EUS-FNA Mutational Analysis in Differentiating Autoimmune Pancreatitis and Pancreatic Cancer

Original Paper Received: March 21, 2011 Accepted after revision: August 8, 2011 Published online: October 11, 2011

Pancreatology 2011;11:482–486 DOI: 10.1159/000331505

EUS-FNA Mutational Analysis in Differentiating Autoimmune Pancreatitis and Pancreatic Cancer Asif Khalid a, b John Dewitt c N. Paul Ohori a Jey-Hsin Chen c Kenneth E. Fasanella a Michael Sanders a Kevin M. McGrath a Marina Nikiforova a  

 

 

 

 

 

 

 

a c

University of Pittsburgh Medical Center and b VA Pittsburgh Health Care System, Pittsburgh, Pa., and Indiana University Medical Center, Indianapolis, Ind., USA

 

 

 

Key Words Pancreatic cancer ⴢ Autoimmune pancreatitis ⴢ Endoscopic ultrasound ⴢ K-ras

Abstract Background/Aims: Autoimmune pancreatitis (AIP) may mimic pancreatic cancer (PC). The detection of DNA mutations in endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) material may improve discrimination between AIP and PC and is the context for this study. Methods: In a retrospective study, archived EUS-FNA material from patients with AIP and PC at two centers was analyzed for KRAS mutations and loss-of-heterozygosity analysis involving 18 microsatellite markers. KRAS status and the fractional allelic loss (number of affected microsatellites divided by informative ones) were compared for AIP and PC. Results: Thirty-two patients with 33 samples were studied. There were 16 patients with AIP (17 samples) and 16 patients with PC. DNA amplification failed in 7 samples. Of 25 patients (26 samples), 14 had AIP (7 male, age 57 8 17 years; mean 8 SD) and 11 had PC (7 male, age 65 8 14 years; mean 8 SD). Cytology results for AIP were inflammatory = 3, inconclusive = 10, suspicious for malignancy = 2 and for PC were malignant = 5, suspicious for malignancy = 4 and inconclusive = 2, respec-

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tively. KRAS mutation was detected in none of the AIP cases and 10/11 PC cases (91%, Pearson ␹2 = 22.16, p ! 0.001) or 10/16 PC cases (63%) accounting for PC cases with failed DNA amplification. Mean (8SD) fractional allelic loss for the AIP cases (0.16 8 0.15) was not significantly different from the PC cases (0.26 8 0.19). Conclusions: A KRAS mutation in EUS/ FNA material from a pancreatic mass is associated with malignancy and may help discriminate from benign conditions such as AIP. Copyright © 2011 S. Karger AG, Basel and IAP

Introduction

Autoimmune pancreatitis (AIP) is a chronic fibroinflammatory disorder that responds to corticosteroid therapy [1]. Recently, two subtypes of AIP have been described based on distinct clinicopathological features: type I AIP (lymphoplasmacytic sclerosing pancreatitis, LPSP) and type II AIP (idiopathic duct centric pancreatitis, IDCP) [1, 2]. While AIP (type I and II) may have a spectrum of clinical and radiological presenting features [2, 3], the challenge remains differentiation from pancreatic cancer (PC). As such, an accurate diagnosis of AIP is necessary to avoid therapy otherwise reserved for PC, Asif Khalid, MD Division of Gastroenterology, M2, c-wing, PUH 200 Lothrop Street Pittsburgh, PA 15213 (USA) Tel. +1 412 648 9592, E-Mail khalida @ upmc.edu

