Dissecting the biology of hairy cell leukemia variant: Insights from whole exome and RNA sequencing

Abstract

Abstract Introduction: Hairy cell leukemia variant (HCL-v) is a rare aggressive splenic B-cell lymphoma that differs significantly from its classical counterpart (HCL) in both clinical behavior and molecular profile. Unlike classical HCL, HCL-v lacks the hallmark of BRAF V600E mutation while it shares morphologic and immunophenotypic features with other splenic small B-cell lymphomas - namely splenic marginal zone lymphoma (SMZL) and splenic diffuse red pulp lymphoma (SDRPL). The biological mechanisms driving HCL-v remain poorly understood, not least due to the difficulty in accessing robustly diagnosed primary specimens. In fact, published studies are limited by their reliance on peripheral blood rather than splenectomy specimens, which hinders accurate classification and introduces a significant confounder in interpreting molecular findings. To address this gap, we performed whole exome sequencing (WES) and transcriptome analysis (RNA-seq) on a unique collection of hard-to-acquire splenectomy specimens, providing a tissue-based molecular framework to distinguish HCL-v from other splenic B-cell neoplasms. Methods: Tumor-derived genomic DNA was extracted from frozen spleen tissue of 10 HCL-v cases, each with B-cell purity ≥70%. WES was performed to identify somatic mutations, with variant calling and annotation performed using standard bioinformatic pipelines and reference databases. RNA sequencing (RNA-seq) was conducted on 7 HCL-v cases and compared to splenic tissue transcriptomes from 9 SMZL and 5 SDRPL cases. Differential expression and over-representation analysis (ORA) were used to define transcriptional programs specific to HCL-v. Fusion gene analysis was performed on Illumina's BaseSpace platform using the Manta-fusion tool. Results: The most frequently mutated genes in HCL-v were TP53, followed by BRAF and MAP2K1, ARID1A, KMT2D and NOTCH2. All BRAF mutations concerned non-V600E variants - specifically L485W (exon 12), S605N (exon 15), and K499E (exon 12) - located within the kinase domain and previously reported in only a limited number of tumors, primarily melanoma. KLF2 mutations were absent in HCL-v cases, despite being the second most frequently altered gene in HCL, further emphasizing a key genomic distinction between these entities. Several patterns of mutual exclusivity were observed, with no co-occurrence of mutations between BRAF and MAP2K1, KMT2D and NOTCH2, or NOTCH2 and KMT2D, suggesting separation of patients into distinct subgroups with different pathogenic trajectories. Tumor mutation burden was calculated using both total and non-synonymous mutations, with median values of 4.54 and 2.54 mutations/Mb, respectively. RNA-seq documented that HCL-v shows a distinct transcriptional signature compared to SMZL and SDRPL. In particular, ORA revealed that HCL-v is enriched for immune-related signaling pathways, (B-cell receptor, NF-κB, TNF, IL-17 pathways), and pathogen-response programs, supporting the idea that HCL-v is more immunologically “alert”. Notably, both comparisons (HCL-v vs SMZL and HCL-v vs SDRPL) revealed consistent and significant upregulation of canonical MAPK pathway effectors, including FOS, JUN, EGR1, ATF3, NR4A1, and NR4A2 (padj < 0.003, log₂FC > 3.4), exclusively in HCL-v - the only entity harboring MAP2K1 mutations. This results supports that MAP2K1 mutations are functionally activating and define a distinct MAPK-driven molecular phenotype in HCL-v. Fusion transcript analysis uncovered a recurrent KANSL1–ARL17 fusion in 4/7 HCL-v cases, compared to only 1/9 SMZL and 1/5 SDRPL cases. Although this fusion has been reported in a limited number of cancer studies, its precise role in tumorigenesis remains poorly understood. KANSL1 encodes a nuclear protein involved in epigenetic processes while ARL17 encodes a regulatory protein of the ARF family. KANSARL fusion, derived from the 17q21.31 inversion polymorphism, may be cancer-predisposing and tumor-maintaining, despite its detection in normal tissues, since it may impact cell growth and motility. Its higher prevalence in HCL-v warrants further investigation into its precise contribution to the unique biology of this lymphoma subtype. Conclusion:We report that HCL-v displays a distinct molecular profile with mutually exclusive mutations, and a transcriptional program enriched for immune-related and MAPK pathway activation. These novel insights hold translational potential towards implementing precision interventions in HCL-v.