identification of subsets of patients with graves’disease by cluster analysis

Clinical Endocrinology (1983) 18, 335-345

IDENTIFICATION OF SUBSETS OF PATIENTS WITH GRAVES’ DISEASE BY CLUSTER ANALYSIS

VALERIA STENSZKY, CS. BALAZS, L. KOZMA, SZ. ROCHLITZ, J. C. BEAR AND NADIR R . FARID

Blood Transfusion Centre, 111 Department of Medicine, University Medicaf School, Computer Centre of Kossuth University, Debrecen, Hungary and Faculty of Medicine,

Memorial University of Newfoundland, St John’s, Neufoundland, Canada AIB 3V6

(Received 16 February 1982; revised 28 September 1982: accepted 28 September 1982)

SUMMARY

We have applied cluster analysis methods to forty-nine laboratory and clinical characteristics (including 26 HLA-A, B antigens) observed in 196 Graves’ disease patients. Three subgroups could be identified: group I (seventy-nine patients) had small goitres, low indices of autoimmunity and a tendency to remission with medical treatment; group IIa (twenty-nine patients) had clinical and laboratory features of ‘Hashitoxicosis’; Group IIb (eighty-four patients) had a high incidence of ophthalmopathy, familial aggregation, marked evidence of autoaggression and a tendency to relapsing hyperthyroidism. The prevalence of HLA-B8 was 8.9% in group 1, 20.7% in group IIa and 86.9% in group IIb. This study demonstrates that Graves’ disease can be subdivided using cluster analysis into clinically relevant subgroups which are further distinguished by their correlation with HLA-B8. Possible immunological bases for these observed patterns are discussed.

Graves’ disease is generally accepted to be an autoimmune disorder characterized by hyperthyroidism, diffuse goitre and, more variably, ophthalmopathy and localized myxoedema. In the individual patient, the clinical picture depends upon the variable appearance of these symptoms and signs, which may precede, accompany or follow each other. Attempts have been made to distinguish different forms of Graves’ disease using clinical and laboratory features (Liddle et al., 1965;Bowden & Rose, 1969; Solomon et al., 1977; Volpi, 1978). In recent years, subdivision has also been attempted based on the association of various features of the disease with HLA antigens, for instance the tendency of Graves’ disease to recur in patients with HLA-B8 and particularly those with HLA-DR3, and the association of ophthalmopathy with this antigen (reviewed in Farid & Bear, 1981). The present study investigates:

Correspondence: Dr Valeria Stenszky, Blood Transfusion Centre, 111 Department of Medicine, Debrecen, Hungary.

0300-0664/83/0400-0335f02.00 0 1983 Blackwell Scientific Publications

335

336 V. Stenszkqs et al.

1 whether different subgroups of Graves' disease can be distinguished using the mathematical method of cluster analysis, applied to clinical, laboratory and genetic features; 2 whether the subgroups distinguished by cluster analysis have diagnostic or prognostic significance; 3 whether the subgroups distinguished differ in HLA associations.

SUBJECTS A N D METHODS

The study comprises 196 consecutive patients with Graves' disease who were referred to the Female Section of the First Department of Medicine, Debrecen between 1971 and 1981. Patients included 170 females and 26 males; age at onset ranged between 13 and 62 years, averaging 37.9 years. Diagnosis was based on clinical criteria and confirmed by elevated serum T3 levels, suppressed serum TSH, radioiodine uptakes and rectilinear scanning of the thyroid gland. In the 179 patients in which it was examined [I3'I] thyroid uptake did not suppress after 7 days of treatment with T3. Over the period of study, serum T4 assays were not available. The size of the goitre was estimated by palpation as described by Perez et a/. (1960). All patients were scored upon presentation as described by Crooks er a/. (1959).

Ophthalmopathy was classified by the criteria of the American Thyroid Association (Werner, 1969, 1977) as follows:

0 = no physical signs or symptoms; + = only signs, no symptoms (signs limited to upper lid retraction, stare and lid

lag); + + = soft tissue involvement, proptosis;

+ + + = soft tissue involvement and extraocular muscle lesion.

