The race for early cancer detection is one that physicians have been running for millennia. In one of humanity’s oldest surviving medical texts, the ancient Egyptian physician Imhotep described 46 ailments and how they might be cured. For the treatment of malignant tumors, he dedicated only nine glyphs, conveying in his brevity a familiar anguish as it reads simply, “There is nothing.”1
Physicians like Imhotep had little hope of treating cancer, in part because the disease was only discoverable to them in its most advanced stages. Even with little understanding of tumor biology, physicians knew that early detection and intervention were key to patient survival. But cancer is an insidious disease that hides well among healthy cells, possessing few distinguishing features that might signal its presence. Because of this, cancer has been able to outpace our early detection efforts for thousands of years.
Fortunately, times are changing. Advances in DNA sequencing technology have exposed a previously overlooked feature of many cancer cells, one that appears in the earliest stages of disease development. A growing body of studies suggests that DNA methylation sequencing may be the edge physicians have sought since the days of Imhotep, enabling tumors to be detected before they can grow beyond the reach of our therapeutics.2-4
DNA methylation is one of the ways in which cells organize distinct identities despite inheriting the same genome. The addition of a methyl group to the fifth carbon on cytosine (forming 5-methylcytosine or 5mC) alters how the surrounding DNA interacts with transcriptional effectors, often decreasing its accessibility and silencing the region’s influence over cell behavior. Precise hypermethylation of specific genes or regulators can thus enable cells to control which genes are used to construct their identity.
Tom Charlesworth, PhD
5mC demethylation is also a useful tool for this. Upon oxidation of 5mC, the resulting 5-hydroxymethylcytosine (5hmC) intermediate increases DNA accessibility and is associated with the re-awakening of silenced genes, enabling epigenetic gene regulation to be a dynamic process.
Through the selective application of 5mC and 5hmC, cells establish precise and chemically stable gene expression profiles that are remarkably consistent within cell types. When methylation patterns are abnormally disrupted, however, cell behavior can become erratic and potentially malignant.
Abnormal methylation patterns are recognized as a hallmark of cancer.4 Genes responsible for regulating cell cycle progression, DNA damage repair, and cell adhesion, for example, are frequently hypermethylated in tumor cells, while oncogenic transcription factors, retrotransposons, and epigenetic regulators may be hypomethylated.3 The combination of silencing tumor suppressors and waking oncogenic genes is believed to not only play an important role in disease biology, but may be critical for the earliest stages of tumor development.
A recent study by Sepulveda and colleagues underscores this point.5 By analyzing mouse embryonic stem cells lacking a regulatory enzyme called OGT, the researchers observed a genome-wide shift in cytosine modification: levels of 5hmC surged while 5mC plummeted. This imbalance coincided with the awakening of transposable elements—dormant stretches of viral DNA embedded in the genome—and the upregulation of interferon-stimulated genes, both characteristic of cellular stress and early oncogenic activity.
Crucially, these changes were most evident in heterochromatin, the tightly packed, transcriptionally silent regions of the genome long thought to be inert. The study suggests that a rise in 5hmC within these regions may foreshadow loss of methylation control and the destabilization of the genome itself—a kind of molecular tremor before the quake.
Results like these imply that disruption of methylation patterns can drive malignant processes and may represent an early—and measurable—marker of malignant transformation.
Next-generation sequencing (NGS) has proven to be a powerful tool for early cancer detection. Among its many uses, NGS can be used to interrogate fragments of circulating cell-free DNA—leaked into the bloodstream from healthy and malignant cells alike—for the molecular hallmarks of cancer.
Whereas tumor-identifying mutations may only affect a small fraction of bases in the genome, altered methylation patterns can span hundreds of bases to multiple kilobase regions. This creates many more opportunities to recognize tumor-specific patterns among cell-free DNA, even when no genomic mutation is present. [Thana Prasongsin/Getty Images]
In theory, fragments of DNA bearing cancer-associated mutations can be leaked from individual tumor cells and used to detect tumors long before they become recognizable through conventional means. This is supported by several studies demonstrating the sensitive detection of tumor-derived DNA fragments (ctDNA) in patients that otherwise appear cancer-free.6 However, these tests cannot recognize the majority of tumor-derived DNA. Instead, they can only positively identify ctDNA if the fragment contains specific mutations. Such fragments can be exceedingly rare and may be easily overlooked in the milieu of non-malignant circulating DNA.
