VEXAS (Vacuoles, E1 enzyme, X-linked, Autoinflammatory, Somatic) syndrome is a recently described syndrome associated with adult-onset inflammatory disease, hematologic abnormalities, and somatic mutations in the UBA1 (ubiquitin-activating enzyme 1) gene, which occurs predominantly in male patients.¹ Common hematologic manifestations of VEXAS include macrocytic anemia, progressive cytopenias, and an increased risk of developing myelodysplastic syndrome (MDS) and/or plasma cell neoplasms. The characteristic bone marrow (BM) finding in VEXAS syndrome is cytoplasmic vacuolization in myeloid and erythroid precursors, with relatively small, round, well-demarcated vacuoles (Figure 1A–C), which can be subtle in some cases.² In addition, patients with VEXAS syndrome typically show a hypercellular BM with myeloid hyperplasia, which may progress over time. Finally, mild dyspoiesis in any lineage may be seen, while a subset of patients with more extensive dysplasia meets diagnostic criteria for MDS.

The UBA1 gene is located on the X chromosome and encodes the E1 ubiquitin-activating enzyme, which is required for the initiation of ubiquitylation. Most reported cases of VEXAS syndrome have been associated with missense mutations involving methionine 41 (p.Met41) in exon 3 of UBA1, which leads to diminished expression of the cytoplasmic isoform of UBA1, UBA1b.³ However, non-p.Met41 variants have been reported,^4-8 including splice site mutations affecting the intron 2/exon 3 junction of UBA1, which are thought to cause loss of UBA1b expression. Additionally, three cases harboring a UBA1 p.Ser56Phe variant have been previously reported.^{5, 6, 9} This variant is thought to act through a distinct mechanism, by decreasing the catalytic activity of the UBA1 protein rather than expression of the cytoplasmic UBA1b isoform.⁶ Furthermore, the initial report of this variant by Poulter et al. suggested that this variant may be associated with distinct BM morphology. Here, we report the fourth case of a VEXAS patient with a UBA1 p.Ser56Phe variant, including the most complete pathologic review to date, and confirm the atypical pathologic findings associated with this noncanonical UBA1 variant.

A 41-year-old male initially presented in 2016 with a 9-month history of isolated normocytic anemia. In addition, he reported intermittent fevers, fatigue, weight loss, migratory arthralgias, and painful cutaneous nodules. An extensive laboratory evaluation was performed (Table S1), with results notable for elevated inflammatory markers and low serum iron. A BM biopsy and aspirate smear (Figure 1D–F) showed a hypercellular marrow (80% cellular) with erythroid hyperplasia (M:E ratio 0.5:1) and atypical megakaryocytes, with small hypolobated forms and widely separated nuclear lobes. Myeloid and erythroid precursors showed mild atypia with rare, scattered forms containing cytoplasmic vacuoles, including ~15% of myeloid precursors (myeloblasts, promyelocytes, and myelocytes). Blasts were not increased. Cytogenetic analysis showed a normal male karyotype, and a myeloid next-generation sequencing (NGS) panel covering 37 genes commonly mutated in myeloid malignancies was negative for pathogenic variants. Given the overall clinical and laboratory findings, the anemia and morphologic atypia in the BM were favored to be secondary to underlying rheumatologic disease, which was difficult to classify but was thought to represent rheumatoid arthritis. Over the next 6 years, the patient was refractory to multiple therapies, including prednisone, hydroxychloroquine, methotrexate, and interleukin (IL)-6, IL-1, and tumor necrosis factor α (TNF-α) inhibitors (Figure S1). Peripheral blood counts progressed to pancytopenia.

Due to clinical concern for the recently described VEXAS syndrome, UBA1 single gene sequencing analysis, utilizing an NGS assay covering the entire UBA1 gene (Invitae, San Francisco, CA) was performed on peripheral blood. Testing revealed a c.167C > T (p.Ser56Phe) variant at a variant allele frequency (VAF) range of 18%–28% (the germline testing platform utilized was not validated to report a precise VAF for this somatic variant). A repeat BM biopsy (Figure 1G–I) showed progressive hypercellularity (90% cellular) and erythroid hyperplasia, with a further decrease in the M:E ratio to 0.2:1. There was marked megakaryocytic dysplasia, rare ring sideroblasts were present, and cytoplasmic vacuolization was somewhat more evident, now present in ~35% of myeloid precursors. Blasts were not increased. Cytogenetic analysis showed a normal male karyotype. A myeloid NGS panel, which did not cover the UBA1 gene, identified a pathogenic variant in EZH2 (p.Tyr731His; 8%) and a variant of uncertain significance in KMT2A (p.His1456Tyr; 4%). The patient was diagnosed with MDS with multilineage dysplasia in the setting of VEXAS syndrome.

