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

VEXAS中的细胞质空泡主要见于髓系和红系前体细胞。用流式细胞仪测量的SSC强度反映了细胞内部的复杂性，可以预见，空泡的存在会增加SSC光的强度。我们的结果证实了这一点。我们的分析表明，中性粒细胞、单核细胞和红系前体细胞的SSC比率显著较高。这种差异在中性粒细胞前体细胞中最为显著，而在单核细胞前体细胞中不那么显著，这可能是由于在许多情况下单核细胞中观察到频繁的细胞质空泡化。相比之下，当使用成熟的中性粒细胞而不是前体细胞进行测量时，SSC比率没有差异。根据整个VEXAS和对照患者队列的ROC曲线(AUC=0.97)，以中性粒细胞前体的SSC比值为11作为界值，检测VEXAS病例的阳性预测值(PPV)为91.7%，阴性预测值(NPV)为96.3%。另外，我们使用成熟的中性粒细胞作为内对照，计算了中性粒细胞前体的SSC强度。这一前体/成熟中性粒细胞SSC比率在VEXAS骨髓中也显示出明显更高的值。然而，成熟的中性粒细胞可能不是理想的对照，因为这些细胞中的低颗粒可能导致SSC强度降低。 除VEXAS外，髓系和红系前体细胞中的细胞质空泡也可见。在这项研究中，我们发现VEXAS患者中性粒细胞前体细胞的SSC比率也明显高于非VEXAS患者，而非VEXAS患者的中性粒细胞前体细胞带有细胞质空泡。VEXAS患者通常表现为特征性的大细胞贫血，并可能发展为进行性全血细胞减少和骨髓衰竭。与再生障碍性贫血患者相比，VEXAS患者骨髓前体细胞中的SSC比例也显著升高，VEXAS患者与其他疾病患者的骨髓相比也是如此。 VEXAS综合征患者常伴有骨髓高细胞增多症和异型增生，这些表现常与非VEXAS MDS的表现重叠。我们比较了非VEXAS(UBA1-WT)患者的VEXAS骨髓和MDS患者的骨髓。VEXAS患者骨髓中性粒细胞前体细胞比例明显高于MDS UBA1-WT患者。在患有MDS UBA1-WT的VEXAS患者的MDS病例中也观察到了类似的结果。VEXAS队列中非MDS患者与MDS患者相比，中性粒细胞前体细胞比例差异无统计学意义。伴有UBA1-WT的MDS髓系细胞可见空泡化。然而，在3例伴有空泡化的MDS UBA1-WT骨髓中，中性粒细胞前体细胞的SSC强度低于VEXAS病例，提示MDS(UBA1-WT)病例的空泡可能比VEXAS病例更少或更小。 值得注意的是，VEXAS患者因并发浆细胞肿瘤而移植的骨髓前体细胞的SSC强度明显低于VEXAS合并空泡化的患者，并与对照组相当，进一步表明VEXAS患者中性粒细胞和红系前体细胞的SSC升高确实是由于空泡化所致。 细胞质空泡不是VEXAS综合征的特异症，因为它在其他疾病中也有报道。然而，大量明显空泡化的髓系前体细胞的存在与VEXAS综合征相关，具有极好的敏感性和特异性。因此，在适当的临床和实验室背景下，检测到来自髓系或红系前体的高SSC信号应引起对VEXAS的高度怀疑，这一诊断必须通过适当的分子检测来确认。此外，量化细胞空泡化的能力可能有助于评估VEXAS患者的疾病负担。此外，用流式细胞术鉴定VEXAS综合征中的空泡细胞也有助于突变细胞的分类，这可能有助于该疾病的功能和生化研究。

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.