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Abstract

Background:

Outcomes after thymectomy differ greatly between non-thymomatous and thymomatous myasthenia gravis (MG), meriting an in-depth exploration.

Objective:

To examine the treatment and prognosis of non-thymomatous and thymomatous MG patients after thymectomy.

Design:

A multicenter, retrospective, case–control study focused on MG patients following thymectomy from November 2010 to January 2024. After propensity score matching, 284 patients (142 with non-thymomatous MG and 142 with thymomatous MG) were included, with a median follow-up of 2.94 years.

Methods:

Four outcomes were examined: minimal manifestations status (MMS) or better at the final visit, sustained clinical response, postoperative myasthenic crisis, and long-term mortality. Kaplan–Meier, logistic regression, cox regression, nomogram, receiver operating characteristic curve, decision curve, and calibration curve analyses were used for assessment.

Results:

Non-thymoma patients had a lower proportion of postoperative myasthenic crisis (5.6% vs 13.4%, p = 0.026) and long-term mortality (1.4% vs 9.9%, p = 0.002) but a higher proportion of sustained clinical response (66.2% vs 52.1%, p = 0.016) than thymoma patients. For both non-thymomatous and thymomatous MG, anti-acetylcholine receptor antibody (AChR-Ab) positivity was the independent predictor for MMS or better at the final visit (p = 0.048; p = 0.016) and sustained clinical response (p = 0.035; p = 0.037). Most severe Myasthenia Gravis Foundation of America (MGFA) classification and high-grade Masaoka histopathology were independent predictors for postoperative myasthenic crisis (p < 0.001; p = 0.010) and long-term mortality (p = 0.006; p = 0.014) for thymomatous MG. Postoperative prednisone combined with tacrolimus (Pred + TAC) was associated with achieving sustained clinical response (p = 0.026; p = 0.030) and prednisone tapering for both groups.

Conclusion:

Non-thymomatous MG exhibited a more benign course with better outcomes. AChR-Ab positivity indicated a better prognosis for both groups, while thymomatous MG with severe MGFA classification and high-grade histopathology requires close monitoring and follow-up. Postoperative Pred + TAC could be an effective immunotherapy option for beneficial outcomes.

Plain language summary

Comparing outcomes and postoperative treatment after thymectomy for two types of myasthenia gravis

Why was the study done? Myasthenia gravis (MG) is an immune-mediated disorder characterized by muscle fatigue and fluctuating weakness. MG can be classified into two types: non-thymomatous MG (without thymus tumors) and thymomatous MG (with thymus tumors). This study aimed to examine the treatment and prognosis of these two types of MG and see if certain interventions after thymectomy can help improve recovery.

What did the researchers do? The researchers conducted a study that included 284 patients (142 with non-thymomatous MG and 142 with thymomatous MG) who underwent thymectomy between November 2010 and January 2024. The study compared the postoperative prognoses between the two groups of patients following thymectomy, with a particular focus on evaluating the role of postoperative immunosuppressive therapy.

What did the researchers find? The study found that non-thymomatous MG had better outcomes after thymectomy, as evidenced by fewer severe episodes (called myasthenic crisis), lower long-term mortality, and a higher rate of sustained improvement than thymomatous MG. Both non-thymomatous and thymomatous MG patients who tested positive for a specific antibody (AChR-Ab) had better recovery. However, thymoma patients with more severe MG before surgery and high-grade histopathology were at higher risk of problems after surgery. The combination of prednisone and tacrolimus after surgery was associated with better long-term outcomes for both groups of patients.

What do the findings mean? The findings suggested that non-thymomatous MG exhibited a more benign course with better outcomes. AChR-Ab positivity indicated a more favorable prognosis for both groups. Thymomatous MG patients with severe conditions and high-grade histopathology require enhanced management and follow-up. Additionally, using prednisone and tacrolimus could be a good treatment option for MG after thymectomy.

Keywords

immunotherapy myasthenia gravis non-thymoma thymectomy thymoma

Introduction

Myasthenia gravis (MG) is a rare and clinically heterogeneous autoimmune neuromuscular disorder characterized by weakness and fatigability of muscles.^1,2 In patients with MG, 75%–90% of them have thymic abnormalities, among which thymoma accounts for 20% and thymic hyperplasia accounts for 70%.^3,4 The prognostic outcomes following thymectomy can vary significantly between non-thymomatous and thymomatous MG, necessitating a thorough investigation into these differences.^5,6

Severe generalized MG showed satisfactory long-term remission following thymectomy, particularly in younger patients and those with anti-acetylcholine receptor antibody (AChR-Ab) positivity.⁷ A previous study indicated that MG patients with thymoma had a higher probability of neurological improvement compared to those with thymic lymphoid hyperplasia after extended thymectomy.⁸ To assess the individual prognosis of MG efficiently, it is necessary to develop predictive models for patients with thymoma versus those without, specifically focusing on critical factors that influence postoperative clinical outcomes.⁹

As revealed by the MGTX trial, thymectomy offers significant benefits to MG patients, prominently increasing the probability of reaching sustained minimal manifestations status (MMS) and prednisone (Pred) withdrawal.¹⁰ Thymectomy is often accompanied by postoperative treatment with immunosuppressants, such as Pred, tacrolimus (TAC), and mycophenolate mofetil (MMF), aimed at controlling the MG condition after surgery.¹¹ However, differences in the effect of these immunosuppressants for MG patients following thymectomy remain poorly understood.^12,13 Therefore, it is essential to analyze the clinical efficacy of postoperative immunosuppressants and their potential impact on steroid dosage for both non-thymoma and thymoma patients.

This study aims to compare the postoperative prognoses of patients with non-thymomatous and thymomatous MG undergoing thymectomy, with a particular focus on evaluating the role of postoperative immunosuppressive therapy. By evaluating the effectiveness of predicting models on neurological outcomes, including MMS or better at the final visit, sustained clinical response, postoperative myasthenic crisis, and long-term mortality, we hope to provide valuable insights for clinical decision-making and optimize individualized treatment strategies.

