Article Text

Original research
Rate of change in acetylcholine receptor antibody levels predicts myasthenia gravis outcome
  1. Yuta Kojima1,2,
  2. Akiyuki Uzawa1,
  3. Yukiko Ozawa1,
  4. Manato Yasuda1,
  5. Yosuke Onishi1,
  6. Hiroyuki Akamine1,
  7. Naoki Kawaguchi1,3,
  8. Keiichi Himuro1,4,
  9. Yu-ichi Noto2,
  10. Toshiki Mizuno2,
  11. Satoshi Kuwabara1
  1. 1 Neurology, Graduate School of Medicine, Chiba University, Chiba, Japan
  2. 2 Neurology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan
  3. 3 Neurology, Dowa Institute of Clinical Neuroscience, Neurology Clinic Chiba, Chiba, Japan
  4. 4 Neurology, Matsudo Neurology Clinic, Chiba, Japan
  1. Correspondence to Dr Akiyuki Uzawa, Neurology, Graduate School of Medicine, Chiba University, Chiba, Japan; auzawa{at}chiba-u.jp

Abstract

Objective To investigate the association between changes in anti-acetylcholine receptor antibody (AChR Ab) levels induced by immunosuppressive treatment and myasthenia gravis (MG) prognosis at 1-year post-treatment in patients with MG.

Methods We included 53 consecutive AChR Ab-positive patients with MG whose AChR Ab levels were remeasured within 100 days of initiating immunosuppressive treatment (median remeasuring time post-treatment: 71 (55–84) days). The AChR Ab level reduction rate (RR-AChRAb, %/day) adjusted for the time between treatment initiation, and AChR Ab level remeasurement was calculated as follows: (pretreatment–post-treatment AChR Ab level)/pretreatment AChR Ab level/days between therapy initiation and AChR Ab level remeasurement ×100. Participants were divided into two groups based on the cut-off value of RR-AChR Ab, determined using receiver operating characteristic analyses for achieving minimal manifestation (MM) or better status at 1-year postimmunosuppressive treatment. The Myasthenia Gravis Foundation of America postintervention status and MG activity of daily living (MG-ADL) score at 1-year post-treatment were compared between the two groups.

Results The RR-AChRAb cut-off value was 0.64%/day. The high RR-AChRAb group had a higher ratio of MM or better status (90% vs 65%, p=0.03) and lower MG-ADL score (median; 1 vs 2, p=0.04) than the low RR-AChRAb group. Kaplan-Meier analyses showed the early MM achievement in the high RR-AChRAb group (p=0.002, log-rank test).

Conclusions High RR-AChRAb is associated with a favourable outcome at 1-year post-treatment. AChR Ab remeasurement within 100 days of therapy may be useful for predicting AChR Ab-positive MG outcomes at 1-year post-treatment.

Data availability statement

Data are available upon reasonable request.

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INTRODUCTION

Myasthenia gravis (MG) is an autoimmune disease affecting the postsynaptic membrane of neuromuscular junctions. About 80% of patients with MG have anti-acetylcholine receptor antibodies (AChR Ab) in their sera.1 The pathogenesis of impaired neuromuscular transmission in AChR Ab-positive MG is considered to be the impairment of the binding of acetylcholine to its receptor, the simplification of the postsynaptic membrane by complement activation resulting in the formation of the membrane attack complex and acetylcholine receptor internalisation by cross-binding of the autoantibody to the receptor.2–6 The detection of this pathognomonic AChR Ab in the patient’s serum is very useful for the diagnosis of MG due to its high specificity in patients with MG and its commercially available testing.7 In addition to being a diagnostic marker for MG, the utility of serum AChR Ab levels as a disease biomarker has been investigated in several studies.8–10 However, the correlation between the clinical status and AChR Ab levels in individual patients remained unclear, because there were previous conflicting results. Some studies reported that a reduction of AChR Ab levels through plasma exchange was associated with marked clinical improvement,8 9 while Sanders et al 10 identified only a weak correlation between changes in AChR Ab levels and clinical severity, because AChR Ab levels fell in 63% of patients with MG who did not show improvement after treatment.10 Regarding the long-term prognosis of MG, the utility of AChR Ab levels as a prognostic marker has rarely been reported so far. In this study, we aimed to determine whether the rate of reduction of AChR Ab levels (RR-AChRAb), which was newly developed in the present study, measured before and after immunosuppressive treatment reflects the treatment response and predicts the outcome in MG.

