A novel sensitive detection method for DNA methylation in circulating free DNA of pancreatic cancer.

Keiko Shinjo,Genta Nagae,Hidemi Ito,Kenji Yamao,Hiroyuki Aburatani,Satoru Takahashi,Keisuke Katsushima,Kazuo Hara,Yoshiteru Murofushi,Shoji Tajima,Miho Suzuki,Yuta Umezu,Yutaka Kondo,Yusuke Okuno,Ichiro Takeuchi,Takayoshi Umeda,Keitaro Matsuo,Hiromu Suzuki

doi:10.1371/journal.pone.0233782

Abstract

Despite recent advances in clinical treatment, pancreatic cancer remains a highly lethal malignancy. In order to improve the survival rate of patients with pancreatic cancer, the development of non-invasive diagnostic methods using effective biomarkers is urgently needed. Here, we developed a highly sensitive method to detect DNA methylation in cell-free (cf)DNA samples based on the enrichment of methyl-CpG binding (MBD) protein coupled with a digital PCR method (MBD–ddPCR). Five DNA methylation markers for the diagnosis of pancreatic cancer were identified through DNA methylation microarray analysis in 37 pancreatic cancers. The sensitivity and specificity of the five markers were validated in another independent cohort of pancreatic cancers (100% and 100%, respectively; n = 46) as well as in The Cancer Genome Atlas data set (96% and 90%, respectively; n = 137). MBD–ddPCR analysis revealed that DNA methylation in at least one of the five markers was detected in 23 (49%) samples of cfDNA from 47 patients with pancreatic cancer. Further, a combination of DNA methylation markers and the KRAS mutation status improved the diagnostic capability of this method (sensitivity and specificity, 68% and 86%, respectively). Genome-wide MBD-sequencing analysis in cancer tissues and corresponding cfDNA revealed that more than 80% of methylated regions were overlapping; DNA methylation profiles of cancerous tissues and cfDNA significantly correlated with each other (R = 0.97). Our data indicate that newly developed MBD–ddPCR is a sensitive method to detect cfDNA methylation and that using five marker genes plus KRAS mutations may be useful for the detection of pancreatic cancers.

Highlights

Pancreatic cancer is the fourth leading cause of cancer deaths, of which the five-year survival rate is less than 5%, with the number of cases predicted to increase worldwide [1]
Of 485,577 Ilumina Infinium probes, we identified 5,575 probes that were highly methylated in pancreatic cancers but not methylated in histologically normal pancreatic tissues (n = 3)
Cell-free DNA is released from tumors into circulating blood and contains tumor specific mutations, methylation sites, and miRNAs

Summary

Introduction

Pancreatic cancer is the fourth leading cause of cancer deaths, of which the five-year survival rate is less than 5%, with the number of cases predicted to increase worldwide [1]. Serum carbohydrate antigen 19–9 (CA19-9) is a classical biomarker that is widely used for detecting pancreatic cancer It is inadequate because of its low sensitivity (41–86%) and specificity (33–100%) [2]. With regard to pancreatic cancer, DNA methylation markers, cyclin-dependent kinase inhibitor 2A (CDKN2A/p16), ras-associated domain family member 1 (RASSF1A) and neuronal pentraxin 2 (NPTX2) in cfDNA are considered diagnostic markers. None of these are sufficiently powerful in diagnosing pancreatic cancer since a relatively insensitive method (methylation-specific PCR) is used and/or these genes are frequently found methylated in other types of cancers [9, 10]

Methods

Results

Conclusion