Myasthenic and Nonmyasthenic Thymoma

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Group of Great Britain, King Gustav V Jubilee Fund, and the Karolinska Institute. ... Neurological Science, RoyalFree Hospital School of Medi- cine, Rowland Hill Street, .... lymphocytes'0 ranged from very high ("lymphoepi- thelial") (Figure 3A ...
American Journal of Pathology, Vol. 127, No. 3, June 1987 Copyright X American Association of Pathologists

Myasthenic and Nonmyasthenic Thymoma An Expansion of a Minor Cortical Epithelial Cell Subset?

N. WILLCOX, M. SCHLUEP, M. A. RITTER, H. J. SCHUURMAN, J. NEWSOM-DAVIS, and B. CHRISTENSSON

From the Department of Neurological Science, Royal Free Hospital, Department ofImmunology, Royal Postgraduate Medical School, and Institute of Neurology, London, England; the Division of Internal Medicine and Institutefor Pathology, University Hospital Utrecht, Utrecht, The Netherlands; and the Department ofPathology, Karolinska Hospital, Stockholm, Sweden

The authors report an immunohistologic study of primary thymomas from 23 cases with myasthenia gravis (MG) and 7 without. Typical T6+ cortical thymocytes were usually abundant. Most epithelial cells initially appeared to be of cortical type, too, though many bore subcapsular inarkers in most samples. However, two-color immunofluorescence revealed unexpected heterogeneity, numerous epithelial cells simultaneously expressing some or all of the markers ofboth these subsets (even in two pleural metastases). It is in-

ferred that there is a common tumor stem cell whose normal counterpart may be related to the rare patches of similar phenotype in the cortex in control samples. The authors could detect no major differences in 5 of 7 samples from nonmyasthenics; thus, most thymoma cases may risk the development of MG. Finally, thymomas from 6 of 7 further MG cases pretreated with corticosteroids showed very few cortical thymocytes, and the (phenotypically similar) epithelium was more obvious. (AmJ Pathol 1987, 127:447-460)

THYMIC tumors are associated with a variety of probably autoimmune disorders but especially with myasthenia gravis (MG), which occurs in 30-40% of all thymoma patients.'-3 MG is a particularly well characterized disease mediated by autoantibodies to the acetylcholine receptor (AChR) of muscle.4 The 10% of MG patients that have a thymoma almost invariably have other autoantibodies to striational antigen(s) in muscle5 that apparently also react with some membrane constituent of the thymoma cells.6 Similarly, AChR-like material appears to be present in both the normal and MG thymus.7'8 The coincident occurrence in thymoma cases of autoantibodies to these two different muscle antigens present in the thymus suggests that autosensitization may be initiated there,9 and the same may also be true in nonthymoma cases. Because thymomas are epithelial in origin,'0 we have used monoclonal antibodies (MAbs) to various thymic epithelial (and lymphoid) cell types, and have also typed them by nuclear morphology, "I in MG and non-MG cases in order to identify the neoplastic cell type and look for clues as to the pathogenesis of MG. The MAbs have defined at least three major epithelial

subsets in the normal thymus,'2'14 namely, cortical ("phenotype A," Figure 1), subcapsular ("phenotype B"), and medullary ("phenotype C"). In addition, Hassall's corpuscles have a distinctive phenotype13 (MR13+, IP3+, IP4+, HLA-DR+, and keratin+). Others have recently concluded (from small series) that the epithelial cells in some MG thymomas are principally cortical in type, though with variable HLA-DR expression,'5 but in other cases are mainly medullary.16 Our present analysis of 25 MG samples (including 2 pleural metastases) reveals a more complex picture, with epithelial cell populations often simultaneously expressing cortical and subcapsular markers in unusual combinations, and we now propose an explanation that also accounts for the heterogeneity found previously by others.'0" 5"16

447

Supported by grants from the MRC, the Sir Jules Thorn, Welton, Wellcome (travel) Trusts, the Muscular Dystrophy Group of Great Britain, King Gustav V Jubilee Fund, and the Karolinska Institute. Accepted for publication January 13, 1987. Address reprint requests to Nick Willcox, Department of Neurological Science, Royal Free Hospital School of Medicine, Rowland Hill Street, London NW3 2PF.

