Molecular clustering on ctDNA improves the prognostic stratification of DLBCL patients compared to ctDNA levels

• Molecular clustering on ctDNA recapitulates molecular clusters identified on tissue biopsy in DLBCL

• Compared to ctDNA levels only, the addition of molecular clustering improved outcome prediction in DLBCL patients

Circulating tumor DNA (ctDNA) levels can help predict outcomes in diffuse large B-cell lymphoma (DLBCL), but its integration with DLBCL molecular clusters remains unexplored. Using the LymphGen tool in 77 DLBCL with both ctDNA and tissue biopsy, a 95.8% concordance rate in molecular cluster assignment was observed, showing the reproducibility of molecular clustering on ctDNA. A multicenter, prospective cohort of 166 newly diagnosed DLBCL was analyzed for ctDNA levels and molecular clusters using CAPP-seq. Patients with ctDNA levels < 2.5 log10hGE/mL had a 4-year progression-free survival (PFS) and overall survival (OS) of 71.7% and 85.7%, respectively, compared to 50.3% and 61.0% for those with higher ctDNA levels (p=0.0018 and p=0.0017). Recursive partitioning showed that patients with ctDNA levels > 2.5 log10hGE/mL were further stratified by clusters ST2/BN2. In this group, ST2/BN2 patients associated with a favorable outcome with a 4-year PFS and OS of 87.5% and 100%, respectively, compared to 38.0% and 47.1% for other clusters (p=0.003 and p=0.001). Combining ctDNA levels and ST2/BN2 clusters improved outcome prediction. Low-risk patients (N=51), characterized by ctDNA levels < 2.5 log10hGE and/or BN2/ST2 cluster, had a 4-year PFS and OS of 75.3% and 87.8%. High-risk patients (N=115), with ctDNA levels > 2.5 log10hGE and no BN2/ST2 cluster, had a 4-year PFS and OS of 38.0% and 47.1%. Adding cluster assignment to ctDNA levels improved the model's C statistics (0.63 vs. 0.59 for PFS and 0.68 vs. 0.63 for OS). Liquid biopsy thus provides a multi-layered approach for outcome prediction in DLBCL.