Following three cycles of azacitidine, the patient underwent a reduced intensity conditioning, matched unrelated donor peripheral blood stem cell transplant. A BM biopsy performed 30 days after transplant showed mild myeloid and erythroid atypia, including occasional myeloid elements with vacuolization. However, NGS using an expanded panel covering UBA1 was negative for the previously identified UBA1, EZH2, and KMT2A variants, and engraftment studies showed 99% donor chimerism. At 6 months following transplant, a BM biopsy showed no evidence of dysplasia, and engraftment studies showed 100% donor chimerism. The patient currently reports no significant rheumatologic symptoms.

Past reports of the BM morphology associated with VEXAS syndrome have described myeloid hyperplasia as a stereotypic finding. However, we describe here the BM findings in a second case of VEXAS with a noncanonical UBA1 p.Ser56Phe variant and confirm the presence of marked erythroid hyperplasia in the context of this variant, as was described by Poulter et al.⁶ Interestingly, Sakuma et al.⁷ recently described a patient with MDS with a UBA1 p.Tyr55His variant; although not described in the manuscript, a supplemental figure with an image from this patient's BM aspirate also showed marked relative erythroid hyperplasia. The distinct BM morphology associated with the p.Ser56Phe variant may be due to the alternative mechanism of disease proposed for this variant, involving reduced function rather than reduced protein expression of cytoplasmic UBA1. A similar mechanism may also explain the pathology and BM morphology associated with the p.Tyr55His variant. Importantly, Poulter et al. were unable to evaluate for cytoplasmic vacuoles in the patient with a p.Ser56Phe variant. Here, we show that, similar to cases carrying a canonical p.Met41 variant, the p.Ser56Phe is also associated with cytoplasmic vacuolization. Taken together, these findings suggest that, in the appropriate clinical setting, the presence of cytoplasmic vacuolization associated with either myeloid or erythroid hyperplasia may warrant testing for a UBA1 mutation. Cytoplasmic vacuolization in VEXAS is typically most apparent in immature myeloid and erythroid elements. The cytoplasmic vacuolization was somewhat subtle in our case, possibly due to the erythroid hyperplasia and the abundance of more mature erythroid elements, with a relative paucity of immature myeloid and erythroid elements. However, a threshold of ≥10% of myeloid precursors with >1 vacuole has been proposed as a sensitive and specific diagnostic feature of VEXAS.¹⁰ Indeed, our patient met this threshold at both time points, and as the M:E ratio and absolute number of myeloid precursors in the marrow decreased over time, the percentage of myeloid precursors meeting this threshold increased.

Furthermore, our findings highlight the fact that UBA1 assays that test only for Met41 variants are insufficient to identify all patients with VEXAS. While UBA1 will likely increasingly be incorporated into myeloid NGS panels, redesign of NGS panels takes time. In the interim, single gene UBA1 assays are available, through academic and commercial labs, and some institutions may choose to develop a UBA1 assay. Met41 is present in exon 3 of UBA1, along with Tyr55 and Ser56. Given the well-described presence of splice site variants affecting the intron 2/exon 3 junction, a UBA1 assay sequencing this splice junction as well as the entirety of exon 3 would be required to cover the vast majority of reported pathogenic UBA1 variants associated with myeloid disease. Ultimately, as the spectrum of pathogenic UBA1 variants continues to expand, sequencing of all coding regions via NGS may become standard in the future.

