Integrated DNA Methylation and Multiclock Epigenetic Aging Analysis Reveals Distinct Biological Aging Signatures in Responders and Nonresponders

Background: Deoxyribonucleic acid (DNA) methylation-driven epigenetic aging is increasingly recognized as a biomarker of disease progression and therapeutic response. This study integrates genome-wide methylation profiling with multiclock epigenetic age estimation to investigate differences between responder (R) and nonresponder (NR) groups. Methods: Sixteen samples (8 R, 8 NR) were analyzed using the Infinium Human Methylation 850 (EPIC) array, interrogating over 935,000 CpG sites. Data preprocessing and quality control were performed using minfi, ChAMP, and RnBeads, ensuring high-quality data with > 935,000 detected CpG sites per sample. Normalization and background correction were applied in GenomeStudio, followed by differential methylation analysis using the Illumina Custom Model. Significant differentially methylated regions were identified using DiffScore (> 13/< −13) and Δβ (> 0.2/< −0.2). Epigenetic aging was assessed using multiple clocks, including PCGrimAge, DunedinPACE, Hannum, Horvath, and PhenoAge. Results: Differential methylation analysis identified distinct CpG signatures separating R and NR groups, with key genes such as SPDEF, NOL4, FN3K, ATF2, and CD300E showing significant alterations. Epigenetic aging analysis revealed a consistent trend of biological age deceleration in responders, with negative age acceleration observed across multiple clocks (up to −15% in PCGrimAge and −25% in Horvath). In contrast, nonresponders exhibited increased biological aging and greater variability, including positive age acceleration up to +12%. DunedinPACE analysis demonstrated faster aging rates in nonresponders (up to +24%), whereas responders maintained values closer to expected or slower aging trajectories. PhenoAge further supported increased aging heterogeneity in nonresponders. Conclusion: This integrative analysis highlights a strong association between epigenetic aging dynamics and treatment response, where responders exhibit slower biological aging compared to nonresponders. These findings underscore the potential of combining DNA methylation and epigenetic clocks for biomarker discovery and precision medicine. Publication History Article published online: 15 July 2026 © 2026. The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution License, permitting unrestricted use, distribution, and reproduction so long as the original work is properly cited. (https://creativecommons.org/licenses/by/4.0/) Thieme Medical and Scientific Publishers Private Limited A-13A, Graphix Tower 1, 6th floor, Sector 62, Noida 201309, Uttar Pradesh, India