Correlation of Clonal Hematopoiesis in Vexas Syndrome with Immunophenotype Alterations in CD34+ HSPCs (Hematopoietic Stem Progenitors Cells): Multiparametric Flow Cytometry Heterogeneity in CD34+CD38- Stem Cells Fraction and Upregulation of CD90 Thy-1 By Unsupervised Strategy Using PCA, Tsnee and Flowsom

Introduction: VEXAS (vacuoles,E1enzyme,X-linked, autoinflammatory,somatic) syndrome is caused by somatic UBA1mutations affecting myeloid compartment of hematopoietic stem and progenitor cells (HSPC).VEXAS syndrome is a unique systemic autoinflammatory disease associated with cytopenias with marrow dysplasia(1).There is currently no published data concerning the immunophenotypic characteristics pattern of CD34+ HSC in UBA1 mutated patients w/wo MDS.The identification of an easily-accessible profile in CD34+CD38- most immature HSC for UBA1 mutations might help to identify UBA1 patients beside molecular testing and potential target of therapeutic interest. Methods: Here we report flow cytometry of bone marrow CD34+ HSPCs in a cohort of 11 UBA1 mutated patients from the Lyon University Hospital. Our routine 2-tubes panel used in the follow-up of AML MRD, were applied with minimal mandatory 8 markers per tube, according to ELN recommendations (to identify the pattern of LAIP/DfN in bulk and LSC in CD34+CD38- fraction). A «backbone» of CD34/CD38/CD45/CD117 , and CD7,CD56, CD13, CD33, CD19 for the 1^st tube and CD90,CLL1+TIM3+CD97, CD123, CD45RA for the 2^nd one. Acquired data in BD Canto and Lyric cytometers were analysed using DIVA/FlowJo and KALUZA softwares for classical strategy completed with Tsnee and FlowSom software for unsupervised analyse. Control group was healthy donor or regenerating marrow and MDS patients UBA1 negative. A total of minimal 500000 to one million of cells was acquired in each tube. Results: The clinical and biological characteristics of the 11 UBA1 mutated patients are summarized in Fig1A. Parameters as MFI in cell lineage and LSC markers, percentage of targeted populations (lympho-myeloid primed progenitor (LMPP), Multipotential Progenitor (MPP), Common myeloid progenitor (CMP), granulocyte/monocyte progenitor (GMP) and megakaryocyte/erythroid progenitor (MEP), were integrated in principal component analysis (PCA) (Fig1B). PCA analysed compared our VEXAS cohort with normal marrow (n=10) and MDS patients without UBA1 mutation (n=23), and clusterized properly UBA1 mutated patients from normal marrowand MDS patients. The most contributive variables for appropriate clusterizing were LSC/progenitors markers CD90 Thy1/ CD45RA and CLL1/TIM3 /CD97, CD123 and GMP, MEP, MPP, LMPP-like populations. Interestingly, all normal marrow clusterized together, highly separated from UBA1 mutated cases. However, MDS UBA1 negative patients were heterogeneously distributed, suggesting a strong variability in CD34+ fraction. No lineage marker (i.e. CD13) or aberrant markers (i.e. CD7) was involved in PCA clustering, suggesting that alteration in LSC:HSCP fractions are mostly relevant for distinction between clonal haematopoiesis in UBA1 mutated patients compared to MDS UBA1 negative and normal marrow. A correlation matrix showed a close correlation between LSC/progenitors markers and CD34+CD38- frequency, mostly based on CD90/Thy-1 overexpressed in clonal hematopoiesis observed in UBA1 mutated patients (Fig 1C). We merged data from 2 UBA1 mutated, 2 MDS UBA1 negative and 4 healthy donors to perform unsupervised analysis Tsnee and FlowSom (Fig1D). Interestingly, both UBA1 mutated patients showed different pattern in CD34+ compared to MDS and normal healthy donors suggesting most primitive alteration in CD90 Thy-1 expression in CD34+CD38-cells. Flowsom map allowed to identify nodes of stem cell with abnormal phenotype clearly different in UBA1 mutated patients, backgating of this node showed the overexpression of CD90 in CD34+CD38- cells in UBA1 pos. Conclusions: This is the first large immunophenotypic characterisation by flow of clonal haematopoiesis in UBA1 mutated patients. Our results showed that VEXAS syndrome is characterized by overexpression of CD90 Thy-1 primitive Stem cell markers in CD34+ HSPCs and in most immature CD34+CD38- fraction. Altogether, we can hypothesize that such phenotype might represent specific disease hallmark involved in physiopathology of niche deregulation, which should be confirmed in a larger cohort of patients in a future multicentric clinical trials to improve biological diagnosis and comprehension of kinetics of clonal haematopoiesis in VEXAS patients.

1) Beck DB, Ferrada MA, Sikora KA, et al. Somatic mutations in UBA1 and severe adult-onset autoinflammatory disease. N Engl J Med. 2020;383:2628-2638.

Ghesquieres:Gilead: Consultancy, Honoraria; Roche: Consultancy, Honoraria; BMS: Honoraria; Abbvie: Honoraria. Sujobert:Astrazeneca: Research Funding; Janssen: Research Funding; Gilead/Kyte: Consultancy; Astellas: Consultancy; Daichi Sankyi: Consultancy.