Methods

Study subjects

This was a retrospective, multicenter, case–control study performed at the neurological centers from the First Affiliated Hospital of Zhengzhou University, First Affiliated Hospital of Xi’an Jiaotong University, and Tongji Hospital of Huazhong University of Science and Technology. MG patients who accepted thymectomy from November 2010 to January 2024 were enrolled. This article adheres to the STROBE guidelines.¹⁴ MG was diagnosed with fluctuating muscle weakness with one or more of the following criteria: positive neostigmine testing, seropositive AChR-Ab or muscle-specific tyrosine kinase antibody (MuSK-Ab), and the presence of a minimum decrement of 10% following repetitive nerve stimulation.¹⁵ Postoperative treatment was defined as the initiated medication regimens before the patients were discharged from the hospital following thymectomy. The exclusion criteria were: (1) Follow-up duration <1 year or the outcome was unknown. (2) Thymectomy prior to MG onset. (3) Inconsistent or missing data. (4) Only completed the biopsy. (5) Postoperative immunosuppressants were not maintained at steady doses for at least 3 months unless the patients died. Steady doses were defined as azathioprine (AZA) 2–3 mg/kg/day, TAC 2–4 mg/day, cyclosporine A (CsA) 3–5 mg/kg/day, methotrexate (MTX) 7.5–25 mg/qw, MMF 1–3 g/day, and/or Pred maximum 40 mg/day followed by gradual tapering.^16–18 (6) Postoperative myasthenic crisis before immunotherapy is initiated. The flow diagram depicting the inclusion and exclusion criteria for this study is detailed in Figure 1.

Figure 1.

Flow diagram of inclusion and exclusion criteria for enrolling patients.

Baseline characteristics and potential predictive variables

Clinical data were extracted through a comprehensive review of electronic medical records, including demographic information, clinical history, laboratory findings, and treatment regimens. MG severity was evaluated by Myasthenia Gravis Foundation of America (MGFA) clinical classification.¹⁹ According to the onset age of the disease, MG has been divided into four subtypes: juvenile-onset MG (JMG, onset age <18 years), early-onset MG (EOMG, onset age ⩾18 and <50 years), late-onset MG (LOMG, onset ⩾50 and <65 years), and very-late-onset MG (VLOMG, onset ⩾65 years).²⁰ Thymic epithelial tumors (i.e., thymomas) were histologically classified according to the World Health Organization criteria and modified Masaoka staging system.²¹ Patients were defined as having refractory MG if they responded insufficiently to Pred and at least one immunosuppressive agent over at least 1 year.¹⁵

Outcome measure

The primary outcomes were MMS or better at the final visit and sustained clinical response. MMS or better includes complete stable remission, pharmacological remission, and MMS according to MGFA postintervention status (MGFA-PIS).¹⁹ Sustained clinical response was defined as consistently achieving MMS or better without relapse.²² Relapse is defined as the reoccurrence of one or more MG symptoms for more than 24 h in patients who have achieved MMS or better.²² No response is defined as no unchanged, worse, or exacerbated clinical symptoms according to MGFA-PIS, requiring the addition of new drugs.²² The duration of sustained clinical response was calculated from the start of thymectomy to the date of relapse or no response. The following secondary clinical outcomes were investigated: (1) Postoperative myasthenic crisis, defined as respiratory failure requiring noninvasive ventilation or intubation for more than 24 h within 30 days after thymectomy.²³ (2) Long-term mortality, encompassing all-cause mortality with specific attention to MG crisis-related mortality.

Statistical analysis

Missing values were visualized with “VIM” package and participants with complete data were included in the analysis. Categorical variables are presented as n (%), whereas continuous variables are described by mean ± SD or median (interquartile range). To compare variables between different groups, t tests, Chi-squared tests, Mann–Whitney U tests, one-way ANOVA, or Kruskal–Wallis tests were performed accordingly. Propensity score matching (PSM) was performed to adjust for confounders. Collinearity diagnostics were examined for variables, and logistic regression and Cox regression analysis were performed for noncollinearity variables. A variance inflation factor >10 was considered collinearity between variables. Factors that were significant in the univariate analysis were included in the multivariate analysis with stepwise forward selection. Nomograms were constructed by “rms” package to facilitate and visualize the models generated. Calibration curves, receiver operating characteristic (ROC) curves, and decision curve analysis (DCA) curves were performed to assess the model performance. Model performance was quantified by C-index and area under the ROC curve (AUC) values. The value of AUC is equal to the C-index in the logistic regression model. C-index and AUC values >0.5 were considered significant, while >0.7 were considered good.²⁴ Nomograms based on the Cox regression and Kaplan–Meier curves visualize the cumulative probability of sustained clinical response and survival for MG,^25–27 which take a value of 100% at baseline and gradually decrease with the increase of time. Time-to-event comparisons were analyzed by log-rank tests. Multiple-comparison analysis was applied with the Bonferroni correction. Statistical significance was set at p < 0.05 for two-sided tests. Statistical analyses and figure preparation were conducted using SPSS 24.0 (IBM, Armonk, NY, USA), Origin 2021 (OriginLab, Northampton, MA, USA), GraphPad Prism 8.01 (GraphPad Prism, San Diego, CA, USA), and R 4.4.1 (R Foundation for Statistical Computing, Vienna, Austria).

Results

Baseline characteristics of patients

Patient characteristics before and after PSM are shown in Table 1. First, the clinical characteristics of the 603 patients who met all the criteria were analyzed before PSM. The results demonstrated that thymoma patients were older age at thymectomy (p < 0.001) and disease onset (p < 0.001), shorter disease duration (p < 0.001), more often in males (p = 0.006), and had higher frequency of AChR-Ab positivity (p < 0.001), lower frequencies of JMG subtype (p < 0.001), concomitant autoimmune diseases (AIDs; p < 0.001), and preoperative refractory MG (p = 0.005) compared with non-thymoma patients. Furthermore, thymoma patients need more aggressive treatments, with more intravenous immunoglobulin/plasma exchange (IVIG/PLEX) perioperatively (p < 0.001), and substantially fewer of those can stop the drug completely at the final visit (p < 0.001). After PSM, the above covariates were well-balanced between the two groups. In total, 142 non-thymomatous MG patients and 142 thymomatous MG patients were available for all the following analyses. The difference in varieties of AIDs and tumors between the two groups was not significant (Figure 2(a) and (b)). Detailed pathological types of thymoma and non-thymoma are shown in Figure 2(c). Patients with non-thymomatous MG had a better prognosis, manifested by a higher rate of sustained clinical response (p = 0.016) and lower rates of postoperative crisis (p = 0.026) and long-term mortality (p = 0.002). No remarkable difference was detected in MGFA-PIS from presurgery to the last follow-up (Figure 2(d)). Kaplan–Meier curves and the pie chart also indicated better outcomes for non-thymomatous MG (Figure 2(e)–(g)).

