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

Clinical Center

Investigators

Abstract

Cytoplasmic vacuoles in VEXAS are predominantly found in myeloid and erythroid precursors. SSC intensity measured by flow cytometry reflects the internal complexity of cells and it would be anticipated that the presence of vacuoles increases the intensity of the SSC light. This was confirmed by our results. Our analysis demonstrated a significantly higher SSC ratio for neutrophil, monocytic and erythroid precursors. The difference was most striking among neutrophil precursors and less prominent for monocyte precursors, probably due to the frequent cytoplasmic vacuolization observed in monocytes in many conditions. In contrast, there was no difference in SSC ratios when the measurements were made using mature neutrophils instead of precursors. Based on the ROC curve for the entire VEXAS and control patient cohort (AUC = 0.97), using a SSC ratio cutoff for neutrophil precursors of 11, the positive predictive value (PPV) for detecting VEXAS cases is 91.7% while the negative predictive value (NPV) is 96.3%. We additionally calculated the SSC intensity of neutrophil precursors using mature neutrophils as internal controls. This precursor/mature neutrophil SSC ratio also demonstrated a significantly higher value in VEXAS marrows. However, mature neutrophils may not be ideal controls, since hypogranularity in these cells may result in decreased SSC intensity. Cytoplasmic vacuoles in myeloid and erythroid precursors can be found in conditions other than VEXAS. In this study, we found significantly higher SSC ratios for neutrophil precursors in VEXAS cases also when compared to non VEXAS marrows containing precursor cells with cytoplasmic vacuoles. VEXAS patients usually present with characteristic macrocytic anemia and may develop progressive pancytopenia and bone marrow failure. When compared with marrows with aplastic anemia patients, the SSC ratio in marrow precursors was also significantly higher in VEXAS cases and the same was observed when VEXAS cases were compared to marrows with other conditions. Bone marrow hypercellularity with dysplasia is frequently present in VEXAS syndrome and these findings often overlap with those seen in non-VEXAS MDS. We compared the VEXAS marrows with those with MDS in non-VEXAS (UBA1-WT) patients. The neutrophil precursors in the marrows from VEXAS patients showed significantly higher SSC ratios than in marrows with MDS UBA1-WT. Similar findings were observed in MDS cases of VEXAS patients with MDS UBA1-WT. There was no significant difference in SSC ratio of neutrophil precursors when non-MDS were compared with MDS cases within the VEXAS cohort. Vacuolization may be found in myeloid cells in MDS with UBA1-WT. However, in the three cases of MDS UBA1-WT marrow with vacuolization, the SSC intensity in neutrophil precursors was lower than that in the VEXAS cases, suggesting that the vacuoles in the MDS (UBA1-WT) cases may be less numerous or smaller compared with VEXAS cases4. Of interest, the SSC intensity of marrow precursors in a VEXAS patient who was transplanted due to a concurrent plasma cell neoplasm and did not show vacuoles in the marrow cells was much lower than in VEXAS cases with vacuolization, and comparable to that of the control group, further suggesting that the higher SSC in neutrophil and erythroid precursors in VEXAS patients is indeed due to vacuolization. Cytoplasmic vacuoles are not specific of VEXAS syndrome since they have been reported in other disorders. However, the presence of a high number of significantly vacuolated myeloid precursors has been associated with VEXAS syndrome with an excellent sensitivity and specificity. Thus, in the appropriate clinical and laboratory context, the detection of high SSC intensity signals from myeloid or erythroid precursors should raise high suspicion for VEXAS, a diagnosis that would have to be confirmed by the appropriate molecular testing. Furthermore, the ability to quantify cellular vacuolization may potentially be helpful in assessing disease burden in VEXAS patients. Additionally, the identification of vacuolated cells by flow cytometry in VEXAS syndrome could also facilitate the sorting of mutated cells, which may be useful for functional and biochemical studies in this disease.