Identification of VEXAS syndrome in patients with myeloid malignancies may influence clinical decision-making, as allogeneic hematopoietic stem cell transplant (allo-HCT) is currently the only potentially curative therapy with respect to VEXAS-associated rheumatologic disease. Given the morbidity associated with allo-HCT, it is currently reserved for patients with severe, treatment-refractory VEXAS or secondary myeloid malignancies.¹¹ A recent multicenter cohort of 13 patients who underwent allo-HCT for refractory VEXAS or related myeloid malignancy showed 2-year overall survival of 82%, with resolution of the VEXAS clinical phenotype in all patients with prolonged follow-up.¹²

VEXAS（空泡、E1 酶、X 连锁、自身炎症、体细胞）综合征是最近描述的一种与成人发病的炎症性疾病、血液学异常和 UBA1 （泛素激活酶 1）基因体细胞突变相关的综合征，该病主要发生在男性患者中。¹ VEXAS 的常见血液学表现包括大细胞性贫血、进行性血细胞减少以及发生骨髓增生异常综合征 (MDS) 和/或浆细胞肿瘤的风险增加。 VEXAS 综合征的特征性骨髓 (BM) 发现是髓系和红系前体中的细胞质空泡化，具有相对较小、圆形、界限清楚的空泡（图 1A-C），在某些情况下可能很微妙。²此外，VEXAS 综合征患者通常表现出细胞增多性骨髓增生，并可能随着时间的推移而进展。最后，任何谱系都可能出现轻度发育不良，而一部分具有更广泛发育不良的患者符合 MDS 的诊断标准。

UBA1基因位于X染色体上，编码E1泛素激活酶，这是启动泛素化所必需的。大多数报道的 VEXAS 综合征病例与UBA1外显子 3 中蛋氨酸 41 (p.Met41) 的错义突变有关，这导致 UBA1、UBA1b 细胞质亚型的表达减少。³然而，已报道非 p.Met41 变体^4-8包括影响UBA1内含子 2/外显子 3 连接的剪接位点突变，这被认为会导致 UBA1b 表达丧失。此外，此前已报道过三例携带UBA1 p.Ser56Phe 变体的病例。 ^{5, 6, 9}该变体被认为通过独特的机制发挥作用，即降低 UBA1 蛋白的催化活性，而不是降低细胞质 UBA1b 同工型的表达。⁶此外，Poulter 等人对该变体的初步报告。表明该变异可能与不同的骨髓形态有关。在这里，我们报告了第四例患有UBA1 p.Ser56Phe 变异的 VEXAS 患者，包括迄今为止最完整的病理学检查，并确认了与这种非典型UBA1变异相关的非典型病理结果。

一名 41 岁男性最初于 2016 年就诊，有 9 个月的孤立性正细胞性贫血病史。此外，他还报告有间歇性发烧、疲劳、体重减轻、游走性关节痛和皮肤结节疼痛。进行了广泛的实验室评估（表 S1），结果显着的是炎症标志物升高和血清铁低。 BM 活检和抽吸涂片（图 1D-F）显示细胞过多的骨髓（80% 细胞）伴红系增生（M:E 比例 0.5:1）和非典型巨核细胞，具有小分叶形式和广泛分离的核叶。髓系和红系前体细胞表现出轻度异型性，具有罕见、分散的形式，含有细胞质空泡，包括约 15% 的髓系前体细胞（成髓细胞、早幼粒细胞和骨髓细胞）。爆炸没有增加。细胞遗传学分析显示男性核型正常，覆盖骨髓恶性肿瘤中常见突变的 37 个基因的骨髓下一代测序 (NGS) 检测结果显示致病性变异呈阴性。考虑到总体的临床和实验室检查结果，骨髓中的贫血和形态异型性被认为是继发于潜在的风湿性疾病，这种疾病很难分类，但被认为代表类风湿性关节炎。在接下来的 6 年里，该患者对多种疗法均无效，包括泼尼松、羟氯喹、甲氨蝶呤、白细胞介素 (IL)-6、IL-1 和肿瘤坏死因子 α (TNF-α) 抑制剂（图 S1）。外周血计数进展为全血细胞减少。

由于临床对最近描述的 VEXAS 综合征的关注，利用覆盖整个UBA1基因的 NGS 检测（Invitae，旧金山，加利福尼亚州）对外周血进行了UBA1单基因测序分析。测试显示 c.167C > T (p.Ser56Phe) 变体，其变体等位基因频率 (VAF) 范围为 18%–28%（所使用的种系测试平台未经验证，无法报告该体细胞变体的精确 VAF）。重复的 BM 活检（图 1G-I）显示进行性细胞增多（90% 细胞）和红系增生，M:E 比率进一步下降至 0.2:1。存在明显的巨核细胞发育不良，存在罕见的环状铁粒幼细胞，细胞质空泡化更为明显，目前存在于约 35% 的骨髓前体细胞中。爆炸没有增加。细胞遗传学分析显示男性核型正常。未覆盖UBA1基因的骨髓 NGS 检测组鉴定出EZH2中的致病性变异（p.Tyr731His；8%）和KMT2A中的意义不确定的变异（p.His1456Tyr；4%）。该患者在 VEXAS 综合征的情况下被诊断为 MDS 合并多系发育不良。