Table 1.

Baseline characteristics.

Variable	Before PSM			After PSM
Variable	Non-thymomatous MG (n = 263)	Thymomatous MG (n = 340)	p Value	Non-thymomatous MG (n = 142)	Thymomatous MG (n = 142)	p Value
Age at thymectomy, years	34.98 ± 16.13	47.16 ± 11.41	<0.001	43.72 ± 10.85	42.07 ± 14.32	0.274
Age at onset, years	31.28 ± 18.27	45.47 ± 12.04	<0.001	41.57 ± 11.42	39.93 ± 15.15	0.303
Duration from MG onset to thymectomy, years	1.50 (0.40–5.00)	0.45 (0.20–1.50)	<0.001	0.95 (0.30–2.50)	0.60 (0.25–2.50)	0.349
MG subtype
JMG	70 (26.6%)	6 (1.8%)	<0.001	7 (4.9%)	4 (2.8%)	0.319
EOMG	142 (54.0%)	200 (58.8%)		95 (66.9%)	104 (73.2%)
LOMG	43 (16.3%)	125 (36.8%)		32 (22.5%)	31 (21.8%)
VLOMG	8 (3.0%)	9 (2.6%)		8 (5.6%)	3 (2.1%)
Sex
Males	91 (34.6%)	155 (45.6%)	0.006	50 (35.2%)	92 (64.8%)	0.223
Females	172 (65.4%)	185 (54.4%)		60 (42.3%)	82 (57.7%)
Symptoms at onset
Ocular MG	154 (58.6%)	216 (63.5%)	0.213	77 (54.2%)	86 (60.6%)	0.280
Generalized MG	109 (41.4%)	124 (36.5%)		65 (45.8%)	56 (39.4%)
Serum antibody
AChR-Ab positive	229 (87.1%)	325 (95.6%)	<0.001	130 (91.5%)	135 (95.1%)	0.235
MuSK-Ab positive	0 (0.0%)	0 (0.0%)		0 (0.0%)	0 (0.0%)
Seronegative	34 (12.9%)	15 (4.4%)		12 (8.5%)	7 (4.9%)
Surgical approach
OPEN	124 (47.1%)	147 (43.2%)	0.338	64 (45.1%)	69 (48.6%)	0.552
VATS	139 (52.9%)	193 (56.8%)		78 (54.9%)	73 (51.4%)	0.552
Concomitant other AIDs	56 (21.3%)	34 (10.0%)	<0.001	22 (15.5%)	15 (10.6%)	0.217
Concomitant other tumors	11 (4.2%)	21 (6.2%)	0.279	8 (5.6%)	10 (7.0%)	0.626
Most severe MGFA classification before thymectomy
I–III	228 (86.7%)	291 (85.6%)	0.698	122 (85.9%)	121 (85.2%)	0.866
IV–V	35 (13.3%)	49 (14.4%)		20 (14.1%)	21 (14.8%)
Non-thymomatous histopathology
Normal	27 (10.3%)	–	–	17 (12.0%)	–	–
Thymic hyperplasia	175 (66.5%)		–	89 (62.7%)	–
Thymolipoma	25 (9.5%)		–	11 (7.7%)	–
Thymic cyst	24 (9.1%)		–	16 (11.2%)	–
Others	12 (4.6%)		–	9 (6.3%)	–
Thymomatous WHO histopathology
A + AB	–	72 (21.2%)	–		29 (20.4%)	–
B1	–	68 (20.0%)			22 (15.5%)
B2/B3/C	–	200 (58.8%)			91 (64.1%)
Thymomatous Masaoka histopathology
I	–	167 (49.2%)	–		63 (44.4%)	–
II–III	–	129 (37.9%)			56 (39.4%)
IV	–	44 (12.9%)			23 (16.2%)
Preoperative refractory MG	39 (14.8%)	26 (7.6%)	0.005	13 (9.2%)	11 (7.7%)	0.670
Postoperative treatment
No medicine	5 (1.9%)	9 (2.6%)	0.588	4 (2.8%)	2 (1.4%)	0.456
Pyridostigmine alone	64 (24.3%)	84 (24.7%)		34 (23.9%)	35 (24.6%)
Pred	113 (43.0%)	152 (44.7%)		51 (35.9%)	53 (37.3%)
AZA	0 (0.0%)	2 (0.6%)		0 (0.0%)	2 (1.4%)
MMF	1 (0.4%)	1 (0.3%)		0 (0.0%)	1 (0.7%)
TAC	5 (2.0%)	3 (0.9%)		4 (2.8%)	1 (0.7%)
Pred + AZA	6 (2.4%)	5 (1.5%)		4 (2.8%)	1 (0.7%)
Pred + MMF	19 (7.2%)	16 (4.7%)		11 (7.7%)	8 (5.6%)
Pred + TAC	49 (18.6%)	62 (18.2%)		34 (23.9%)	36 (25.4%)
Pred + MTX	1 (0.4%)	2 (0.6%)		0 (0.0%)	1 (0.7%)
Pred + CsA	0 (0.0%)	1 (0.3%)		0 (0.0%)	1 (0.7%)
Targeted biologics^a	0 (0.0%)	4 (1.2%)		0 (0.0%)	1 (0.7%)
Perioperative IVIg/PLEX	45 (17.1%)	128 (37.6%)	<0.001	24 (16.9%)	29 (20.4%)	0.446
Postoperative Pred dosage, mg/d	10.00(0.00–20.00)	15.00(0.00–25.00)	0.075	10.00(0.00–20.00)	15.00(0.00–25.00)	0.161
Follow-up, years	3.50 (2.00–6.20)	2.25 (1.00–5.48)	<0.001	3.00 (1.77–5.81)	2.86 (1.01–5.82)	0.146
Treatment at the final visit
No medicine	48 (18.3%)	22 (6.5%)	<0.001	24 (16.9%)	9 (6.3%)	0.227
Pyridostigmine alone	62 (23.6%)	101 (29.7%)		34 (23.9%)	40 (28.2%)
Pred	57 (21.7%)	84 (24.7%)		27 (19.0%)	37 (26.1%)
AZA	4 (1.5%)	2 (0.6%)		2 (1.4%)	1 (0.7%)
MMF	8 (3.0%)	8 (2.4%)		4 (2.8%)	2 (1.4%)
TAC	38 (14.4%)	52 (15.3%)		23 (16.2%)	27 (19.0%)
Pred + AZA	1 (0.4%)	6 (1.8%)		1 (0.7%)	1 (0.7%)
Pred + MMF	13 (4.9%)	7 (2.1%)		7 (4.9%)	4 (2.8%)
Pred + TAC	27 (10.3%)	45 (13.2%)		17 (12.0%)	16.0 (11.3%)
Pred + MTX	4 (1.5%)	3 (0.9%)		2 (1.4%)	1 (0.7%)
Pred + CsA	0 (0.0%)	1 (0.3%)		0 (0.0%)	1 (0.7%)
Targeted biologics^b	1 (0.4%)	9 (2.6%)		1 (0.7%)	3 (2.1%)
IVIg/PLEX at the final visit	3 (1.1%)	11 (3.2%)	0.090	2 (1.4%)	7 (4.9%)	0.175
Postoperative refractory MG	20 (7.6%)	16 (4.7%)	0.136	7 (4.9%)	5 (3.5%)	0.555
Final prednisone dose, mg/d	0.00(0.00–5.00)	0.00(0.00–10.00)	0.133	0.00(0.00–7.50)	0.00(0.00–10.00)	0.651
Outcomes
MMS or better the final visit	208 (79.1%)	267 (78.5%)	0.868	118 (83.1%)	114 (80.3%)	0.539
Sustained clinical response	157 (59.7%)	173 (50.9%)	0.031	94 (66.2%)	74 (52.1%)	0.016
Postoperative myasthenic crisis	11 (4.2%)	30 (8.8%)	0.025	8 (5.6%)	19 (13.4%)	0.026
Long-term mortality	2 (0.08%)	22 (6.5%)	<0.001	2 (1.4%)	14 (9.9%)	0.002