448

WILLCOX ET AL HLA-DR A

MR3

AJP * June 1987

Keratin

MR19 RFD4

Sections (5 u) from formaldehyde-fixed paraffinembedded tissue blocks were stained with hematoxylin and eosin (H&E); samples were classified according to lymphocyte content'0 (Table 1). Some blocks were reembedded in plastic, and 1-u sections were stained with pararosaniline for classification into Types 1-6 by nuclear morphologic features. " Cells of Type 1 are found normally in subcapsular and perivascular areas, Type 2 (pale-staining nuclei) mainly in the outer cortex, and Types 3 (intermediate) and 4 (dark) in the mid and inner cortex and medulla; Type 5 cells are undifferentiated cells seen at the corticomedullary junction and medulla, and Type 6 cells are large medullary cells around Hassall's corpuscles."I Other (unfixed) blocks (in Ames OCT) were snapfrozen (in isopentane cooled in liquid nitrogen) and stored at -70 C; cryostat sections (6,u) were air-dried for 24 hours, fixed in chloroform/acetone and stained by indirect immunoperoxidase (IP)'2 or immunofluorescence (IF)12" 3 techniques. Two separate blocks were analyzed by IP from 18 ofthe cases, but only one was available in the others. Thymus cell suspensions were prepared (and cultured) on the day of thymectomy and were stained (by IF) fresh or after storage in liquid nitrogen.'7

Leu7

I

normal cortical-rnajor

B normal subcapsular

c

normal medullary D normal cortical-minor

E thymoma F thymoma

G

thymoma

Figure 1-Epithelial phenotypes in human thymus and thymoma. Leu-7+ epithelial labeling is typically strong on subcapsular epithelium in the fetus, and often in adulthood, too, but is usually absent in childhood.12 For simplicity, the Hassall body phenotype has been omitted; it is typically keratin+, HLA-DR+, IP3+, IP4+, MR13+,13 and accounts for almost all the HLA-DR+ epithelial labeling in the medulla.

Materials and Methods Patients We obtained adequately preserved specimens of primary thymomas from 30 patients, 23 with and 7 without MG, and from pleural metastases from two other MG patients. The patients' clinical details are summarized in Table 1. "Normal" control samples were taken from adults and children undergoing cardiac surgery.

Reagents and Analysis Each block was normally studied with all the antibodies listed in Table 2 by IP staining. For two-color IF we routinely used either 1) mouse MAbs (followed

Table 1 -Thymoma Patients Studied

MG symptoms (mean + SD ancJ range) Age at onset

Case number Myasthenics Untreated 1-15 6M 9F

(years)

Duration (months)

Mg grade*

Serum anti-AChR (nmol) Geometric mean

Anti-striatedt muscle antibody

Tumor bulk (cu cm) Mean ± SD (range)

10 4 (1 ND)

162 ± 133

(3.9-250) 7.6X2.9 (2.2-43.6)

5 2 (1 ND)

25 ± 28

IIA lB III XSD

Primary thymomas

41.3 + 10.1 (26-60)

7.9 + 6.5

16-23 3M 5F

47.4 + 10.8

44.1 ± 57.3

(37-64)

(37-149)

24-25 2F

33.5+4.9 (30,37)

(3-28)

5 3 6 (1 ND)

lmmunosuppressedt 1

3 4

20.4X.3.6

(4-500) (5-90)

Pleural metastases

Nonmyasthenics 26-32 6M 1F

38+42 (9, 68)

Primary thymomas 58.6 ± 14.2 Not applicable

(35-72)

