Use of flow cytometric light scattering to recognize the characteristic vacuolated marrow cells in VEXAS syndrome

Abstract

VEXAS (Vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome is a systemic autoinflammatory disease caused by somatic mutations in the UBA1 gene and presents with adult-onset, long-term refractory inflammatory symptoms, associated with macrocytic anemia that may progress to pancytopenia, progressive bone marrow (BM) failure, myelodysplastic syndrome, and plasma cell neoplasm.1-3 UBA1 mutations are detected in hematopoietic stem and progenitor cells and peripheral-blood myeloid cells but not in mature lymphocytes or fibroblasts.2 The characteristic morphologic features in this syndrome are cytoplasmic vacuoles in myeloid and erythroid precursors.2,4-6 Figure 1A depicts a marrow from a patient with VEXAS syndrome, demonstrating cytoplasmic vacuoles in erythroid and myeloid precursors. Detection of a pathogenic UBA1 mutation is required for the diagnosis of VEXAS syndrome. Immunophenotypic alterations detected via flow cytometry have been described,7 but, to our knowledge, there are no specific markers for the identification of this disorder in the BM. Flow cytometry is a useful technology for the diagnosis of lymphohematopoietic diseases. Light scattering, together with fluorescence signals, is routinely used for the characterization of hemopoietic and lymphoid cells. The intensity of the light scattered at different angles from the light excitation point provides information regarding cellular and physical properties. Forward and side scatters (SSCs) are the 2 flow cytometric nonfluorescent light measurements that are informative of cell size and internal complexity, respectively. SSC intensity increases with increase in the quantity of cytoplasmic granules9 and also reflects the presence of vacuole-like structures within the cytoplasm of neutrophils producing reactive oxygen species.10 Here, we report a simple flow cytometric analysis strategy to recognize likely vacuolated neutrophil, monocytic, and erythroid precursors in the BMs of patients with VEXAS syndrome through SSC intensity measurements. We studied 27 patients with VEXAS syndrome with confirmed UBA1 mutations. Patients were all male, with a median age of 68 years. Concurrent myelodysplastic syndrome was diagnosed in 8 of these patients. Cytoplasmic vacuoles in marrow cell precursors were observed in 26 patients. One patient received an autologous stem cell transplant, and his marrow cells after HSCT showed no vacuoles. A group of 106 randomly selected patients without VEXAS syndrome served as controls. Their diagnoses included aplastic anemia (n = 34), myelodysplastic syndrome with wild-type UBA1 gene (MDS UBA1-WT) (n = 32), non-VEXAS syndrome marrows with cytoplasmic vacuoles in precursors (n = 13), and other conditions, possibly associated with stressed marrow (n = 27). Detailed information on the control cases is shown in supplemental Table 1. All patients were enrolled in institutional review board–approved National Institutes of Health protocols.