of 5 patients with AIP (GAT, n = 3; TGT, n = 1; and GCT/TGT, n = 1; Fig. 2B) and in the gallbladder epithelium of 4 patients with AIP (GAT, n = 1; TGT, n = 1; GCT, n = 1; and GTT, n = 1; Fig. 2C). The 4 patients with high-frequency K-ras mutation in the gallbladder also showed high-frequency K-ras mutation in the common bile duct, but the mutations in the ras gene were different. Low-frequency K-ras mutation (+ or +/−) was detected in the common bile duct of 3 patients and in the gallbladder of 3 patients (Table 3).
Relationship Between Serum IgG4 Levels and Infiltration of IgG4-Positive Plasma Cells and K-ras Mutation in the Common Bile Duct and the Gallbladder
Serum IgG4 levels were elevated in 6 patients (>135 mg/dL; Table 3). The scores for the infiltration of IgG4-positive plasma cells in the common bile duct (3,3,3,3,3) and in the gallbladder (3,3,3,3,2) of patients with AIP with high serum IgG4 levels were significantly higher than the scores in the common bile duct (2,2,2) and in the gallbladder (2,1,1) of patients with normal serum IgG4 levels (P = 0.05 and P = 0.04, respectively).
The scores for the K-ras mutation in the common bile duct (3,3,3,3,3) and in the gallbladder (3,3,3,2) of patients with AIP with high serum IgG4 levels were significantly higher than the scores in the common bile duct (0.5, 0.5) and in the gallbladder (0.5,0.5,0.5) of patients with normal serum IgG4 levels (P = 0.02 and P = 0.04, respectively).
This study revealed 3 important, new findings regarding AIP.
First, high-frequency K-ras mutation was detected in the pancreas of all 8 patients with AIP and in the pancreatic juice of the other 2 patients with AIP. The mutant type of ras gene was GAT in all the 10 patients. On the other hand, in the patients with chronic alcoholic pancreatitis, high-frequency K-ras mutations in the resected pancreas and the pancreatic juice were only detected in 40% and 10% of the samples, respectively.
K-ras mutation is believed to occur at a relatively early stage during the multistep carcinogenesis process. K-ras mutation was found in mucous cell hyperplasia in chronic pancreatitis, a condition that is considered to be a risk factor for the development of pancreatic cancer.11-13 The incidence of K-ras mutation in chronic pancreatitis was reported to be 27%.22 According to Tada et al,23 K-ras mutation was detected in 19 (24%) of 79 cases of hyperplastic epithelium of the pancreas and in all 30 cases of pancreatic cancer. K-ras mutation was not detected in a normal pancreatic duct epithelium. Six mutant types of ras gene were detected in the hyperplastic epithelium of the pancreas including GAT (21%), GTT (21%), CGT (5%), TGT (37%), and AGT (16%), and the mutant types detected in pancreatic cancer were GAT (53%), GTT (33%), and CGT (14%).23 The frequencies and types of K-ras mutation detected in the pancreas of patients with AIP were quite different from those of the hyperplastic epithelium.
The cumulative risk of pancreatic cancer in subjects with chronic pancreatitis was reported to be 1.8% after 10 years and 4.0% after 20 years.13 Regarding AIP, 4 cases of pancreatic cancer associated with AIP have been reported in the English literature.5-8 Of these 4 cases, pancreatic cancer was diagnosed simultaneously with AIP in 2 cases,5,8 1 case of pancreatic cancer developed 5 years after pancreatoduodenectomy for AIP6 and the other developed 3 years after starting steroid therapy.7 We have experienced no cases of pancreatic cancer associated with AIP, but an additional 3 cases were reported in the Japanese literature.24-26 Localizations of these 7 pancreatic cancers were in the pancreatic head (n = 1), the body (n = 3), and the tail (n = 4). Recently, elevation of serum IgG4 levels was detected in 7.0%27 to 9.6%28 of patients with pancreatic cancer. Because AIP is a relatively rare and newly described type of pancreatitis, the prevalence of developing pancreatic cancer in AIP is currently unclear. However, in contrast to pancreatic cancer that typically occurs decades after the onset of chronic pancreatitis, pancreatic cancer associated with AIP developed during a much shorter time frame. This may be because K-ras mutation occurred far more frequently and significantly in the pancreatic ducts of patients with AIP compared with patients with chronic pancreatitis.
