Department of Experimental Medicine, University of Genova, Genova, Italy.
Laboratory of Molecular Biology, IRCCS Istituto Giannina Gaslini, Genova, Italy.
J Immunother Cancer. 2021 Apr;9(4). doi: 10.1136/jitc-2020-002293.
High-risk neuroblastomas (HR-NBs) are rare, aggressive pediatric cancers characterized by resistance to therapy and relapse in more than 30% of cases, despite using an aggressive therapeutic protocol including targeting of GD2. The mechanisms responsible for therapy resistance are unclear and might include the presence of GD2neg/low NB variants and/or the expression of immune checkpoint ligands such as B7-H3.
Here, we describe a multiparametric flow cytometry (MFC) combining the acquisition of 10 nucleated singlets, Syto16pos CD45neg CD56pos cells, and the analysis of GD2 and B7-H3 surface expression. 41 bone marrow (BM) aspirates from 25 patients with NB, at the onset or relapse, are analyzed, comparing results with cytomorphological analysis (CA) and/or immunohistochemistry (IHC). Spike in experiments assesses the sensitivity of MFC. Kaplan-Meier analysis on 498 primary NBs selects novel prognostic markers possibly integrating the MFC panel.
No false positive are detected, and MFC shows high sensitivity (0.0005%). Optimized MFC identifies CD45negCD56pos NB cells in 11 out of 12 (91.6%) of BM indicated as infiltrated by CA, 7 of which coexpress high levels of GD2 and B7-H3. MFC detects CD45negCD56posGD2neg/low NB variants expressing high surface levels of B7-H3 in two patients with HR-NB (stage M) diagnosed at 53 and 139 months of age. One of them has a non-MYCN amplified tumor with unusual THpos PHOX2Bneg phenotype, which relapsed 141 months post-diagnosis with BM infiltration and a humerus lesion. All GD2neg/low NB variants are detected in patients at relapse. Kaplan-Meier analysis highlights an interesting dichotomous prognostic value of MML5, ULBPs, PVR, B7-H6, and CD47, ligands involved in NB recognition by the immune system.
Our study validates a sensitive MFC analysis providing information on GD2 and B7-H3 surface expression and allowing fast, specific and sensitive evaluation of BM tumor burden. With other routinely used diagnostic and prognostic tools, MFC can improve diagnosis, prognosis, orienting novel personalized treatments in patients with GD2low/neg NB, who might benefit from innovative therapies combining B7-H3 targeting.
高危神经母细胞瘤（HR-NB）是一种罕见的侵袭性小儿癌症，其特征是尽管采用了包括针对 GD2 的靶向治疗在内的积极治疗方案，但仍有超过 30%的病例出现耐药和复发。耐药的机制尚不清楚，可能包括存在 GD2neg/low NB 变体和/或表达免疫检查点配体，如 B7-H3。
在这里，我们描述了一种多参数流式细胞术（MFC），结合了 10 个有核单细胞的采集、Syto16pos CD45neg CD56pos 细胞，以及 GD2 和 B7-H3 表面表达的分析。分析了 25 名 NB 患者在初诊或复发时的 41 份骨髓（BM）抽吸物，将结果与细胞形态学分析（CA）和/或免疫组织化学（IHC）进行比较。在 498 例原发性 NB 中进行的生存分析选择了新的可能整合 MFC 面板的预后标志物。
未检测到假阳性，MFC 显示出高灵敏度（0.0005%）。优化的 MFC 在 12 份 BM 中有 11 份（91.6%）被 CA 指示为浸润的情况下识别出 CD45negCD56pos NB 细胞，其中 7 份同时表达高水平的 GD2 和 B7-H3。MFC 在两名诊断为 HR-NB（M 期）的患者中检测到 CD45negCD56posGD2neg/low NB 变体，这些变体表面表达高水平的 B7-H3，他们的年龄分别为 53 岁和 139 岁。其中一名患者的肿瘤非 MYCN 扩增，表现出不寻常的 THpos PHOX2Bneg 表型，在诊断后 141 个月出现 BM 浸润和肱骨病变复发。所有 GD2neg/low NB 变体均在患者复发时检测到。生存分析突出了 MML5、ULBPs、PVR、B7-H6 和 CD47 的有趣二分预后价值，这些配体涉及到 NB 被免疫系统识别。
我们的研究验证了一种敏感的 MFC 分析方法，该方法提供了关于 GD2 和 B7-H3 表面表达的信息，并能够快速、特异性和敏感地评估 BM 肿瘤负荷。与其他常用的诊断和预后工具结合使用，MFC 可以改善诊断、预后，为 GD2low/neg NB 患者提供新的个性化治疗方案，并可能受益于结合 B7-H3 靶向的创新治疗方法。
