Genomic characteristics of diffuse large B-cell lymphoma associated with Wiskott-Aldrich syndrome

Abstract Introduction Wiskott-Aldrich syndrome (WAS) is a rare X-linked inborn error of immunity (IEI) characterized by thrombocytopenia, eczema, and immune dysregulation. Patients with WAS have an increased risk of malignancies in younger ages than sporadic cases. Most of the malignant diseases are non-Hodgkin's lymphoma, particularly diffuse large B cell lymphoma (DLBCL), in patients with WAS, which is distinct from the T cell immunophenotypes reported in murine models. Impaired immunosurveillance by T and NK cells, a characteristic feature of classical WAS, may contribute to lymphomagenesis in these patients. In addition, the WAS gene itself has been reported to function as a tumor suppressor gene by suppressing DNA double-strand breaks and facilitating homologous recombination repair, further supporting its role in preventing malignant transformation. Despite its high incidence and limited survival outcomes, the genomic mechanisms underlying lymphomagenesis in patients with WAS remain largely unknown. Methods We analyzed four tumor samples from three Japanese WAS patients (P1_L1, P1_L2, P2, and P3) diagnosed with DLBCL during long-term follow-up. One patient (P1) developed DLBCL twice. All patients harbored pathogenic germline variants in the WAS gene: P1 (c.11delG; p.Gly4fs40), P2(c.395insGGAGAT; p.Asp121insGD), and P3 (c.257G>A; p.Arg86His). Histopathological and immunophenotypic evaluations were performed by an experienced hematopathologist. Genomic DNA was extracted from formalin-fixed, paraffin-embedded (FFPE) samples and pre-treated with uracil-DNA glycosylase to remove FFPE-induced deamination artifacts. Genomic profiling was conducted using the Oncomine™ Tumor Mutation Load Assay, which targeted 409 cancer-related genes and enabled detection of somatic single nucleotide variants (SNVs), insertions and deletions, and tumor mutational burden (TMB). It also supported copy number variation (CNV) analysis by evaluating the gene-level read depth relative to internal references. Results All patients developed DLBCL between 16 and 22 years of age. All tumors were CD20-positive, with high Ki-67 proliferation indices (70–90%). P2 tested positive for Epstein-Barr virus (EBV). According to the Hans' classification, P1_L1, P1_L2 and P2 were Germinal Center B-cell-like (GCB)-type, whereas P3 was classified as a non-GCB-type. Tumor-normal paired analyses were conducted using matched tumor and normal genomes, with an average sequencing coverage of 1,821× and 1,414× per base, respectively. After applying the filtering algorithms, an average of 14.5 somatic variants were identified per sample. TMB values calculated from somatic variants ranged from 5.01 to 24.4 variants/Mb: P1_L1 (11.75), P1_L2 (7.51), P2 (5.01), and P3 (24.4). These levels were substantially elevated compared to the TMB in sporadic DLBCLs (around 3 variants/Mb), strongly supporting the role of WAS as a tumor suppressor gene. SNVs in genes associated with NF-κB pathway were observed in three out of four tumors, excluding the EBV-positive P2. These included a nonsense variant of PTEN (p.Lys13Ter, variant allele frequency [VAF] 37.5%) in P1_L1, a missense variant of CARD11 (p.Thr128Met, VAF 27.5%) in P1_L2, and a nonsense variant of FAS (p.Tyr232Ter, VAF 22.2%) in P3. In the EBV-positive P2, a missense variant of TNFRSF14 (p.Gly60Val, VAF 12.8%), which was associated with follicular lymphoma, was identified. CNV analysis revealed the amplification of REL in P1_L1 and P1_L2, MALT1 in P1_L1 and P2, and IRF4 in P3, suggesting recurrent NF-κB activation. Validation experiments for the SNVs and CNVs are currently ongoing. Conclusions This is the first comprehensive analysis of the histopathological and genomic features of DLBCL in patients with WAS. NF-κB activating variants, including the amplification of REL, were partly common to DLBCLs in patients with IL10RA deficiency. Unlike typical immunodeficiency-associated B-cell lymphoproliferative disorders, which often display Activated B-cell-like (ABC)-type DLBCL with NF-κB pathway dependency, three of our cases were GCB-type DLBCL. These findings suggest that WAS-associated DLBCL may arise via distinct oncogenic mechanisms probably due to genomic instability and impaired immunosurveillance, potentially bridging the features of both GCB and ABC subtypes. Further molecular investigations in larger cohorts are warranted to refine the classification and therapeutic targets of lymphomas arising from IEIs, such as WAS.