The Implications of Heterogeneous DNA Methylation for The Accurate Quantification of Methylation

Abstract

DNA methylation based biomarkers have considerable potential for molecular diagnostics, both as tumor specific biomarkers for the early detection or post-therapeutic monitoring of cancer as well as prognostic and predictive biomarkers for therapeutic stratification. Particularly in the former, the accurate estimation of DNA methylation is of compelling importance. However, quantification of DNA methylation has many traps for the unwary, especially when heterogeneous methylation comprising multiple alleles with varied DNA methylation patterns (epialleles) is present. The frequent occurrence of heterogeneous methylation as distinct from a simple mixture of fully methylated and unmethylated alleles is generally not taken into account when DNA methylation is considered as a cancer biomarker. When heterogeneous DNA methylation is present, the proportion of methylated molecules is difficult to quantify without a method that allows the measurement of individual epialleles. In this article, we critically assess the methodologies frequently used to investigate DNA methylation, with an emphasis on the detection and measurement of heterogeneous DNA methylation. The adoption of digital approaches will enable the effective use of heterogeneous DNA methylation as a cancer biomarker.

KEYWORDS::

• biomarker
• cancer
• CDKN2B
• digital PCR
• high-resolution melting
• MGMT
• minimal residual disease
• molecular diagnostics

Acknowledgements

The authors thank Denise O‘Keefe, Nick Wong and Jeff Craig for critical reading and helpful comments on this manuscript.

Financial & competing interests disclosure

Alexander Dobrovic is the coinventor of intellectual property on MS-HRM and SMART-MSP held by the Peter MacCallum Cancer Centre (Australia) and the University of Aarhus (Denmark). We acknowledge the National Breast Cancer Foundation (Australia), the Cancer Council of Victoria, the Victorian Cancer Agency and the US Department of Defense for funding epigenetics projects which led directly to the consideration of the issues discussed in this manuscript. Views and opinions of, and endorsements by the authors do not reflect those of the US Army or the US Department of Defense. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Additional information

Funding

Alexander Dobrovic is the coinventor of intellectual property on MS-HRM and SMART-MSP held by the Peter MacCallum Cancer Centre (Australia) and the University of Aarhus (Denmark). We acknowledge the National Breast Cancer Foundation (Australia), the Cancer Council of Victoria, the Victorian Cancer Agency and the US Department of Defense for funding epigenetics projects which led directly to the consideration of the issues discussed in this manuscript. Views and opinions of, and endorsements by the authors do not reflect those of the US Army or the US Department of Defense. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.