Vacuoles in circulating immature myeloid cells in <scp>VEXAS</scp> syndrome and comparison with <scp>UBA1</scp>‐wild type patients
Authors: Valentin Lacombe, Franck Genevieve, Floris Chabrun
Journal: American Journal of Hematology (2023)

We did not identify any vacuole-related differences in circulating immature myeloid cells between VEXAS patients and UBA1-WT 'VEXAS-like' patients. The similar vacuolization of circulating immature myeloid cells between VEXAS and UBA1-WT patients is explained by the main bloodstream passage of late precursors, in which the vacuolization is already similar in bone marrow in both cases. It was with great interest that we read the article by Tosato et al. recently published in this journal.1 The authors presented images of vacuoles in peripheral blood cells of patients with VEXAS syndrome (Vacuoles, Enzyme E1, X-linked, Autoinflammatory, Somatic). VEXAS syndrome is an auto-inflammatory disorder related to somatic UBA1 mutations in hematopoietic stem cells.2 The presence of vacuoles in marrow myeloid and erythroid precursor cells is a key feature of this disease, and an intense marrow vacuolization is highly specific to VEXAS syndrome.3, 4 However, as bone marrow aspiration involves an invasive procedure, it is not a suitable screening tool for use in diagnosis. Finding a cytological abnormality specific to VEXAS syndrome and detectable in peripheral blood would therefore be of great interest. To this end, we previously used deep learning to distinguish VEXAS patients from UBA1-wild type patients.5 We demonstrated that subtle morphological changes in mature myeloid cells were identified by deep learning to detect VEXAS patients. The morphological singularity of the mature myeloid cells of VEXAS patients was not, however, evident for human analysis and did not consist of a higher presence of vacuoles in the mature myeloid cells of VEXAS patients. The bloodstream passage of marrow precursors is common in various blood diseases and in situations of intense inflammation. VEXAS syndrome could therefore be a typical condition leading to the bloodstream passage of marrow precursors. As VEXAS myeloid precursors have the specific trait of being highly vacuolated, we were very enthusiastic when reading Tosato et al.'s description of vacuoles in circulating immature precursor cells in patients with VEXAS syndrome. Indeed, the identification of typical vacuolated precursors in peripheral blood, also described by another research team,6 could have served as an interesting screening tool for VEXAS syndrome. We therefore compared the vacuolization of circulating immature myeloid cells between VEXAS and UBA1-wild type (UBA1-WT, "VEXAS-like"). In case we found a significant difference between them, we planned to evaluate its diagnostic performance for VEXAS syndrome diagnosis. We included VEXAS and UBA1-WT patients who had similar clinical and biological features leading to suspect VEXAS syndrome seen at Angers University Hospital between September 2020 and August 2023. We restricted our analyses to patients presenting circulating immature myeloid cells on blood smears. We only considered the blood smears taken within 3 months of the UBA1 sequencing in order to avoid the inclusion of tests performed before the emergence of the mutation and to limit the hypothetical effect of treatments on the vacuolization. UBA1 gene was sequenced using Sanger analysis and/or high throughput sequencing, to further define the VEXAS and UBA1-WT groups according to the results. Images of individual cells on blood smears were obtained by scanning peripheral blood smears stained with May-Grunwald Giemsa with a DM96 Digital Microscope (CellaVision AB, Lund, Sweden). Each immature myeloid cell was reviewed and labeled by a hematologist and categorized according to cell type (myelocyte, metamyelocyte), presence of vacuole(s), number of vacuole(s), and the vacuoles' area. Cells were classified as myelocytes or metamyelocytes, whereas promyelocytes represented a negligible minority of cells in rare patients and were not considered. Quantitative data were compared by Student's t-test and alpha risk was set at 5%. We included 5 VEXAS patients in whom immature circulating precursors were identified on blood smears within 3 months of UBA1 sequencing, and 10 UBA1-WT patients satisfying the same criteria. All 15 included patients presented with anemia, persistent C-reactive