Diagnosis of pancreatic neoplasms using a novel method of DNA methylation analysis of mucin expression in pancreatic juice.

Seiya Yokoyama,Kyoichi Takaori,Toru Niihara,Hiroto Nishimata,Dai Ikebe,Michiyo Higashi,Surinder K Batra,Yuko Goto,Taketo Yamaguchi,Taro Hara,Sho Kitamoto,Suguru Yonezawa,Yoshifumi Arisaka,Sadao Tanaka

doi:10.1371/journal.pone.0093760

Abstract

Mucins (MUC) play crucial roles in carcinogenesis and tumor invasion in pancreatic ductal adenocarcinoma (PDAC) and intraductal papillary mucinous neoplasms (IPMNs). Our immunohistochemistry (IHC) studies have shown a consensus position on mucin expression profiles in pancreatic neoplasms as follows: MUC1-positive but MUC2-negative expression in PDACs; MUC1-negative but MUC2-positive expression in intestinal-type IPMNs (dangerous type); MUC1-negative and MUC2-negative expression in gastric-type IPMNs (safe type); High MUC4 expression in PDAC patients with a poor outcome; and MUC4-positive expression in intestinal-type IPMNs. We also showed that three mucin genes (MUC1, MUC2 and MUC4) expression in cancer cell line was regulated by DNA methylation. We have developed a novel ‘methylation-specific electrophoresis (MSE)’ method to analyze the DNA methylation status of mucin genes by high sensitivity and resolution. By using the MSE method, we evaluated pancreatic juice samples from 45 patients with various pancreatic lesions. The results were compared with final diagnosis of the pancreatic lesions including IHC of mucin expression in the paired pancreatic tissues. The results indicated that the DNA methylation status of MUC1, MUC2 and MUC4 in pancreatic juice matched with the mucin expression in tissue. Analyses of the DNA methylation status of MUC1, MUC2 and MUC4 were useful for differential diagnosis of human pancreatic neoplasms, with specificity and sensitivity of 87% and 80% for PDAC; 100% and 88% for intestinal-type IPMN; and 88% and 77% for gastric-type IPMN, respectively. In conclusion, MSE analysis of human pancreatic juice may provide useful information for selection of treatment for pancreatic neoplasms.

Highlights

Patients with pancreatic ductal adenocarcinoma (PDAC) have a poor clinical outcome, despite improvements in diagnosis and treatment methods
A plot of the unmethylation index (U-index) for MUC1 against the mRNA level for MUC1 showed a significant correlation (R2 = 0.406, P,0.001) (Figure 1). This result indicates that the extent of DNA methylation status of MUC1 is a trigger for regulation of expression of MUC1 mRNA in pancreatic tissue
Analyses of the DNA methylation status of MUC1, MUC2 and MUC4 in pancreatic juices were useful for differential diagnosis of human pancreatic neoplasms i.e. PDAC, intestinal-type intraductal papillary mucinous neoplasms (IPMNs) and gastrictype IPMN, with high specificity and sensitivity

Summary

Introduction

Patients with pancreatic ductal adenocarcinoma (PDAC) have a poor clinical outcome, despite improvements in diagnosis and treatment methods. Resection at an early stage gives a relatively favorable outcome, but PDACs are diagnosed in an advanced stage in most cases [1]. Indolent neoplasms such as intraductal papillary mucinous neoplasms (IPMNs) occur in the pancreas [2] and sometimes transform into lesions with an invasive character and a poor outcome [3,4]. IPMNs are the most common cystic neoplasm of the pancreas, and are classified into gastric, intestinal, pancreatobiliary, and oncocytic types [2,6]. A recent study showed that the morphological subtype of IPMN is an independent prognostic factor: i.e. patients with gastric-type IPMN have a fair prognosis, those with intestinal-type or oncocytic-type IPMN have a relatively less favorable prognosis, and those with pancreatobiliary-type IPMN have the poorest prognosis [6]

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