Targeted, Low Cost Epigenetic Microarray for AML and MDS Detection
Authors: Neaman Miriam , Bell Mika , Deek Jasline , Haldar Rita , Manor Amit , Moshe Yakir , Avivi Irit Mazza , Mittelman Moshe , Vitkon Roy , Levi Mor , Gat Roi , Fridberg Gil , Birov Natalia , Daukėlaitė Guoda , Sablauskas Karolis , Černovas Andrejus , Zucenka Andrius , Vaitekėnaitė Vilmantė , Kastritis Efstathios , Papajik Tomas , Kriegova Eva , Cernan Martin , Dyskova Tereza , Ebenstein Yuval 
Published: December 2024

Introduction: Aberrant DNA methylation patterns in Acute Myeloid Leukemia (AML) are well documented. However, the oxidized form of methylation, 5hmC, indicating active methylation erasure, is difficult to detect and has been less studied. We introduce a novel biochemical labeling procedure that allows optical detection of both epigenetic marks on a custom microarray. The custom-designed microarray targets genomic regions prone to epigenetic alterations in AML. This research is part of a multicenter clinical trial funded by the Horizon Cancer Mission (NCT05735704). Specifically, this study focuses on AML and Myelodysplastic Syndrome (MDS), aiming to identify differentially methylated (5mC) and hydroxymethylated (5hmC) genomic regions between these conditions and healthy control samples. These regions could serve as biomarkers for classifying samples as AML, MDS, or healthy states.
Methods: Peripheral blood samples from 45 newly diagnosed AML patients, 14 MDS patients, and 46 healthy volunteers were collected across four European clinical centers. All samples were processed in our lab 7-10 days after collection. We designed a custom microarray targeting 13,000 genomic loci based on a comprehensive literature review of epigenetic modulation in AML. The new microarray slides accommodate 24 samples per slide, enabling low-cost and high-volume analysis.
Genomic DNA was extracted, and both 5hmC and 5mC were fluorescently labeled. The DNA was then hybridized to the custom microarray. The array was scanned to provide the degree of epigenetic modulation at each genomic loci via the fluorescence intensity recorded. Data were extracted and analyzed using a novel computational pipeline that enables internal normalization by calculating pairwise ratios across the array.
Results: The median age of AML patients was 65 years (IQR 50-74), compared to 66 years (IQR 55-74) of healthy controls. According to the 2022 ELN risk stratification, 18% of AML patients were classified as favorable, 36% as intermediate, and 36% as adverse. NPM1 and FLT3 mutations were identified in 35% and 27% of patients, respectively. NGS myeloid panel testing, conducted in two-thirds of patients, revealed mutations in epigenetic modifiers (DNMT3A, TET2, ASXL1) in 25% (n=12).
The MDS cohort comprised 14 patients, median age was 72 (IQR 65-77): eight patients had low-risk MDS under observation and six patients required therapeutic intervention.
Our analysis revealed distinct methylation and hydroxymethylation patterns differentiating AML and MDS patients from healthy controls. 5mC analysis identified 187 differential biomarkers discriminating between AML and control samples with 91% sensitivity and 100% specificity. For 5hmC, 176 identified biomarkers achieved 91% sensitivity and 92% specificity.
Preliminary results for MDS patients allowed distinguishing them from healthy individuals with 100% sensitivity for the 5mC biomarkers and 67% sensitivity for the 5hmC markers. Both markers showed 100% specificity.
Additionally, we analyzed the ability to distinguish between MDS and AML patients using differential epigenetic markers. 5mC analysis yielded 67% sensitivity and 100% specificity, while 5hmC analysis showed 100% sensitivity and 89% specificity, for identifying MDS patients.
Conclusion: This study introduces a newly developed, minimally invasive tool for accurately detecting AML and MDS using peripheral blood samples. Our method offers an opportunity to detect these myeloid malignancies without performing bone marrow aspiration, an advantage particularly important for diagnosing MDS. The epigenetic biomarkers we identified provide an additional layer of data to complement routine diagnostic tests, especially valuable in cases lacking specific markers, such as normal karyotype AML without common prognostic mutations. Our approach successfully distinguishes between MDS and AML, offering the possibility of early detection of MDS progression to AML through blood testing whenever progression is suspected. Lastly, the identified epigenetic signatures may provide crucial insights into disease mechanisms, guiding future discovery of novel therapeutic targets in AML and MDS.