Pan-cancer analysis of DNA epigenetic modifications by hydrophilic interaction liquid chromatography-tandem mass spectrometry

Accumulating evidence in recent years indicates that DNA methylation (5-methyl-2'-deoxycytidine, 5-mdC) and hydroxymethylation (5-hydroxymethyl-2'-deoxycytidine, 5-hmdC) have been implicated in various biological processes, and the aberrations of these DNA cytosine modifications is tightly associ-ated with cancer. N6-methyl-2'-deoxyadenosine (m6dA), as a newly discovered epigenetic modification in genome of mammals, has been demonstrated to play vital regulatory roles in tumorigenesis. How-ever, the content information of m6dA in human tumor tissues is still limited and pan-cancer analysis of these DNA epigenetic modifications is lacked. Herein, we developed a sensitive and robust stable isotope-diluted hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method for accurate quantification of m6dA, 5-mdC and 5-hmdC in genomic DNA from 82 pairs of human tumor tissues and matched tumor-adjacent normal tissues. The types of tumors included esophagus cancer, lung cancer, breast cancer, liver cancer, pancreatic cancer, gastric cancer, stromal tumor and colorectal cancer. Compared to the normal tissues, we revealed the level of m6dA was increased in tumor tissues of esoph-agus cancer, lung cancer and liver cancer, whereas the level of m6dA was diminished in tumor tissues of pancreatic cancer and gastric cancer; while the contents of 5-mdC and 5-hmdC exhibited significant decrease in tumor tissues of most types of cancer. It is worth noting that we revealed, for the first time, the content of genomic m6dA in pancreatic cancer, stromal tumor and colorectal cancer. The significant changes of these DNA epigenetic modifications indicate they may serve as indicators of cancers. In ad-dition, this study will benefit for better understanding of the regulatory roles of these DNA epigenetic modifications in cancers.(c) 2023 Published by Elsevier B.V. on behalf of Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences.

Automated summary

Accumulating evidence suggests that DNA methylation, hydroxymethylation, and m6dA play important regulatory roles in tumorigenesis and their aberrations are closely associated with cancer. This study developed a method for accurate quantification of these DNA modifications in human tumor tissues and revealed significant changes in m6dA, 5-mdC, and 5-hmdC levels in different types of cancer.