Anti-acetylcholine receptor antibodies in myasthenia gravis

Journal of the Neurological Sciences, 1985, 69:335-343

335

Elsevier

Anti-acetylcholine Receptor Antibodies in Myasthenia Gravis Part 3. The Effect of Thymectomy H . J . G . H . Oosterhuis 1, P.C. Limburg 2, E. Hummel-Tappel 2, W. Van den Burg 3 and T.H. The 2 Departments of INeurology, 2ClinicalImmunology and 3Neuropsychology, UniversityHospital, Groningen (The Netherlands) (Received 16 January, 1985) (Revised, received 25 March, 1985) (Accepted 25 March, 1985)

SUMMARY

The clinical condition and the serum levels of antibodies to acetylcholine receptor protein were followed in 30 patients with myasthenia gravis before and in a period varying from 2 to 4 (mean 3) yr after thymectomy. Twenty-five patients improved in the 2 yr following thymectomy. A highly significant correlation was found between the change in clinical condition and the change in antibody.levels. Only 3 patients improved without a fall of antibody level. Prethymectomy antibody levels were positively correlated with the severity of the clinical condition and with the degree of thymus hypertrophy.

Key words: Antibodies to A C h R receptors - Myasthenia gravis - T h y m e c t o m y

INTRODUCTION

In patients suffering from generalized myasthenia gravis (MG) removal of a non-tumoural thymus is generally considered to be potentially effective, especially for patients younger than 45 years old and in the 3-5 years after onset. The reason for the Correspondence to: Prof. Dr. H. J. G. H. Oosterhuis, Department of Neurology, University Hospital, P.O. Box 30.001, 9700 RB Groningen, The Netherlands.

0022-510X/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

336 success in (young) patients with a normal or hyperplastic thymus and the failure in (older) patients with a thymoma is not well known. The two main hypotheses offering an explanation are, first, the presence of abnormal antigens, e.g. myoid cells bearing acetylcholine receptors (Kao and Drachman 1977; Wekerle and Ketelsen 1977) inside the normal thymus, which trigger an immune response, and, second, a primary fault in immunoregulation mediated by the thymus, at least in young individuals. Because antibodies to the acetylcholine receptor protein (anti-AChR) can be detected in the serum of 80-90 ~o of the patients with a generalized MG (Lindstrom et al. 1976; Lefvert et al. 1978; Limburg et al. 1983), attempts have been made to correlate the concentrations of anti-AChR before and after thymectomy with changes in the clinical state (Bartoccini et al. 1980; Tindall 1981a; Olanow et al. 1982; Vincent et al. 1983). We have extended our clinical and serological follow-up of individual patients (Oosterhuis et al. 1983) and report the results in 30 patients followed up in a 2-4 years period (mean 3.0 years) after thymectomy. METHODS

Patients. We selected 30 patients (24 women, 6 men) with a generalized MG who underwent thymectomy between 1978 and 1982 and who fulfilled the following criteria: serum samples with detectable anti-AChR were available at least once before thymectomy, the patients were examined at least once by the first author, further details of their clinical state were available at the moments of serum sampling, and the histological picture of their thymus could be studied in the original preparates. Details of the data are presented in Table 1. The assessment of the clinical state, without knowledge of the serological data, was based on a 6-point scale of disability (class 0: complete clinical remission without therapy; class 1: minor symptoms; class 2: mild disability; class 3: moderate disability; class 4: severe disability; class 5: respiratory support necessary) described in detail in our previous paper (Oosterhuis et al. 1983). Quantified tests of muscle strength performed with a dynamometer, vital capacity and tests of muscle fatigue (e.g. arm stretched-out time) were used when possible. About half of the patients were treated by other neurologists and their data were used in combination with the patients' own reports to classify the clinical state. The prethymectomy classification was: class 1: 3 patients; class 2:12 patients; class 3: 9 patients; class 4: 2 patients; class 5:4 patients. Graphs were drawn of the changes in clinical state and of the anti-AChR titers at the moments of clinical evaluation (See Figs. 1 and 2) in each individual patient. Thymectomy was performed in the 1st year of the disease in 11 patients, in the second year in 9 patients, in the third year in 3 patients, in the 4th year in 6 patients and later in 2 patients. Ages at the time of thymectomy varied from 8.5 to 44 years (mean 26 years). The histological picture of the thymus was classified as normal (cortex area >i medulla, no germinal centres), mild hypertrophic (medulla > cortex, germinal centres 1 per field of vision, magnification 25 x ), severe hypertrophic (medulla > cortex, germinal centres 2-5 per field of vision), atrophic (remnants of thymus in fat tissue). Antibodies to acetylcholine receptor protein (anti-AChR) were measured by the