All patients were treated with a combination of lithium and methimazole for at least 3 months after they had been rendered clinically and biochemically euthyroid. Drug treatment was then discontinued and patients seen on a weekly basis. Prognosis was judged to be poor when relapse occured in less than 1 year or when two or more relapses followed medical therapy which had been effective in rendering the patient euthyroid.

Laboratory tests All patients underwent needle biopsy of the thyroid. The aspirates were spread and

stained with Maya-Griinwald-Giemsa (Einhorn & Franzer, 1962; Folendorf et a/., 1975). Lymphocytic infiltration was considered positive if the criterion:

I 2 was met. Number of acinar cells

Number of lymphoid cells All patients were studied for at least 26 HLA-A and -B antigens, using NIH lymphocytotoxicity tests involving I 12 well-characterized antisera (Amos, 1974).

The level of circulating immune complexes (IC) was determined by thrombocyte aggregation techniques (Penttinen et a/ . , 1973; Palosou & Leikola, 1975). The number of active T lymphocytes was tested by the method of Wybran et al. (1975), Wybran & Fudenberg (1973) and Yu (1975), modified as follows: cells were incubated for 5 min at 37 C and subsequently centrifuged at 40 C.

Subsets of Graves' Diseuse 337

Short lived suppressor T cell function and Concanavalin A (Con A)-generated suppressor T cell function were measured by methods modified (Balazs et al., 1979a) from those of Bresnihan & Jasin (1977). Briefly, mononuclear cells were separated from 20 ml of heparinized peripheral blood by hypaque-ficoll gradient centrifugation; washed twice in tissue culture medium (TCM) 199; resuspended in TCM 199 supplemented with L-glutamine lo%, fetal calf serum (FCS) previously heat inactivated, and penicillin 100 pg/ml; and adjusted to 2 x lo6 cells/ml. Cells were cultured in triplicate at 37°C in an atmosphere containing 9S% air and 5% COZ. Control tubes were cultured in the absence of mitogen. Test tubes were stimulated at 0 h or after 24 h of pre-incubation with Con A (Pharmacia) at concentrations of 0, 5 and 10 pg/ml. Cell viability was determined with trypan blue at 24 h and at a termination of the cultures. The average viability of unstimulated cells was 78% up to 24 h incubation and 65% at culture termination. Culturing was concluded after 72 h. Two hours prior to harvesting, 2 pCi of [3H]-thymidine were added to each tube; radioactivity was subsequently measured by liquid scintillation counting (Nuclear Chicago Isocap 300). The activity of the short-lived suppressor cells was calculated as:

stimulation after 24 h pre-incubation, in ct/min stimulation at 0 h, in ct/min

Suppressor index =

Con A activated suppressor cell activity was studied by the modified method of Hubert et al. (1975). 5 x lo6 mononuclear cells were incubated as described above, with or without 5.0 pg/ml of Con A at 37°C for 24 h. Cells were then washed three times in tissue culture medium (TCM) 199 and 1 x lo6 cell amounts resuspended in 1 ml of fresh culture medium (TCM 199, FCS and antibiotics). To measure suppressor activity, autologous cells were obtained from the same donor 24 h later and 2 x lo6 freshly prepared and 1 x loh previously activated cells were cultured in the presence of Con A at a suboptimal concentration (2 pg/ml) at 37°C for 72 h. DNA synthesis in the second culture was measured as above. Percentage suppression was calculated as:

100 x ct/min in activated cells ct/min in non-activated cells

Antinuclear antibodies, rheumatoid factor, and antithyroglobulin antibodies were determined by passive haemaglutination techniques. Serum T3 and TSH were measured by radioimmunoassays (Abbott Laboratories Kit). For the cluster analysis, quantitative as well as qualitative measures were reduced to integer scores. Table 1 shows the patients' attributes used in the cluster analysis, the number of patients evaluated, and the distribution of scores.

