Dissecting clonal hematopoiesis in diffuse large B-cell lymphoma through multi-compartment genomic profiling Free

Background: Clonal hematopoiesis (CH) in diffuse large B-cell lymphoma (DLBCL) has been investigated in a limited number of studies, most of which focused exclusively on genomic DNA from peripheral blood. However, comprehensive multi-compartment profiling including genomic DNA, plasma cell-free DNA (cfDNA), and tumor-infiltrating CH (TI-CH) remains largely unexplored. This gap hinders a full understanding of the dynamics and clinical relevance of CH in DLBCL. This study aims to: i) characterize CH from the myeloid compartment and plasma cfDNA; ii) evaluate the clinical impact of CH in newly diagnosed DLBCL and its association with disease molecular subtypes; iii) assess the clonal dynamics of CH at baseline and end of treatment (EOT); iv) determine the prevalence and clinical significance of TI-CH clones in DLBCL.

Methods: A total of 366 samples from 169 patients with newly diagnosed DLBCL treated with R-CHOP–like regimens were analyzed for CH using CAPP-Seq–based NGS. Sample types included granulocytes (n=126), plasma cfDNA (n=93 at baseline and n=90 at end of treatment), and fresh-frozen or FFPE lymph node biopsies (n=57). A custom panel targeting 28 genes commonly mutated in CH was used for analysis.

Results: Median age of the cohort was 67 years and all patients were treated with R-CHOP–based regimens. At a median follow-up of 49.5 months, overall survival (OS) was 71.6%, and progression-free survival (PFS) was 61.1%. CH was detected in the granulocyte compartment in 29.4% of the cohort, with DNMT3A (19.8%) and TET2 (10.3%) as the most frequently mutated genes. Notably, cfDNA analysis identified a higher prevalence of CH, detecting mutations in 38.5% of cases at baseline, most commonly in DNMT3A (22.0%) and TET2 (18.7%). Given its sensitivity and broader detection, cfDNA-derived CH was subsequently utilized to evaluate the clinical impact of CH in the study population. CH⁺ patients had a significantly higher median age of 74 years compared to 61 for CH^- patients (P < 0.001). Among CH⁺ patients, DNMT3Amutations were significantly enriched in the C3 molecular subtypeof DLBCL (P = 0.009), suggesting a potential biological link between CH-related epigenetic alterations and specific tumor subtypes. This association remained consistent in samples collected at EOT, indicating a stable relationship over the disease course. Furthermore, the presence of CH was significantly associated with an increased risk ofsecond malignancies, observed in 33.3% of CH⁺patients compared to 9.4% of CH^- cases (P = 0.007). In contrast, CH did not predict for a higher incidence of therapy related hematologic toxicities (P > 0.050). CH⁺ patients demonstrated marginally reduced PFS (55.8% vs. 64.1%) and OS (69.8% vs. 72.4%) relative to CH^- patients; however, these differences did not reach statistical significance (P = 0.547 and P = 0.466, respectively), indicating that CH, though associated with older age, does not negatively impact survival outcomes. The dynamics of CH was analyzed in 90 sequential plasma cfDNA samples collected at both baseline and EOT. The prevalence of CH increased from 36.0% at baseline to 55.0% in EOT samples (P < 0.001). The median VAF also rose significantly, from 1.6% to 2.1% (P = 0.011). Notably, mutations ofPPM1D, a DNA damage response gene, were mostly undetectable prior to therapy but emerged following chemoimmunotherapy, increasing from 1.1% (1/93 ) baseline cases to14.4% (13/90) EOT cases. Fifty-seven DLBCL lymph nodes were sequenced to determine the prevalence of TI-CH, which was identified in 19.3% (11/57) of patients, most commonly involving mutations of DNMT3A (10.5%, VAF range 3.2–27.0) and TET2 (7.0%, VAF range 2.45–27.4). Similar to CH in other compartments, also TI-CH status did not significantly influence clinical outcomes, with median PFS of 66.8% in TI-CH⁺ patients versus 61.4% in TI-CH^-patients (P=0.799), and median OS of 70.7% versus 81.1%, respectively (P = 0.410).

Conclusions: Multi-compartment profiling of CH in DLBCL reveals that CH affects multiple anatomic compartments, preferentially targets specific disease molecular subtypes and associates with increased risk of second malignancies. R-CHOP treatment significantly expands CH clones in prevalence and VAF, including de novo emergence of PPM1D mutations. Both CH and TI-CH did not impact clinical outcomes and therapy related toxicities of DLBCL patients when treated with R-CHOP-based regimens.