337 radioimmunoprecipitation test with antigen from muscles of amputated human legs (for details see Limburg et al. 1983). The values used per individual patients were obtained by serial measurements with the same sample of antigen and expressed as a percentage of the last prethymectomy value. The absolute values ranged from 3 to 975 nmol/l (positive > 2 nmol/1) and the mean number of determinations was 8.8 (5-20) per patient. Total serum immunoglobulin (IgG) was measured in each serum sample by a nephelometer using a Beckman ICS analyser II and the commercial reference sample. Other therapies. Five patients with a hyperplastic thymus received prednisone and/or azathioprine during some period postoperatively; data of anti-AChR were not included in the calculations unless prednisone was omitted completely for at least 0.5 year and azathioprine for 1 year. Statistics. Wilcoxon's signed-ranks test was used for measuring differences between means of paired observations. Kendall's rank correlation coefficient was used for assessing relationships. Both tests were one-sided. RESULTS

The histological features of the thymus were as follows: atrophy, 1: normal, 9: mild hypertrophy, 9: severe hypertrophy, 11. There was no significant relation between the degree of thymic abnormalities and age, or the duration of the disease. The titers of anti-AChR before thymectomy were related to the degree of hypertrophy (z = 0.29, P < 0.05) and to clinical severity (z = 0.33, P < 0.01). In the period ranging from 3 to 17 months prior to thymectomy 2 or 3 anti-AChR measurements were done in 16patients. Antibodies rose (>25% of the initial value) in 7patients; remained constant in 8 and fell in one (No. 11). Degrees of clinical improvement in patients without prednisone are given in Table 2. In general both the mean disability score of the patients and the mean antibody levels, as a percentage of prethymectomy values, fell significantly (Table 3). The degree of improvement and the change of anti-AChR were correlated after 1, 2 and 3 years, and the correlation became more apparent as the time following thymectomy increased. The greatest proportional fall of antibody titers occurred after a half year in 14 patients, after 1 year in 2 patients, after 2 years in 4 patients. In 2 patients a further fall in antibody levels was observed in the years after a complete clinical remission was reached (Figs. 1 and 2). In only 1 patient who went into a complete remission could no anti-AChR be detected at the end of the observation. Four patients (Table 1, Nos. 27-30) treated with prednisone during part of the postthymectomyperiod improved while their antibody levels fell steeply. After cessation of prednisone 2 patients remained in remission (Nos. 27 and 28), 1 patient continued to improve with a rise in antibody level (No. 29) and 1 patient who was in a clinical remission deteriorated with a slight rise in antibody levels but improved when prednisone was started again (No. 30). The data of these patients during prednisone treatment are not given in Table 2. In all patients the concentration of total IgG in the serum was measured and the quotient of anti-AChR/IgG was calculated to determine if the change in anti-AChR was

338 TABLE 1 PATIENTS' DATA

Patient No.

Sex

Age at onset of M G (yr)

Duration of MG to thymectomy (mo)

Thymus histology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

F M F F F F F M M M F M F F F F F F F F F F F F F F F M F F

22 19 22 21 11 28 20 22 30 43 36 38 33 26 16 26 26 11 14 31 33 16 19 8 28 19 26 15 27 16

168 10 15 27 44 40 23 33 12 9 13 38 10 5 8 19 48 9 15 12 48 13 8 14 80 5 13 3 7 5

mH sH mH sH sH mH mH mH sH A mH sH sH N sH N mH mH mH N N sH N sH N sH sH N N N

Thymus histology. N = normal; mH = mild hypertrophy; sH = severe hypertrophy; A = atrophy; antiAChR = antibodies to acetylcholine receptor protein. Prethymectomy in nmol/l; postthymectomy in % of prethymectomy values.

the effect of a concomitant change in total IgG. Although spontaneous fluctuations in IgG level occurred, the general trend in the change of anti-AChR was not influenced. In only 1 patient who improved in the first week after thymectomy did total IgG and anti-AChR show the same proportional fall but this phenomenon was also seen in other patients who did not improve. DISCUSSION

