Thymoma

Eighty-one cases of thymoma were studied immunohistologically with the use of three mouse monoclonal antibodies: one was specific for subcapsular-cortical, one for intra-cortical, and one for medullary epithelial cells. Twenty-eight (60.9%) of 46 polygonal cell thymomas were of the cortical type and 1 (2.2%) was of the medullary type. Ten (55.6%) of 18 spindle cell thymomas and 7 (41.2%) of 17 mixed cell thymomas were of the medullary type, and 1 (5.6%) of 18 spindle cell thymomas was of the cortical type. Fourteen (17.3%) of 81 thymomas were composed of epithelial cells that were triple positive immunologically; although these are unusual, they also may be present in the normal thymus, Based on these findings, triple-positive epithelium in the normal thymus consists of common stem cells that can differentiate into subcapsular-cortical, intra-cortical, and medullary epithelium: these cells may be the target cells for tumorigenesis. Epithelium in polygonal cell thymoma tends to differentiate into cortical epithelium, whereas epithelium in spindle and mixed cell thymomas differentiates into medullary epithelium. Cancer 1992; 69:2463-2468.

lary-can be distinguished based on their reactivity to various markers.'-6 Because thymomas are epithelial in t rig in,^ many researchers have studied thymomas by using monoclonal antibodies directed against different thymic epithelial components to classify thymomas as cortical or medullary.6*8-1' Some authors have reported that thymomas can be classified as cortical or medullary.8-11 Others have concluded that the subdivision of thymomas into cortical or medullary types is not s u b~t a n t i a t e d .~~~~'~ Various monoclonal antibodies directed against different thymic epithelial cells were discussed in these studies; however, few studies used both cortical epithelial marker and medullary epithelial marker, and, in earlier studies, almost all monoclonal antibodies used as medullary epithelial recognized both medullary epithelium and subcapsular-cortical epithelium. We used two monoclonal antibodies: UH-1, which recognized intra-cortical epithelium," and PE-35, which recognized thymic medullary epithe1i~m.l~ By using these two markers along with anti-Leu-7, a marker for subcapsular-cortical epitheli~rn,'~,'~-'~ we were able to conduct an immunohistologic study of 81 cases of thymoma.

Tissues
Tissue samples were collected from 81 patients with thymoma who were seen at various hospitals in Japan from 1984 to 1990. These thymomas occurred in 35 men and 46 women whose ages ranged from 28 to 79 years (mean, 53.8 years). Myasthenia gravis (MG) was present in 29 patients. During surgery for funnel chest, normal thymuses were collected from five children (age range, 8 months to 7 years). Fresh specimens of thymomas and normal thymuses were snap frozen in OCT medium (Miles Incorporated, Elkhart, IN) and stored at -75 "C for immunoperoxidase staining. Portions of the specimens fixed in 10% formalin were embedded in paraffin for routine histologic studies. Based on the shape of the neoplastic epithelial cell and the criteria of Rosai and L e~i n e ,~ the 81 thymomas were classified as follows: polygonal, 46; spindle, 18; and mixed cell, 17 (Fig. 1).

Staining Procedure
Frozen serial sections (4 pm) were fixed in acetone at 4°C for 10 minutes, air-dried, washed in phosphatebuffered saline (pH 7.2), and then stained according to immunoperoxidase staining techniques. Sections were stained according to the streptoavidin-biotin-peroxi-dase complex method using the Histofine streptoavidin-biotin-peroxidase complex kit (Nichirei, Tokyo, Japan). Anti-Leu-7, UH-1, and PE-35 were applied at dilutions of l : l O , 1:900, and 1:1000, respectively. The sections were reacted in a solution consisting of 20 mg of 3,3'-diaminobenzidine-4 hydrogen chloride (HC1) in 100 ml of 0.05 mol/l tris-HC1 buffer (pH 7.6) containing 0.005 YO hydrogen peroxide (H202); this reaction was complete in 10 minutes. Next, the sections were washed in phosphate-buffered saline, counterstained with methyl green, dehydrated in a graded alcohol series and xylene, and mounted with a synthetic resin.

Reactivity of Antithymic Epithelial Antibodies W i t h the Normal Thymus
UH-1 reacted with cortical epithelium by forming a network ( Fig. 2, top left). Subcapsular-cortical epithelium, which was negative for UH-1 (Fig. 2, top left), was positive for anti-Leu-7. A small group of Leu-7-positive epithelium also was observed in the outer cortex in a

Reactivity of Antithymic Epithelial Antibodies W i t h Thymomas of Three Different Histologic Types
The staining patterns of individual thymomas fell into two major categories: thymomas with epithelial networks that only stained for one marker and thymomas with epithelial networks that were positive for two or three markers. Because we used serial sections in the current study, three markers were found to be expressed in various combinations on the same neoplastic epithelium (Fig. 3). We recognized eight immunologic phenotypes of neoplastic epithelium: U (UH-1+, PE-3 5 , Leu-7-); P (UH-1-, PE-35+, Leu-7-); L (UH-1-, PE-35-, Leu-7+); UP (UH-1+, PE-35+, Leu-7-); UL (UH-1+, PE-35-, Leu-7+); PL (UH-1-, PE-35+, Leu-7+); UPL (UH-1+, PE-35+, Leu-7+); and N (UH-1-, PE-35-, Leu-7-). The relationship between the histologic types and the immunologic phenotypes is summarized in Table 1. Epithelium of phenotypes U, P, and L is common in the normal thymus, whereas epithelium of phenotypes UP, UL, PL, UPL, and N is not. Epithelium of phenotype UL appears to be localized in the cortex. Therefore, epithelium of phenotypes U, L, and UL can be classified as the immunologically cortical type. Twenty-eight (60.9%) of 46 polygonal cell thymomas were of the cortical type and consisted of the cortical type of epithelium, whereas 10 (55.6%) of 18 spindle and 7 (41.2%) of 17 mixed cell thymomas were of the medullary type and consisted of the medullary type of epithelium, i.e., epithelium of phenotype P. Of 46 polygonal cell thymomas, only 1 (2.2%) was of the medullary type. Of 18 spindle cell thymomas, only 1 (5.6%) was of the cortical type. There was no cortical type in 17 mixed cell thymomas.