and to permit timely treatment of PC. Unfortunately, proposed diagnostic criteria, imaging and serum markers remain imperfect tools in distinguishing AIP and PC [4–8]. A recent comparison [6] of the approaches utilizing Japanese [7] and the Mayo Clinic criteria [8] highlighted these weaknesses and suggested that ⬃30% of AIP cases require either a trial of steroid therapy or pathological confirmation for diagnosis. Endoscopic ultrasound (EUS)-guided core biopsy has been used in some centers to obtain a sufficiently large specimen for pathological diagnosis of AIP but the expertise is not widely available [9, 10]. EUS-fine needle aspiration (FNA) is an established technique to make a malignant cytological diagnosis in suspected PC. The accuracy of EUS-FNA cytology for PC is approximately 80% [11]. A variety of ancillary techniques have been used to increase the sensitivity of EUSFNA cytology with variable results including the detection of DNA mutations. A number of DNA abnormalities including KRAS oncogene mutation, tumor suppressor gene losses and telomerase activity have been detected in malignant pancreatic EUS-FNA samples and used to differentiate from benign or inflammatory pancreatic conditions [12–16]. The role of detecting these DNA abnormalities in differentiating AIP from PC has not been studied.

DNA Analysis All molecular analyses were performed at the Molecular Anatomic Pathology Lab at the University of Pittsburgh. All the EUSFNA cytology material from the pancreatic abnormality of each patient was evaluated and manually microdissected by one expert cytologist under a microdissection microscope targeting the most representative cells to avoid blood and luminal contaminant cells. DNA was isolated from the microdissected cells using a special kit designed for DNA extraction from small samples. DNA PCR amplification was performed for (1) direct sequencing for KRAS codon 12 and 13 mutations and, (2) loss-of-heterozygosity (LOH) analysis targeting tumor suppressor genes known to be affected in PC [12, 16]. Eighteen microsatellite markers on chromosomal arms 1, 3, 5, 9, 10, 11, 17, 21 and 22 were selected and are detailed in table 2. To detect LOH, mean of previously validated LOH peak ratios from normal blood lymphocytes or normal tissues for each microsatellite marker was used. Each microsatellite marker was detected individually. Fluorescent capillary electrophoresis was utilized to measure LOH, as described previously [16]. As an estimate of the amount of allelic imbalance in a sample, the fractional allelic loss was calculated by dividing the number of microsatellites affected by the total number of informative microsatellite markers [12]. Statistical Analysis Continuous variables such as patient age are presented as mean 8 SD. Differences in categorical variables in the PC and AIP groups were compared using the ␹2 test. The Student’s unpaired t test was utilized in case of continuous variables. A twotailed p value of !0.05 was considered statistically significant. All analyses were performed using the STATA statistical software package (STATA, version 9.0; Stata Corp., College Station, Tex., USA).

Methods and Materials This retrospective study was undertaken at two tertiary care academic medical centers (The University of Pittsburgh Medical Center and Indiana University Medical Center) with approval from the respective institutional review boards. Patients The EUS database at the two participating centers was used to identify patients undergoing EUS-FNA for suspicion of PC that were ultimately proven to have AIP. For the inclusion of patients with AIP, at least one of the following two criteria had to be met: (1) confirmatory histology (resection or core biopsy), and/or (2) resolution of radiographic abnormality and symptoms with steroid therapy. Patients with confirmed PC and available EUSFNA cytology were included as controls. Effort was made to include the same number of patients with PC that had malignant and inconclusive cytology results. Data on the clinical presentation, lab and imaging findings, therapy and follow-up were included. AIP Pathological Diagnosis AIP resection and core biopsy specimens were reviewed by an expert pathologist at each institution to (1) confirm the diagnosis, and (2) classify the case as LPSP or IDCP based on recently published criteria [1–3].

Molecular Analysis in AIP and Pancreatic Cancer

Results

Patients A total of 33 FNA samples from 32 patients were included in the study. Sixteen patients (17 samples) had AIP and 16 PC. DNA amplification failed in 7 samples. Therefore, results are presented on the remaining 26 samples from 25 patients. Of the 25 patients, 14 patients had AIP and 11 had PC. The mean age and sex ratio for patients in the AIP group (age 57 8 17 years; mean 8 SD; 7 males) and PC group (age 65 8 14 years; mean 8 SD; 7 males) were not significantly different (table 1). AIP Patients Key features of the patients with AIP are presented in table  1. Nine of 14 patients reached a diagnosis of AIP based on pathological evaluation of resection specimens (6 patients) or EUS-guided core biopsy (2 patients) or laparoscopic-guided core biopsy (1 patient). The remaining 5 patients responded to corticosteroid therapy with resoPancreatology 2011;11:482–486

