A new era for oncology testing: Early detection with 5hmC profiles

Clinical laboratory teams have been familiar with methylation analysis for years, particularly for diagnosing imprinting disorders such as Prader-Willi and Angelman syndrome.¹ But more recently, epigenetic profiling has become quite useful for early detection of cancer. Key advances in epigenomic analysis and machine learning tools have enabled new tests that can detect some of the earliest biological signs of cancer from a simple blood sample.

These epigenetic assays add to the growing universe of liquid biopsy tests. Unlike their predecessors, which aimed to analyze genetic variants in circulating tumor cells or DNA, epigenetic blood tests scan for the regulatory instructions governing the underlying genome. Methylation pattern changes can reveal which genes are turned off and which are activated, providing useful information that may be more directly linked to the biology of cancer activity than variants in the otherwise static genome.

As these tests have been developed, they have largely been commercialized in two broad categories: single-cancer detection tests or multi-cancer detection tests. Some are intended for screening, while others aim to provide early detection of cancer in individuals with high risk factors. In some cases, these blood-based tests have been developed specifically for cancers for which no widely available early detection technique exists, particularly for high-mortality cancers, including pancreatic and ovarian.

The 5hmC difference: A new view of methylation

Traditional methylation analysis targets 5-methylcytosine (5mC), the regulatory mechanism that silences gene activation and is found widely in CpG dinucleotides across the human genome. It is the most common type of methylation and consequently the most comprehensively characterized.

In the past decade, scientists have turned their attention to a different type of methylation known as 5-hydroxymethylcytosine (5hmC). This is intriguing for disease detection and classification purposes because it doesn’t silence genes; in fact, it marks the genes that are turned on. Given that more of the genome is silenced than activated, the ability to focus on active genes through 5hmC signals offers two key advantages. First, it is a better measure of active biology — especially the upregulated biological changes associated with growing cancer — than methylation designed to repress transcription. Second, it dramatically narrows the amount of epigenomic data needed for analysis, so any biological signal is less likely to be drowned out by noise.

On a technical note, 5hmC analysis does not require the bisulfite conversion process used to detect 5mC; as a result, it does not damage DNA during analysis. Indeed, the bisulfite conversion process converts 5hmC and 5mC identically, making it impossible to distinguish 5hmC signals in DNA prepared this way.

Nearly a decade ago, scientists at Stanford University reported that they had successfully tracked 5hmC profiles in circulating cell-free DNA.² Their interest in 5hmC stemmed from its previously established links to the onset of cancer; in this proof-of-concept work, the epigenetic mark was analyzed in samples representing several different types of cancer. The team identified 5hmC signatures associated with specific types and even stages of cancer — data that subsequently informed the development of early detection tests based on 5hmC profiles. Since then, this epigenetic data has also been shown to enable the identification of a tumor’s tissue of origin.³ Taken together, 5hmC analysis makes it possible to detect cancer very early — even earlier than many imaging modalities — and to determine its location and stage for optimal clinical utility.

Single-cancer tests

There are now a number of methylation-based tests available for detecting individual cancers, including tests focused on cancers of the lung, bladder, liver, colon, and pancreas, among others.^4-6 5mC analysis continues to be of interest in the biomedical research community, with techniques that have been used for a variety of cancer types.⁷

But mounting evidence demonstrates the added value of an approach based on 5hmC. For instance, researchers in the UK compared 5mC and 5hmC data for colorectal cancer, finding that changes in 5hmC were commonly found at stage 1, suggesting that this type of methylation could be more powerful than 5mC for enabling early detection of cancer.⁸

In a larger study focused on pancreatic cancer, scientists analyzed 5hmC data from cell-free DNA, comparing results from a cancer cohort to a non-cancer cohort.⁹ They identified specific genes for which 5hmC epigenetic patterns were relevant to early detection of pancreatic cancer and used tissue-derived 5hmC information to further classify results found in cell-free DNA.

