Serological Markers of Clinical Improvement in MuSK Myasthenia Gravis

Abstract

Background and Objectives

In this retrospective longitudinal study, we aimed at exploring the role of (a) MuSKimmunoglobulin G (IgG) levels, (b) predominant MuSK-IgG subclasses, and (c) antibody affinity as candidate biomarkers of severity and outcomes in MuSK-MG, using and comparing different antibody testing techniques.

Methods

Total MuSK-IgGs were quantified with radioimmunoassay (RIA), ELISA, flow cytometry, and cell-based assay (CBA) serial dilutions using HEK293 cells transfected with MuSK-eGFP. MuSK-IgG subclasses were measured by flow cytometry. SAffCon assay was used for determining MuSK-IgG affinity.

Results

Forty-three serum samples were obtained at different time points from 20 patients withMuSK-MG (median age at onset: 48 years, interquartile range = 27.5–72.5; women, 16/20), with 9 of 20 (45%) treated with rituximab. A strong correlation between MuSK-IgG levelsmeasured by flow cytometry and RIA titers was found (rs = 0.74, 95% CI 0.41–0.89, p = 0.0003), as well as a moderate correlation between CBA end-point titers and RIA titers (rs = 0.47, 95% CI 0.01–0.77, p = 0.0414). A significant correlation was found between MuSK-IgG flow cytometry levels and disease severity (rs = 0.39, 95% CI 0.06–0.64, p = 0.0175; mixed-effects model estimate: 2.296e-06, std. error: 1.024e-06, t = 2.243, p = 0.032). In individual patients, clinical improvement was associated with decrease in MuSK-IgG levels, as measured by either flow cytometry or CBA end-point titration. In all samples,MuSK-IgG4 was themost frequent isotype (mean ± SD: 90.95% ± 13.89). A significant reduction ofMuSK-IgG4 and, to a lesser extent, of MuSK-IgG2, was seen in patients with favorable clinical outcomes. A similar trend was confirmed in the subgroup of rituximab-treated patients. In a single patient,MuSKIgG affinity increased during symptom exacerbation (KD values: 62 nM vs 0.6 nM) while total MuSK-IgG and IgG4 levels remained stable, suggesting that affinity maturation may be a driver of clinical worsening.

Discussion

Our data support the quantification of MuSK antibodies by flow cytometry. Through a multimodal investigational approach, we showed that total MuSK-IgG levels, MuSK-IgG4 and MuSK-IgG2 levels, and MuSK-IgG affinity may represent promising biomarkers of disease outcomes in MuSK-MG.