The relationship between the degree of myasthenic weakness and anti-AChR titers is still a matter of debate. The great individual variability in antibody titers is probably the main reason why these do not correlate well with the clinical condition

339

Clinical condition (yr after thymectomy)

Anti-AChR (yr after thymectomy)

0

0.5

1

2

3

3 2.5 2.5 5 3 3 2

2 1.5 2.5 4 2 3 0.5

1.5 1.5 2.5 3.5 1.5 2 0.5

1 1

1.5

1.5

1

2.5 3

1.5 2.5

1 2

-1.5 2.5 3.5 1 2 0 0.5 1 1.5

1.5

1.5

1

1

2 2 3 4 2 2.5 4.5 3 2.5 2 3 1.5 5 3.5 2.5 5 3 2.5 b'¢ 5

2 -2.5 2.5 1.5 2 1.5 2.5 2 1.5 2 0 1.5 3 -2b 0b 2c 1b

2 0 2 2.5 1.5 2.5 1 2 1.5 1.5 1 0 0 3 2 1b 0b 1c 0.5 b

2 0 1 2.5 1 1.5 1 1.5 1 0 0 0 0 3.5 2 1b 0 1.5 0b

3.5 1 2 0 0.5 1 1.5

4

3 2

2 0, 2 1 1 1 0.5 0

0.5 0

0 0 a 2.5 Ib 0 1 2

0

1.5 0

0

0.5

1

2

3

72 23 160 540 142 57 20 12 18 8 4 59 14 14 478 41 20 97 30 17 20 28 26 64 975 61 58

-126 112 60 66 120 80 100 83 62 60 116 -91 99 78 70 50 41 35 55 89 -40 121 -16 b 0b 60 ~ 7b

70 130 91 40 62 120 65 92 88 62 58 117 85 64 64 51 91 30 37 20 45 82 54 22 124 79 5b 0b 60 ~ 7b

-130 118 32 66 100 25 66 72 30 55 100 61 8 57 54 100 17 18 8 30 79 23 12 115 115 4b

14 125

8 0.5 b

37 14

4

50 71 100 22 66 66 36

54 119

102 61 40 38 75 15 0 25

11 7

19 9

15

106 4b

98 5

0 98 7b

106 13

14 b

Clinical condition: class 0 - 5 (see text). a Died in myasthenic crisis. b Treated with prednisone. c Treated with azathioprine.

when groups of patients in various clinical conditions are compared. In the present study we found that before thymectomy the most severely affected patients had higher antibody titers than the mildly affected ones. Our follow-up of individual patients in the period of 2-4 years after thymectomy showed that 11 of the 14 patients who finally improved 2 degrees or more had a fall in antibody titers of > 50 ~o of their preoperative values, while none of the 5 patients who did not improve and only 5 of the 11 patients who improved 1 degree showed a > 50% fall. The same tendency was seen 1 and 2 years after thymectomy. The correlation between the clinical improvement and the proportional decrease in anti-AChR became more apparent with the lapse of time. In some patients antibody titers continued to fall several years after a complete clinical remission

340 TABLE 2 DEGREES OF CLINICAL IMPROVEMENT IN PATIENTS WITHOUT PREDNISONE Improvement expressed as a change in clinical classes (see Methods and Table 1); the number of patients in complete clinical remission in parentheses.

Improvement

After 1 yr

After 2 yr

After 3-4 yr

0-0.5 1-1.5 >~2

9 13 (1) 4 (2)

5 13 (1) 9 (6)

5 10 (1) 11 (7)

26 (3)

27 (7)

26 (8)

MG

W2e

W 14

MG

' ii i~ :~iiiiiiiiiiiiiiiiiiiiiiii:: ! : :iiiiiiiiiiiiiii~::ii: :!iiiil • an,,iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiH!i A Ch R iiiiiiiiiiiiiiiiiiiii~;;:i~:.~:

eanti

AChR 25 ~

301

-\

"[01 thymec~t

;'gG25 ~'e

lO~

YEARS

...........

iiiii~i~:°

\

1 "~.