METHODS

Subjects

A total of 53 patients with AChR Ab-positive MG, who visited the Chiba University Hospital (Chiba, Japan) between January 2013 and December 2018 and underwent AChR Ab testing within 100 days after immunosuppressive treatment, were retrospectively enrolled (figure 1). MG was diagnosed on the basis of a typical clinical history of MG presenting fluctuating muscle weakness with fatigability, AChR Ab positivity and abnormalities in an edrophonium test or electrophysiological tests.11 Patients who already received immunosuppressive treatment before the first visit underwent no immunosuppressive treatment or only supportive therapy such as high-dose intravenous methylprednisolone, intravenous immunoglobulin and plasmapheresis without oral prednisolone or immunosuppressants were excluded. Also, patients who could not be followed up until 1-year postimmunosuppressive treatment were excluded from the study. Serum AChR Ab levels were measured at each time point by using a commercially available radioimmunoassay (SRL, Tokyo, Japan), and levels ≥ 0.5 nmol/L were considered positive.

Figure 1

Flow diagram showing the process of deciding participants to be analysed. Consecutive 117 patients with AChR-Ab positive MG visiting our institution were assessed for eligibility. Sixty-four patients were excluded due to the following reasons; immunosuppressive treatment before the first visit (n=11), temporary treatment for MG (n=10), not performing antibody measurement (n=38), no 1 year follow-up (n=5). AChR Ab, acetylcholine receptor antibody; MG, myasthenia gravis.

The study was approved by the ethics committee of the hospital (IRB No. 2266, Chiba University Hospital); patients were enrolled via an opt-out methodology.

Data collection

The data of the subjects such as age at onset, sex, MG Foundation of America clinical classification (MGFA class)12 at initial visit, MG-ADL score13 at initial visit, the second measurement of AChR Ab level and 1 year after immunosuppressive treatment, postintervention status as defined by MGFA,12 daily oral prednisolone dose at 1-year postimmunosuppressive treatment, AChR Ab levels at initial visit and the second measurement after immunosuppressive treatment, and the time from the initiation of immunosuppressive treatment to the second measurement of AChR Ab levels were obtained from medical records. (online supplemental figure 1). Patients with MG were classified into three groups (ELT classification; early-onset MG (EOMG) with age at onset ≤ 49 years without thymoma, late-onset MG (LOMG) with age at onset ≥ 50 years without thymoma, and thymoma-associated MG (TAMG)).14 ΔMG-ADL score was calculated as follows: ΔMG-ADL score=MG-ADL score at initial visit−MG-ADL score at the second measurement of AChR Ab levels. We also calculated the RR-AChRAb, which was newly developed for this study, in each subject as an indicator of how many percentages AChR Ab reduce in a day after immunosuppressive treatment. The formula used was as follows: RR-AChRAb (%/day)=(pretreatment AChR Ab level−post-treatment AChR Ab level)/pretreatment AChR Ab level/days between the treatment initiation and the remeasurement of AChR Ab levels ×100. The data on MG exacerbation after a 1-year follow-up were collected to investigate the association of AChR Ab reduction in short-term follow-up with MG exacerbation in long-term follow-up.

