5-hydroxymethylcytosine (5-hmC) is a modified form of the DNA base cytosine. It was shown to have a functional role in gene-expression regulation, and its global levels were found to be predominantly stable and highly tissue-specific. A significant global reduction in 5-hmC level was reported for various human cancers, such as melanoma, colorectal, pancreatic, breast, liver, lung, prostate, brain and blood cancers. This reduction suggests that 5-hmC may serve as a powerful cancer biomarker, potentially enabling early-stage detection. Several methods for 5-hmC detection and quantification have been developed in recent years. However, they suffer from limitations such as low sensitivity, experimental complexity, and high costs, and therefore, none meet the requirements for routine clinical diagnostics. To address this, we have developed a fluorescence-based platform for high-throughput and cost-effective quantification of global genomic 5-hmC levels. It employs a robust chemo-enzymatic reaction for fluorescently tagging the 5-hmC residues and a chemically-activated multi-well slide for analysis of multiple samples simultaneously. The assay was optimized to address the high sensitivity required to analyze tissues demonstrating very low levels of 5-hmC. The low noise level of this protocol enables the detection of extremely low 5-hmC levels (0.0035%), lower than the level found in blood or various cancer tissues. We utilized the assay to characterize the 5-hmC content in colorectal cancer (CRC) and colon tissue adjacent to the tumor relative to healthy control colon tissues. We observed a significant reduction in 5-hmC for CRC tissue relative to a healthy colon. We also show that in some cases, the adjacent tissue already shows a molecular reduction of 5-hmC relative to the healthy baseline average. This assay may be integrated into existing cancer screening tests such as colonoscopy, potentially providing additional molecular information. Furthermore, this highly sensitive method is not limited to a specific tumor type and can be employed to various types of cancers, including those expressing very low levels of 5-hmC such as blood cancers.
