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

Kazuhiro Yoshida, Takeshi Nagasaka, Yuzo Umeda, Takehiro Tanaka, Keisuke Kimura, Fumitaka Taniguchi, Tomokazu Fuji, Kunitoshi Shigeyasu, Yoshiko Mori, Hiroyuki Yanai, Takahito Yagi, Ajay Goel, Toshiyoshi Fujiwara

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2 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)1-13
Number of pages13
JournalJournal of Cancer Research and Clinical Oncology
DOIs
Publication statusAccepted/In press - Apr 19 2016

Fingerprint

Epigenomics
Methylation
Neoplasms
Mutation
Genetic Promoter Regions
Genes
DNA
DNA Methylation
Disease-Free Survival
Extracellular Matrix
Carcinogenesis
Immunohistochemistry
Staining and Labeling
Carcinoma

Keywords

  • Dysplasia
  • EFEMP1
  • Epigenetics
  • Invasive carcinoma
  • Methylation
  • Mucinous neoplasms

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

Cite this

@article{a35d78f3317c4dbda445f503d7057363,
title = "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",
author = "Kazuhiro Yoshida and Takeshi Nagasaka and Yuzo Umeda and Takehiro Tanaka and Keisuke Kimura and Fumitaka Taniguchi and Tomokazu Fuji and Kunitoshi Shigeyasu and Yoshiko Mori and Hiroyuki Yanai and Takahito Yagi and Ajay Goel and Toshiyoshi Fujiwara",
year = "2016",
month = "4",
day = "19",
doi = "10.1007/s00432-016-2164-x",
language = "English",
pages = "1--13",
journal = "Journal of Cancer Research and Clinical Oncology",
issn = "0171-5216",
publisher = "Springer Verlag",

}

TY - JOUR

T1 - Expansion of epigenetic alterations in EFEMP1 promoter predicts malignant formation in pancreatobiliary intraductal papillary mucinous neoplasms

AU - Yoshida, Kazuhiro

AU - Nagasaka, Takeshi

AU - Umeda, Yuzo

AU - Tanaka, Takehiro

AU - Kimura, Keisuke

AU - Taniguchi, Fumitaka

AU - Fuji, Tomokazu

AU - Shigeyasu, Kunitoshi

AU - Mori, Yoshiko

AU - Yanai, Hiroyuki

AU - Yagi, Takahito

AU - Goel, Ajay

AU - Fujiwara, Toshiyoshi

PY - 2016/4/19

Y1 - 2016/4/19

N2 - 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.

AB - 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.

KW - Dysplasia

KW - EFEMP1

KW - Epigenetics

KW - Invasive carcinoma

KW - Methylation

KW - Mucinous neoplasms

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U2 - 10.1007/s00432-016-2164-x

DO - 10.1007/s00432-016-2164-x

M3 - Article

C2 - 27095449

AN - SCOPUS:84964381118

SP - 1

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JO - Journal of Cancer Research and Clinical Oncology

JF - Journal of Cancer Research and Clinical Oncology

SN - 0171-5216

ER -