Supplemental material

Statistical analysis

All statistical analyses were performed by using JMP software V.14.0 (SAS Institute, Cary, North Carolina, USA). To assess the association between two variables, Spearman’s rank correlation was used. The receiver operating characteristic (ROC) curve was generated to determine an appropriate cut-off value for RR-AChRAb in order to achieve the minimal manifestation (MM) or better clarity (MM better) of the postintervention status at 1-year postimmunosuppressive treatment. The cut-off value was calculated using the Youden index (sensitivity+specificity–1).15 In order to clarify the clinical characteristics of the patients with MG and high or low RR-AChRAb, comparisons of clinical findings between the high and low RR-AChRAb groups were performed using χ2 test or Mann-Whitney U test. Additionally, multivariable logistic regression analysis, adjusting for age, sex and disease duration, was performed to investigate the association of MM achievement and the high and low RR-AChRAb groups. The cumulative incidence of MM achievement within 1 year after immunosuppressive treatment was analysed by using the Kaplan-Meier method and the log-rank test in comparison between the high and low RR-AChRAb groups. Values of p<0.05 were considered statistically significant.

RESULTS

Clinical characteristics

The characteristics of the 53 patients with MG are summarised in table 1. The MG-ADL score tended to decrease gradually at each point. Seventy-nine per cent of participants achieved MM or better status within 1-year post immunosuppressive treatment. Forty-nine patients took oral prednisolone as immunosuppressive treatment for MG and the median dose at 1 year after the initiation was 9 mg/day. Regarding immunosuppressants, 26 patients took tacrolimus (22 patients received tacrolimus plus PSL and 4 received only tacrolimus). Median AChR Ab levels decreased between the first and second measurement, and median time from the initiation of therapy to the second measurement of AChR Ab level was 71 days (IQR: 55–84 days). The changes of absolute values of AChR Ab levels and MG-ADL scores in ELT classification are shown in figure 2. As shown in online supplemental figure 2, a tendency of higher AChR Ab levels at second measurement and lower RR-AChRAb in EOMG than in LOMG and TAMG was identified, although there was no significant difference of initial AChR Ab levels between three groups. There were five patients who underwent plasmapheresis or intravenous immunoglobulin due to MG exacerbation after a 1-year follow-up. Among these, four patients had elevated AChR Ab levels at exacerbation. In addition, four of five patients had low RR-AChRAb (<0.64%/day).

Supplemental material

Table 1

Demographic data of patients with myasthenia gravis

Figure 2

Change of absolute values of acetylcholine receptor antibody levels and MG-ADL scores between each measurement in ELT classification. X axis showed the days between the initiation of immunosuppressive treatment and the second measurement of AChR antibody. In the upper row, AChR antibody level at each point was plotted. AChR antibody levels were indicated in Y axis as logarithms. In the lower row, MG-ADL scores at initial visit and the second measurement of AChR antibody were plotted. These plots were presented separately by ELT classification. AChR, acetylcholine receptor; ELT, early-onset MG, late-onset MG and thymoma-associated MG; MG-ADL, myasthenia gravis activity of daily living.

Correlation between RR-AChRAb levels and clinical findings

As shown in table 2, RR-AChRAb correlated strongly with ΔMG-ADL scores (ρ=0.50, p=0.0005). High RR-AChRAb was associated with older age, severe disease severity at initial visit, high initial PSL dose and low PSL dose 1 year after immunosuppressive treatment.

Table 2

Correlation of reduction rate of AChR antibody titers with clinical findings

ROC analyses to determine the cut-off value of RR-AChRAb

ROC curve of RR-AChRAb for achieving MM or better status of postintervention status at 1 year after immunosuppressive treatment was shown in online supplemental figure 3. The cut-off value of RR-AChRAb determined by the corresponding Youden index was 0.64%/day. The area under the curve was 0.70 and moderately accurate for predicting MM better achievement.