The cluster analysis used the unweighted variable group method of Sokal & Sneath (1963). In principle, this involves the construction of a mathematical distance model. The distance between two patients identical for all characteristics scored is 0 and this distance increases with the divergence between the two patients in the characteristics. Distances are then transformed into values resembling matrix correlation coefficients such that identity for all values results in d*i, = 1.0 and maximum difference d*i, = - 1 . Distance matrix values are calculated as:

d.. - Z'(Ki-Kji) " - Z'maxl

338 V . Stenszkj, et nl.

where:

d,, K, , K,, maxi = maximum possible attribute score for attribute 1

and summation is over all attributes.

= distance between patients i and j; = Ith attribute score of patient i ; = lth attribute score of patient j ;

Then d*,, = 1 - I d , and ranges from - I to + 1.

Using these quantities patients can be grouped together using pre-set criteria of similarity. With the method used, patients must fall into discrete categories; that is, the clusters formed are not allowed to overlap. On the other hand, clusters will not be found ifpatients show little or no similarity to one another for the characteristics evaluated. The large number of patients and characteristics evaluated precludes the possibility of a few cases unduly influencing the overall result. This is a reasonable safeguard since on clinical grounds it is only probable, not certain, that a patient in a particular category will exhibit a particular characteristic. The method makes no prior assumptions as to the classificatory importance of any characteristic; all are weighted equally. These properties of the mathematical method make it appropriate for present purposes of exploratory analysis. The data of each patient results in one point in a space of thirty-one dimensions, assuming all characteristics are scored. That is, each characteristic contributes one

Table 1 . The scoring of patients for each attribute evaluated In the cluster analysis

Scores Distribution of scoring

Attribute 0 I 2 0 1 2 Number tested

I 2 i 4 5 6

7 8 9

10 1 1 12 13 14 15 I6 17 18 19 20 21

23 7 3 --

Sex Female Age at time of prcsentation Goitre size 1-2 Crooks test 1 &20 Family accumulation N o Association with other autoimmune N O

Infiltrative ophthalmopathy 0. +

- < 29

disease

Tendency to recurrence Good I 3 ' I uptake < 40",,

Serum T3 (nmolll) 5 3.5 Serum TSH (pu:ml) - < 6-0 Lymphocytic infiltration of thyroid gland No Htg antibody titre 2-16 Anti-nuclcar antibody titrc 2-16 Rheumatoid Factor 2-16 Circulating 1C 2-8 Active E-rosettes 0-19

T3 non-suppressibility No

Short lived suppressor T cell function 2 3.0 Con A activated suppressor T cell function 2 75",, Lymphocyte transformation index 1-39 Maximal acid output of stomach 0-4.9

0-1.0 Basal acid output of stomach

Male 30 5 3 4 1 21- 30 Yes Yes

++.+++ Bad r4lQ, Yes 3 . 6 5 6.1-10 Yes 31-64 32-64 32-64 I 6 3 2 20-30 2.9-1.0 40-74"" 4 1 5-16 1.1-3.9

170 26 ~ 196 66 130 - 196

13 120 53 186 163 33 - 196 150 46 - 196

99 97 - 196 100 96 - 196 31 161 - 192 0 179 - 179

27 69 86 182 175 3 I 179 151 35 - 186 105 76 15 I96 189 6 1 196 190 5 1 196 71 64 48 183 3 189 4 196

27 24 16 67 31 33 3 67

142 54 ~ 196 60 24 12 96 62 I X 16 . 96

175 21 - 196

24-49 26 HLA antigens (see text) 196

Subsets oj‘ Graves’ Disease 339

dimension, except for HLA antigen resemblances, which were evaluated taking into consideration the fact that dissimilarity of two patients could not be by more than eight antigens (individual consideration of all typed antigens would have decreased excessively the distance between patients due to matching for absence of antigens).

If observations for a particular attribute are not available for a particular patient, that patient cannot be compared to others with respect to the attribute. The occurrence of such missing observations should not bias conclusions.

The results of the cluster analysis are presented in the form of binary tree, drawn using a Digigraf 1008 plotter.

RESULTS

Fig. 1 presents the results of the cluster analysis. Individual patients are represented on the abscissa and their linkage level or similarity, which is analogous to a correlation coefficient, is indicated on the ordinate.The smaller the linkage level between patients or patient groups, the weaker is their resemblance.

