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Picking Apart the Epigenome To Advance Cancer Research

DNA double helix with 5mC and 5hmC methylation modifications indicated.
Credit: biomodal.
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While the importance of genetics in health and disease has long been known, the role of epigenetic changes, which are not reflected in our DNA but nonetheless impact gene expression, is an area of great interest and growing research. DNA methylation is one such important way in which the genome can be modified. In most cases, methylation occurs at the C5 position of cytosine in CpG dinucleotides. Further research subsequently identified that this modification comes in two different forms — DNA methylation at the fifth position of cytosine (5mC) and hydroxymethylation (5hmC).

To get the full picture, it is important to determine both the genetic and epigenetic landscape within samples. This has typically necessitated the use of multiple techniques to obtain the necessary data and laborious downstream data processing. Detail is also lost when techniques such as bisulfite sequencing (bis-seq) are used, as these look at total methylation and readouts from 5mC and 5hmC cannot be picked apart (collectively denoted 5modC). However, biomodal has developed a solution that addresses these issues. The six-base genome produced by biomodal’s duet multiomics solution evoC not only provides data on the traditional four DNA bases but also 5mC and 5hmC separately, in one workflow.

We recently caught up with Dr. Tom Charlesworth, director of market strategy and corporate development at biomodal, at the American Association for Cancer Research (AACR) annual conference 2024 to talk about why six-base genome sequencing is such an important development for the field of genetics and especially epigenetics, how it may help cancer research and their goals for further development of the technology.

Karen Steward (KS): Why is the development of six-base genome sequencing such an important advancement for those working in epigenetics?

Tom Charlesworth (TC): Since 2008, it has been known that biologically important epigenetic DNA modifications consist of both 5mC and 5hmC. Prior to this, it was believed that only 5mC was relevant to mammalian biology. However, the most popular tool to assess DNA methylation, bisulfite sequencing, is unable to distinguish between these two modifications. Tools have been developed to distinguish the two modifications and have enabled epigeneticists to understand at a broad level that the two modifications have different and opposing biological functions. However, these tools have significant limitations. For example, they may require multiple experiments and subtractive analysis or be based on pulldown approaches that do not provide single base resolution or are low accuracy. With the advent of duet evoC, an epigeneticist is able to distinguish both modifications in a single experiment from a low input sample. Not only this, but it is known that there are functional and biological interactions between the two modifications and their genetic context, for instance when a genetic variant changes the methylation pattern. With the advent of the six-base genome, these interactions can be explored in full without confounding factors that arise from combining lower fidelity datasets.

KS: How is six-base sequencing being applied to cancer research? Are there any areas you’d like to highlight where it is particularly helpful or solves previous challenges?

TC: We’ve had great interest from translational oncology researchers who understand that there are both genetic and epigenetic drivers of cancer. Hematologic malignancies, for example, often involve the combination of mutations in the epigenetic machinery that result in widespread changes in the distribution of 5mC and 5hmC. With the six-base genome, researchers are finally able to generate data to help them fully understand the relationships between those genetic and epigenetic alterations. We’re also seeing a lot of interest from researchers interested in deriving the most information possible from precious cell-free DNA (cfDNA) samples. The six-base genome provides genetics, epigenetics and fragment information and we see our customers using this rich multiomic information to improve understanding of the biology of cancer development, treatment response minimal residual disease (MRD) monitoring and to discover biomarkers that would otherwise be completely invisible using current technologies.

KS: Given the additional granularity that six-base sequencing provides over 5-base, does this advance offer diagnostic potential?

TC: As alluded to above, we’ve seen in our own data that with the addition of the 5hmC signal you can uncover otherwise invisible biomarkers, and we’ve highlighted examples of enhancers where the addition of 5hmC allows you to see differences between a cfDNA sample from a healthy individual, versus someone with early-stage colorectal cancer (CRC). More recently at AACR, we demonstrated the power of six-base data to generate classifiers for early CRC detection that were far more performative than using 5mC of 5hmC alone, or a conflated 5modC signal. We saw an increase in the receiver operating characteristic (ROC) curve from ~ 0.7 to ~ 0.9 when distinguishing between healthy and stage I CRC.

KS: Formalin fixation is commonly used to preserve samples but can cause damage to sample DNA. How does this impact six-base sequencing and how does it compare to alternative techniques?

TC: We also released data at AACR comparing six-base data generated from lung cancer and CRC clinical fresh frozen and formalin-fixed, paraffin-embedded (FFPE) samples. We saw high concordance between modified base calls between the two sample types, and that concordance was significantly higher than that published for other epigenetic sequencing technologies.

KS: What are your goals for the short- and longer-term future in terms of developing this system further?

TC: We’re excited for our customers to apply the technology to neuronal applications where there are elevated levels of 5hmC and no one really knows why, or what it’s doing there. We’ve also seen some really exciting observations with our data and with early customers that the six-base genome can be used to predict other functional aspects of the genome like gene expression and chromatin accessibility. We’re releasing tools to help customers analyze their methylation data quickly and explore these predictive powers and I can’t wait to build on these early observations and better understand how we can support customers in getting the most from the richness of data the six-base genome provides. What else can they predict? How can we build generalizable predictive models? Can the predictions be applied to cfDNA or other low input applications? There’s unlimited potential.

Dr. Tom Charlesworth was speaking to Dr. Karen Steward, Senior Scientific Specialist for Technology Networks.

About the interviewee

Head shot of Dr. Tom Charlesworth

Tom Charlesworth, PhD, is the director of marketing strategy and corporate development at biomodal. A dynamic leader, Tom has a wealth of experience vital to defining the long-term vision and strategy for biomodal’s products. His focus on customer needs, market trends and key partnerships supported the launch of duet multiomics solutions, revealing the power of the five- and six-base genome. Tom is highly effective in managing cross-functional teams, overseeing all elements of the product development lifecycle and delivering finely tuned product marketing strategies designed to improve customer experience and grow market share. Tom has over 10 years of experience in the life sciences industry and holds a PhD in biochemistry from the University of Cambridge.