三个周期的阿扎胞苷后，患者接受了强度降低的调节，匹配的无关供体外周血干细胞移植。移植后 30 天进行的骨髓活检显示轻度髓系和红系异型性，包括偶尔出现空泡化的髓系成分。然而，使用覆盖UBA1 的扩展面板进行的 NGS对于先前鉴定的UBA1、EZH2和KMT2A变体呈阴性，并且植入研究显示 99% 的供体嵌合。移植后 6 个月，BM 活检显示没有发育不良的证据，移植研究显示 100% 供体嵌合。患者目前报告没有明显的风湿病症状。

过去有关与 VEXAS 综合征相关的 BM 形态的报告将骨髓增生描述为一种刻板的发现。然而，我们在此描述了第二例具有非典型UBA1 p.Ser56Phe 变异的 VEXAS 病例的 BM 研究结果，并证实了在该变异的背景下存在明显的红细胞增生，正如 Poulter 等人所描述的那样。⁶有趣的是，Sakuma 等人。⁷最近描述了一位患有 MDS 且具有UBA1 p.Tyr55His 变异的患者；尽管手稿中没有描述，但带有该患者骨髓抽吸图像的补充图也显示出明显的相对红细胞增生。与 p.Ser56Phe 变体相关的独特 BM 形态可能是由于针对该变体提出的替代疾病机制，涉及细胞质 UBA1 的功能减少而不是蛋白质表达减少。类似的机制也可以解释与 p.Tyr55His 变体相关的病理学和 BM 形态。重要的是，保尔特等人。无法评估 p.Ser56Phe 变异患者的细胞质空泡。在这里，我们表明，与携带典型 p.Met41 变体的病例类似，p.Ser56Phe 也与细胞质空泡化有关。总而言之，这些发现表明，在适当的临床环境中，与骨髓或红系增生相关的细胞质空泡化的存在可能需要检测UBA1突变。 VEXAS 中的细胞质空泡化通常在未成熟的骨髓和红细胞成分中最为明显。在我们的病例中，细胞质空泡化有些微妙，可能是由于红系增生和较成熟的红系元素丰富，而未成熟的髓系和红系元素相对较少。然而，具有 >1 个液泡的骨髓前体细胞≥10% 的阈值已被提议作为 VEXAS 的敏感且特异的诊断特征。¹⁰事实上，我们的患者在两个时间点都达到了这个阈值，并且随着骨髓中 M:E 比率和髓系前体细胞的绝对数量随着时间的推移而减少，满足这个阈值的髓系前体细胞的百分比增加了。

此外，我们的研究结果强调了这样一个事实：仅检测 Met41 变异的UBA1检测不足以识别所有 VEXAS 患者。虽然UBA1可能会越来越多地被纳入骨髓 NGS 组合中，但 NGS 组合的重新设计需要时间。在此期间，可以通过学术和商业实验室进行单基因UBA1检测，一些机构可能会选择开发UBA1检测。 Met41与 Tyr55 和 Ser56 一起存在于UBA1的外显子 3 中。鉴于影响内含子 2/外显子 3 连接的剪接位点变异的存在已得到充分描述，因此需要对该剪接连接以及整个外显子 3 进行测序的UBA1测定法才能涵盖绝大多数报道的与骨髓相关的致病性UBA1变异疾病。最终，随着致病性UBA1变异谱的不断扩大，通过 NGS 对所有编码区进行测序可能会成为未来的标准。

骨髓恶性肿瘤患者中 VEXAS 综合征的识别可能会影响临床决策，因为同种异体造血干细胞移植 (allo-HCT) 是目前 VEXAS 相关风湿病唯一可能治愈的疗法。考虑到与同种异体 HCT 相关的发病率，它目前仅用于患有严重、难治性 VEXAS 或继发性骨髓恶性肿瘤的患者。¹¹最近一项由 13 名因难治性 VEXAS 或相关骨髓恶性肿瘤接受同种异体 HCT 治疗的患者组成的多中心队列显示，2 年总生存率为 82%，所有经过长期随访的患者的 VEXAS 临床表型均得到解决。¹²