Aberrant methylation scanning by quantitative DNA melting analysis with hybridization probes as exemplified by liquid biopsy of SEPT9 and HIST1H4F in colorectal cancer

Objective: The generally accepted method of quantifying hypermethylated DNA by qPCR using methylation-specific primers has the risk of underestimating DNA methylation and requires data normalization. This makes the analysis complicated and less reliable.Methods: The end-point PCR method, called qDMA-HP (for quantitative DNA Melting Analysis with hybridization probes), which excludes the normalization procedure, is multiplexed and quantitative, has been proposed. qDMA-HP is characterized by the following features: (i) asymmetric PCR with methylation-independent primers; (ii) fluorescent dual-labeled, self-quenched probes (commonly known as TaqMan probes) covering several interrogated CpGs; (iii) post-PCR melting analysis of amplicon/probe hybrids; (iv) quantitation of unmethylated and methylated DNA alleles by measuring the areas under the corresponding melt peaks.Results: qDMA-HP was tested in liquid biopsy of colorectal cancer by evaluating SEPT9 and HIST1H4F methylations simultaneously in the single-tube reaction. Differences in the methylation levels in healthy donors versus cancer patients were statistically significant (p < 0.0001), AUCROC values were 0.795-0.921 for various marker combinations.Conclusions: This proof-of-concept study shows that qDMA-HP is a simple, normalization-independent, quanti-tative, multiplex and closed tube method easily adapted to clinical settings. It is demonstrated, for the first time, that HIST1H4F is a perspective marker for liquid biopsy of colorectal cancer.

Automated summary

This study presents a new PCR method, qDMA-HP, for quantitative analysis of methylated DNA in liquid biopsy without the need for data normalization. The method was successfully tested for colorectal cancer liquid biopsy and showed promising results.