Alternative splicing patterns in genetic subtypes of diffuse large B-cell lymphoma.

backgroundDiffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease with distinct genetic subtypes, each exhibiting unique molecular characteristics. Alternative splicing (AS) plays a critical role in hematologic malignancies, but its role in DLBCL progression remains less understood. Recent studies have focused extensively on genetic variants, but systematic exploration of AS in DLBCL is limited.

methodsWe analyzed whole-exome and RNA sequencing data from 620 DLBCL patient samples obtained from the European Genome-phenome Archive. RNA-seq data were aligned using STAR and quantified with RSEM to calculate TPM-normalized expression values. Subtype classification was performed using the LymphGen framework, integrating somatic variants identified with GATK Mutect2, annotated with Annovar, and copy number variations, as well as BCL2 and BCL6 fusion events detected using Arriba and validated with FISH. Alternative splicing events were identified with rMATS, and isoform-level differences were analyzed using DRIMSeq. Splicing-related pathway activity was assessed using MSigDB gene sets, and splicing factor expression was evaluated across subtypes.

resultsThe BN2 and MCD subtypes demonstrated higher activity in AS-related pathways, including spliceosome assembly and mRNA splice site recognition, accompanied by a significant increase in non-canonical isoforms. In the BN2 subtype, differential splicing was observed, especially in genes like ACTG1, RPLP0, CD74, and ACTB. These genes showed significant changes in their isoforms, suggesting that alternative splicing may play a role in driving the aggressive behavior associated with this subtype. Notably, these genes include essential housekeeping components, suggesting that targeting their splicing variants could disrupt critical cellular processes in cancer cells while sparing normal tissues. In addition, splicing factor analysis identified RBM39 as significantly downregulated in MCD, suggesting its role in aberrant splicing regulation. RBM39, an RNA-binding protein involved in the regulation of splicing, may contribute to DLBCL pathogenesis through its reduced expression, potentially leading to abnormal splicing patterns that promote malignancy in the MCD subtype.

conclusionsIntegrated analysis using rMATS and DRIMSeq consistently identified subtype-specific AS patterns, consolidating our findings. Future research should focus on validating the functional impact of these splicing alterations and assessing their potential as biomarkers for more tailored therapeutic approaches in DLBCL.