Our second significant finding is that high-frequency K-ras mutation was detected in the common bile duct epithelium of 5 patients with AIP and in the gallbladder epithelium of 4 patients with AIP. Four mutant types were detected in the biliary tract, although only GAT was detected in the pancreas. The incidence rates of K-ras mutation in patients with bile duct cancer and patients with gallbladder cancer was reported to be 20%29 to 100%30 and 38%31 to 55%,30 respectively. The mutant type in the bile duct and the gallbladder cancers was reported to be GAT in 50%32 to 80%30 and 86%30 of mutation-positive cases, respectively.
Sclerosing cholangitis is the most frequent extrapancreatic lesion seen in patients with AIP (40/55; 73% in our series). However, sclerosing cholangitis associated with AIP is quite different from primary sclerosing cholangitis, which sometimes develops into bile duct cancer, because it is responsive to steroid therapy and has abundant infiltrating IgG4-positive plasma cells in the intrahepatic bile duct wall in contrast to primary sclerosing cholangitis.33,34 Sclerosing cholecystitis was found in 24% of our patients with AIP. It is recently suggested that sclerosing cholangitis and cholecystitis occur via the same mechanism as AIP (called as IgG4-related sclerosing disease) because the histopathological findings are similar and patients with either condition respond well to steroid therapy.34,35 Recently, early bile duct cancer in a patient with sclerosing cholangitis with abundant infiltration of IgG4-positive plasma cells associated with AIP was histologically confirmed in the resected specimen.36 Because high-frequency K-ras mutation was found in the biliary tract of half of the patients with AIP we studied, sclerosing cholangitis and sclerosing cholecystitis associated with AIP may be a risk factor for biliary cancer.
A third important observation from our study is that the degrees of K-ras mutation in the bile duct and the gallbladder of patients with AIP were correlated with the degree of fibroinflammation with infiltration of IgG4-positive plasma cells. Pancreatic intraepithelial neoplasias, ductal precursor lesions giving rise to invasive pancreatic adenocarcinoma, are classified from low grade (PanIN-1) to high grade (PanIN-3).12,21 K-ras mutation is thought to be the initiating mutation responsible for PanIN-1 lesions.12 In cancer-associated PanIN lesions, there was a stepwise increase in K-ras mutation that correlated with the grade of dysplasia.37 However, abnormal changes in the pancreatic duct epithelium were less frequent in patients with AIP than in patients with chronic pancreatitis. The epithelia of the bile duct and the gallbladder of all 9 patients with AIP also showed no hyperplastic or dysplastic changes. Increased proliferation of cells and p53 overexpression were not detected in any specimen. K-ras mutation in patients with AIP may occur in a mechanism different from chronic pancreatitis.
Zen et al38 reported that forkhead box P3 (Foxp3)-positive regulatory T cells, producing interleukin 10 and transforming growth factor β, which was followed by IgG4 class switching and fibroplasias, were increasingly detected in the pancreas and the biliary tract of patients with AIP. The present study also demonstrated increasing infiltration of Foxp3-positive cells in the fibroinflammatory lesions with many IgG4-positive plasma cells. On the other hand, Foxp3-positive regulatory T cells were increased locally in pancreatic cancer.39 Furthermore, it was recently reported that Foxp3-positive regulatory T cells and inflammation played an essentially important role for K-ras-mediated lung tumorigenesis in mice.40
In our series, spontaneous improvement occurred in 3 patients with AIP, and 4 asymptomatic patients with AIP with segmental pancreatic enlargement have demonstrated no changes without steroid therapy during 1 to 2.5 years. Autoimmune pancreatitis is usually diagnosed at active stage, but IgG4-related fibroinflammation may have persisted subclinically in the pancreatobiliary regions for a long time. In patients with AIP, K-ras mutation may occur in the persistent fibroinflammatory pancreas and the biliary tract with Foxp3-positive cells.
This study is the first to reveal frequent and significant K-ras mutation in the pancreatobiliary regions of patients with AIP. The analyzed cases in this study are too small to be conclusive, but these findings suggest that AIP may be a risk factor of pancreatobiliary cancer. Further study of more cases and other sites of the pancreas including the mechanism of K-ras mutation should be necessary. However, because AIP may predispose patients to development of pancreatic or biliary cancer, diagnosis and follow-up of AIP should be conducted with careful attention to the possibility of pancreatobiliary cancer occurring simultaneously or within a short time after AIP diagnosis.
In conclusion, significant K-ras mutation was detected in the pancreas of all patients with AIP. The detection of K-ras mutation in the bile duct and the gallbladder was correlated with IgG4-related fibroinflammation with Foxp3-positive cells. Autoimmune pancreatitis may be a risk factor of pancreatobiliary cancer.
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