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Background: Patients with higher-risk (HR) myelodysplastic syndrome (MDS), ineligible for allogeneic hematopoietic stem cell transplantation (alloHSCT), require prompt therapeutic interventions, such as treatment with hypomethylating agents (HMAs) to restore normal DNA methylation patterns, mainly of oncosuppressor genes, and consequently to delay disease progression and increase overall survival (OS). However, response assessment to HMA treatment relies on conventional methods with limited capacity to uncover a wide spectrum of underlying molecular events., Methods: We implemented liquid chromatography-tandem mass spectrometry (LC-MS/MS) to assess 5' methyl-2' deoxycytidine (5mdC), 5' hydroxy-methyl-2'-deoxycytidine (5hmdC) levels and global adenosine/thymidine ([dA]/[T]) ratio in bone marrow aspirates from twenty-one HR MDS patients, pre- and post-HMA treatment. Additionally, targeted methylation analysis was performed by interpretation of NGS-methylation (MeD-seq) data obtained from the same patient cohort., Results: LC/MS-MS analysis revealed a significant hypomethylation status in responders (Rs), already established at baseline and a trend for further DNA methylation reduction post-HMA treatment. Non-responders (NRs) reached statistical significance for DNA hypomethylation only post-HMA treatment. The 5hmdC epigenetic mark was approximately detected at 37.5-40% among NRs and Rs, implying the impairment of the natural active demethylation pathway, mediated by the ten-eleven (TET) 5mdC dioxygenases. R and NR subgroups displayed a [dA]/[T] ratio < 1 (0.727 - 0.633), supporting high frequences of 5mdC transition to thymidine. Response to treatment, according to whole genome MeD-seq data analysis, was associated with specific, scattered hypomethylated DMRs, rather than presenting a global effect across genome. MeD-seq analysis identified divergent epigenetic effects along chromosomes 7, 9, 12, 16, 18, 21, 22, X and Y. Within statistically significant selected chromosomal bins, genes encoding for proteins and non-coding RNAs with reversed methylation profiles between Rs and NRs, were highlighted., Conclusions: Implementation of powerful analytical tools to identify the dynamic DNA methylation changes in HR MDS patients undergoing HMA therapy demonstrated that LC-MS/MS exerts high efficiency as a broad-based but rapid and cost-effective methodology (compared to MeD-seq) to decode different perspectives of the epigenetic background of HR MDS patients and possess discriminative efficacy of the response phenotype to HMA treatment., Competing Interests: Declarations. Ethics approval and consent to participate: All patients and healthy participants provided their written informed consent according to the Declaration of Helsinki, being informed about both clinical and translational investigations and the study was approved by the University General Hospital of Patras Ethics Committee (approval number 33807/24.12.2020). Consent for publication: All authors contributed to the article and approved the submitted version. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Background: As new treatment options for patients with higher-risk myelodysplastic syndromes are emerging, identification of prognostic markers for hypomethylating agent (HMA) treatment and understanding mechanisms of their delayed and short-term responses are essential. Early fetal hemoglobin (HbF) induction has been suggested as a prognostic indicator for decitabine-treated patients. Although epigenetic mechanisms are assumed, responding patients' epigenomes have not been thoroughly examined. We aimed to clarify HbF kinetics and prognostic value for azacytidine treated patients, as well as the epigenetic landscape that might influence HbF re-expression and its clinical relevance., Results: Serial HbF measurements by high-performance liquid chromatography (n = 20) showed induction of HbF only among responders (p = 0.030). Moreover, HbF increase immediately after the first azacytidine cycle demonstrated prognostic value for progression-free survival (PFS) (p = 0.032, HR = 0.19, CI 0.24-1.63). Changes in methylation patterns were revealed with methylated DNA genome-wide sequencing analysis (n = 7) for FOG-1, RCOR-1, ZBTB7A and genes of the NuRD-complex components. Targeted pyrosequencing methodology (n = 28) revealed a strong inverse correlation between the degree of γ-globin gene (HBG2) promoter methylation and baseline HbF levels (p = 0.003, r s  =  - 0.663). A potential epigenetic mechanism of HbF re-expression in azacytidine responders was enlightened by targeted methylation analysis, through hypomethylation of site -53 of HBG2 promoter (p = 0.039, r s  =  - 0.504), which corresponds to MBD2-NuRD binding site, and to hypermethylation of the CpG326 island of ZBTB7A (p = 0.05, r s  = 0.482), a known HbF repressor. These changes were associated to blast cell clearance (p HBG2  = 0.011, r s  = 0.480/p ZBTB7A  = 0.026, r s  = 0.427) and showed prognostic value for PFS (p ZBTB7A  = 0.037, HR = 1.14, CI 0.34-3.8)., Conclusions: Early HbF induction is featured as an accessible prognostic indicator for HMA treatment and the proposed potential epigenetic mechanism of HbF re-expression in azacytidine responders includes hypomethylation of the γ-globin gene promoter region and hypermethylation of the CpG326 island of ZBTB7A. The association of these methylation patterns with blast clearance and their prognostic value for PFS paves the way to discuss in-depth azacytidine epigenetic mechanism of action., (© 2024. The Author(s).)