I

YEARS i

Figs. 1 and 2. q~-----------o:Anti-AChR in nmol/l; • •: IgG in g/l; shaded area: severity of MG in clinical degrees (see text). Therapy with anticholinesterases not indicated. Fig. 1. Patient 23, woman aged 20 years. Mild generalized MG in complete clinical remission 1.5 years after thymectomy. Anti-AChR decreased gradually until 5 years after thymectomy. IgG levels did not change. Fig. 2. Patient 19, woman aged 14 years. Moderate generalized MG, slightly improved by anticholinesterases before thymectomy and improved gradually in the 5 years after thymectomy. Anti-AChR fell steeply in the first months after thymectomy and continued to fall more gradually in the next years. IgG levels did not change.

was achieved. W i t h o n l y 2 exceptions, p a t i e n t s w h o d i d n o t exhibit a fall in a n t i b o d y titers i m p r o v e d o n l y mildly or n o t at all. Clinical i m p r o v e m e n t w a s n o t correlated with the histological picture o f the t h y m u s , with the d u r a t i o n o f the disease, n o r with the p r e o p e r a t i v e level o f antibodies. H o w e v e r , the m o s t impressive i m p r o v e m e n t was seen in j u v e n i l e p a t i e n t s with serious signs. T h i s lack o f correlation b e t w e e n t h y m i c p a t h o l o g y a n d p r e o p e r a t i v e severity was also f o u n d in a previous s t u d y ( O o s t e r h u i s 1977).

341 TABLE 3 CLINICAL IMPROVEMENT EXPRESSED AS THE DIFFERENCE WITH PRETHYMECTOMY RATINGS (MEANS) Anti-AChR levels in ~ ofprethymectomy values. ~-correlations are used to compare the changes in clinical and serological parameters. Time after thymectomy (years)

Clinical improvement M -

0

-

Anti-AChR levels z-correlations - -

F0

M 100

P < 0.0005 /

7 / P < 0.005

L 0.83

0.22

1

1.23

0.25

70.0

2

1.53

0.51 (P < 0.001)

59.6 n / P < 0.05

1.71

0.56 (P < 0.001)

52.0

0.5

<000 I P < 0.08

3

,<00

97.7

A

1<000, 1

J

Previous studies concerning anti-AChR after thymectomy either report a fall in antibody titers with concomitant clinical improvement (Bartoccini et al. 1980; Tindall 1981a; Vincent et al. 1983), clinical improvement without change in antibody titers (Roses et al. 1981; Olanow et al. 1982) or no change in antibody titers without details about the clinical state (Komfeld et al. 1981). Most of these studies however either had a short follow-up period or included few patients. The study of Olanow et al. (1982) which concluded that no correlation existed between anti-AChR levels and the clinical state after thymectomy is not comparable to ours in several aspects: 22 of their 47 patients used steroids before thymectomy and 15 thereafter; 10 patients had antiAChR < 1 nmol/l and 18 patients were thymectomized at high age (>55 years); 8 patients with a thymoma were included in their series. Of their 11 patients with anti-AChR levels > 1 nmol/l and not treated with steroids, 10 showed a considerable fall (mean 4 6 ~ ) and 1 an increase (to 174~o) concomitant with clinical improvement. Thus reduced are their data in agreement with ours. A similar correlation between the decrease in antibodies and clinical improvement has been reported by Vincent et al. (1983); their study is quite comparable to ours in terms of patient characteristics, thymus pathology and the scoring of the clinical condition. In addition they determined antibodies against the g-bungarotoxin binding

342 site (i.e. the acetylcholine receptorsite itself) in 15 patients, but these antibodies only represented 3.1~o of the total anti-AChR and showed no particular trend after thymectomy. The same lack of correlation between these "blocking" antibodies and the clinical state was demonstrated by Tindall (1981b) and by Besinger et al. (1983). This probably indicates that in most patients disease severity is more indicated by the total amount of anti-AChR than by the more specific blocking subclass. It is obvious that the residual neuromuscular function is primarily related to the fraction of the still functioning acetylcholine receptors, but by which factors the actual number of functioning ACh receptors is determined (amount of ACh, anti-AChR?) is unknown. The improvement after thymectomy must be due to an increase in functioning receptors and, in most patients, this improvement is accompanied by a considerable fall in their antibody levels. However, a few patients form an exception to this rule and thymectomy may even improve MG in the absence of detectable anti-AChR in the serum (Olanow et al. 1982). The data from this study confn'm our previous findings (Oosterhuis et al. 1983) and those of others (Besinger et al. 1983; Vincent et al. 1983) that in most myasthenic patients a clear change in clinical condition is associated with a definite change in anti-AChR titers. ACKNOWLEDGEMENTS

