Four thymomas, two from patients with myasthenia gravis, were studied using a panel of monoclonal antibodies. Almost all the associated lymphocytes were ...
Thymoma: An Immunohistochemical Study WING C. CHAN, M.B., B.S., GHAZI S. ZAATARI, M.D., SEYED TABEI, M.D., MARTHA BIBB, MT(ASCP), AND RUSSELL K. BRYNES, M.D.
Four thymomas, two from patients with myasthenia gravis, were studied using a panel of monoclonal antibodies. Almost all the associated lymphocytes were T-cells with the common thymic lymphocyte phenotype, suggesting that the neoplastic epithelium can provide a microenvironment that retains or perhaps attracts immature T-cells. HLA-Dr antigens were expressed by some of the epithelial cells and many of the lymphoid cells in the perivascular space. The Leu-7 antigen was present on a subset of epithelial cells and had a highly variable expression in different thymomas. The percentage of lymphocytes bearing the OKT8defined antigen appeared to be lower in patients with myasthenia gravis, while the expression of other markers did not appear to be consistently or significantly different. There did not appear to be marked differences in the lymphocyte subpopulations of thymomas with different morphology, and there was no evidence of differentiation of the thymomas into cortical and medullary areas. (Key words: Thymoma; Myasthenia gravis; T-lymphocytes) Am J Clin Pathol 1984; 82: 160-166 A THYMOMA CONSISTS of two components: thymic epithelial cells, which are the neoplastic component, and small lymphocytes, which are nonneoplastic.15 In the normal thymus, the epithelial cells carry HLA-Dr determinants, 4 connect with each other by desmosomes, and contain bundles of tonofilaments that can be demonstrated by antikeratin antibodies.10 The epithelial cell component of a thymoma is ultrastructurally similar to that in the normal thymus 15 and reacts also with antikeratin antibodies. 310 Normal thymic lymphocytes are precursors of peripheral blood T-cells and these two populations of cells have different surface characteristics and functional properties. There are multiple subpopulations within the thymic lymphocytes as defined by a panel of monoclonal antibodies, and a maturation sequence has been postulated.13 The most mature cells preferentially are found in the medulla, while the cortex is dominated by more immature elements.4,7 The lymphocytes in a thymoma have been shown to be T-cells.6,9'1' Their surface characteristics and functional properties suggest that the majority are immature lymphocytes. A recent study of a thymoma 12 has shown that over 70% of the isolated lymphocytes expressed Received October 25, 1983; received revised manuscript and accepted for publication January 30, 1984. Address reprint requests to Dr. Chan: Department of Pathology and Laboratory Medicine, Emory University Hospital, 1364 Clifton Road, Atlanta, Georgia 30322.
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Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia
the OKT6 defined antigen and formed stable sheep red blood cell rosettes, both of which are characteristics of immature lymphocytes. We conducted an immunohistochemical study of four thymomas in order to determine, (1) the types of lymphocytes generally associated with thymomas; (2) their distribution within the tumor; and (3) the differences, if any, between tumors of different histologic appearances and those with or without associated myasthenia gravis. We also studied the expression of HLA-Dr antigen and the Leu-7 antigen1 on the neoplastic epithelial cells. Materials and Methods Tissue blocks were fixed in 10% neutral buffered formalin and also in B5 fixative. Sections were stained with hematoxylin and eosin. Representative areas of the tumors also were frozen in O.C.T. (Lab-Tek Products, Naperville, IL) using isopentane cooled in solid carbon dioxide. The frozen tissue then was stored at - 7 0 ° C . Frozen sections were prepared for immunohistochemical studies, which were performed within two months. A panel of monoclonal antibodies was used for these studies. Their origin and specificities are summarized in Table 1. Monoclonal antibodies from different sources may vary in their reactivity in immunohistochemical assays, and the most suitable ones for our assay system were determined empirically. The tissue sections were fixed in cold acetone for 10 minutes and incubated with 10% horse serum in 0.05 M TRIS buffer at pH 7.6 for 30 minutes to diminish subsequent nonspecific antibody binding. The reagents then were incubated with the appropriate tissue sections at room temperature for 30 minutes in the following sequence: monoclonal antibody, rabbit antimouse IgG, swine antirabbit IgG, and rabbit antiperoxidaseperoxidase complex (Accurate Chemical and Scientific Corp., Westbury, NY). The sections were rinsed with TRIS saline and washed with TRIS buffer containing 1% horse serum between each incubation step. Peroxidase activity was demonstrated using diaminobenzidine te-
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Table 1. Monoclonal Antibodies Used in This Study Designation and Source
Specificity
References
OK.T6 (Ortho Diagnostics, Raritan, NJ) OKT3 (Ortho Diagnostics) Til (Coulter Immunology, Hialeah, FL) T8 (Coulter Immunology) Anti-Leu-3a (Becton-Dickinson, Mountain View, CA) Anti-Leu-7 (Becton-Dickinson)
Immature T-lymphocytes Mature T-lymphocytes Sheep red blood cell receptor T-suppressor/cytotoxic cells T-helper cells Natural killer cells and cells involved in antibody dependent cell-mediated cytotoxicity B-lymphocytes, Activated T-lymphocytes, monocytes and some epithelial cells
Reinherz and Schlossman13-14 Reinherz and Schlossman1314 Reinherz and Schlossman141314 Reinherz and Schlossman 5 Evans and associates 1 Abo and Balch
Anti-HLA-Dr (Becton-Dickinson)
Lampson and Levy8
The table lists the major specificities of these antibodies and is not an exhaustive list of all reactivities.
