Identification of differential DNA methylation alterations of ovarian cancer in peripheral whole blood based on within-sample relative methylation orderings

Leukocyte cell proportion changes affect the detection of cancer-associated aberrant DNA methylation alterations in peripheral blood samples. We aimed to detect cellular DNA methylation changes in ovarian cancer (OVC) blood samples avoiding the above-mentioned cell-composition effects. Based on the within-sample relative methylation orderings (RMOs) of CpG loci in leukocyte subtypes, we developed the Ref-RMO method to detect aberrant methylation alterations from OVC blood samples. Stable CpG pairs with consistent RMOs in different leukocyte subtypes were determined, more than 99% of which retained their RMO patterns in peripheral whole blood (PWB) in independent datasets. Based on the stable CpG pairs, significantly reversed CpG pairs were detected from OVC PWB samples, which were relative to clinical information such as age, subtype, grade, stage, or CA125 level. Results showed 439 CpG loci were determined to be significant differential DNA methylations between OVC and healthy blood samples. They were mainly enriched in KEGG pathways, such as cytokine-cytokine receptor interaction, apoptosis, proteoglycans in cancer, and immune-associated Gene Ontology terms. STRING analysis showed that they tended to have functional interactions with cancer-associated genes recorded in the COSMIC database. Leukocyte cellular differential DNA methylations could be identified by the proposed RMO-based method from OVC PWB samples, which were cancer-associated aberrant signals against cell-composition effects.

白细胞比例变化会干扰外周血样本中癌症相关异常DNA甲基化改变的检测。本研究旨在规避前述细胞组成效应，实现卵巢癌（OVC）血液样本中细胞DNA甲基化变化的精准检测。基于白细胞亚型内CpG位点的样本内相对甲基化排序（RMOs），本研究开发了Ref-RMO方法，用于从OVC血液样本中识别异常甲基化改变。本研究筛选得到在不同白细胞亚型中具有一致RMO的稳定CpG位点对，其中超过99%的位点对在独立数据集的外周全血（PWB）中保留了其RMO特征模式。基于上述稳定CpG位点对，本研究从OVC PWB样本中检测到显著反转的CpG位点对，这类位点对与患者年龄、亚型、分级、分期或CA125水平等临床信息显著相关。研究结果显示，共鉴定出439个在OVC与健康对照血液样本间存在显著差异的DNA甲基化位点。这些差异甲基化位点主要富集于细胞因子-细胞因子受体相互作用、细胞凋亡、癌症相关蛋白聚糖等京都基因与基因组百科全书（KEGG）通路，以及免疫相关基因本体（Gene Ontology）术语。STRING蛋白质相互作用数据库（STRING）分析表明，这些差异甲基化位点倾向于与癌症体细胞突变目录（COSMIC）中收录的癌症相关基因存在功能互作。本研究所提出的基于RMO的方法，可从OVC PWB样本中识别出白细胞源性差异DNA甲基化，这类信号属于可对抗细胞组成效应的癌症相关异常甲基化信号。