CLONAL HEMATOPOIESIS IN DIFFUSE LARGE B-CELL LYMPHOMA

Abstract

D. Piffaretti and I. Romano equally contributing authors.

Introduction: Clonal Hematopoiesis (CH) may promote diffuse large B-cell lymphoma (DLBCL) in two ways: by seeding mutations in the B-cells progenitors, potentially contributing to malignant transformation. Alternatively, lymphoma may be promoted by the clonal and pro-inflammatory tumor microenvironment derived from CH. In this study we aimed to determine: (i) CH prevalence in newly diagnosed DLBCL; (ii) clinical impact of CH; (iii) correlation between CH and DLBCL genetic lesions; (iv) co-occurrence frequency of DLBCL driver and CH mutations at the single cell level; (v) enrichment of CH in cells of the lymphoma microenvironment compared to blood.

Methods: Patients (n = 387) from the IOSI-EMA003 and SAKK38/19 trials were analyzed. CH mutations were identified in genomic DNA using a myeloid panel, while a lymphoid panel detected DLBCL mutations, somatic copy number abnormalities, and BCL6 fusions in plasma cfDNA. Multiomic scDNA-seq and immunophenotyping of paired peripheral blood (PB) or bone marrow (BM) and disaggregated lymph nodes of 6 patients were performed. A custom Tapestri (MissionBio) panel targeted CH mutations and barcoded the dominant DLBCL clone by covering trunk oncogene mutations, enabling simultaneous genotyping and phenotyping of B cells, T cell subtypes, and myeloid cells. To assess whether CH-bearing myeloid cells support DLBCL in vitro: (i) BM cells from Tet2 knockout (KO) and control mice were differentiated into macrophages and co-cultured with a Tp53 KO murine B-cell lymphoma line; (ii) biallelic TET2-KO human THP1 monocytic cells, were differentiated into macrophages and co-cultured with DLBCL cell lines. Additionally, lymphoma-prone Klf2^fl/fl/Notch2IC^fl/+/Cd19Cre^+/−/Cd45.2^+/+ oncogenic and Cd19Cre^+/−/Cd45.2^+/− control mice were adoptively transplanted with BM cells from Tet2^+/+, Tet2^+/−, and Tet2^−/− C57BL/6 Cd45.1^+/− mice without conditioning.

Results: The analysis was done including patients per the CONSORT diagram (Figure 1A). CH mutations (VAF > 1%) were found in 38% of patients, primarily in DNMT3A and TET2, and correlated with age (Figure 1B). However, CH showed no association with features of lymphoma aggressiveness (clinical stage, B-symptoms, IPI) or DLBCL subtypes (cell of origin, C1-C5). Cox analyses (univariate/multivariate, adjusted for IPI), revealed no impact of CH on progression-free survival or lymphoma-specific survival. In vitro, lymphoma cell survival was limitedly affected by TET2 status in macrophages. Engraftment in control mice mirrored the typical load of CH in humans (∼1% PB leukocytes at 3 months), whereas Tet2^+/− and Tet2^−/− cells failed to engraft in the lymphoma-prone Klf2l^fl/fl/Notch2IC^fl/+/Cd19Cre^+/−/Cd45.2^+/+ mice, suggesting elimination of Tet2-deficient cells in an oncogenic setting. Single-cell analysis showed no co-occurrence of CH and DLBCL mutations, and CH-derived cells were not enriched in the lymphoma microenvironment.

Conclusions: CH mutations likely have a limited role in DLBCL promotion.

Research funding declaration: The study was supported by ISREC Foundation, Helmut Horten Foundation, ETH Zurich Lymphoma Challenge.

Keywords: tumor biology and heterogeneity; aggressive B-cell non-Hodgkin lymphoma; diagnostic and prognostic biomarkers

No potential sources of conflict of interest.