protein elevation, and at least 1 clinical inflammatory sign found in VEXAS syndrome.2 In particular, arthritis was noted in 4 of 10 UBA1-WT patients and 2 of 5 VEXAS patients, lung or pleural involvement in 4 of 10 UBA1-WT and 4 of 5 VEXAS patients, and neutrophilic dermatosis in 2 of 10 UBA1-WT and 1 of 5 VEXAS patients. Significant cytological signs of dysplasia in bone marrow aspirate were observed in 7 of 10 UBA1-WT patients and 2 of 5 VEXAS patients, but a high level of vacuolated marrow precursor cells was only observed in the 5 VEXAS patients. Clinical, biological, and bone marrow features of the included patients are detailed in Supplementary Table S1. We analyzed 99 blood smears available in the period of interest and including immature myeloid cells (15 among VEXAS patients, 84 among UBA1-WT patients), with a total of 458 images of circulating precursors. Regardless of the type of peripheral circulating precursor considered, neither the proportion of vacuolated cells nor the number of vacuoles per vacuolated cell, nor the surface area of vacuoles per vacuolated cell differed between the two groups (Table 1). Notably, the proportion of vacuolated circulating immature cells did not differ between the groups: 30.6% (±33.4) in VEXAS patients and 22.6% (±30.2) in UBA1-WT patients (p = 0.36). In 14 of 15 patients, the presence of vacuoles in circulating immature cells was observed in the absence of an active infection: 6 patients (4 UBA1-WT and 2 VEXAS patients) presented no infection during the period of interest, and 8 patients (5 and 3, respectively) had an infection but presented with vacuolated circulating immature cells before and/or after this infection (Supplementary Table S1). In 1 UBA1-WT patient, vacuolated circulating immature cells were only observed during an infection (dermohypodermitis and COVID-19). In bone marrow aspirations, the highly vacuolated myeloid cells in VEXAS syndrome are the early precursors (myeloblasts and promyelocytes), whereas the proportion of vacuolated cells in the more mature marrow precursors (myelocytes and metamyelocytes) did not differ between UBA1-mutated or UBA1-WT patients.4 As the circulating precursors are in a very large majority late myeloid precursors (myelocytes and metamyelocytes), our results are in line with marrow descriptions demonstrating no phenotypical differences in these cells between VEXAS patients and controls. This therefore raises questions regarding the real link between VEXAS syndrome and the peripheral vacuolated cells described by some authors like Tosato et al.1 Indeed, we demonstrated that vacuolated immature circulating cells were also observed in other conditions of systemic inflammation (UBA1-WT group) and were not specifically associated with the diagnosis of VEXAS syndrome. As a consequence, the observation of a few vacuolated precursors on blood smears from VEXAS patients would be neither specific to nor pathognomonic of the presence of UBA1 mutation, but rather related to the intense inflammation. The identification of circulating vacuolated myeloid precursors cannot therefore be used as a diagnostic tool in screening for VEXAS syndrome. In conclusion, we did not identify any vacuole-related differences in circulating immature myeloid cells between VEXAS patients and UBA1-WT 'VEXAS-like' patients. This suggests that when vacuolated circulating myelocytes and metamyelocytes had previously been observed in blood smears, these were related to the systemic inflammation but were not specific to VEXAS syndrome. The similar vacuolization of circulating immature myeloid cells between VEXAS and UBA1-WT patients is explained by the main bloodstream passage of late precursors, in which the vacuolization is already similar in bone marrow in both cases. The rare bloodstream passage of early precursors that would have vacuoles specific to VEXAS is possible, but the analysis of the vacuolization of circulating immature myeloid cells, constituted of late precursors, is an ineffective means of screening for VEXAS syndrome. We are grateful to Dr. Anne Bouvier for performing the genetic analyses used in this study. None. None. The data that support the findings of this study are available from the corresponding author upon reasonable request. Supplementary Table S1. Patients' features and infections during the period of interest. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.