Laboratory Assessment of Patients with Hypereosinophilic Syndrome

嗜酸性粒细胞增多综合征是一组以血液或组织中显著的嗜酸性粒细胞增多和嗜酸性粒细胞相关的临床表现为特征的疾病。人嗜酸性粒细胞表达白细胞介素-5受体α（IL 5 RA）。Benralizumab（MEDI-563; Fasenra，MedImmune/AstraZeneca）是一种针对IL 5 RA的人源化、无岩藻糖基化单克隆抗体，其靶向携带IL 5 RA的细胞，以增强抗体依赖性细胞毒性。贝那利珠单抗用于一项随机、双盲、安慰剂对照的2期临床试验。在20例有症状的患者中进行了一系列三个月的贝那利珠单抗或安慰剂皮下注射，这些患者患有PDGFRA阴性嗜酸性粒细胞增多综合征，并且绝对嗜酸性粒细胞计数至少为1000个细胞/mL 3;所有患者都接受了这种疾病的稳定治疗（药物或饮食改变）。该方案之后是一个开放标签期，在此期间，患者的背景治疗可以根据耐受性逐渐减少，以及一个扩展期。随机化阶段的主要终点是第12周时嗜酸性粒细胞绝对计数减少至少50%。探索性终点包括对临床和实验室反应预测因素的评估。 在随机化阶段，贝那利珠单抗组发生主要终点的患者多于安慰剂组（9/10例患者90% vs. 3/10例患者30%，P=0.02）。在基线和第12周获得骨髓穿刺和活检样本。在贝那利珠单抗组的所有患者中，骨髓嗜酸性粒细胞、嗜酸性粒细胞前体以及血液和骨髓嗜碱性粒细胞的数量在第12周显著降低。有趣的是，肥大细胞的数量和血清类胰蛋白酶水平没有变化。在开放期，19例患者中有17例（89%）观察到临床和血液学缓解，19例患者中有14例（74%）持续48周;在后一组中，14例患者中有9例（64%）的背景治疗可以逐渐减少。这些患者的骨髓和组织嗜酸性粒细胞增多也受到抑制。最常见的药物相关不良事件，头痛和乳酸脱氢酶水平升高，发生在32%的患者第一剂贝那利珠单抗后，所有患者在48小时内消退。其他不良事件的发生频率在两组中相似。在所检查的许多潜在的反应预测因子中，只有临床疾病亚型似乎与初始反应或复发相关。 在另一项研究中，我们研究了嗜酸性粒细胞疾病患者中唾液酸结合免疫球蛋白样凝集素（Siglec）8的表达。唾液酸结合免疫球蛋白样凝集素（Siglec）8选择性地在嗜酸性粒细胞、肥大细胞和嗜碱性粒细胞上表达，当与嗜酸性粒细胞结合时，可导致细胞死亡。我们试图表征正常供体（ND）和嗜酸性粒细胞供体（EO）中的表面和可溶性Siglec-8（sSiglec-8）水平，并评估抗Siglec-8抗体在体外诱导嗜酸性粒细胞死亡中的功效。通过使用流式细胞术和定量PCR评估Siglec-8的嗜酸性粒细胞表达。通过ELISA测量血清sSiglec-8水平。通过使用流式细胞术在体外和在人源化小鼠中在体内评价IgG 4（嵌合2 E2 IgG 4）和无岩藻糖基化IgGl（嵌合2 E2 IgGl c2 E2 IgGl）抗Siglec-8抗体对嗜酸性粒细胞死亡的诱导。Siglec-8在ND和EO的嗜酸性粒细胞上一致表达，与绝对嗜酸性粒细胞计数或疾病活动性无关。sSiglec-8水平在来自大多数供体的血清中是可测量的，与绝对嗜酸性粒细胞计数或Siglec-8表面表达无关。c2 E2 IgG 1和嵌合2 E2 IgG 4在诱导来自ND和EO的纯化的嗜酸性粒细胞在过夜IL-5引发后的细胞死亡（膜联蛋白-V阳性）方面同样有效。相反，在没有IL-5的情况下，仅在EO中观察到纯化的嗜酸性粒细胞的杀伤，并且仅在c2 E2 IgG 1中观察到自然杀伤细胞介导的嗜酸性粒细胞杀伤。最后，用抗Siglec抗体处理人源化小鼠导致体内IL-5诱导的嗜酸性粒细胞增多症的稳健消耗。Siglec-8在来自EO和ND的血液嗜酸性粒细胞上高度表达，并且代表嗜酸性粒细胞病症的潜在治疗靶标。在IL-5存在的情况下，增强对嗜酸性粒细胞的杀伤可能会导致IL-5驱动的嗜酸性粒细胞增多症患者的疗效增加。

Hypereosinophilic syndrome is a group of diseases defined by marked eosinophilia in blood or tissue and eosinophil-related clinical manifestations. Human eosinophils express interleukin-5 receptor alpha (IL5RA). Benralizumab (MEDI-563; Fasenra, MedImmune/AstraZeneca) is a humanized, afucosylated monoclonal antibody against IL5RA that targets IL5RA-bearing cells for enhanced antibody-dependent cellular cytotoxicity. Benralizumab was utilized in a randomized, double-blinded, placebo-controlled, phase 2 clinical trial. A series of three monthly subcutaneous injections of either benralizumab or placebo in 20 symptomatic patients who had PDGFRA-negative hypereosinophilic syndrome and an absolute eosinophil count of at least 1000 cells/mL3 was performed; all the patients were receiving stable therapy (drugs or dietary changes) for this disease. This regimen was followed by an open-label phase, during which the patient's background therapy could be tapered as tolerated, and an extension phase. The primary end point of the randomized phase was a reduction of at least 50% in the absolute eosinophil count at week 12. The exploratory end points included an assessment of clinical and laboratory predictors of response. During the randomized phase, the primary end point occurred in more patients in the benralizumab group than in the placebo group (9 of 10 patients 90% vs. 3 of 