Supplemental material

Comparison between high and low RR-AChRAb groups

As shown in table 3, participants were divided into the high RR-AChRAb group (>0.64, n=30) and the low RR-AChRAb group ( ≤ 0.64, n=23) by the cut-off value of the ROC analysis. The high RR-AChRAb group showed a higher age and a higher proportion of LOMG than did the low RR-AChRAb group. Although MGFA class was higher in the high RR-AChRAb group at initial visit, there was no difference in the MGFA class between two groups at the second measurement of AChR Ab levels. Regarding the MG-ADL score, at initial visit, the high RR-AChRAb group showed higher scores, while at the second measurement of AChR Ab and 1 year after the immunosuppressive treatment, the high RR-AChRAb group showed lower scores than the low RR-AChRAb group. As a result, ΔMG-ADL score was higher in the high RR-AChRAb group. Although AChR Ab levels were not different between two groups at the initial visit, the high RR-AChRAb group showed lower levels of AChR Ab at the second measurement. Intial prednisolone dose was higher, and daily oral prednisolone doses at 1-year postimmunosuppressive treatment were lower in the high RR-AChRAb group. The multivariable analysis showed that a high RR-AChRAb was associated with MM achievement after adjustment for age, sex and disease duration (OR=4.6 (CI 0.9 to 22.3), p<0.05).

Table 3

Comparison between high and low groups of reduction rate of AChR antibody levels

Kaplan-Meier analysis for MM achievement

All participants in this study were included in the log-rank analysis and figure 3 shows the result of the Kaplan-Meier analysis as a cumulative incidence format. The numbers of patients who achieved MM status within 1-year postimmunosuppressive treatment were 27 (90%) in the high RR-AChRAb group and 15 (65%) in the low RR-AChRAb group, respectively (table 3). The median time from the initiation of immunosuppressive treatment to achieving MM status was significantly lower (126 days) in the high RR-AChRAb group than in the low RR-AChRAb group (340 days) (p=0.002, log rank).

Figure 3

Cumulative incidence of minimal manifestation achievement between the high and low groups of the rate of reduction of acetylcholine receptor antibody levels. The high anti-acetylcholinereceptor antibody level reduction rate (RR-AChRAb group showed a higher rate of minimal manifestation (MM) achievement within 1 year after immunosuppressive treatment than the low RR-AChRAb group (p=0.002, Log-rank test).

DISCUSSION

This observational study retrospectively investigated the association between the rate of change of AChR Ab levels before and after immunosuppressive treatment and clinical outcome at 1-year post-treatment in patients with MG. The results showed that high RR-AChRAb levels were associated with great improvements of MG symptoms and favourable outcomes at 1-year post-treatment, and patients with high RR-AChRAb levels achieved MM or better status earlier than patients with MG in the low RR-AChRAb group. RR-AChRAb was correlated not only with the change of MG severity but also with daily oral prednisolone dose at 1-year postimmunosuppressive treatment. Namely, RR-AChRAb could predict 1-year outcome and steroid responsiveness in MG.

The absolute value of AChR Ab levels was previously reported to have no correlation with the clinical status of individuals, which was explained mainly by the affinity of AChR Ab to the receptor, not reflecting the serum antibody pool,16 17 whereas it has been seldom reported whether AChR Ab levels within individual patients correlated with the clinical severity or prognosis. However, patients with MG whose AChR Ab levels paralleled clinical improvement or relapse are often encountered in clinical practice. Thus, we chose not the absolute value but the rate of reduction AChR Ab levels as a main object in this study. Additionally, the rate was adjusted for the time from the start of immunosuppressive treatment to post-therapy measurement because AChR Ab levels did not reach the plateau and changed day by day several months after the therapy.

Sanders et al 10 reported that there were no significant correlations of the absolute amount of change in AChR Ab levels with any severity measures. In addition, the per cent change in AChR Ab levels correlated only the change in manual muscle testing scores. However, correlations of RR-AChRAb not only with change in disease severity, but also with future prednisolone doses were identified in this study. The difference in study setting between the previous study10 and this study leading to the conflicting results was unclear, but the difference in treatment methods such as prednisolone dose or usage of immunosuppressants may be one of the causes of the conflicting results. For MG treatment, oral prednisolone was generally used at a high dose at the early stage and tapered gradually according to the patients’ condition. Altogether, RR-AChRAb could reflect steroid responsiveness in MG, and a high RR-AChRAb may predict steroid responsiveness. This was in line with our results. The greater improvement of symptoms was confirmed in the high RR-AChRAb group, where 93% of patients underwent oral prednisolone therapy.