This is where DNA methylation is advancing the field. Whereas tumor-identifying mutations may only affect a small fraction of bases in the genome, altered methylation patterns can span hundreds of bases to multiple kilobase regions.2 This creates many more opportunities to recognize tumor-specific patterns among cell-free DNA, even when no genomic mutation is present. Increasing your odds of recognizing tumor-derived DNA is particularly important for early cancer detection, when the rarity of tumor cells translates to few detectable ctDNA fragments.
But, while methylation sequencing has been shown to detect late-stage cancers with high sensitivity, results in early cancer detection have been less promising. This is likely due to a key limitation of methods like whole-genome bisulfite sequencing, which fail to distinguish between 5mC and 5hmC—often flattening the two into a single, conflated readout of modified cytosine (modC). As described above, 5mC and 5hmC have distinct influences over gene expression and appear to form temporally distinct patterns, with 5hmC potentially foreshadowing the loss of 5mC in the early stages of cancer.2,5 When we fail to distinguish between them, we lose a biomarker that is increasingly associated with early-stage disease.
This was highlighted in a recent liquid biopsy study focused on detecting early-stage colorectal cancer (CRC) in cfDNA.2 In this study, plasma from stage I and stage IV CRC samples was analyzed using 6-base sequencing technology, which enabled base-level resolution of 5mC, 5hmC, and the four standard DNA bases. The team showed that changes to the methylation landscape in stage I samples were mostly limited to 5hmC, and that these changes often occurred where 5mC is lost in stage IV samples.
Most striking among the team’s observations was the finding that stage I CRC could be detected with 85% sensitivity and a 95% specificity—meeting or exceeding clinical thresholds in blood-based cancer screening—only when both 5mC and 5hmC patterns were considered together. In effect, the team’s use of 6-base sequencing technology enabled them to interrogate cfDNA fragments for previously unseen methylation patterns, which in turn opened the door to sensitive, specific, and early CRC detection.
Methylation sequencing technology is still maturing. Its incorporation into clinical workflows will require continued validation, thoughtful interpretation, and scalable workflows. But the direction of growth is clear. As we gain the ability to look beyond genomics into the realm of epigenomics, our understanding of disease biology becomes richer, more dynamic, and more precise, giving us a critical leg up in the race for early detection.
Tom Charlesworth, PhD, is the director of marketing strategy and corporate development at biomodal.
References
1. Breasted JH, Smith E. The Edwin Smith Surgical Papyrus Published in Facsimile and Hieroglyphic Transliteration. 1930.
2. Puddu F, Johansson A, Modat A, et al. 5-methylcytosine and 5-hydroxymethylcytosine are synergistic biomarkers for early detection of colorectal cancer. bioRxiv (Cold Spring Harbor Laboratory). October 31, 2024. doi:https://doi.org/10.1101/2024.10.30.621123
3. Nishiyama A, Nakanishi M. Navigating the DNA methylation landscape of cancer. Trends in genetics: TIG. June 10, 2021;37(11):1012-1027. doi:https://doi.org/10.1016/j.tig.2021.05.002
4. Esteller M, Dawson MA, Kadoch C, et al. The epigenetic hallmarks of cancer. Cancer Discovery. October 4, 2024;14(10):1783-1809. doi:https://doi.org/10.1158/2159-8290.cd-24-0296
5. Sepulveda H, Li X, Arteaga-Vazquez LJ, et al. OGT prevents DNA demethylation and suppresses the expression of transposable elements in heterochromatin by restraining TET activity genome-wide. Nature Structural & Molecular Biology. March 28, 2025. doi:https://doi.org/10.1038/s41594-025-01505-9
6. Peng Y, Mei W, Ma K, Zeng C. Circulating tumor DNA and minimal residual disease (MRD) in solid tumors: current horizons and future perspectives. Frontiers in Oncology. November 17, 2021;11. doi:https://doi.org/10.3389/fonc.2021.763790
The post Cracking Cancer’s Code as Methylation Sequencing Expands the Window for Early Detection appeared first on GEN - Genetic Engineering and Biotechnology News.
