Expansion of epigenetic alterations in EFEMP1 promoter predicts malignant formation in pancreatobiliary intraductal papillary mucinous neoplasms

Abstract

Purpose: Although limited understanding exists for the presence of specific genetic mutations and aberrantly methylated genes in pancreatobiliary intraductal papillary mucinous neoplasms (IPMNs), the fundamental understanding of the dynamics of methylation expansion across CpG dinucleotides in specific gene promoters during carcinogenesis remains unexplored. Expansion of DNA methylation in some gene promoter regions, such as EFEMP1, one of the fibulin family, with tumor progression has been reported in several malignancies. We hypothesized that DNA hypermethylation in EFEMP1 promoter would expand with the tumor grade of IPMN.

Methods: A sample of 65 IPMNs and 30 normal pancreatic tissues was analyzed. IPMNs were divided into the following three subsets according to pathological findings: 31 with low-grade dysplasia (low grade), 11 with high-grade dysplasia (high grade), and 23 with associated invasive carcinoma (invasive Ca). Mutations in the KRAS or GNAS genes were analyzed by Sanger sequencing, and methylation status of two discrete regions within the EFEMP1 promoter, namely region 1 and region 2, was analyzed by bisulfite sequencing and fluorescent high-sensitive assay for bisulfite DNA (Hi-SA). Expression status of EFEMP1 was investigated by immunohistochemistry (IHC).

Results: KRAS mutations were detected in 39, 55, and 70 % of low-grade, high-grade, and invasive Ca, respectively. GNAS mutations were observed in 32, 55, and 22 % of low-grade, high-grade, and invasive Ca, respectively. The methylation of individual regions (region 1 or 2) in the EFEMP1 promoter was observed in 84, 91, and 87 % of low-grade, high-grade, and invasive Ca, respectively. However, simultaneous methylation of both regions (extensive methylation) was exclusively detected in 35 % of invasive Ca (p = 0.001) and five of eight IPMNs (63 %) with extensive methylation, whereas 20 of 57 (35.1 %) tumors of unmethylation or partial methylation of the EFEMP1 promoter region showed weak staining EFEMP1 in extracellular matrix (p = 0.422). In addition, extensive EFEMP1 methylation was particularly present in malignant tumors without GNAS mutations and associated with disease-free survival of patients with IPMNs (p < 0.0001).

Conclusions: Extensive methylation of the EFEMP1 gene promoter can discriminate invasive from benign IPMNs with superior accuracy owing to their stepwise accumulation of tumor progression.

Keywords: Dysplasia; EFEMP1; Epigenetics; Invasive carcinoma; Methylation; Mucinous neoplasms.

Fig. 1

Bisulfite sequencing of the discrete EFEMP1 gene promoter regions. a Schematic representation of the location of discrete EFEMP1 gene promoter regions and the result of bisulfite sequencing. The white and gray boxes denote untranslated and translated exon in the EFEMP1 gene, respectively. The red allow indicates the location of the EMBL_M1 motifs ‘TGACATCTGTTGGG’, a candidate of CTCF biding site. The black arrow indicates the transcriptional starting site. The blue box indicates the regions of which methylation status was analyzed by Nomoto et al. (2010). The green box indicates the regions of which methylation status was analyzed by Yue et al. (2007) and Sadr-Nabavi et al. (2009). Vertical lines indicate CpG sites; white circles represent unmethylated CpGs; and gray circles represent methylated and unmethylated CpGs observed by bisulfite direct sequencing. b Examples of bisulfite sequencing in region 1 and 2. Each CpG was categorized as unmethylated or methylated CpG. TpG denotes the CpG site consisting of unmethylated CpG only. C/TpG denotes the CpG site consisting of both methylated (CpG) and unmethylated CpGs (TpG)

Fig. 2

Methylation analysis of EFEMP1. a Results of EFEMP1 methylation by fluorescent Hi-SA. Blue arrows represent PCR fragments with non-methylation in HhaI sites. Red arrows represent methylated PCR fragments cleaved by HhaI. b Frequencies of EFEMP1 methylation according to pathological findings. Kaplan–Meier survival curves for disease-free survival (c), excluding a patient with remaining cancer at the resected margin, and overall survival (d) according to EFEMP1 promoter methylation status

Fig. 3

Expression analysis of EFEMP1. IHC staining of EFEMP1 in intraductal papillary mucinous neoplasms with strong staining (a), moderate staining (b), and weak staining (c). Association between EFEMP1 methylation status and IHC staining (d). EFEMP1, epidermal growth factor-containing fibulin-like extracellular matrix protein 1; IHC immunohistochemical; NA not available