The cases can be considered as falling into two main groups, one showing two subdivisions, and a residuum of four cases (extreme left of plot) showing little resemblance to the others, or to each other. Table 2 compares the groups, with respect to the attributes scored.

Group I comprises seventy-nine patients who generally had a favourable course of hyperthyroidism (a tendency to remission following antithyroid mediation), small goitre, lack of ophthalmopathy and lymphocytic infiltration of the thyroid gland, low titres of human thyroglobulin (Htg) antibody and circulating immune complexes, and relatively low proliferative reactivity of lymphocytes to thyroglobulin.

Group I1 as a whole is characterized by presence of infiltrative ophthalmopathy, familial aggregation, severe course (two or more relapses of hyperthyroidism), increased titres of Htg antibody and immune complexes and markedly decreased suppressor T cell function.

Group IIa comprises twenty-nine patients in whom hyperthyroidism was associated with lymphocytic infiltration of the thyroid gland. Patients developed high titres of thyroglobulin antibodies, varying degrees of histologic chronic thyroiditis or both. These patients would fit the category of ‘Hashitoxicosis’ (Buchanan et nl., 1961; Fatourechi et nl., 1971; Jackson, 1975).

Group IIb comprises eighty-four patients in whom hyperthyroidism is associated with opthalmopathy, a refractory disease course, circulating immune complexes and decreased suppressor T cell function. A further subdivision of group IIb is suggested by the analysis (Fig. 1 and Table 2), group IIbl being distinguished from Group IIb2 and Group IIa by relatively low Htg antibody levels, frequency of other autoimmune disease and frequency of lymphocytic infiltration of the thyroid.

On the extreme left hand side of Fig. 1 can be seen a residuum of four patients. Each has Graves’ disease and clinical diabetes mellitus, but these patients show little other similarity to one another.

The prevalence of HLA-B8 was 9% in Group I, 21% in Group IIa, 84% in Group IIbl and 91% in Group IIb2 (Table 2). This gradient clearly suggests an association of B8 with Graves’ disease severity, B8 being infrequently associated with relatively benign disease (indeed Group I patients were B8 less often than were 450 Hungarian controls, among

340 V. Stenszky et al.

I4 z a a

W

H a 3 O

W

Subsets of Graves' Disease 34 1

Table 2. Mean values or frequencies of attributes and measures evaluated, by cluster groups

Attributes

Subgroups Non-linked Total

Group I Group I1 IIa IIbl IIbz cases patients (n=79) (n=113) (n=29) (n=49) (n=35) (n=4) (n=196)

1 Sex (% male) 2 Age at the time of manifestation 3 Goitre size 4 Crooks test 5 Family accumulation (%) 6 Association with other auto-

immune disease (%) 7 Infiltrative ophthalmopathy 8 Tendency to recurrence (%) 9 I3'I uptake

10 T3 non-suppressibility (%) 11 Serum T3 (nmolil) 12 Serum TSH (puiml) 13 Lymphocytic infiltration of

thyroid gland (%) 14 Htg antibody titre 15 Anti-nuclear antibody titre 16 Rheumatoid factor 17 Circulating IC 18 Active E-rosettes 19 Short lived suppressor T cell

function 20 Con. A activated suppressor T cell

function (%) 2 I Lymphocyte transformation index 22 Maximal acid output of stomach 23 Basal acid output of stomach 24-49 Frequency of HLA-B8 (%)