Histologic Types and Immunologic Phenotypes of Thymoma W i t h MG
MG was observed in 29 (35.8%) of 81 thymomas. The histologic types and immunologic phenotypes of these 29 thymomas are summarized in Table 2. They consisted of 24 polygonal and 5 mixed cell thymomas. Six- teen (55.1%) thymomas were of the cortical type and 3 (10.3%) were of the medullary type.

Discussion
In the normal human thymus, three distinct epithelial cells-subcapsular-cortical, intra-cortical, and medullary-can be distinguished based on their localization and reactivity to various markers. Thus, thymomas may be classified into at least three types depending on which epithelial cells they are derived from (subcapsular-cortical, intra-cortical, or medullary). However, an analysis of 81 thymomas (29 were from patients with MG) using anti-Leu-7, UH-I, and PE-35, each of which is specific for subcapsular-cortical, intra-cortical, and medullary epithelial cells in the normal thymus, respectively, showed 8 immunologic phenotypes. Epithelium of phenotypes U, P, and L is common in the normal thymus, whereas phenotypes UP, UL, PL, UPL, and N are not.
Ring and Addis reported Leu-7-positive epithelium in both the cortico-medullary junction area and cortex, as well as in the subcapsular-cortex in the normal human thy mu^.^ Hirokawa et al. reported that medullary epithelium was usually negative for UH-1, but a few positive cells were occasionally observed. Consequently, phenotypes UP, UL, PL, and UPL also may be present in the normal thymus as minor subpopulations.
The heterogeneity of normal thymic epithelium, according to the reactivity to monoclonal antibodies specific for different types of keratin polypeptides, reflects different stages in epithelial maturation, i.e., thymic epithelium is capable of undergoing sequential stages of diff erentiati~n.'~,'~ Therefore, each epithelial cell of the eight phenotypes may represent different stages of the 0 0 2 (2.5) 3 (6.5) 2 (11.1) 3 (17.6) 8 (9.9) 11 (23.9) 0 0 11 (13.6) l ( 2 . 2 ) 3 (16.7) 4 (23.5) 8 (9.9) 11 (23.9) l ( 5 . 6 ) 2 (11.8) 14 (17.3) 2 (4.3) 1 (5.6) 1 (5.9) 4 (4.9) Total 46    differentiation in the normal thymus. Recently, some authors suggested that thymic epithelial cells can be derived from common stem cells.6,20,21 Thus, because 14 (1 7.3%) of the 81 thymomas were predominantly composed of phenotype UPL epithelial cells and because these cells may be present in the normal thymus as a minor subpopulation, we conclude that phenotype UPL epithelium in the normal thymus consists of common stem cells that can differentiate into subcapsularcortical, intra-cortical, and medullary epithelial cells. These cells may be the target cells for tumorigenesis. Also, we deduced that the epithelial cells of the unusual phenotypes UP, UL, and PL in the normal thymus and in thymoma may represent the intermediate stages of differentiation into epithelium of usual phenotypes. In our study, 28 (60.9%) of 46 polygonal cell thymomas were of the cortical type and 1 (2.2%) was of the medullary type. Ten (55.6%) of 18 spindle cell thymomas and 7 (41.2°/~) of 17 mixed cell thymomas were of the medullary type, and 1 (5.6%) of 18 spindle thymomas was of the cortical type. Epithelium in polygonal cell thymoma tends to differentiate into cortical epithelial cells, whereas epithelium in spindle and mixed cell thymomas tends to differentiate into medullary epithelial cells. The unusual phenotype epithelium in each histologic type represents intermediate stages of differentiation into cortical and medullary type epithelial cells.
Morphologically, polygonal, spindle, and mixed cell thymomas appear to correspond to the cortical, medullary, and mixed thymomas, respectively, of   are not a mixture of cortical type epithelium and medullary type epithelium, but are tumors with characteristics similar to medullary (spindle cell) thymomas that show a radically heterogenous composition. This morphologic heterogeneity can be explained by cells showing a different degree of maturation within the same t~m o r~~,~~,~~ while exhibiting an immunologically homogenous composition.
We compared the tumor invasiveness of thymomas of usual phenotypes with those with unusual phenotypes. Because the degree of differentiation of unusual phenotype epithelium seemed to be lower than that of usual phenotype epithelium, we thought that the thymoma composed of unusual phenotype epithelium would be more invasive; however, no significant difference was detected.
MG was observed in 29 (35.8%) of 81 cases of thymoma. Histologically, 24 (82.6%) of 29 thymomas were of the polygonal cell type and the rest were of the mixed cell type. Sixteen (55.2%) of 29 tumors were composed of cortical epithelial cells, ie., epithelium of phenotypes U, L, and UL, and 3 (10.3%) were composed of medullary epithelial cells. The remaining ten tumors were composed of unusual phenotype epithelial cells. Thus, thymomas with MG showed a wide spectrum of immunologic phenotypes. The pathogenesis of MG associated with thymoma is heterogenous and complex.