Postoperative targeted biologics include efgartigimod (n = 3), telitacicept (n = 1) before PSM, while include efgartigimod (n = 1) after PSM.

At the final visit, targeted biologics include efgartigimod (n = 7), telitacicept (n = 2), and rituximab (n = 1) before PSM, while include efgartigimod (n = 3) and telitacicept (n = 1) after PSM.

AChR-Ab, acetylcholine receptor antibody; AIDs, autoimmune diseases; AZA, azathioprine; CsA, cyclosporin A; EOMG, early-onset MG (onset ⩾18 and <50 years); IVIg, intravenous immunoglobulin; JMG, Juvenile-onset MG (onset <18 years); LOMG, late-onset MG (onset ⩾50 and <65 years); MG, myasthenia gravis; MGFA, Myasthenia Gravis Foundation of America; MMF, mycophenolate mofetil; MMS, minimal manifestations status; MTX, methotrexate; MuSK-Ab, muscle-specific tyrosine kinase antibody; PLEX, plasmapheresis; Pred, prednisone; PSM, propensity score matching; TAC, tacrolimus; VATS, video-assisted thoracoscopic surgery; VLOMG, very-late-onset MG (onset ⩾65 years); WHO, World Health Organization.

p Value < 0.05 are marked in bold.

Figure 2.

Comparison of clinical characteristics and outcomes between thymomatous and non-thymomatous MG patients following thymectomy. (a–c) Features of concomitant AIDs, concomitant tumors, and pathological type. (d) Assessment of MGFA-PIS from preoperative to the last follow-up. (e) Kaplan–Meier curve of sustained clinical response. (f) Pie chart representing the occurrence of postoperative myasthenic crisis. (g) Kaplan–Meier curve of long-term mortality.

Risk factors and prediction model for MMS or better at the final visit and the evaluation

Univariate logistic regression analysis for MMS or better at the final visit is shown in Supplemental Table 1. Multivariate logistic regression analysis revealed that short disease duration (odds ratio (OR) = 0.91, B = −0.095, p = 0.016) and AChR-Ab positivity (OR = 4.50, B = 1.505, p = 0.002) were independent risk variables for all MG patients (Figure 3(a)). A nomogram was subsequently developed and revealed a relatively good accuracy with an AUC value of 0.702 (Figure 3(b) and (c)). The calibration curve and DCA curve also demonstrated the reliability of the nomogram (Figure 3(d) and (e)).

Figure 3.

Multivariate logistic regression analysis and nomograms to predict MMS or better at the final visit for MG patients following thymectomy. (a–e) Forest plot, nomogram, ROC curve, calibration curve, and DCA curve for all MG. (f–j) Forest plot, nomogram, ROC curve, calibration curve, and DCA curve for non-thymomatous MG. (k–o) Forest plot, nomogram, ROC curve, calibration curve, and DCA curve for thymomatous MG.

For non-thymomatous MG, short disease duration (OR = 0.86, B = −0.148, p = 0.026) and AChR-Ab positivity (OR = 3.64, B = 1.293, p = 0.048) were independent risk factors and integrated into the nomogram (Figure 3(f) and (g)). The ROC curve, calibration curve, and DCA curve showed good discrimination for non-thymomatous MG with an AUC value of 0.720 (Figure 3(h)–(j)). For thymomatous MG, AChR-Ab positivity (OR = 7.50, B = 2.015, p = 0.016) and Masaoka histopathology (IV vs I, OR = 0.14, B = −1.951, p = 0.014) were included in the final risk model (Figure 3(k) and (l)). The nomogram for predicting MMS or better in thymoma patients indicated a good predictive performance with an AUC value of 0.731 (Figure 3(m)–(o)).

Risk factors and prediction model for sustained clinical response and the evaluation

The cumulative probability of relapse or no response increased monotonically over time, thus, the cumulative probability of sustained clinical response showed a monotonically decreasing trend. The univariate Cox regression analysis of risk factors for relapse or no response is presented in Supplemental Table 2. Among postoperative immunosuppressive treatments, only Pred, Pred + TAC, and Pred + MMF were considered for the analysis, as other immunotherapy options account for a smaller proportion. For all MG, multivariate analysis showed that the independent factors affecting cumulative relapse or no response were thymoma (hazard ratio (HR) = 1.57, B = 0.454, p = 0.037), AChR-Ab positivity (HR = 0.42, B = −0.880, p = 0.005), postoperative Pred + TAC (vs Pred, HR = 0.37, B = −0.994, p < 0.001), and Pred + MMF (vs Pred, HR = 0.34, B = −1.070, p = 0.012; Figure 4(a)). The above independent predictors were used to construct a nomogram for predicting sustained clinical response (Figure 4(b)). The AUC values were 0.709 at 1 year, 0.684 at 3 years, and 0.639 at 5 years (Figure 4(c)). The calibration curve showed poor discriminative ability, while the DCA curves indicated the nomogram generated a clinical net benefit (Figure 4(d) and (e)).