16.5 36.50

1 .oo 24.60 5.06 7.59

0.84 8.86

60.06

3.90 3.25 9.46

100

17.54 4.8 1 7.29 9.25

20.41 3.24

48.68

2.50 8.20 2.38 8.9

1 1.47 38.90

1.74 29.36 23.90 28.31

1.66 77.00 63.23

7.10 2.73

24.50

100

69.41 6.67 7.46

44.77 17.02 1.65

28.66

4.22 5.15 1.64

69.90

3.45 37.80

1.55 29.00 17.20 44.80

0.93 27.60 61.40

7.40 2.75

50.00

100

62.50 7.79

10.80 32.30 20.40 2.00

34.85

3.07 5.00 0.63

20.70

12.20 17.10 42.90 34.10

1.75 1.88 28.70 30.60 24.50 28.60 10.20 40.00

2.18 1.54 89.80 100.00 64.51 62.97

6.49 7.60 2.75 2.69 6.50 28.60

100 100

26.44 135.30 6.70 5.71 5.96 6.80

40.10 61.64 16.20 15.67 1.54 1.51

28.60 23.60

4.51 4.77 4.62 6.01 2.07 1.86

83.70 91.40

0.0 36.25

1.25 26.50 25.00

100~00

1.50 50.00 57.00 100

5-70 15.35 25.00

259.00 9.50

19.00 54.00 17.40

1.53

29.60

3.75 12-50 4.80

50.00

13.26 37.87

1.43 27.38 16.33 2 1.42

1.33 48.98 61.82 100

5.78 3.20

18.45

52.37 5.98 7.63

30.64 18.39 2.29

36.75

3.52 6.53 2.00

44.90

whom B8 frequency was 18.8%), but very common among those with relapsing disease associated with ophthalmopathy and stigmata of autoimmunity, and of intermediate frequency among cases of intermediate severity.

DISCUSSION

Cluster analysis has developed in the context of numerical taxonomy with the goal of delineating objective classifications of individuals evaluated for a number of attributes. The more attributes considered, the more generally applicable a classification is likely to be; for purposes of initial and general classification attributes are best considered ignoring preconceptions as to their relative significance (Sokal, 1974). To that end, we analyzed all available clinical and laboratory data on each patient studied, without regard to any proposed relevance of particular attributes to pathogenesis, severity or natural history of disease (Miyahara, 1976). The clinical significance of many laboratory correlates of Graves' disease is in any case unclear; thus collective evaluation might aid in subgrouping patients, and aid in associating investigative findings to clinical course of disease as well. Features of little or no relevance in this context should contribute little to the grouping of patients, but are unlikely to change the aggregation of individual patients into groups.

342 V. Stenszkj. et al.

Using this approach, we were able to distinguish three main groups, one clearly separated from the other two, Group I is characterized by small goitres and a tendency to remit following treatment with antithyroid drugs. Surprisingly, the thyroid glands of these patients showed less lymphocytic infiltration than those of patients in the other two groups. Lymphocytic infiltration has been thought to predict greater likelihood of euthyroidism or even hypothyroidism following surgery or other destructive therapy for Graves' disease. Irvine et al. (l977), however, found that titres of antimicrosomal antibodies were higher in patients whose disease did not remit following antithyroid medication than in those whose disease did remit. Significantly, lymphocytes infiltrating Graves' thyroids include a much lower percentage of suppressor T cells than do those infiltrating in autoimmune thyroiditis (Totterman el al., 1979).

Group I1 differs from group I in showing more vigorous immune responses: high titres of antithyroglobulin antibodies, increased T lymphocyte reactivity to thyroglobulin, circulating immune complexes, lymphocytic infiltration and infiltrative ophthalmopathy, and diminished suppressor T cell function. These patients tended to relapse and commonly reported a positive family history; they had other autoimmune disease more commonly than group I patients. Both Con A-induced suppressor T cell and short lived suppressor cell indices were significantly (Balazs el al., 1979a, b) lower in group I1 compared to group I, as was to a lesser extent the percentage of active sheep red cell rosettes. which correlates closely with cell-mediated immunity (Wybran et al., 1975).