1

1

10.3X1.2

1

1

152 ± 138 55 and 250

1

2

818 ± 723 (12-2000)

(9, 11.9) Case 27-0.6

(2 others negative, 4 others ND)

(4 ND)

*Modified from Osserman and Genkins.37 IA, mild; IIB, moderate; III, acute severe, generalized symptoms. tAnti-striated muscle antibodies assayed by indirect IF on rat muscle. f Patient 16 received azathioprine from about 1 year until 5 months before surgery, and patient 24 for the 5 preoperative months. Patients 17-19 and 23 received corticosteroids for 2-8 months preoperatively, patients 20-22 corticosteroids plus azathioprine for 1-2 months, and patient 25 for 5 years.

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MARKERS/ORIGINS OF TIIYMOMA EPITHELIUM

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Table 2-Monoclonal Antibodies Used MAb To epithelial cells: Leu-7 MR3 RFDR 1 RFDR 2 RFD4 MR19 To T lymphocytes: NA 1/34

RFT8 OKT3

Ig class IgM IgGl lgM | IgG2 J IgM IgM J

Subcapsular epithelial cells (in fetus and adult) Cortical epithelium HLA-DR on cortical epithelium, interdigitating cells, B cells, and macrophages Medullary and subcapsular epithelial cells

IgG2 IgGl IgG2

Cortical thymocytes (T6-like) T8+ T lymphocytes Medullary and peripheral T lymphocytes

by goat anti-mouse Ig-FITC, Department of Immunology, Royal Free Hospital) plus rabbit anti-keratin (followed by swine anti-rabbit Ig-TRITC, all Dako, Copenhagen) or 2) a mouse IgG McAb (followed by goat anti-mouse y-FITC, Southern Biotechnical Associates, Birmingham, Alabama) plus a mouse IgM McAb (followed by goat anti-mouse ,u-TRITC, Southern Biotechnical). The combinations we routinely used are given in Table 3, where the analysis of the IF staining is shown. All the sections were studied independently by two observers and checked by two others.

Table 3-The Different Combinations of MAbs Used in Two-Color Immunofluorescence and the Enumeration of Epithelial Phenotypes They Define (Figure 1) (With Case 7 as an Example)

Group B Rabbit a-keratin MR19 IgM Leu-7 IgM RFDR1 IgM

Group A RFDR2 Rabbit MR3 a-keratin (IgGl) (IgG2) (% of doubly labeled cells) ND 75 50 ND

80 50 50 80

60 50 40 ND

Sections were stained with one antibody from each group followed by the appropriate class- or species-specific FITC- and TRITC-coupled second layers. Each number given is the percentage of the total epithelium stained in the relevant combination that was double positive. Forexample, in this particular case, 80% of the (keratin+) epithelium was MR3+. The 50% of MR19+, HLA-DR+ (RFDR2+) epithelium defines phenotype G. Sometimes there was a higher frequency of MR19+, MR3+ epithelium than that (eg, 65%); the excess (15%) would then correspond to phenotype F; and similarly, an excess of Leu-7+ HLA-DR+ epithelium would define phenotype E (though neither was evident in this case). Phenotype A is defined by the 25% of MR19-, keratin+ (or Leu-7-, keratin+) epithelium, and is cross-checked by the 25% that was MR3+, Leu-7- (not shown). Ukewise, the 20% of MR3-, keratin+ epithelium corresponds to phenotype B and is cross-checked by the MR3-, MR19+ frequency. Table 4 shows the approximate percentages derived in this way. Wherethere was "loss" of HLA-DR labeling, phenotypes E-G could not be fully resolved; and neither could phenotypes D-G where there was "loss" of Leu-7.

