Periodic Reporting for period 1 - U-BiomarCARE (Moving from whole genome cfDNA methylation to a PCR-based liquid biopsy assay for detecting high-risk atherosclerotic plaques)
Période du rapport: 2023-07-01 au 2024-12-31
General practitioners and cardiologists are faced every day with challenging decisions on whom to refer for diagnostics of coronary artery disease (CAD) as sign and symptoms of cardiac ischemia require immediate action. Diagnosis of CAD is key as ischemic heart disease accounts for 45% of deaths in females and 39% in males in the European Union. Yet, diagnosis of CAD in women is regularly delayed and often unrecognized. As the main pathology underlying CAD, it is essential to understand atherosclerotic plaque biology. In recent years, my research group and others have identified clear sex differences in the atherosclerotic plaque morphology and its (epi)genetic regulation. One consequence is that female plaques are more difficult to detect than male plaques, especially those which cause symptoms. As part of the ERC UCARE project, we uncovered a plaque-specific epigenetic signal in cell-free DNA from plasma of women with CAD. This signal consisted of hypomethylated regions specific for plaques that can be detected in plasma cell-free DNA. Our methods thus far have been focused on whole genome sequencing analyses. Yet, for studying the predictive value of these regions in large cohorts of patients with CAD, as well as exploring commercial potential, we need to develop a more specific methodology. Our aim in this current U-BiomarCARE project is to investigate if the methylation status of plaque-specific regions can be detected in plasma with a PCR-based method. Once we prove that this methodology is reproducible and comparable to whole genome DNA methylation sequencing, we can perform large biomarker studies in both women and men suspected of heart disease and explore potential commercialization of plaque-specific cell-free DNA methylation profiles to accurately detect CAD. This will be a game changer for the healthcare sector as it will allow a specific and timely detection of dangerous atherosclerotic plaques both in men and women.
As part of the ERC UCARE project, we identified a plaque-specific epigenetic signal in cell-free DNA (cfDNA) from the plasma of women with CAD. This signal, consisting of hypomethylated regions specific to plaques, can be detected in plasma cfDNA. Our methods thus far have utilized whole genome sequencing, but to study the predictive value of these regions in large cohorts and explore commercial potential, we need to develop a more specific methodology. The current U-BiomarCARE project aims to determine whether the methylation status of plaque-specific regions can be detected in plasma using a droplet digital PCR (ddPCR)-based method. This method targets the most plaque-specific CpG candidate regions, using predesigned methylated and unmethylated probes, allowing us to enrich, amplify, and quantify the methylated and unmethylated signals at these genomic regions.
In previous analyses, we demonstrated that 18-53% of plaque DNA is unmethylated at this CpG site, while other tissues and cell types show less than 5% unmethylation. We confirmed the plaque-specific nature of this signal by applying the ddPCR assay to genomic DNA from 16 plaques, where unmethylated fragments were found in all samples with an average of 46%. These fragments were absent in heart and peripheral blood mononuclear cell (PBMC) DNA, further emphasizing their plaque-specificity. Additionally, we observed that smooth muscle cell (SMC)-derived myofibroblasts were the primary source of this plaque-specific methylation pattern, while endothelial cells showed no detectable unmethylated signal.
To support the role of this CpG in plaque biology, we integrated bulk and single-cell RNA data from the same atherosclerotic plaques. The top CpG is located upstream of MYH10, a well-known vascular SMC gene, and we showed that MYH10 expression in plaques inversely correlates with methylation status. High MYH10 expression was particularly prominent in plaques with a fibrous phenotype. This is further supported by single-cell RNA sequencing (scRNAseq), which revealed that genes negatively associated with this CpG were predominantly expressed in SMCs.
To explore the potential for detecting plaque-specific DNA in circulation, we applied the ddPCR assay to plasma cfDNA from 52 patients with suspected CAD. Among CAD patients, 40% tested positive for an unmethylated signal, although this was not significantly different from the 20% positivity rate observed in patients without significant atherosclerosis. While there was a trend toward higher proportions of unmethylated fragments in CAD patients, the difference did not reach statistical significance.