Physicians like Imhotep had little hope of treating cancer, in part because the disease was only discoverable to them in its most advanced stages. Even with little understanding of tumor biology, physicians knew that early detection and intervention were key to patient survival. But cancer is an insidious disease that hides well among healthy cells, possessing few distinguishing features that might signal its presence. Because of this, cancer has been able to outpace our early detection efforts for thousands of years.
Fortunately, times are changing. Advances in DNA sequencing technology have exposed a previously overlooked feature of many cancer cells, one that appears in the earliest stages of disease development. A growing body of studies suggests that DNA methylation sequencing may be the edge physicians have sought since the days of Imhotep, enabling tumors to be detected before they can grow beyond the reach of our therapeutics.2-4
Transforming epigenetic landscape
DNA methylation is one of the ways in which cells organize distinct identities despite inheriting the same genome. The addition of a methyl group to the fifth carbon on cytosine (forming 5-methylcytosine or 5mC) alters how the surrounding DNA interacts with transcriptional effectors, often decreasing its accessibility and silencing the region’s influence over cell behavior. Precise hypermethylation of specific genes or regulators can thus enable cells to control which genes are used to construct their identity.
Tom Charlesworth, PhD
5mC demethylation is also a useful tool for this. Upon oxidation of 5mC, the resulting 5-hydroxymethylcytosine (5hmC) intermediate increases DNA accessibility and is associated with the re-awakening of silenced genes, enabling epigenetic gene regulation to be a dynamic process.
Through the selective application of 5mC and 5hmC, cells establish precise and chemically stable gene expression profiles that are remarkably consistent within cell types. When methylation patterns are abnormally disrupted, however, cell behavior can become erratic and potentially malignant.
Abnormal methylation patterns are recognized as a hallmark of cancer.4 Genes responsible for regulating cell cycle progression, DNA damage repair, and cell adhesion, for example, are frequently hypermethylated in tumor cells, while oncogenic transcription factors, retrotransposons, and epigenetic regulators may be hypomethylated.3 The combination of silencing tumor suppressors and waking oncogenic genes is believed to not only play an important role in disease biology, but may be critical for the earliest stages of tumor development.
A recent study by Sepulveda and colleagues underscores this point.5 By analyzing mouse embryonic stem cells lacking a regulatory enzyme called OGT, the researchers observed a genome-wide shift in cytosine modification: levels of 5hmC surged while 5mC plummeted. This imbalance coincided with the awakening of transposable elements—dormant stretches of viral DNA embedded in the genome—and the upregulation of interferon-stimulated genes, both characteristic of cellular stress and early oncogenic activity.
Crucially, these changes were most evident in heterochromatin, the tightly packed, transcriptionally silent regions of the genome long thought to be inert. The study suggests that a rise in 5hmC within these regions may foreshadow loss of methylation control and the destabilization of the genome itself—a kind of molecular tremor before the quake.
Results like these imply that disruption of methylation patterns can drive malignant processes and may represent an early—and measurable—marker of malignant transformation.
Signal for early cancer detection
Next-generation sequencing (NGS) has proven to be a powerful tool for early cancer detection. Among its many uses, NGS can be used to interrogate fragments of circulating cell-free DNA—leaked into the bloodstream from healthy and malignant cells alike—for the molecular hallmarks of cancer.
Whereas tumor-identifying mutations may only affect a small fraction of bases in the genome, altered methylation patterns can span hundreds of bases to multiple kilobase regions. This creates many more opportunities to recognize tumor-specific patterns among cell-free DNA, even when no genomic mutation is present. [Thana Prasongsin/Getty Images]
In theory, fragments of DNA bearing cancer-associated mutations can be leaked from individual tumor cells and used to detect tumors long before they become recognizable through conventional means. This is supported by several studies demonstrating the sensitive detection of tumor-derived DNA fragments (ctDNA) in patients that otherwise appear cancer-free.6 However, these tests cannot recognize the majority of tumor-derived DNA. Instead, they can only positively identify ctDNA if the fragment contains specific mutations. Such fragments can be exceedingly rare and may be easily overlooked in the milieu of non-malignant circulating DNA.
