Myasthenia gravis (MG) is an acquired autoimmune disease of the neuromuscular junction (NMJ) characterized by partial or systemic skeletal muscle weakness and fatigability typically worsening after activity (1). It is caused by autoantibodies that target the functional molecules at the postsynaptic membrane of the NMJ. The estimated prevalence of MG is 150–250 per 1,000,000 with an annual incidence of 8–10 per 1,000,000 (2). Nearly 80–90% patients with MG present with antibodies (Ab) against acetylcholine receptor (AChR). 1–10% of patients have antibodies against muscle-specific tyrosine kinase (MuSK). The remaining patients may demonstrate antibodies against lipoprotein-related protein 4 (LRP4) or agrin in the postsynaptic membrane at NMJ (3).

The management of MG aims to improve the symptoms of muscle weakness and quality of life while minimizing the drug side effects (4). Current therapies in MG consists of acetylcholinesterase (AChE) inhibitors, conventional immunosuppressive treatments, short-term fast-acting therapy such as plasma exchange and intravenous immunoglobulin (IVIg) and thymectomy (4). Most patients experience significant relief with AChE inhibitors and traditional oral immunosuppressants. However, to achieve satisfactory therapeutic effect, patients usually require long-term or lifelong immunosuppressive therapy. Moreover, 10–30% of patients are refractory to conventional immunosuppression (3, 5, 6). They have persistent symptoms or frequent relapses, frequently require PE or IVIg therapy, or are intolerable to the side effects of immunosuppressive agents. Therefore, it is of great significance to find out effective and safe treatment options for refractory MG.

Rituximab (RTX) is a chimeric mouse/human monoclonal antibody which specifically deplete CD20 positive B lymphocytes (7). It was approved as therapy for non-Hodgkin B-cell lymphoma (8), as well as some autoimmune diseases such as rheumatoid arthritis (9) and systemic lupus erythematosus (10). In the past two decades, growing evidence have shown that patients with MG may benefit from RTX therapy (11). A previous meta-analysis showed that patients with MG responded well to RTX treatment, regardless of the serotypes of MG (12). However, this meta-analysis only assessed the response rate of RTX therapy. Recently, some new studies have emerged, and evaluated a variety of other outcome indicators such as post intervention status (PIS) defined by the Myasthenia Gravis Foundation of America (MGFA) (13), the dosage of prednisone, quantitative MG (QMG) score. Data about the changes of these outcomes in patients with AChR-MG and MuSK-MG after RTX therapy are still lacking. Therefore, we performed an updated meta-analysis to evaluate the effectiveness and safety of RTX therapy for the treatment of refractory MG, which might allow more extensive using of RTX in treating refractory MG.

RTX was firstly reported to be effective in treating MG in 2000 (44). In the past 20 years, a considerable number of studies reported successful using RTX as a second-line therapy for refractory MG. A previous meta-analysis showed that 83.9% of patients improved PIS after RTX treatment. Here, in our updated meta-analysis, we re-evaluated the effectiveness of RTX from more dimensions. Firstly, we found that 64% of patients achieved MMS or better in PIS following RTX therapy, which is the goal for the treatment of MG (4). Secondly, RTX significantly decreased the QMG score by 1.55 and the GC doses by 1.46. Moreover, 81% patients stopped taking oral immunosuppressants at last visit. In conclusion, RTX could effectively alleviate the symptoms of MG, decrease QMG score and reduce the doses of steroids and non-steroid immunosuppressive agents.

Some studies suggested that the benefits of RTX might depend on the serotype of MG. Patients with MuSK-MG maintained a sustained disease control and withdraw immunosuppressants more quickly compared to their AChR-MG counterparts (18, 21, 22, 36). Our analysis revealed that the proportion of achieving MMS or better and the proportion of stopping oral immunosuppressants in MuSK-MG was higher than those in AChR-MG, but the differences between subgroups were not statistically significant because of the overlapping of the 95% CI. The previous meta-analysis showed that PIS was improved in 80.4% of patients with AChR-MG and 88.8% of patients with MuSK-MG (12). While, in our study, 51% of AChR-MG patients and 79% of MuSK-MG patients achieved MMS or better. The difference between the two subgroups enlarged when the outcome measures become more stringent. Together, these results suggested that MuSK-MG might be a little more sensitive to RTX than AChR-MG and patients with MuSK-MG might achieve the treatment goal more easily compared to AChR-MG patients. Different responses to RTX therapy may be accounted by the pathogenesis discrepancy of AChR-MG and MuSK-MG. Most anti-MuSK Abs are IgG4 and secreted by short-lived plasma cells, the precursors of which are CD20 positive B cells. Depleting CD20 positive B cells by RTX results in dramatical reductions of the population of short-lived plasma cells and the titer of anti-MuSK Abs (45). Nevertheless, anti-AChR Abs, belonging to IgG1 and IgG3 subclasses, are mostly secreted by long-lived plasma cells. Their population size is self-sustaining and not affected by B-cell depletion (43). However, not all outcome measures differed between AChR-MG and MuSK-MG. The two groups were very close in the dose change of GC after RTX therapy. This may be due to the fact that patients with MuSK-MG was steroids-dependent, thus counteracting the superiority of MuSK-MG over AChR-MG during RTX therapy. Different response to RTX might also be related to disease duration and severity. A retrospective study revealed that time to remission after RTX treatment was shorter in newly onset patients compared with patients with refractory MG (46). Therefore, more studies should be carried out to identify the characteristics of patients who benefit significantly from RTX treatment.

