Patients were identified through the Peking Union Medical College Hospital (PUMCH) MG registry platform. The study was approved by the regional ethics committee of PUMCH, and participants provided written consent to registration in the MG registry and the use of recorded data for research purposes. The study was conducted from January 1, 2016 to November 30, 2021. This study followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline for cohort studies.

All patients included were diagnosed with MG. We prospectively collected demographic information and data on the course of illness, medication, neurological physical examinations, MGFA classification, MGC, MG-ADL scores, and Repetitive Nerve Stimulation (RNS) results. Patients' serum samples were acquired prospectively at enrolment. Serum AChR antibody titers and MuSK antibody were estimated by the immunoprecipitation methods using ¹²⁵I-alpha-bungarotoxin and ¹²⁵I-MuSK, respectively (RSR Limited, Cardiff, UK). All MuSK antibody-positive patients with MG were enrolled in the cohort.

In this study, participating patients with MuSK-MG, either received or did not receive IS after a complete discussion with the neurologists. Financial burden and potential AEs are the main concerns for patients who refused IS. We defined the IS+ group as treatment with one or more non-steroid IS for at least 6 months and the IS- group as treatment with only steroids during the follow-up. The following exclusion criteria were applied: the duration of observation was < 6 months, < 2 follow-up visits, concurrent neurologic diseases interfering with assessment, and immunosuppressive therapy for other indications during the observation.

Non-steroid IS dosing range was as follows: AZA 100–150 mg/day, TAC 3–5 mg/day, and MMF 1,000–3,000 mg/day. In the long-term follow-up, specialists set the achievement of MMS and better as the treatment goal and reduced the steroids to the minimum maintenance dose according to the long-term side effects of the steroids. All IS+ and IS- patients were treated with rescue therapy after an exacerbation, including intravenous methylprednisolone pulse therapy, intravenous immunoglobin injections (IVIG), and plasma exchange (PLEX).

Patients were followed up by the specialist group every 6 month. Medication, MGC score, MG-ADL score, MG-PIS classification, and MuSK antibody results were recorded at each follow-up visit. Drug side effects were regularly monitored.

The study's primary outcome was relapsed, defined as a physician-confirmed exacerbation of MG in a previously stable state, except for other possible contributors to the exacerbation of weakness, such as electrolyte disturbance, infection, etc. A general ΔMGC score of > or equal to 3, treatment with rescue therapy, and/or hospitalization was considered clinically significant (17). The interval between the first relapse and time 0 after enrolment was recorded in days. We also assessed whether the association between IS therapy and relapse differed between the following subgroups: age at disease onset, high vs. low MuSK antibody titer, onset type, high vs. low MGFA subtype, and with vs. without relapse history before enrollment.

The primary endpoint of this study was to calculate whether the use of IS significantly reduced the risk of relapse, and the results were calculated according to Cox risk proportional model, with Power calculated based on reference to Rosner and Freedman et al. The relapse rate of MuSK-MG in the IS+ group was 15% according to Evoli et al. (6) and 56% according to Guptill et al. (15), in the IS- group treated with steroids alone. Assuming IS+ and IS- patients were divided in a 1:1 ratio, at an expected dropout rate of 5%, 26 patients were required in each group. Then, we further assumed a postulated hazard ratio (HR) of 0.25 and α (two-sided) of 0.05, this study had more than 80% power to detect an HR of 0.25 or lower.

Continuous variables that were not normally distributed were expressed as the median and interquartile range (median, IQR) and categorical variables were expressed as frequencies and percentages (%). The χ² and Mann-Whitney U tests were used for the comparison of categorical and continuous variables that were not normally distributed between the IS+ and IS- groups, respectively. Group differences in relapse risk were assessed using Kaplan-Meier curves, while univariate and multivariate hazard ratios (HRs) were assessed from Cox proportional hazards regression. The association of IS application with relapse of MG was compared in each subgroup: gender, age of onset, first onset muscle group, highest MGFA, relapse before time 0, initial MuSK antibody titters, and different disease duration subgroups. Stratified Cox risk proportional regression models were applied for subgroup analysis. All statistical results were significant by taking an α <0.05, two-sided test. Prism 7 (GraphPad Software) was used for unadjusted statistical tests. SPSS 28.0 (IBM) was used for statistical analysis.