The high RR-AChAb group achieved MM status earlier after immunosuppressive treatment, presumably because high RR-AChAb reflected good steroid responsiveness. High RR-AChRAb was associated with older age at onset and EOMG had the lowest RR-AChRAb values of ELT classification, consistent with previous reports that the only independent predictor of an optimal outcome in MG identified through multivariable analyses was onset after the age of 50 years and that LOMG achieved MM status earlier than EOMG.18 19 A multicentre cross-sectional study in Japan revealed that in generalised MG, low-dose prednisolone regimen, whose prednisolone was maintained at a maximum dose of 20 mg/day, may lead to earlier achievement of MM or better status on prednisolone ≤ 5 mg/day.20 In contrast, in our study, there was not a difference in maximum prednisolone dose between the two groups of RR-AChRAb. This inconsistency may be accounted for by the difference in other therapies accompanying steroid treatment or the initial dose and de-escalation schedule of prednisolone. Our result suggested that even if a high initial dose of prednisolone was used, steroids could be de-escalated enough and a favourable outcome could be achieved in the case of a steroid-responsive patient.

Early fast-acting treatment (EFT) strategies, which use plasmapheresis, intravenous immunoglobulin or intravenous high-dose methylprednisolone treatment aggressively at the initiation of the treatment, were recommended as one of the effective treatment approaches for MG, based on the idea that the initial treatment goal is maintaining MM better status with an oral prednisolone dose of 5 mg/day or less.21 22 However, there is no consensus on what kind of patients is eligible for EFT. Our results suggested that RR-AChRAb may help select an appropriate treatment for each patient with MG, because RR-AChRAb can be calculated at a relatively early stage of treatment, and it could reflect steroid responsiveness.

However, this study has several limitations. First, the retrospective study design may affect the results via selection bias, several confounding factors and some missing data on MG-ADL scores. There may be an influence on the results by factors that prompted the clinician to repeat antibody testing in patients. Although there was no difference of MM achievement 1 year after immunosuppressive treatment between the included patients and the excluded patients due to no antibody follow-up, the use of less than half of the population for the analysis may influence the results. Second, the number of patients enrolled in this study was small, since it was carried out in a single institution. This reduced the power and robustness of the statistical analysis. Third, the radioimmunoassay for measuring AChR Ab level in this study used a single concentration in each sample. It may affect the accuracy of AChR Ab level for comparison between each time point. Fourth, the clinical severity during the initial visit and the age at onset were different between the low and high RR-AChRAb groups, which might have influenced the outcome. Finally, the analysis in patients under the same kind and intensity of treatment for MG and duration from immunosuppressive treatment to the second measurement of AChR Ab levels could not be performed. To address these concerns, the clinical utility of RR-AChRAb should be explored by multicentre-prospective study with large numbers in future.

In conclusion, we retrospectively investigated the association between the changes in AChR Ab levels with outcome at 1-year postimmunosuppressive treatment in patients with MG. A high RR-AChRAb was associated with favourable prognosis and low dose of prednisolone, presumably because a change in AChR Ab levels reflects steroid responsiveness. Therefore, remeasurement of AChR Ab levels within 100 days after immunosuppressive treatment was recommended for choosing the treatment and predicting the outcome in AChR Ab-positive MG.

Data availability statement

Data are available upon reasonable request.

Ethics statements

References

Supplementary materials

Footnotes

  • Contributors YK, AU and SK contributed to the study conception, design and writing of the manuscript. YO, MY, YO, HA, NK and KH contributed to the data collection and analysis. YN and TM contributed to the interpretation of data and revision of the manuscript. All authors approved the final version.

  • Funding This work was supported in part by the Health and Labour Sciences Research Grant on Intractable Diseases (Neuroimmunological Diseases) from the Ministry of Health, Labour and Welfare of Japan (20FC1030).

  • Competing interests None declared.

  • Provenance and peer review Not commissioned; externally peer reviewed.

  • Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.

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