Ribosomal DNA methylation as stable biomarkers for detection of cancer in plasma

Abstract

BackgroundRecently, liquid biopsy for cancer detection has pursued great progress. However, there are still a lack of high quality markers. It is a challenge to detect cancer stably and accurately in plasma cell free DNA (cfDNA), when the ratio of cancer signal is low. Repetitive genes or elements may improve the robustness of signals. In this study, we focused on ribosomal DNA which repeats hundreds of times in human diploid genome and investigated performances for cancer detection in plasma. ResultsWe collected bisulfite sequencing samples including normal tissues and 4 cancer types and found that intergenic spacer (IGS) of rDNA has high methylation levels and low variation in normal tissues and plasma. Strikingly, IGS of rDNA shows significant hypo-methylation in tumors compared with normal tissues. Further, we collected plasma bisulfite sequencing data from 224 healthy subjects and cancer patients. Means of AUC in testing set were 0.96 (liver cancer), 0.94 (lung cancer and), 0.92 (colon cancer) with classifiers using only 10 CpG sites. Due to the feature of high copy number, when liver cancer plasma WGBS was down-sampled to 10 million raw reads (0.25× whole genome coverage), the prediction performance decreased only a bit (mean AUC=0.93). Finally, methylation of rDNA could also be used for monitor cancer progression and treatment. ConclusionTaken together, we provided the high-resolution map of rDNA methylation in tumors and supported that methylation of rDNA was a competitive and robust marker for detecting cancer and monitoring cancer progression in plasma.

Methylation of 45S Ribosomal DNA (rDNA) Is Associated with Cancer and Aging in Humans

“…They found that the methylation levels of a majority of the CpG sites within Zone 2 (upstream of 5′IGS) and Zone 4 (downstream of 3′IGS) regions were significantly hypomethylated in four types of cancers including the liver, lung, prostate, and colon in comparison with the normal tissues. Besides, the variation trend of methylation levels in Zones 2 and 4 in the above four cancer types was overall consistent with that in the normal tissues, contrasting with the highly varied methylation levels in Zones 1, 3, and 5 [ 30 ]. The authors thus suggested that the methylation status of Zones 2 and 4, i.e., partial sequences of IGS, could serve as a useful marker for certain cancer detection in plasma [ 30 ].…”

“…Besides, the variation trend of methylation levels in Zones 2 and 4 in the above four cancer types was overall consistent with that in the normal tissues, contrasting with the highly varied methylation levels in Zones 1, 3, and 5 [ 30 ]. The authors thus suggested that the methylation status of Zones 2 and 4, i.e., partial sequences of IGS, could serve as a useful marker for certain cancer detection in plasma [ 30 ].…”

“…Moreover, Zhang et al [ 30 ] analyzed the overall rDNA methylation levels in different cancers and normal tissues. The rDNA region was divided into five zones, i.e., Zone 1 (including the 5′ETS, transcribed regions, and 3′ETS) and Zones 2-5 (all belonging to the IGS region), without overlaps according to their CpG sites [ 37 ].…”

Human rDNA and Cancer

“…Besides, both hypo- and hypermethylation may be found in the abnormally functioning rDNA locus. Thus in the late-stage ovarian cancer, breast cancer, primary endometrial carcinoma, liver cancer, cervical cancer (cervical intraepithelial neoplasia), and hepatocellular carcinomas, the hypomethylation was found in the promoter [ 41 , 45 , 50 , 51 , 52 , 53 ] but the hypermethylation appeared frequently in some transcribed spacer regions, in IGS, and in the regions coding for 28S and 5.8S rRNA [ 42 ]. On the other hand, in patients with primary oral squamous cell carcinoma and prostate cancer, the locus seemed to be methylated as in norm [ 14 , 54 ].…”