The more frequent occurrence of HLA-B8 in group I1 than in group I is consistent with the tendencies of HLA-B8/DR3-positive patients to exhibit recurring thyrotoxicosis, eye disease and blast transformation in response to thyroglobulin, tendencies which have been previously described individually (Farid & Bear, 1981). I t has also long been believed that a small goitre bodes well for therapeutic response (McKenzie & Zakarija, 1979). This particular characteristic could not previously be related to HLA phenotype (Farid et al., 1976), but group I, into which patients with small goitre tend to fall, has a low frequency of HLA-B8. Conversely, recurrence of hyperthyroidism following adequate treatment with antithyroid drugs is a clear indication of severity in Graves' disease, and i t is also recognized that patients with recurring disease tend to have larger goitres, and that exophthalmos and infiltrative ophthalmopathy are more likely and tend to be more severe when hyperthyroidism is refractory. Immunological investigations indicate a correlation between the presence ofcirculating thyroid stimulating antibodies (TSAbs) at the end of a course of antithyroid treatment and tendency for hyperthyroidism to recur (McKenzie & Zakarija, 1979). Although many investigators have indicated that positivity for HLA-B8 (and more so DR3), may be a useful predictor for exacerbation (reviewed in Farid & Bear, 1981), others have not been able to substantiate this finding (Dahlberg et al., 1981; McKenna et al., 1982). When both TSAb and HLA phenotype are related to disease recurrence in the same patients, TSAb status is found to be a much better predictor of subsequent activity of the disease (McGregor et al., 1980; Davies et al., 1982).

A schema can be proposed to integrate these immunological phenomena. Lawley et al. (I98 1 ) found that lymphocytes from healthy individuals positive for HLA-B8 and DR3 have lower concentrations of Fc receptors, and are less efficient in clearing immune complexes, than those from persons who are negative for those antigens. Irrespective of the composition of the circulating immune complexes in patients with Graves' disease (Calder er al., 1974; Mariotti er al.. 1979) it is apparent that the percentages of patients positive for these complexes, and complex concentrations, is highest in subgroup IIb (1

Subsets of Graves’ Disease 343

and 2 ) in which also the prevalence of HLA-B8 is highest. These immune complexes, by modulating Fc receptors on the surface of immunocompetent cells, may reduce the function of suppressor T cells (Theofilopoulos & Dixon, 1979) and/or reduce their entry into the intravascular compartment. Immunosuppression resulting from hyperthyr- oidism (Wall et al., 1980) cannotper se explain the differences between patient subgroups reported. These gross differences in suppressor function do not negate the suggestion that specific suppressor function (rather than non-specific function; Okita et al., 1981) is deranged in Graves’ disease; a pronounced defect in specific suppressor T cell function is in fact seen.

The presence and titre of circulating immune complexes may reflect immunoregulatory defects mediated by antibody networks (Jerne, 1974; Islam et al., in press). Thus, patients in group I may be capable of producing efficient anti-idiotypic antibody against TSAb (as one possible antigen) which subsequently diminishes production and reduces formation of circulating immune complexes (Van der Heide et al., 1980). Other patients might be unable to make anti-idiotypic antibodies or able to make only anti-idiotypic antibodies with low affinity and little impact on TSAb production. While this study did not include measurement of TSAb, it does show that when patients are categorized on the basis of a large number of characteristics the subgroups with less tendency to remit have a high prevalence of HLA-B8, larger goitres and more severe ophthalmopathy, and a greater prevalence of immunological aberrations. These patients are also likely to have greater genetic predisposition to Graves’ disease as evidenced by more frequently positive family history.

In summary, this cluster analysis of Graves’ disease cases reveals associated increasing gradients of severity of disease, frequency of HLA-B8, frequency of positive family history, and immunological activity, including a decline in suppressor T cell function. This suggests the existence of an underlying continuum (not necessarily smooth) of genetic liability, apparently related to that for Graves’ disease severity, associated with the MHC and mediated through immunoregulatory disturbance.

To our knowledge, this study is the first in which HLA-associated disease has been examined by a cluster technique; it demonstrates the feasibility of subgrouping patients into clinically relevant categories which are, for instance, independently correlated with the possession of a particular HLA phenotype. It is likely that the arguments about disease heterogeneity, for example, with regard to insulin dependent diabetes (Farid & Bear, 1981), based on individual characteristics considered one at a time with different results being found in different series, may put on a firmer basis by applying these methods we have tested in Graves’ disease.

ACKNOWLEDGEMENTS

We wish to thank Dr Marilyn Preus for helpful discussion, Medical Audiovisual Services for the painstaking preparation of the figure, and Miss Gail O’Brien for typing the manuscript.

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