The study flowchart is shown in Figure 1. We identified 59 patients with MuSK antibody-positive in the database, 53 patients fulfilled the inclusion criteria and exclusion criteria (Figure 1). Of the 53 eligible patients, 39 patients (73.60%) were women; the median age of onset was 47 years (IQR 34–55). Fourteen of them received IS and steroid combination therapy [10 (71.40%) women]. The baseline characteristics of the two groups were largely comparable, including age at onset, gender composition, first muscle group affected, highest MGFA, severity as reflected by the most recent MGC/ADL score before time 0, MuSK antibody titers at time 0, RNS results, number of cases with muscle atrophy, or duration of disease before time 0 (Table 1A).

Eleven (78.6%) and 5 (12.8%) patients had a history of MG relapse before enrolment in the two groups, with a statistically significant difference (p < 0.01). Six patients had a history of non-steroid IS therapy before enrolment, four in the IS+ group and two in the IS- group, two patients discontinued AZA treatment due to adverse events (AEs) before enrolment: 1 with drug-related granulocyte deficiency and another with an allergic reaction at the first week. The number of patients accepting steroid therapy at time 0 was 10/14 (71.4%) in the IS+ group and 14/39 (35.9%) in the IS- group, with statistically significant difference. The number of patients with a history of IVIG application within the 3 months prior to time 0 was 5 (35.7%) and 17 (43.6%) in the IS+ group and the IS- group, with no statistical difference (p = 0.74). One patient had undergone thymectomy, and the interval from surgery to enrolment was up to 4 years.

The number of patients receiving steroids in the two groups was 14/14 cases (100%) and 36/39 cases (92.3%), respectively. The median daily dosage of steroids was comparable between the IS+ group and the IS- group at the end of follow-up (10 [9.38–15] mg and 10 [5–12], p = 0.40). The number of patients who successfully discontinued steroids was slightly greater in the IS- group (3/39) than that in the IS+ group (0/14), although the results were not statistically different (p = 0.09). The other three outcomes, including ADL score, MGC score, improving in ADL, and improvement in QMG, did not have statistical differences in the IS+ group and IS- group (Table 1B).

Non-steroid IS medications included AZA (n = 9, 9/14, 48%) and TAC (n = 5, 5/14, 35.7%). No participants discontinued IS therapy owing to severe AEs during follow-up, thus, suggesting good tolerability. None of the patients was maintained on ChE-I at the end of the follow-up.

The median duration of observation was comparable between the IS+ group and the IS- group (780 [352, 915] days vs. 840 [630, 1290] days, p = 0.70). Log-rank tests did not reveal an association between relapse and factors, such as gender, initial symptoms, history of previous relapses, duration of disease before time 0, antibody titer at time 0, highest MGFA classification, and length of steroid use (Table 2).

The IS was also associated with a longer duration of remission than steroid monotherapy (median 210 [0–480] days for steroid monotherapy; data not available for the IS+ group since 12/14 patients remained stable; HR = 0.21, 95% CI,0.05–0.58, p = 0.03, Figure 2). This association remained statistically significant after adjustment for age of onset, gender, relapse history, highest MGFA, and accumulation of steroid application (HR = 0.23, 95% CI,0.05–0.93, p = 0.04: Table 3). The proportion of patients in a clinically stable state at 500 and 1000 days was higher with IS than with steroid monotherapy (500 days: 14/14 [100%] IS treatment vs. 25/39 [64.1%] controls, p < 0.01; 1000 days: 13/14 [92.9%] and 15/39 [38.5%], p < 0.01).

Log-rank test revealed a weak association between age and relapse, which disappeared after adjustment in the multivariable Cox regression model (p = 0.06). The patients were grouped according to antibody titer and a multiple Cox regression model showed an HR = 1.02, (95% CI, 0.43–2.43, p = 0.96) for relapse in the higher antibody titer group after adjustment. There was no statistical difference between the higher and the lower antibody titer group in age at onset, gender, duration of disease, steroid therapy and IS therapy before enrollment (Supplementary Table_e1).

Subgroup analyses are shown in Figure 3, in most of the subgroups, IS use was shown to correlate with a reduction in relapse events. Because of the small sample size in each subgroup, interaction analyses were not performed. IS showed better protection in subgroups with longer disease duration before time 0 (>12 months) and MGFA classifications II-V; however, it needs further study whether these results are statistically significant. Data analysis of other subgroups, such as gender, age at onset, different forms of onset, different relapse histories, and different antibody groups showed no association between IS and relapse.