Figure 4.

Multivariate Cox regression analysis and nomograms to predict sustained clinical response for MG patients following thymectomy. (a–e) Forest plot, nomogram, ROC curve, calibration curve, and DCA curve for all MG. (f–j) Forest plot, nomogram, ROC curve, calibration curve, and DCA curve for non-thymomatous MG. (k–o) Forest plot, nomogram, ROC curve, calibration curve, and DCA curve for thymomatous MG.

For non-thymoma patients, AChR-Ab positivity (HR = 0.41, B = −0.881, p = 0.035), preoperative refractory MG (HR = 2.90, B = 1.066, p = 0.016), postoperative Pred + TAC (vs Pred, HR = 0.40, B = −0.919, p = 0.026), and Pred + MMF (vs Pred, HR = 0.21, B = −1.570, p = 0.020) were independent predictors of multivariate analysis (Figure 4(f)). The nomogram for predicting the 1, 3, and 5-year sustained clinical response showed unsatisfactory discriminative ability with AUC values of 0.665, 0.682, and 0.631, respectively (Figure 4(g)–(j)). For thymoma patients, AChR-Ab positivity (HR = 0.36, B = −1.025, p = 0.037) and postoperative Pred + TAC (vs Pred, HR = 0.35, B = −1.063, p = 0.030) were independent predictors for cumulative relapse or no response (Figure 4(k)). The nomogram for sustained clinical response reflected general predictive performance (Figure 4(l)–(o)), with AUC values of 0.703, 0.674, and 0.647 for 1, 3, and 5 years, respectively. The C-indexes were 0.663, 0.660, and 0.667 for all MG, non-thymomatous MG, and thymomatous MG.

Risk factors and prediction model for postoperative myasthenic crisis and the evaluation

Risk factors for postoperative myasthenic crisis in univariate logistic analysis are shown in Supplemental Table 3. For all MG patients, multivariate regression analysis revealed that older age at thymectomy (OR = 1.06, B = 0.061, p = 0.002), open approach (OR = 5.73, B = −1.745, p = 0.001), and MGFA IV–V under the most severe condition (OR = 8.77, B = 2.171, p < 0.001) were independent risk variables (Figure 5(a)). The nomogram was composed of the above independent risk factors and showed promising predictive capability with an AUC value of 0.832 (Figure 5(b)–(e)).

Figure 5.

Multivariate logistic regression analysis and nomograms to predict postoperative myasthenic crisis for MG patients following thymectomy. (a–e) Forest plot, nomogram, ROC curve, calibration curve, and DCA curve for all MG. (f–j) Forest plot, nomogram, ROC curve, calibration curve, and DCA curve for non-thymomatous MG. (k–o) Forest plot, nomogram, ROC curve, calibration curve, and DCA curve for thymomatous MG.

For non-thymomatous MG, older age at onset (OR = 1.06, B = 0.058, p = 0.032) and MGFA IV–V under the most severe condition (OR = 7.30, B = 1.988, p = 0.012) were integrated into the nomogram (Figure 5(f) and (g)). The ROC curve, calibration curve, and DCA curve exhibit good calibration with an AUC value of 0.774 (Figure 5(h)–(j)). For thymomatous MG, multivariate regression analysis showed that the open approach (OR = 5.73, B = 1.579, p = 0.001), MGFA IV–V under the most severe condition (OR = 10.93, B = 2.391, p < 0.001), and Masaoka histopathology (IV vs I, OR = 6.42, B = 1.860, p = 0.010) remained significant (Figure 5(k) and (l)). The nomogram for predicting postoperative myasthenic crisis in thymoma patients indicated a high predictive performance, with an AUC value of 0.871 (Figure 5(m)–(o)).

Risk factors and prediction model for long-term mortality and the evaluation

The results of the univariate Cox analysis to predict long-term mortality are shown in Supplemental Table 4. No JMG patients experienced death, so only EOMG, LOMG, and VLOMG patients were included in the multivariate analysis. Thymoma (HR = 9.39, B = 2.239, p = 0.003), VLOMG + LOMG (HR = 5.64, B = 1.730, p = 0.002), and MGFA IV–V under the most severe condition (HR = 4.65, B = 1.536, p = 0.003) were independent factors of long-term all-cause mortality for all MG patients (Figure 6(a)). The above three predictors were used to construct a reliable nomogram for predicting overall survival with AUC values of 0.887, 0.884, and 0.853 for 1, 3, and 5 years, respectively (Figure 6(b)–(e)).

Figure 6.

Multivariate Cox regression analysis and nomograms to predict long-term mortality for MG patients following thymectomy. (a–e) Forest plot, nomogram, ROC curve, calibration curve, and DCA curve to predict overall survival for all MG. (f–j) Forest plot, nomogram, ROC curve, calibration curve, and DCA curve to predict overall survival for thymomatous MG. (k–o) Forest plot, nomogram, ROC curve, calibration curve, and DCA curve to predict MG crisis-free survival for thymomatous MG.

As only two non-thymoma patients had died, no significant risk factors have been identified in the univariate or multivariate analysis for non-thymomatous MG. For thymomatous MG, VLOMG + LOMG (HR = 5.64, B = 1.708, p = 0.002), MGFA IV–V under the most severe condition (HR = 3.65, B = 1.294, p = 0.006), and Masaoka histopathology (IV vs I, HR = 7.37, B = 1.997, p = 0.014) were all independent risk factors for all-cause mortality (Figure 6(f)). The nomogram for predicting the 1, 3, and 5-year overall survival was well-calibrated with AUC values of 0.870, 0.831, and 0.867, respectively (Figure 6(g)–(j)). The nomograms showed good clinical utility in predicting overall survival for all MG (C-index = 0.878) and thymomatous MG (C-index = 0.844). During the follow-up period, nine thymomatous MG patients deteriorated and died due to myasthenia crisis, accounting for 64.3% of all-cause deaths. After multivariate adjustment of thymomatous MG, VLOMG + LOMG (HR = 6.73, B = 1.907, p = 0.021) and MGFA IV–V under the most severe condition (HR = 13.17, B = 2.578, p = 0.002) were incorporated into the nomogram to predict long-term MG crisis-related mortality (Figure 6(k) and (l)). The ROC curve, calibration curve, and DCA curve for crisis-free survival indicated that the nomogram was reliable, with AUC values of 0.888, 0.928, and 0.947 for 1, 3, and 5 years, respectively (Figure 6(m)–(o)). The C-index was 0.908, which indicated that the nomogram had excellent discrimination.