In order to clarify whether there were differences in the efficacy of RTX in patients with different disease severity, subgroup was performed. In fact, the proportion of patients achieving MMS or better was higher in mild to moderate patients than severe patients, but the difference was not statistically significant because 95%CI overlapped. This suggested that RTX therapy could be initiated as soon as possible in refractory MG instead of waiting until the disease worsened. The dose of RTX may be another important factor affecting RTX efficacy. Subgroup analysis showed there was no difference in proportion of patients achieving MMS or better between the low dose group and the routine dose group. Our result was similar to a previous meta-analysis in AChR-MG (47). These two studies preliminarily indicated that low dose RTX was as effective as routine dose. Furthermore, some studies suggested that low dose RTX was less likely to have serious side effects (48). Due to the limited number of patients using the low dose regimen, these conclusions were not necessarily reliable. Further clinical studies should be conducted to evaluate the long-term efficacy and safety of low dose RTX in treating refractory MG.

RTX was well-tolerated in patients with MG. Even though most of the patients were treated with GC and immunosuppressants at the same time, adverse effects were only observed in 19.6% of (71 of 363) patients. This is similar to adverse effects rates of RTX in treating other autoimmune diseases (48–50). The most common adverse effects were infusion reactions, which could be easily prevented by antihistamines and steroids. Several studies reported that infusion reactions became less frequently when RTX was reinjected. 5.8% of patients had treatable infections such as pneumonia, herpes zoster, viral gastroenteritis and cholecystitis. Only one patient (0.2%) was diagnosed with PML, which is a fatal infection of brain caused by John Cunningham virus and usually infects immune-compromised patients. This patient received a total dose of 10 g RTX concomitant with prednisone, azathioprine and mycophenolate mofetil (31). Despite the rarity of PML, clinicians should take it into consideration when patients have manifestation of encephalopathy.

There were some limitations in our study. Firstly, most of the studies included in the meta-analysis were observational studies, which might overestimate the effectiveness of treatments compared with controlled trails. Secondly, we could not compare the efficacy of RTX with other drug since most of the included studies were single-arm. Thirdly, the number of patients in each study was relatively small. In subgroup analysis, the number of cases in some studies was no more than 5, which resulted in great randomness of research results. Finally, the heterogeneity between studies was remarkable. There were many reasons for the high heterogeneity. MG is a rare disease with high heterogeneity. Moreover, the RTX regimen, follow up duration and baseline characteristics of patients differed among studies. We could not perform meta-regression because some information was inaccessible in studies. Therefore, a large sample randomized controlled trial is necessary to evaluate the effectiveness and safety of RTX in treating refractory MG.

In conclusion, this systemic review and meta-analysis suggested that RTX therapy could improve the PIS of a considerable number of patients with refractory MG to reach MMS or better with a good safety profile. It also exhibited a steroid-sparing effect. Furthermore, RTX reduced QMG scores and the use of conventional oral immunosuppressants. The efficacy was related to the patient's serotype and disease severity, but not to the doses of RTX. It is necessary to conduct RCTs to compare the efficacy and safety between RTX and other treatments, and to explore the characteristics of patients who are sensitive to RTX therapy.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.

CZ, JG, and GZ conceived the study. CZ and MP did the literature review. CZ, MP, and DC organized the database and performed the statistical analyses. CZ wrote the primary manuscript. JS and ZL critically revised the manuscript. All authors read and approved the final version of the manuscript.

This study was supported by the National Natural Science Foundation of China (Grant No. 81901226).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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