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Objective Universal noninvasive genomic screening to detect cancer and/or fetal DNA in plasma at all stages of development is highly warranted. Since 5-hydroxymethylcytosine (5hmC) emerged as an intermediate metabolite in active DNA demethylation, there have been increasing efforts to elucidate its function as a stable modification of the genome. In the current study, we demonstrate that discrete 5hmC sites within 80 bp hotspot regions exist in a greater proportion of cancer versus normal cells. Result 5hmC was detected in 16 of 17 known hotspots having C to T or G to A mutations. The results show the presence of two characteristically distinct 5hmC groups: Tier 1 Group with 3 to eightfold more 5hmCs detected in tumor-cells than in normal-cell derived DNA (as observed in 6 of 11 CpG sites). Tier 2 group with equal allele frequency of 5hmC among normal and tumor-cell derived DNA at 5 CpG hotspot sites as well as 5 non-CpG hotspots. Thus, detection and quantification of the Tier 1 group of 5hmC sites or its prevalence at or near cancer mutation hot spots in cells may enable early detection, screening and potentially prediction of the likelihood of cancer occurrence or the severity of the cancer.

5-Hydroxymethylcytosine (5hmC) at or near cancer mutation hot spots as potential targets for early cancer detection