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Table 1. Demographic and clinical data on the autoimmune pancreatitis cases

Patient Age

Sex

Presenting complaint

1 2 3 4 5 6 7 8 9

75 70 73 79 70 53 35 32 37

male male female male male female female female male

pain weight loss pain/jaundice/weight loss jaundice/weight loss jaundice pancreatitis/anorexia pain/weight loss pain weight loss

10 11 12 13 14

41 55 65 66 61

female male female male female

pain pain/jaundice pain jaundice/DM/weight loss nausea/vomiting

Serum IgG4a

normal elevated >2! elevated >1! normal normal normal normal

elevated >2! normal

Location of pancreatic FNA

Cytology

Pathologyb

AIP subtypeb

neck/body/tail body/tail head head head tail neck body/tail tail tail head head head head body

benign benign benign suspicious benign benign atypical atypical suspicious atypical benign malignant atypical benign atypical

distal panc. distal panc. Whipple Whipple

LPSP LPSP LPSP LPSP

lap. Bx core biopsy

LPSP LPSP

core biopsy Whipple core biopsy

LPSP LPSP LPSP

DM = Diabetes mellitus; distal panc. = distal pancreatectomy; lap. Bx = laparoscopic biopsy. a Serum IgG4 provided for cases where available. Over 140 mg/dl considered elevated. b The pathology and AIP subtype information provided for cases where available.

lution of symptoms and radiographic abnormality. All patients with available resection pathological specimens and core biopsies were classified as LPSP. A total of 15 separate FNA samples from 14 patients with AIP were submitted for molecular analysis. A majority of patients with AIP had inconclusive cytology results (10 of 15 samples), 3 of 15 cytology samples were read as consistent with an inflammatory process and 2 of 15 samples were read as suspicious for malignancy (table 1). PC Patients All 11 patients with PC reached a diagnosis based on either surgical pathology (9 patients) or positive cytology and typical course (2 patients). Five of 11 cytology samples from PC were read as positive for malignancy, 4 of 11 suspicious for malignancy and 2 of 11 as inconclusive. Molecular Analysis KRAS mutation was detected in none of the AIP cases and 10/11 PC cases (91%, Pearson ␹2 = 22.16, p ! 0.001) with sensitivity 91%, specificity 100%, positive predictive value 100% and negative predictive value 94% for diagnosing PC. The 1 patient with PC and no KRAS mutation also did not manifest any microsatellite loss. The FNA 484

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cytology was read as inconclusive, revealing atypical cells but no malignant cells. If the PC samples that failed to amplify are included in the analysis as a true measure of clinical utility the sensitivity decreases to 63%. Data on individual microsatellite loss in the AIP and PC cases are presented in table 2. The mean (8 SD) fractional allelic loss for the AIP cases (0.16 8 0.15) was not significantly different from the PC cases (0.26 8 0.19, table 2). Discussion

With recent advances in our understanding of AIP, it is evident that patients carrying this diagnosis may have a heterogeneous presentation spectrum with varied organ involvement, different pathological and serological features and nonuniform response to therapy. Proposed diagnostic algorithms continue to remain inaccurate in differentiating AIP from PC with potential for misdiagnosis. Tissue confirmation of AIP in support of the clinical diagnosis followed by response to steroid therapy may be optimum but EUS-guided core biopsy of the pancreas may not always be available or feasible. As such ancillary techniques to improve the accuracy of EUS-FNA cytolKhalid /Dewitt /Ohori /Chen /Fasanella / Sanders /McGrath /Nikiforova  

 

 

 

 

 

 

 

Table 2. Summary of the molecular data categorized by disease

entity Microsatellite

Location/gene

D1S171 1p/CMM/MYC L D1S407 3S1516 3p/VHL D3S1539 D5S615 5q/APC D5S1384 D9S251 9p/CDKN2A/p16 D9S1748 D9S1851 9q/PTCH D10S520 10q/PTEN D10S1173 D11S387 11q/MEN1 D11S916 D17S974 17p/TP53 D17S1289 D17S1877 17q/NM-23 D21S1256 21q/ETS2 D22S268 22q/NF2 KRAS point mutationa Mean FAL 8 SD a

