Epigenetic profiling to identify aggressiveness markers in non-Hodgkin lymphoma via a targeted methylation-based multi-cancer early detection (MCED) blood test.

7018 Background: Non-Hodgkin lymphoma (NHL) encompasses a broad spectrum of malignancies, ranging from highly indolent to highly aggressive subtypes, each requiring distinct clinical management strategies. We developed a targeted methylation-based MCED test, and our prior work showed aggressive solid tumors exhibit elevated cell-free DNA (cfDNA) tumor fractions and distinct methylation signatures characterized by high methylated-to-unmethylated (M/U) ratios. This study focused on NHL subtypes to systematically compare epigenetic biomarkers (cfDNA tumor fraction and methylation signatures) for reliable differentiation of indolent and aggressive NHL. Methods: Pretreatment blood samples from NHL patients enrolled in a MCED study were analyzed using the targeted methylation assay. Patients were stratified into aggressive (diffuse large B-cell lymphoma [DLBCL], n=134; mantle cell lymphoma [MCL], n=18; other aggressive NHL, n=2) and indolent subgroups (follicular lymphoma [FL], n=58; marginal zone lymphoma [MZL], n=38; other indolent NHL, n=16). cfDNA tumor fraction was quantified via a zero-inflated negative binomial model, based on the distribution of methylation signal intensities. Methylation profiles were assessed by computing M/U ratios between subgroups. Intergroup comparisons of these biomarkers were conducted using the Mann-Whitney U test, with adjustments for multiple testing. Additional subgroup analyses were stratified by clinical stage to mitigate confounding factors. Results: MCED testing showed higher sensitivity in identifying aggressive NHL subtypes (78.06% vs 67.54% in indolent subtypes), even in stage I-II cases (62.50% vs 46.34% in indolent subtypes). Aggressive subtypes exhibited significantly higher cfDNA tumor fractions than indolent subtypes (Wilcoxon rank-sum test, p=1.27e- 06 ). A total of 31,075 methylation markers (71.66% hypomethylated) showed elevated M/U ratios in aggressive subtypes. These differences in cfDNA tumor fractions and M/U ratios persisted after clinical stage stratification. Conclusions: The MCED assay's superior sensitivity for aggressive NHL suggests preferential detection of aggressive NHL while minimizing indolent NHL overdiagnosis. Aggressive NHL is characterized by elevated cfDNA tumor fractions and distinct methylation profiles with high M/U ratios. These findings demonstrate cfDNA methylation patterns are promising epigenetic biomarkers, enabling aggressiveness differentiation in both solid tumors and NHL. Collectively, this study establishes a potential non-invasive strategy for NHL risk stratification and optimized clinical management.