By demonstrating that methylation-based circulating plaque markers can be measured using this sequencing-free ddPCR method, we are advancing towards the ability to detect plaque-specific DNA in circulation. Once proven reproducible and comparable to whole genome sequencing, this methodology can expand to a capture-based sequencing method that will enable large biomarker studies in both women and men suspected of heart disease. This would significantly improve early and accurate detection of CAD, potentially revolutionizing the healthcare sector by allowing for specific, timely identification of dangerous atherosclerotic plaques in both women and men. For this, we have applied for a new ERC PoC that will develop a larget set of CpGs.
In previous analyses, we demonstrated that 18-53% of plaque DNA is unmethylated at this CpG site, while other tissues and cell types show less than 5% unmethylation. We confirmed the plaque-specific nature of this signal by applying the ddPCR assay to genomic DNA from 16 plaques, where unmethylated fragments were found in all samples with an average of 46%. These fragments were absent in heart and peripheral blood mononuclear cell (PBMC) DNA, further emphasizing their plaque-specificity. Additionally, we observed that smooth muscle cell (SMC)-derived myofibroblasts were the primary source of this plaque-specific methylation pattern, while endothelial cells showed no detectable unmethylated signal.
To support the role of this CpG in plaque biology, we integrated bulk and single-cell RNA data from the same atherosclerotic plaques. The top CpG is located upstream of MYH10, a well-known vascular SMC gene, and we showed that MYH10 expression in plaques inversely correlates with methylation status. High MYH10 expression was particularly prominent in plaques with a fibrous phenotype. This is further supported by single-cell RNA sequencing (scRNAseq), which revealed that genes negatively associated with this CpG were predominantly expressed in SMCs.
To explore the potential for detecting plaque-specific DNA in circulation, we applied the ddPCR assay to plasma cfDNA from 52 patients with suspected CAD. Among CAD patients, 40% tested positive for an unmethylated signal, although this was not significantly different from the 20% positivity rate observed in patients without significant atherosclerosis. While there was a trend toward higher proportions of unmethylated fragments in CAD patients, the difference did not reach statistical significance.
By demonstrating that methylation-based circulating plaque markers can be measured using this sequencing-free ddPCR method, we are advancing towards the ability to detect plaque-specific DNA in circulation. Once proven reproducible and comparable to whole genome sequencing, this methodology can expand to a capture-based sequencing method that will enable large biomarker studies in both women and men suspected of heart disease. This would significantly improve early and accurate detection of CAD, potentially revolutionizing the healthcare sector by allowing for specific, timely identification of dangerous atherosclerotic plaques in both women and men. For this, we have applied for a new ERC PoC that will develop a larget set of CpGs.
We will file for IPR with our top signals, this will be done in 2025
We applied for the BioStudio program from the BioInnovation Institute, and with that received a report regarding our IP position through Aera Technology and Law that concluded: "the biomarker panel may be patentabe". Also, we received a report from Back Bay (consultants in Boston) which shared their advice towards a start-up company:
• In April 2024, the FDA announced that laboratory-developed tests will be classified as in-vitro diagnostic products, which are
considered medical devices and are now subject to increased FDA oversight
+ Given the increasing regulatory scrutiny in the US for lab-developed tests, an initial launch in Europe can help to validate the
technology and support sufficient data/revenue generation for a US FDA-filling and subsequent launch
• Currently, leading cfDNA companies offer sample collection kits that are ordered by physicians and returned to the company to be
run at the company’s in-house laboratory
+ To handle a large sample volume, the Den Ruijter lab will have to establish its own high throughput testing capabilities.
We applied for the BioStudio program from the BioInnovation Institute, and with that received a report regarding our IP position through Aera Technology and Law that concluded: "the biomarker panel may be patentabe". Also, we received a report from Back Bay (consultants in Boston) which shared their advice towards a start-up company:
• In April 2024, the FDA announced that laboratory-developed tests will be classified as in-vitro diagnostic products, which are
considered medical devices and are now subject to increased FDA oversight
+ Given the increasing regulatory scrutiny in the US for lab-developed tests, an initial launch in Europe can help to validate the
technology and support sufficient data/revenue generation for a US FDA-filling and subsequent launch
• Currently, leading cfDNA companies offer sample collection kits that are ordered by physicians and returned to the company to be
run at the company’s in-house laboratory
+ To handle a large sample volume, the Den Ruijter lab will have to establish its own high throughput testing capabilities.