Mirandari, Amatta (2025) A multi-omics approach to explore the epigenomic landscape of splenic marginal zone lymphoma (SMZL). University of Southampton, Doctoral Thesis, 368pp.
Abstract
Splenic marginal zone lymphoma (SMZL) is a rare, low-grade B-cell malignancy. Whilst often indolent in nature, a significant proportion of patients (70%) will require treatment. The biased usage of the IGHV1-2*04 allele is found in ~30% of SMZL patients and has been associated with histological transformation and poor survival. This subtype features ongoing Somatic Hypermutation (SHM) and frequent mutations in del(7q), KLF2, and NOTCH2, suggesting genetic and antigenic drivers; however, its molecular features remain understudied.
The study aims to comprehensively explore the epigenomic landscape and features of SMZL using an integrated multi-omics analysis, with particular focus on the IGHV1-2*04 sub-type. Using a novel tumour-normal paired whole genome sequencing (WGS) dataset from 25 SMZL patients, somatic aberrations were identified across the genome, which contributed to a larger integrated SMZL cohort (n=294) with WGS, extended panel re-sequencing, methylation, transcriptomic, and chromatin-accessibility data. Integrated analysis with single-cell RNA and VDJ-seq data from SMZL (n=3) and normal spleens (n=2) further revealed intra-tumoral heterogeneity.
The matched WGS validated key driver gene mutations in KLF2, NOTCH2, KMT2D, TNFAIP3, TRAF3, SPEN and TP53. Novel non-coding mutational hotspots in key B-cell regulatory genes were identified and confirmed by the re-sequencing panel (n=76/294), targeting genes such as BCL6 (21%, 16/76), PAX5 (14%, 11/76), BACH2 (13.5%, 10/76). Importantly, enrichment of an SBSSHM (p < 0.05) signature revealed a possible role of aberrant SHM in the generation of key driver regions and genes. Furthermore, the IGHV1-2*04 subtype showed the expected inferior time-to-first treatment (TTFT) (HR=1.63, CI:1.08-2.45, p=0.018) and intermediate levels of IGHV SHM (97-99.9% IGHV identity, p < 0.001). It was further shown that this subtype is enriched for molecular and genetic features associated with more aggressive disease, including: i) KLF2, NOTCH2, and TRAF3 mutations, and del(7q) (p< 0.01), ii) greater mutational burden (n>=2 mutations per case, p< 0.01), iii) elevated proliferative history (epiCMIT from methylation data, p=0.003), iv) deregulation (by RNA-seq and methylation data) of cell-cycle control pathways (G2M pathway ,FDR=0.02,E2F targets, FDR<0.01), v) higher global promoter methylation (High-M) in PRC2 targets (p=0.04), vi) evidence of PRC2 perturbation by over-representation of transcription factor binding sites (EZH2, SUZ12) in hypermethylated CpGs (FDR<.05) and vii) branching clonal trajectories with evidence of ongoing somatic hypermutation, resulting in divergent B-cell clonotypes.
Overall, we present an analysis of novel datasets in SMZL, uncovering key functional pathways and features influencing clinical outcomes of the IGHV1-2*04 subtype. Findings further suggest selective pressures on the B-cell receptor, driven by lesions acquired through aSHM mechanisms. These insights may provide potential avenues for new targeted therapies.
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