Autoimmune pancreatitis

PC

3/15 samples

3/11 samples

4/15 samples

4/11 samples

1/15 samples

3/11 samples

1/15 samples

4/11 samples

0/15 samples 9/15 samples

0/11 samples 5/11 samples

1/15 samples

3/11 samples

5/15 samples

3/11 samples

0/15 samples 1/15 samples 1/15 samples 0/15 samples 0.1680.15

0/11 samples 2/11 samples 3/11 samples 10/11 samples 0.2680.19

p < 0.001.

ogy in differentiating benign versus malignant pancreatic aspirates are desirable. Over the last decade a number of studies have shown the utility of detecting key mutational abnormalities (KRAS oncogene mutations, tumor suppressor gene loss affecting P53, p16/INK, SMAD4, etc.) in improving the yield of cytology in the context of brushings from biliary strictures [16], FNA of pancreatic masses [12] and pancreatic cysts [14, 15]. This is the first study investigating the role of molecular markers in cytological material in differentiating AIP from PC. The importance of studying such approaches is evident in the number of inconclusive (10/15) and even malignant (2/15) cytology interpretations in the AIP group, and the number of patients with AIP that underwent resection due to the concern for PC (6/14), even though this is a highly selected group of patients. The key finding was that all except one PC aspirate manifested a KRAS mutation, which was highly accurate in diagnosing PC. None of the aspirates from the AIP cases manifested a KRAS mutation. This would suggest that cases suspicious for PC but with inconclusive cytology and absent Molecular Analysis in AIP and Pancreatic Cancer

KRAS mutation should serve as a warning that a benign etiology such as AIP may be operative. The 1 patient with PC and without a KRAS mutation had no malignant cells on cytology, therefore the absence of a KRAS mutation may be due to the absence of any neoplastic tissue. The finding of high frequency of KRAS mutations in PC is consistent with reports from prior studies on detecting KRAS mutations in cytological material from PC and chronic pancreatitis [12, 16, 17]. Direct sequencing for KRAS oncogene mutations is relatively simple to perform in most equipped laboratories, is objective and highly sensitive. Further studies assessing the clinical impact of KRAS mutation analysis in distinguishing PC from AIP are needed. For unclear reasons, LOH affecting a number of different microsatellite markers was detected in FNA from AIP cases, and the fractional allelic loss was therefore not significantly different from the PC cases. This pattern contradicts prior studies [12, 16]. Differences in technique may be present, e.g. we did not perform whole genome amplification prior to LOH analysis as in prior studies. Additional reasons like dilution of lower level allelic loss in PC due to contaminating cells and an incomplete knowledge of molecular events that may be occurring in AIP may all be contributing factors leading to these findings. For example, LOH affecting 10q/PTEN and 17p/TP53 in 9 of 15 and 5 of 15 AIP cases, respectively, is unexpected. While pancreatic intraepithelial neoplasia lesions are known to occur in chronic pancreatitis and may lead to the detection of low-level LOH, the data for AIP in this context is not clear. Further larger studies involving other centers may be helpful in answering some of these questions. Limitations of this study include the retrospective design and small number of patients. Additionally, the failure to adequately amplify DNA in 22% of the patients is a major drawback and markedly reduces the clinical utility of this approach. It is possible that DNA amplification of freshly obtained samples may yield a higher rate of successful DNA amplification. Of the patients with histologically proven AIP, all had LPSP (type I). The lack of patients with IDCP (type II) in this study may limit the generalizability of the results. In conclusion, in this small experience, the detection of a KRAS mutation in EUS-FNA cytology supported the diagnosis of PC and its absence was associated with AIP. These results support the use of caution and additional testing in patients suspected to have PC but inconclusive cytology and absent KRAS mutation. This is a relatively simple and objective PCR analysis that should be able to be performed in most pathology departments. Pancreatology 2011;11:482–486

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Khalid /Dewitt /Ohori /Chen /Fasanella / Sanders /McGrath /Nikiforova