Association of sustained clinical response and corticosteroid tapering under different immunosuppressive therapies

Kaplan–Meier survival curves showed that Pred + TAC and Pred + MMF were significantly correlated with the favorable sustained clinical response of all MG compared with postoperative Pred alone. For non-thymomatous MG and thymomatous MG, only Pred + TAC was statistically significantly more effective than Pred (Figure 7(a)–(c)). Alluvial plots were used to visualize the changes in medications over time. Both groups of patients receiving Pred postoperatively need additional immunosuppressive agents throughout follow-up (Figure 7(d)–(f)). There was a significant reduction in the Pred dose for non-thymoma and thymoma patients at 2 years and beyond after thymectomy, especially in the Pred + TAC group (Figure 7(g)–(i)).

Figure 7.

Sustained clinical response and corticosteroid tapering under different immunotherapy groups of MG patients following thymectomy. (a–c) Kaplan–Meier curve of sustained clinical response for all MG, non-thymomatous MG, and thymomatous MG. (d–f) Alluvial plots of treatment changes among the three groups. (g–i) Daily oral Pred dosage variations among the three groups. Error bars denote the interquartile range for the measurement average. The difference between Pred group and Pred + TAC group is marked in blue; Pred group and Pred + MMF group is marked in red.

Discussions

The results of this retrospective case–control study indicate that non-thymomatous MG exhibited a more favorable prognosis than thymomatous MG, characterized by higher sustained clinical response, lower incidence of postoperative crisis, and lower long-term mortality. Moreover, timely postoperative initiation of immunosuppressive therapy can contribute to sustained clinical response and corticosteroid tapering for both non-thymomatous and thymomatous MG.

Similar to the previous study,²⁸ our study suggested that non-thymoma patients tend to have better clinical outcomes. This does not equate to a greater therapeutic benefit for non-thymoma patients following thymectomy since the more stable disease course may explain their favorable outcomes.²⁹ Both groups achieved approximately 80% of MMS or better at the last follow-up, indicating satisfactory clinical remission after comprehensive treatment. Sustained clinical response can reflect the fluctuations in the condition of MG during follow-up.³⁰ Patients with AchR-Ab negativity are associated with difficulty obtaining MMS or better at the final visit and sustained clinical response, regardless of whether they are in the non-thymomatous or thymomatous group. Thymectomy was not associated with clinical improvement in positive MuSK-Ab MG.³¹ In this study, no patient with MuSK-Ab positivity underwent thymectomy, while 19 seronegative (both AchR-Ab and MuSK-Ab negative) patients were included in our final analysis. Multiple reports have demonstrated that seronegative MG patients with thymic abnormalities or poor treatment responses choose to undergo thymectomy to manage their conditions.^32–34 Searching for new pathogenic antibodies and uncovering meaningful clinicopathological features associated with seronegative MG are worthy of further research.³⁵

Compared to EOMG and LOMG, JMG generally presents with normal thymus or thymus hyperplasia and exhibits a more benign clinical course.^36,37 Several retrospective studies have implicated that thymectomy is more effective for JMG patients versus drug therapy alone.^38,39 African and Asian JMG patients are more likely to have treatment-resistant ophthalmoplegia, and it is extremely difficult for refractory JMG patients to achieve MMS or better.^40,41 Our univariate analyses revealed that non-thymomatous JMG received less clinical benefit compared to EOMG and LOMG (Supplemental Tables 1 and 2). A possible reason for this is that the majority of JMG patients in our study who underwent thymectomy are refractory, drug-resistant cases, and their isolated ocular weakness gains limited benefit from thymectomy.⁴²

Some immunosuppressants can affect the efficacy of cancer treatment and lead to poorer oncologic outcomes.⁴³ However, corticosteroids combined with immunosuppressants favor the inhibition of thymoma invasion and metastasis in MG.⁴⁴ Some physicians recommend that patients reduce or discontinue immunosuppressive agents a week or more before surgery to minimize infection risk while optimizing disease control.⁴⁵ A large proportion of patients who have received thymectomy experience MG relapses and deterioration and need comprehensive postoperative immunosuppressants.³⁰ Our retrospective study is the first to demonstrate that postoperative Pred + TAC has improved prognosis significantly for both non-thymomatous and thymomatous MG. The difference in sustained clinical response between Pred + MMF and Pred was less prominent, probably due to the low number of samples. The nomograms to predict sustained clinical response had insufficient accuracy and large prospective cohort studies are required to optimize the models.

This study also verified the risk factors of postoperative myasthenic crisis and long-term mortality. Patients in both groups who received open thymectomy have a higher risk of postoperative crisis, in agreement with previous reports.⁴⁶ Video-assisted thoracoscopic surgery is usually preferred over thoracotomy and is associated with fewer postoperative complications.⁴⁷ The frequency of long-term mortality of EOMG is remarkably lower than VLOMG + LOMG as comorbidities are an emerging challenge with age and can endanger the life of patients.⁴⁸ High-grade Masaoka stage has been correlated with the aggressiveness and easy relapse of thymomatous MG.^49,50 Furthermore, thymoma patients with MGFA IV–V under the most severe condition have high risks of postoperative myasthenic crisis and long-term mortality, requiring enhanced management and follow-up.⁷

There are some limitations to this study. Firstly, our analysis was based on a retrospective observational study. Secondly, the sample size is relatively small, so we cannot perform internal and external validations of the models. More multicenter prospective studies based on large sample sizes are necessary to assess the clinical utility of the postoperative prognostic models. Thirdly, the treatment regimens partially changed because of the large time span of the study. Biologics like FcRn inhibitors and anti-complement drugs are changing the therapeutic algorithm in MG,^51,52 but their high cost and recent market entry limit patient access, making statistical analysis unfeasible. Evaluation of the potential of biologics in MG patients who underwent thymectomy requires extensive future investigations.

