Abstract 7079: DNA methylation profiles classifies dysplasia degree in colorectal adenomas

Abstract Background: Advanced precancerous lesions (APL), precursors to colorectal cancer (CRC), progress from low-grade dysplasia (LGD) to high-grade dysplasia (HGD) through a stepwise accumulation of genomic alterations. Those alterations include mutations, structural variants, and altered methylation patterns, driving dysplastic cells to carcinogenic transformation. Herein, we present a comprehensive analysis of differential DNA methylation profiles to classify dysplasia severity in colorectal adenomas. Methods: Whole-genome methylation sequencing libraries from 9 HGD and 6 LGD tissue samples were prepared using the NEBNext Enzymatic Methyl-seq kit (EM-seq). To identify differentially methylated regions (DMRs) we have employed two distinct computational tools (DMRcaller and MethylKit) and divided the genome into 1kb bins. DMRs identified by both methods were mapped to genomic features such as genes, gene promoters, CpG islands, and enhancers. Gene ontology and pathway enrichment analyses were performed to elucidate affected biological processes. The identified methylation signature was subsequently investigated in plasma-derived whole-genome EM-seq data from 30 adenoma patients (26 LGD and 4 HGD) and 30 normal samples. Principal component analysis (PCA) and statistical tests were employed to assess its discriminatory power for group classification. Results: Differential methylation analysis identified 3, 751 significant DMRs, with 626 overlapping between both strategies, achieving significant separation of samples by dysplasia grade (FDR = 1.73e-05). The analysis revealed epigenetic changes in genomic clusters regulating DNA-binding transcription factors, tissue morphogenesis, and the Wnt signaling pathway, linking methylation disruptions to dysplasia severity. Furthermore, our methylation signature, when applied to independent plasma samples from HGD and LGD patients, significantly separated the adenoma samples according to their dysplasia grade (FDR = 4.93e-34). Conclusions: Our analysis enhances the understanding of the molecular biology underlying high- and low-grade dysplastic adenomas. The identified methylation signature, reflecting epigenetic alterations and disruptions in genomic context, allows for the distinction between HGD and LGD in both tissue and plasma samples. This approach advances risk stratification and personalized management for adenoma patients. Further studies are necessary to validate our findings in larger cohorts and to evaluate the potential of this signature in predicting CRC progression. Citation Format: Juan Carlos Higareda-Almaraz, Francesco Mattia Mancuso, Pol Canal-Noguer, Kristi Kruusmaa, Arianna Bertossi. DNA methylation profiles classifies dysplasia degree in colorectal adenomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7079.