An important advantage of single-cancer tests is that their focused nature allows diagnostic developers to hone their algorithms for one specific type of cancer and its associated epigenetic signals. This typically leads to higher sensitivity and specificity performance. These tests also provide the benefit of a well-established pathway for diagnostic follow-up in clinical settings.

Multi-cancer tests

A new type of early detection test has emerged in cancer diagnostics recently, and that’s the multi-cancer test. Some of these tests are already commercially available and promise to detect dozens of different types of cancer from a single assay. While the tests are based on a variety of underlying biomarkers and technologies, some have incorporated epigenetic data to boost sensitivity, particularly for early stage cancers. With so many alternatives, though, there is considerable range in accuracy and reliability for these tests.

Even in these early days, though, a few things are fairly certain about multi-cancer tests. One is that they are gaining interest among the general public, with more people paying out-of-pocket to get these results. This interest will likely increase as Medicare is set to begin covering multi-cancer tests starting in 2028, thanks to the recently passed Nancy Gardner Sewell Medicare Multi-Cancer Early Detection Screening Coverage Act.

Also worth noting about multi-cancer tests: they may be a much-needed tool for high-mortality cancers that currently have no widely used screening method, such as pancreatic, ovarian, and liver cancer. While multi-cancer tests that claim to detect dozens of cancer types typically struggle with low sensitivity rates, a narrower test that focuses on unscreened cancers could in theory achieve better sensitivity due to a more targeted approach.

In one study, scientists evaluated 5hmC signals for five cancers: breast, colon, lung, ovarian, and pancreatic.³ They analyzed hundreds of tumor samples, comparing them to normal tissues, as well as cell-free DNA samples from more than 1,000 individuals with cancer and nearly 1,700 healthy individuals. In addition to revealing broad-scale redistribution of 5hmC in early-stage tumors — a signal that could be tracked into later-stage tumors as well — they also found tissue-specific epigenetic patterns. Those patterns made it possible to train a classifier that could predict the tissue of tumor origin with excellent accuracy.

Already, multi-cancer tests are at the heart of a study funded by the National Cancer Institute to learn more about how these tests could be implemented. The Vanguard Study, which aims to enroll as many as 24,000 participants between the ages of 45 and 75, will evaluate the performance of two multi-cancer tests.^10,11 Both are based on methylation detection, but one was designed to look for 5mC while the other looks for 5hmC.

Looking ahead

The ability to use 5hmC patterns to detect the earliest signs of cancer — whether that’s focused testing for a single type of cancer or a multi-cancer test designed to look for several types of cancer at once — could significantly alter how high-risk patients are monitored. A simple blood test enabling early cancer detection could make it possible to diagnose cancer sooner, shifting patients to more treatable stages for potentially better outcomes. It is an exciting time for early cancer detection, with new options for clinical laboratories to consider for their own test menus.

References

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8. Puddu F, Johansson A, Modat A, et al. 5-methylcytosine and 5-hydroxymethylcytosine are synergistic biomarkers for early detection of colorectal cancer. Commun Med (Lond). 2026;6(1):15. doi:10.1038/s43856-025-01278-8.
9. Guler GD, Ning Y, Ku CJ, et al. Detection of early stage pancreatic cancer using 5-hydroxymethylcytosine signatures in circulating cell free DNA. Nat Commun. 2020;11(1):5270. doi:10.1038/s41467-020-18965-w.
10. The Vanguard Study: Testing a new way to screen for cancer. ClinicalTrials.gov. April 13, 2026. Accessed April 13, 2026. https://clinicaltrials.gov/study/NCT06995898.
11. NCI selects two assays for the Vanguard Study on multi-cancer detection tests. National Cancer Institute. January 7, 2025. Accessed April 13, 2026. https://prevention.cancer.gov/news-and-events/news/nci-selects-two-assays-vanguard-study-multi-cancer-detection-tests.