Conclusion

Thymectomy should be recommended for both non-thymomatous and thymomatous patients with AChR-Ab positivity. For non-thymoma patients, thymectomy should be carefully considered for refractory JMG patients due to the poor therapeutic effect. Thymoma patients with high-grade Masaoka stage and severe MGFA classification are more exposed to the risk of adverse outcomes. Pred + TAC remains a viable therapeutic option for patients with poor economic conditions after thymectomy.

Supplemental Material

sj-docx-1-tan-10.1177_17562864251343573 – Supplemental material for Comparison of outcomes and postoperative immunotherapy between patients with non-thymomatous and thymomatous myasthenia gravis following thymectomy

Supplemental material, sj-docx-1-tan-10.1177_17562864251343573 for Comparison of outcomes and postoperative immunotherapy between patients with non-thymomatous and thymomatous myasthenia gravis following thymectomy by Qing Zhang, XuanXuan Pan, Zhuajin Bi, Jiayang Zhan, Mengge Yang, Jing Lin, Mengcui Gui, Zhijun Li, Min Zhang, Xue Ma and Bitao Bu in Therapeutic Advances in Neurological Disorders

Supplemental Material

sj-docx-2-tan-10.1177_17562864251343573 – Supplemental material for Comparison of outcomes and postoperative immunotherapy between patients with non-thymomatous and thymomatous myasthenia gravis following thymectomy

Supplemental material, sj-docx-2-tan-10.1177_17562864251343573 for Comparison of outcomes and postoperative immunotherapy between patients with non-thymomatous and thymomatous myasthenia gravis following thymectomy by Qing Zhang, XuanXuan Pan, Zhuajin Bi, Jiayang Zhan, Mengge Yang, Jing Lin, Mengcui Gui, Zhijun Li, Min Zhang, Xue Ma and Bitao Bu in Therapeutic Advances in Neurological Disorders

Footnotes

Acknowledgements

The authors cordially thank the participants and their families, as well as the support from Hangzhou Zhongmei Huadong Pharmaceutical Co.. Ltd.

Declarations

ORCID iDs

Qing Zhang

Zhuajin Bi

Zhijun Li

Supplemental material

Supplemental material for this article is available online.

References

Conti-Fine

Milani

Kaminski

HJ.

Myasthenia gravis: past, present, and future. J Clin Invest 2006; 116(11): 2843–2854.

Van Enkhuizen

Binns

Betts

, et al. A retrospective observational study on characteristics, treatment patterns, and healthcare resource use of patients with myasthenia gravis in England. Ther Adv Neurol Disord 2024; 17: 17562864241237495.

Salahoru

Grigorescu

Hinganu

, et al. Thymus surgery prospectives and perspectives in myasthenia gravis. J Pers Med 2024; 14(3): 241.

Tuan

Vien

Dong

, et al. The value of CT and MRI for determining thymoma in patients with myasthenia gravis. Cancer Control 2019; 26(1): 1073274819865281.

Huang

Lee

, et al. Analysis of outcomes following surgical treatment of thymolipomatous myasthenia gravis: comparison with thymomatous and non-thymomatous myasthenia gravis. Interact Cardiovasc Thorac Surg 2014; 18(4): 475–481.

Chung

Kim

Lee

, et al. Thymectomy and disease duration in non-thymomatous acetylcholine receptor antibody-positive myasthenia gravis: a single-centre, cross-sectional study. J Neurol Neurosurg Psychiatry 2023; 94(4): 328–330.

Brascia

Lucchi

Aprile

, et al. Thymectomy in severe (Myasthenia Gravis Foundation of America classes IV–V) generalized myasthenia gravis: is the game really worth the candle? Eur J Cardiothorac Surg 2023; 63(5): ezad179.

Zheng

Cai

Shi

, et al. Different neurologic outcomes of myasthenia gravis with thymic hyperplasia and thymoma after extended thymectomy: a single center experience. J Neurol Sci 2017; 383: 93–98.

Marcuse

Hoeijmakers

JGJ

Hochstenbag

, et al. Outcomes after robotic thymectomy in nonthymomatous versus thymomatous patients with acetylcholine-receptor-antibody-associated myasthenia gravis. Neuromuscul Disord 2023; 33(5): 417–424.

10.

Lee

Kuo

Aban

, et al. Minimal manifestation status and prednisone withdrawal in the MGTX trial. Neurology 2020; 95(6): e755–e766.

11.

Dalakas

MC.

Progress in the therapy of myasthenia gravis: getting closer to effective targeted immunotherapies. Curr Opin Neurol 2020; 33(5): 545–552.

12.

Kim

Lim

Lee

, et al. Factors predicting remission in thymectomized patients with acetylcholine receptor antibody-positive myasthenia gravis. Muscle Nerve 2018; 58(6): 796–800.

13.

Wolfe

Kaminski

Aban

, et al. Long-term effect of thymectomy plus prednisone versus prednisone alone in patients with non-thymomatous myasthenia gravis: 2-year extension of the MGTX randomised trial. Lancet Neurol 2019; 18(3): 259–268.

14.

Von Elm

Altman

Egger

, et al. The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement: guidelines for reporting observational studies. Ann Intern Med 2007; 147(8): 573–577.

15.

Choi

Hong

Ahn

, et al. Repeated low-dose rituximab treatment based on the assessment of circulating B cells in patients with refractory myasthenia gravis. Ther Adv Neurol Disord 2019; 12: 1756286419871187.

16.

Chen

Gao

, et al. Clinical and pathological features of idiopathic membranous nephropathy with focal segmental sclerosis. BMC Nephrol 2019; 20(1): 467.

17.

Zhang

Yang

, et al. Immunotherapy choice and maintenance for generalized myasthenia gravis in China. CNS Neurosci Ther 2020; 26(12): 1241–1254.

18.

Cassinotti

Batticciotto

Parravicini

, et al. Evidence-based efficacy of methotrexate in adult Crohn’s disease in different intestinal and extraintestinal indications. Therap Adv Gastroenterol 2022; 15: 17562848221085889.

19.

Barohn

RJ.

Standards of measurements in myasthenia gravis. Ann N Y Acad Sci 2003; 998: 432–439.

20.

Cortes-Vicente

Alvarez-Velasco

Segovia

, et al. Clinical and therapeutic features of myasthenia gravis in adults based on age at onset. Neurology 2020; 94(11): e1171–e1180.