Source or reference

Reactivity in thymus

Becton, Dickinson 13

38 38 12 13 CD1 CD8 CD3

39 40 Ortho Pharmaceutical

Specificity Controls In a control adult thymus the distinct staining patterns given by rabbit antikeratin and RFDR2 (Figure 2A and B) confirm the specificity of the first- and second-layer antibodies, as do other controls omitting the first layer antibodies (not shown). In a section double-stained with MR3 (IgG) and MRl9 (IgM) the labeling is again selective, and there is essentially no cross-over between the two second layers, even with strong staining (Figures 2C and D). Note that, in this infant sample, double labeling was confined to sporadic small groups of cells just deep to the subcapsular region.'2'18 Again, no double staining was observed when either first layer was omitted (not shown), further confirming the specificity of the anti-y-FITC and anti-,u-TRITC.

Results Histologic Analysis All samples contained numerous epithelial cells'0"'9 (Figure 3A), often divided by septa into lobules of variable size that sometimes had an outer "subcapsular"' row of epithelial cells.'0 The numbers ofadmixed lymphocytes'0 ranged from very high ("lymphoepithelial") (Figure 3A, Table 4) through moderate ("lymphodepleted"), to very low or absent ("epithelial," Figure 3E). Perivascular spaces were usually prominent and were often filled with T3+ lymphocytes and HLA-DR+ macrophages. Spindle (epithelial) cells constituted almost the entire tumor in Case 20 and were seen in isolated bundles in some others (Table 4). Spindle cells aside, the majority ofthe thymomatous epithelial cells had large round to oval nuclei (about 10-15 ,u in diameter), with a low to moderate affinity for stains, and usually prominent nucleoli (Figure 3E), features of the so-

h; arsa~~~~~~~~~~~~..

.j_ . _. -' 1|l

C

Figure 2-IF staining of "normal" control infant thymic cortex. The subcapsular epithelium is strongly keratin+ (asterisks), and the main cortical epithelium (solid arrow) is weakly positive (A, TRITC, X294). Only the latter labels for HLA-DR (B, FITC, a separate field, X588). The subcapsular but not the cortical epithelium is MR1 9+ (C, anti-,u TRITC, X588). The reverse is true with MR3 staining of the same field (D, anti-y FITC, X588). This field was selected to show a rare "island" of double labeling (solid arrow). Above it are two false-positive granular cells (open arrows).

B

D

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A

B

C

D

Figure 3A-D-Sections of MG thymoma, Case 16 (all X 175). H&E staining shows typical lymphoepithelial appearances (A). By IP staining (B), most of the lymphocytes are T6+, and there is an extensive MR3+ epithelial network (C), much of which is also Leu-7+ (D). E-MG Case 14, an epithelial field selected to show nuclear morphologic Types 2, 3, and 4. (Pararosaniline, Xi 000).

E

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WILLCOX ET AL

AJP

called "Type 2" cells, the major epithelial component of the normal thymic cortex." In some patients (Table 4 and Figure 3E), there were also sporadic cells with smaller, more intensely staining nuclei like those of Type 3 and 4 cells in the deeper cortex and medulla of the normal thymus." In addition, some samples (Table 4) contained scattered cells with very large very weakly staining, and sometimes irregular nuclei, and

*

June 1997

these "hypertrophic" cells resembled Type 6 cells in the normal medulla." A few cases showed rare (keratin') epithelial clusters resembling small Hassall's corpuscles (Table 4). Variable numbers of HLA-DR+ macrophages were present both in the epithelial areas and the septa. In 4 cases there were germinal centers within the thymoma. The adjacent uninvolved tissue (available in 6

Table 4-Epithelial Cell Phenotypes and Nuclear Morphology in Thymoma

A

Case number

Approximate frequencies of different subtypes Phenotype (defined in Figure 1) Nuclear morphologic Types 1-6 Major MTumor F E G B Minor pathology (% of total epithelium) Major Sporadic

Myasthenics 1 2 3 4 5

6 7 8 9 10 11 12 13 14 15 Immunosuppressed 16 17 18 19 20 21 22 23 Metastases 24 25 Nonmyasthenics 26 27 28 29 30 31 32

70 30* 10* 30 20*