This is where DNA methylation is advancing the field. Whereas tumor-identifying mutations may only affect a small fraction of bases in the genome, altered methylation patterns can span hundreds of bases to multiple kilobase regions.2 This creates many more opportunities to recognize tumor-specific patterns among cell-free DNA, even when no genomic mutation is present. Increasing your odds of recognizing tumor-derived DNA is particularly important for early cancer detection, when the rarity of tumor cells translates to few detectable ctDNA fragments.
But, while methylation sequencing has been shown to detect late-stage cancers with high sensitivity, results in early cancer detection have been less promising. This is likely due to a key limitation of methods like whole-genome bisulfite sequencing, which fail to distinguish between 5mC and 5hmC—often flattening the two into a single, conflated readout of modified cytosine (modC). As described above, 5mC and 5hmC have distinct influences over gene expression and appear to form temporally distinct patterns, with 5hmC potentially foreshadowing the loss of 5mC in the early stages of cancer.2,5 When we fail to distinguish between them, we lose a biomarker that is increasingly associated with early-stage disease.
This was highlighted in a recent liquid biopsy study focused on detecting early-stage colorectal cancer (CRC) in cfDNA.2 In this study, plasma from stage I and stage IV CRC samples was analyzed using 6-base sequencing technology, which enabled base-level resolution of 5mC, 5hmC, and the four standard DNA bases. The team showed that changes to the methylation landscape in stage I samples were mostly limited to 5hmC, and that these changes often occurred where 5mC is lost in stage IV samples.
Most striking among the team’s observations was the finding that stage I CRC could be detected with 85% sensitivity and a 95% specificity—meeting or exceeding clinical thresholds in blood-based cancer screening—only when both 5mC and 5hmC patterns were considered together. In effect, the team’s use of 6-base sequencing technology enabled them to interrogate cfDNA fragments for previously unseen methylation patterns, which in turn opened the door to sensitive, specific, and early CRC detection.
Methylation sequencing technology is still maturing. Its incorporation into clinical workflows will require continued validation, thoughtful interpretation, and scalable workflows. But the direction of growth is clear. As we gain the ability to look beyond genomics into the realm of epigenomics, our understanding of disease biology becomes richer, more dynamic, and more precise, giving us a critical leg up in the race for early detection.
Tom Charlesworth, PhD, is the director of marketing strategy and corporate development at biomodal.
References
1. Breasted JH, Smith E. The Edwin Smith Surgical Papyrus Published in Facsimile and Hieroglyphic Transliteration. 1930.
2. Puddu F, Johansson A, Modat A, et al. 5-methylcytosine and 5-hydroxymethylcytosine are synergistic biomarkers for early detection of colorectal cancer. bioRxiv (Cold Spring Harbor Laboratory). October 31, 2024. doi:https://doi.org/10.1101/2024.10.30.621123
3. Nishiyama A, Nakanishi M. Navigating the DNA methylation landscape of cancer. Trends in genetics: TIG. June 10, 2021;37(11):1012-1027. doi:https://doi.org/10.1016/j.tig.2021.05.002
4. Esteller M, Dawson MA, Kadoch C, et al. The epigenetic hallmarks of cancer. Cancer Discovery. October 4, 2024;14(10):1783-1809. doi:https://doi.org/10.1158/2159-8290.cd-24-0296
5. Sepulveda H, Li X, Arteaga-Vazquez LJ, et al. OGT prevents DNA demethylation and suppresses the expression of transposable elements in heterochromatin by restraining TET activity genome-wide. Nature Structural & Molecular Biology. March 28, 2025. doi:https://doi.org/10.1038/s41594-025-01505-9
6. Peng Y, Mei W, Ma K, Zeng C. Circulating tumor DNA and minimal residual disease (MRD) in solid tumors: current horizons and future perspectives. Frontiers in Oncology. November 17, 2021;11. doi:https://doi.org/10.3389/fonc.2021.763790
The post Cracking Cancer’s Code as Methylation Sequencing Expands the Window for Early Detection appeared first on GEN - Genetic Engineering and Biotechnology News.