Multiparametric flow cytometry highlights B7-H3 as a novel diagnostic/therapeutic target in GD2neg/low neuroblastoma variants

Abstract

BackgroundHigh-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.MethodHere, we describe a multiparametric flow cytometry (MFC) combining the acquisition of 106 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.ResultsNo 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.ConclusionsOur 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.

CD47-SIRPα Checkpoint Inhibition Enhances Neutrophil-Mediated Killing of Dinutuximab-Opsonized Neuroblastoma Cells

“…This member of the B7 family of immunomodulatory regulators is homogeneously expressed in both primary and metastatic neuroblastomas, as well as in a large variety of solid cancers, while it shows low or null protein surface expression in most normal tissues [54,55]. More specifically, a recent study found neuroblastoma patients with GD2-negative/low variants to still express B7-H3 molecule in high levels, suggesting that B7-H3 might represent an optimal alternative targetable molecule for these patients in particular [56]. At least one anti-B7-H3 monoclonal antibody has already been developed, enoblituzumab, which showed potent antitumor activity by peripheral blood mononuclear cells towards B7-H3-expressing tumors [55,57] and has been recently clinically tested in a phase I trial for solid pediatric tumors, including neuroblastoma (www.clinicaltrials.gov: NCT02982941, accessed on 21 April 2021).…”

An Overview of Long Non-Coding (lnc)RNAs in Neuroblastoma

“…At the moment, the genetic and molecular mechanisms leading to NB development are largely unknown. In recent years, some new therapeutic approaches were developed, such as GD2 monoclonal antibodies or B7-H3 IgG antibodies, but these are not applicable in all cases [11,13,15].…”

“…Anti-GD2 monoclonal antibodies are currently used in therapy to improve standard treatments for HR-NBs, but further studies are necessary to confirm the effectiveness of this immunotherapy and to optimize it [11]. However, in 12% of patients with bone marrow relapse, NB cells lose GD2 expression, thus rendering the use of this treatment impossible [12]; for this reason, Dondero et al [13] developed a multiparametric flow cytometry to observe GD2 surface expression, suggesting B7-H3 targeting therapy for those patients in which GD2 is missed. B7-H3 is a transmembrane glycoprotein overexpressed in NB cells (particularly in bone marrow aspirates) [14], as well as in melanomas, gliomas and breast and pancreatic cancers [15].…”

Tumor Microenvironment and Hydrogel-Based 3D Cancer Models for In Vitro Testing Immunotherapies

“…Intriguingly, we observed molecular characteristics more similar to immunophenotypic variances occurring in vivo and not fully comprehended in traditional 2D culture conditions, such as the IFN-γ-induced negative regulation of PVR (CD155) expression on tumor cells after 7 days of 3D culture [ 23 ]. Moreover, it was possible to appreciate an IFN-γ-dependent upregulation of the immune checkpoint ligand B7-H3, a molecule deeply discussed above [ 179 ].…”