We wish to thank the neurologists H. Busch, H. Dammers, P. Fleury, H. Van Luyk, P. Van der Lugt, J. Van Manen, D. De Most van Spijk, E. Ossentjuk, H.J. Troelstra, J. Troost, J. Vos and L.L. Van Woerden for their cooperation in the follow-up of their patients. J. Marrink supervised the IgG measurements. P. Limburg received a grant from the Prinses Beatrix Fonds. REFERENCES Bartoccini, E., F. Scuderi, C. Scopetta, A. Evoli, P. Tonali, L. Guidi, C. Bartoloni and T. Terranova (1980) Myasthenia gravis, thymectomy and antiaeetyleholine receptor antibody, J. Neurol., 224: 9-15. Besinger, U.A., K.V. Toyka, M. H6mberg, K. Heininger, R. Hohlfeld and A. Fateh-Moghadam (1983) Myasthenia gravis - - Long term correlation of binding and bungarotoxin blocking antibodies against acetylcholine receptors with changes in disease severity, Neurology (Cleveland), 33:1316-1321. Kao, I. and D.P. Drachman (1977)Thymic muscle cells bear acetylcholine receptors - - Possible relation to myasthenia gravis, Science, 195: 74-75. Kornfeld, P., J. NaU, H. Smith, Th.W. Mittag, A.N. Bender, E.P. Ambinder, S.H. Horowitz, A.E. Papatestas, H. Gross and G. Genkins (1981) Acetylcholine receptor antibodies in myasthenia gravis, Muscle & Nerve, 4: 513-419. Lefvert, A. K., K. BergstrOm, G. Matell, P.O. Osterman and R. Pirskanen (1978) Determination of acetylcholine receptor antibody in myasthenia gravis - - Clinical usefulness and pathogenetic implications, J. Neurol. Neurosurg. Psychiat., 41: 394-403. Limburg, P., E. Hummel-Tappel, H. The and H.J.G.H. Oosterhuis (1983) Antiacetylcholine receptor antibodies in myasthenia gravis, Part 1 (Their relation to the clinical parameters in 250 patients), J. Neurol. Sci., 58: 357-370. Lindstrom, J.M., M.E. Seybold, V.A. Lennon, S. Whittingham and D.D. Duane (1976) Antibody to acetylcholine receptor in myasthenia gravis, Neurology, 26: 1054-1059. Olanow, C.W., A.S. Wechsler and A.D. Roses (1982) A prospective study of thymectomy and serum acetylcholine receptor antibodies in myasthenia gravis, Ann. Surg., 196:113-121.

343 Oosterhuis, H.J.G.H. (1977) Ergebnisse der Thymektomie in Beziehung zur Thymushistologie. In: G. Hertel et al. (Eds.), Myasthenia Gravis, G. Thieme Verlag, Stuttgart, pp. 237-241. Oosterhuis, H.J.G.H., P.C. Limburg, E. Hummel-Tappel and T. H. The (1983) Antiacetyicholine receptor antibodies in myasthenia gravis, Part 2 (Clinical and serological follow-up of individual patients), J. Neurol. Sci., 58: 371-385. Roses, A. D., C. W. Oianow, M. W. McAdams and R. J. M. Lane (1981) No direct correlation between serum antiacetylcholine receptor antibody levels and clinical state of individual patients with myasthenia gravis, Neurology, 31: 220-224. Tindall, R.S.A. (1981a) Humoral immunity in myasthenia gravis - - Effects of steroids and thymectomy, Neurology, 30: 554-557. Tindall, R.S.A. (1981b) Humoral immunity in myasthenia gravis - - Biochemical characterization of acquired anti-receptor antibodies and clinical correlations, Ann. Neurol., 10: 437-447. Vincent, A., J. Newsom Davis, P. Newton and N. Beck (1983) Acetylcholine receptor antibody and clinical response to thymectomy in myasthenia gravis, Neurology (Cleveland), 33: 1276-1282. Wekerle, H. and U.P. Ketelsen (1977) Intrathymic pathogenesis and dual genetic control of myasthenia gravis, Lancet, i: 678-680.