Somatic mutation discovery in idiopathic multicentric castleman disease (iMCD) lymph node tissue identifies dysregulated intracellular calcium signaling as a potential disease mechanism and therapeutic target Free

Background: Idiopathic multicentric Castleman disease (iMCD) is a rare, atypical lymphoproliferative disorder with significant morbidity and mortality and unknown etiology. Given the clinico-pathologic similarities between iMCD and several lymphomas, the field has long hypothesized that a rare population of somatically mutated clonal cells may be involved in iMCD pathogenesis. Recently, somatic mutations were described in a similar disease called unicentric Castleman disease (UCD), including a recurrent PDGFRB mutation. Additionally, other potential somatic variants in iMCD have been identified, but the studies to date have had limited sample sizes and other methodological limitations. These findings may suggest a potential neoplastic process driven by somatically mutated cells in iMCD. Here, we used a paired tissue strategy to determine the presence and functional significance of somatic variants in lymph node (LN) tissue in a large cohort of iMCD patients.

Methods: To explore the genetic etiology of iMCD, we used whole-exome sequencing from 62 iMCD patients, 6 diffuse large B-cell lymphoma (DLBCL) patients, and 6 healthy controls. LN tissue was paired with peripheral blood mononuclear cells as germline reference control tissue to detect somatic variants. Sequencing reads were mapped to GRCh38 using the BWA aligner, and variants were called using the Mutect2 pipeline within the Genome Analysis Toolkit (GATK). ANNOVAR was used to annotate variants, and high-impact somatic variants were identified using several filtering criteria, including sequencing depth, variant allele fraction (VAF>5%), allele frequency (<=0.01% in gnomAD), amino acid change, and predicted damaging in at least 5 of 7 predictive software that included SIFT, Polyphen2, LRT, FATHMM, M-CAP, ClinPred, and AlphaMissense. Somatic variants passing these thresholds were evaluated for functional significance in iMCD. For validation, variants were generated by site-directed mutagenesis of an ITPR3 construct and transiently transfected into a HEK-293 cell line engineered to be devoid of all endogenous ITPR genes. Channel function was investigated using a fluorescence Ca²⁺ imaging assay in fura-2 loaded cells.

Results: To evaluate assay precision and filtering criteria, we investigated somatic changes in the DLBCL LN tissue. In each of the 6 DLBCL patients, somatic variants were identified in genes known to be functional drivers of DLBCL. Importantly, these genes were not identified in the iMCD and healthy control cohorts, suggesting differing pathological mechanisms in iMCD. In all, 6,914 somatic variants were identified in 4,680 genes in iMCD LN tissue (n=62). We ranked each gene by the number of rare, predicted deleterious somatic mutations and the top 50 somatically altered genes in iMCD included genes involved in calcium signaling (RYR1, RYR3, ITPR3, PIEZO1), PI3K-AKT-MTOR pathway (LRP1, LRP2, LRP5), and immune cell trafficking (C3, RHBDF1, ADCY5, PLXNB1), among others. Interestingly, 4 of the top 5 somatically altered genes were involved in the maintenance of intracellular calcium levels, which was notable given the role of activated T cells in iMCD. Collectively, 20 (32%) iMCD patients had a rare, predicted deleterious somatic variant in one of these 4 genes. We next prioritized the functional validation of ITPR3 due to its expression in lymphocytes, including T cells and macrophages, both of which have been implicated in iMCD pathogenesis. We selected 3 observed somatic variants (D203N, V210G, V432M) and transiently transfected them into a null model for ITPR3. We are currently using this model to validate the functional impact of these variants on Ca²⁺ flux.

Conclusions: Predicted deleterious somatic changes (>5% VAF) were present in several genes with potential functional relevance to iMCD. These genes had diverse immunologic functions, including several with integral roles in gene pathways known to be involved in iMCD. The most common somatically mutated genes in iMCD were intracellular calcium channels that mediate intracellular calcium concentrations. These channels play a crucial role in immune cell function and cellular processes such as proliferation, differentiation, and cytokine secretion. These data suggest the possible involvement of intracellular calcium maintenance in the etiology of iMCD, especially in immune cells within the LN. The validation and functional investigation of these promising gene candidates is currently ongoing.