GENOMIC AND TRANSCRIPTIONAL SINGLE-CELL HETEROGENEITY IN GERMINAL-CENTER LYMPHOMAS: INSIGHTS INTO FOLLICULAR LYMPHOMA TRANSFORMATION

Abstract

B. Ariceta equally contributing author.

Introduction: The diversity of germinal centers has a significant role in the transformation of follicular lymphoma (FL). This heterogeneity in FL is driven by a combination of genetic and epigenetic modifications, and interactions with the tumor microenvironment (TME). Understanding how these mechanisms lead disease progression is crucial for identifying therapeutic targets and prognostic markers.

Methods: We performed single-cell DNA sequencing (scDNA-seq) (Mission Bio Tapestri Platform), single-cell RNA sequencing (scRNA-seq), and spatial transcriptomics (10X Genomics) analysis on 5 lymph node samples at diagnosis: 3 DLBCL (1 GCB, 2 ABC) and 2 FL (1 transformed—tFL- and 1 non-transformed—ntFL-).

Results: In the scRNA-seq analysis, malignant B cells clustered into 6 clusters. Light-zone (LZ) cells were specific to ntF, whereas tFL and GCB were enriched in dark zone/light zone (DZ/LZ) cells. Pre-memory B (pre-M) and pre-plasma cells predominated in ABC (Figure 1a). Differential expression analysis identified BCR activation (DZ-LZ), cytokine signaling (LZ), and pro-tumor pathways activation, including NF-kB (pre-M). Transcriptional similarities between tFL and GCB suggest a common precursor driven by BCR activation. However, GCB revealed a dominant cell-cycle dysregulation signature, while tFL showed an immune-evasion one.

T cell subclusters varied significantly across patients. ntFL was enriched in naïve CD4⁻CD8⁻ and CD8⁺ central memory T cells, while tFL and GCB were enriched in CD4⁺ T cells. CD4⁺ and CD8⁺Teff cells were predominant in ABC samples. CD4⁺ T cells promoted T cell tolerance (IL6/STAT3, PD-1), while CD8⁺Teff cells exhibited high exhaustion marker expression. CD8⁺Teff cells from DLBCL and tFL showed stronger exhaustion profiles than ntFL. CD4⁺ Tfh cells expressed genes involved in adhesion with malignant B cells, with significantly higher expression in DLBCL and tFL (Figure 1b).

In the scDNA-seq analysis, patients harbored mutations in chromatin-modifying genes (KMT2D and EZH2) and oncogenic genes (NOTCH2). In GCB and tFL samples, KMT2D variants were identified as early events, while EZH2 (tFL) and ATM (GCB) mutations emerged as secondary events. A nonsense mutation in TET2 was detected in non-B cells, suggesting the presence of clonal hematopoiesis (CH). A second scDNA-seq analysis was performed to investigate CH further, focusing on CH-related variants in 3 samples. All harbored 2 or 3 mutations in epigenetic modifier genes (TET2, ASXL1, and DNMT3A).

In the spatial transcriptomics analysis, 28,387 spots were examined. Deconvolution using paired scRNA-seq data confirmed an adequate representation of all cell types.

Conclusions: These findings provide an integrated view of cellular heterogeneity in GC lymphomas. Transcriptional similarities between tFL and GCB may aid in identifying transformation predictors. Moreover, these advanced techniques suggest that CH may be more prevalent in B-cell lymphoma than previously describe.

Keywords: genomics, epigenomics, and other -omics; microenvironment; indolent non-Hodgkin lymphoma

Potential sources of conflict of interest:

C. Grande

Consultant or advisory role: Membership on an entity's Board of Directors or advisory committees (AbbVie)

M. Canales

Consultant or advisory role: Consultancy (Beigene, BMS, Incyte, Janssen, Karyopharm, Kite, Kyowa, Lilly, Roche, Takeda)

Honoraria: Speakers Bureau (Incyte, Janssen, Kite, Kyowa, Roche, Takeda)