21.

Romano

Zirafa

Ceccarelli

, et al. Robotic thymectomy for thymoma in patients with myasthenia gravis: neurological and oncological outcomes. Eur J Cardiothorac Surg 2021; 60(4): 890–895.

22.

Cao

Liu

, et al. Remission and relapses of myasthenia gravis on long-term tacrolimus: a retrospective cross-sectional study of a Chinese cohort. Ther Adv Chronic Dis 2022; 13: 20406223221122538.

23.

Ruan

Tang

, et al. Nomogram for predicting the risk of postoperative myasthenic crisis in patients with thymectomy. Ann Clin Transl Neurol 2023; 10(4): 644–655.

24.

Duran-Frigola

Rossell

Aloy

A chemo-centric view of human health and disease. Nat Commun 2014; 5: 5676.

25.

Habib

Benatar

, et al. Time to response with ravulizumab, a long-acting terminal complement inhibitor, in adults with anti-acetylcholine receptor antibody-positive generalized myasthenia gravis. Eur J Neurol 2024; 31(12): e16490.

26.

Wang

Shi

Wang

, et al. The impact of immune markers on thymectomy prognosis in thymoma-myasthenia gravis. J Thorac Dis 2024; 16(10): 6634–6643.

27.

Peeler

De Lott

Nagia

, et al. Clinical utility of acetylcholine receptor antibody testing in ocular myasthenia gravis. JAMA Neurol 2015; 72(10): 1170–1174.

28.

Chen

, et al. Eight-year follow-up of patients with myasthenia gravis after thymectomy. Acta Neurol Scand 2015; 131(2): 94–101.

29.

Fujii

Thymus, thymoma and myasthenia gravis. Surg Today 2013; 43(5): 461–466.

30.

Rath

Taborsky

Moser

, et al. Short-term and sustained clinical response following thymectomy in patients with myasthenia gravis. Eur J Neurol 2022; 29(8): 2453–2462.

31.

Clifford

Hobson-Webb

Benatar

, et al. Thymectomy may not be associated with clinical improvement in MuSK myasthenia gravis. Muscle Nerve 2019; 59(4): 404–410.

32.

Chigurupati

Gadhinglajkar

Sreedhar

, et al. Criteria for postoperative mechanical ventilation after thymectomy in patients with myasthenia gravis: a retrospective analysis. J Cardiothorac Vasc Anesth 2018; 32(1): 325–330.

33.

Narayanaswami

Sanders

Wolfe

, et al. International consensus guidance for management of myasthenia gravis: 2020 update. Neurology 2021; 96(3): 114–122.

34.

Derderian

Potter

Bansal

, et al. Open versus thoracoscopic thymectomy for juvenile myasthenia gravis. J Pediatr Surg 2020; 55(9): 1850–1853.

35.

Marx

Pfister

Schalke

, et al. The different roles of the thymus in the pathogenesis of the various myasthenia gravis subtypes. Autoimmun Rev 2013; 12(9): 875–884.

36.

Pasqualin

Guidoni

Ermani

, et al. Outcome measures and treatment effectiveness in late onset myasthenia gravis. Neurol Res Pract 2020; 2: 45.

37.

Yan

Zhao

Song

, et al. Comparison of anti-acetylcholine receptor profiles between Chinese cases of adult- and juvenile-onset myasthenia gravis using cell-based assays. J Neuroimmunol 2020; 349: 577403.

38.

Zhang

Cao

, et al. Childhood-onset myasthenia gravis patients benefited from thymectomy in a long-term follow-up observation. Eur J Pediatr Surg 2022; 32(6): 543–549.

39.

Carter

Ungerleider

Goldstein

SD.

Thymectomy for juvenile myasthenia gravis: a narrative review. Mediastinum 2024; 8: 35.

40.

Heckmann

Europa

Soni

, et al. The epidemiology and phenotypes of ocular manifestations in childhood and juvenile myasthenia gravis: a review. Front Neurol 2022; 13: 834212.

41.

Nel

Buys

Rautenbach

, et al. The African-387 C > T TGFB1 variant is functional and associates with the ophthalmoplegic complication in juvenile myasthenia gravis. J Hum Genet 2016; 61(4): 307–316.

42.

Gui

Luo

Lin

, et al. Long-term outcome of 424 childhood-onset myasthenia gravis patients. J Neurol 2015; 262(4): 823–830.

43.

Krisl

Doan

VP.

Chemotherapy and transplantation: the role of immunosuppression in malignancy and a review of antineoplastic agents in solid organ transplant recipients. Am J Transplant 2017; 17(8): 1974–1991.

44.

Miura

Azuma

Yamada

, et al. Combined treatment with prednisolone and tacrolimus for myasthenia gravis with invasive thymoma. Acta Neurol Belg 2010; 110(1): 107–109.

45.

Vasavada

Jazrawi

Samuels

Perioperative management of immunosuppressive medications in rheumatic disease patients undergoing arthroscopy. Curr Rev Musculoskelet Med 2021; 14(6): 421–428.

46.

Wang

, et al. Video-assisted thoracoscopic surgery thymectomy versus open thymectomy in patients with myasthenia gravis: a meta-analysis. Acta Chir Belg 2016; 116(5): 282–288.

47.

Tantai

J-C

X-X

, et al. Surgical techniques for early-stage thymoma: video-assisted thoracoscopic thymectomy versus transsternal thymectomy. J Thorac Cardiovasc Surg 2014; 147(5): 1599–1603.

48.

Alkhawajah

Oger

Late-onset myasthenia gravis: a review when incidence in older adults keeps increasing. Muscle Nerve 2013; 48(5): 705–710.

49.

Weis

Yao

Deng

, et al. The impact of thymoma histotype on prognosis in a worldwide database. J Thorac Oncol 2015; 10(2): 367–372.

50.

Zhang

Liu

Qiu

Development of a competing risk nomogram for the prediction of cause-specific mortality in patients with thymoma: a population-based analysis. J Thorac Dis 2021; 13(12): 6838–6847.

51.

Dalakas

MC.

Advances in the therapeutic algorithm for myasthenia gravis. Nat Rev Neurol 2023; 19(7): 393–394.

52.

Dalakas

MC.

Immunotherapy in myasthenia gravis in the era of biologics. Nat Rev Neurol 2019; 15(2): 113–124.

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