10 patients 30%, P=0.02). Bone marrow aspirates and biopsy samples were obtained at baseline and at week 12. The numbers of bone marrow eosinophils, eosinophil precursors, and blood and bone marrow basophils were significantly decreased at week 12 in all the patients in the benralizumab group. Interestingly, the number of mast cells and serum tryptase levels were unchanged. During the open-label phase, clinical and hematologic responses were observed in 17 of 19 patients (89%) and were sustained for 48 weeks in 14 of 19 patients (74%); in the latter group, in 9 of 14 patients (64%), background therapies could be tapered. Bone marrow and tissue eosinophilia were also suppressed in these patients. The most common drug-related adverse events, headache and an elevated lactate dehydrogenase level, occurred in 32% of the patients after the first dose of benralizumab and resolved within 48 hours in all patients. Other adverse events occurred with similar frequency in the two groups. Of the many potential predictors of response that were examined, only clinical disease subtype appeared to be associated with the initial response or relapse. In a separate study, we investigated Sialic acid-binding immunoglobulin-like lectin (Siglec) 8 expression in patients with eosinophilic disorders. Sialic acid-binding immunoglobulin-like lectin (Siglec) 8 is selectively expressed on eosinophils, mast cells, and basophils and, when engaged on eosinophils, can cause cell death. We sought to characterize surface and soluble Siglec-8 (sSiglec-8) levels in normal donors (NDs) and eosinophilic donors (EOs) and assess the efficacy of anti-Siglec-8 antibodies in inducing eosinophil cell death in vitro. Eosinophil expression of Siglec-8 was assessed by using flow cytometry and quantitative PCR. Serum sSiglec-8 levels were measured by means of ELISA. Induction of eosinophil death by IgG4 (chimeric 2E2 IgG4) and afucosylated IgG1 (chimeric 2E2 IgG1 c2E2 IgG1) anti-Siglec-8 antibodies was evaluated in vitro by using flow cytometry and in vivo in humanized mice. Siglec-8 was consistently expressed on eosinophils from NDs and EOs and did not correlate with absolute eosinophil count or disease activity. sSiglec-8 levels were measurable in sera from most donors unrelated to absolute eosinophil counts or Siglec-8 surface expression. c2E2 IgG1 and chimeric 2E2 IgG4 were equally effective at inducing cell death (Annexin-V positivity) of purified eosinophils from NDs and EOs after overnight IL-5 priming. In contrast, killing of purified eosinophils without IL-5 was only seen in EOs, and natural killer cell-mediated eosinophil killing was seen only with c2E2 IgG1. Finally, treatment of humanized mice with anti-Siglec antibody led to robust depletion of IL-5-induced eosinophilia in vivo. Siglec-8 is highly expressed on blood eosinophils from EOs and NDs and represents a potential therapeutic target for eosinophilic disorders. Enhanced killing of eosinophils in the presence of IL-5 might lead to increased efficacy in patients with IL-5-driven eosinophilia.