Integrating Tumor and Organoid DNA Methylation Profiles Reveals Robust Predictors of Chemotherapy Response in Rectal Cancer.

Rectal cancer patients display heterogeneous responses to neoadjuvant treatment-including the intensive total neoadjuvant therapy (TNT)-and reliable biomarkers are lacking to guide which tumors will benefit most from these regimens. Here, we profiled DNA methylation in tumor tissue and matched patient-derived organoids (PDOs) from 18 rectal cancer cases (50 total samples), leveraging the Illumina MethylationEPIC array and quality control filters that retained 771,964 CpG sites. Analyses used linear models (for tissue-only or PDO-only) and a joint linear mixed-effects approach (accounting for patient-level random effects) to identify significant CpGs associated with log-transformed FOLFOX IC50. We found that PDOs faithfully recapitulate patient-tumor methylation patterns (Spearman's correlation >0.95 among replicate organoids), and the joint model uncovered 745 CpGs tied to FOLFOX sensitivity, many of which were missed in tissue-only analyses. Differentially methylated regions reinforced that broader epigenetic blocks near TSS or enhancer regions may modulate chemo-resistance, while pathway enrichment pinpointed focal adhesion, ECM-receptor interaction, calcium signaling, and folate metabolism as key processes. A methylation risk score derived from these CpGs significantly predicted progression-free survival in an independent colorectal cancer cohort (p=0.019), outperforming single-sample-based signatures. These findings suggest that combining methylation profiles from both tumors and PDOs can expose robust epigenetic drivers of therapy response, aiding the development of clinically actionable biomarkers for rectal cancer TNT.