trahydrochloride (Polyscience Inc., Warrington, PA) as substrate (3 mg substrate in 5 mL of 0.03% hydrogen peroxide). Cells that reacted with an antibody had a rim of brown reaction product at their periphery. The number of lymphocytes reacting with a particular antibody in a tissue section was quantitated by counting ten representative fields under X400 magnification. The eyepiece of the microscope was fitted with a micrometer displaying a grid of 25 squares. In each field, the number of positive cells in the central and four corner squares was counted. A lymphocyte was considered positive when half or more of its circumference was covered by the brown reaction product. To be counted, at least one half of a cell had to lie within the boundary of a square. The number of positive cells stained with each antibody was expressed as a ratio to the cells positively stained with T i l (Tl 1 + cells). The slides were evaluated independently by three pathologists, and the mean of the three ratios ± one standard error of the mean was calculated. Terminal deoxynucleotidyl transferase (TdT) was determined on frozen tissue sections by an immunoperoxidase technic similar to that used for the cell surface marker study. The primary antibody was a rabbit anti-TdT (courtesy, Dr. David Gordon) and the second antibody was a biotinylated goat antirabbit IgG. A peroxidase-conjugated avidin-biotin complex (Vector Laboratories, Burlingame, CA) was used instead of rabbit peroxidase-antiperoxidase complex. The same substrate was used, and the reaction product was
seen in the nuclei of cells containing TdT. Thymuses removed from children during cardiac surgery served as controls for this study. Results Clinical Summary Relevant clinical data on the four patients studied are summarized in Table 2. Pathologic Examination A mass measuring 10.0 X 5.0 X 3.5 cm arid weighing 135 g was removed from patient 1. Its surface was smooth, glistening, and tan. Cut surfaces were tan and lobulated. Microscopically, the tumor consisted of a mixture of small round lymphocytes and epithelial cells, which had polygonal or oval nuclei with finely dispersed chromatin and often a small distinct nucleolus. The proportion of lymphocytes and epithelial cells varied from area to area but both were well represented in all areas. Focal microcystic change was seen. The mass excised from patient 2 measured 5.5 X 4.5 X 4.0 cm. It was completely encapsulated, and cut surfaces were tan and lobulated. Its overall microscopic appearance was similar to that of the first tumor, except that epithelial cells were focally more prominent. Perivascular spaces were wide and conspic-
Table 2. Clinical and Laboratory Findings in the Patients Patient
Age/Sex
Hb (g/dL)
WBC (cells/mm3)
Myasthenia Gravis
Other Significant Findings
1 2
53/M 44/M
16.3 15.1
9,700 7,000
Absent Mild, localized
3 4
19/F 49/F
12.1 12.7
5,000 9,100
Generalized Absent
None Positive antibody to acetylcholine receptor None None
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broad bundles containing elongated epithelial cells in a more fibrous stroma. Perivascular spaces and microcystic change were not seen. The adjacent thymus showed scattered prominent germinal centers in its medulla. Immunohistochemical
Studies
Results of immunohistochemical studies of the thymomas are summarized in Table 3 and illustrated in Figures 2 to 4. Most of the lymphocytes in the tissue sections were T-cells (T11 +). From the percentages of cells expressing the various T-cell markers, it was concluded that practically all of the T1 i + cells also expressed the OKT6 defined surface antigen. Most cells also reacted with OKT3 and anti-Leu 3a, while a smaller proportion of cells also were T8 positive. Scattered epithelial cells and their processes demonstrated a high concentration of HLA-Dr antigens, but their number appeared to be too small to account for all the epithelial cells seen in the hematoxylin and eosin sections. This was illustrated especially well in case 4 (Fig. 3). Many of the lymphocytes in the perivascular spaces were HLA-Dr+, while those elsewhere were largely negative. Endothelial cells also expressed HLA-Dr antigens. The anti-Leu-7 antibody reacted with the periphery of lobules in normal thymus (Fig. 4), but the reactivity of anti-Leu-7 with various thymomas was highly variable. FIG. 1. Thymoma from Case 2 showing a mixture of small lymphocytes Case 3 showed a diffuse linear-reticular pattern of staining. and epithelial cells. The perivascular spaces are very prominent. HeThe epithelial lining of perivascular spaces was also posmatoxylin and eosin (X400). itive (Fig. 4). Case 2 showed a similar staining pattern, although it was generally weaker and fewer cells appeared to be positive. In case 1, only focal areas of the thymoma uous (Fig. 1). The tumor from patient 3 measured 3.0 were reactive. In case 4, positive reactions again were X 3.0 X 2.5 cm and was similar macroscopically and obtained focally, but reactivity tended to concentrate at microscopically to that from patient 2. Perivascular spaces also were very prominent. The adjacent thymus showed the boundary between the more cellular nodules and the follicular hyperplasia. The tumor from patient 4 was large, more fibrous bundles of epithelial cells, creating an completely encapsulated with a bosselated surface. It appearance analogous to the staining of the periphery of measured 10.0 X 5.0 X 5.0 cm, and the cut surfaces were the lobules in normal thymus (Fig. 4). lobulated and tan. The epithelial cells were spindle shaped, The vast majority of lymphoid cells in all thymomas and the tumor consisted of cellular nodules of epithelial contained TdT, although the intensity of staining was cells with intermixed small lymphocytes separated by quite variable.
Table 3. T-cell and Subset Distributions in the Thymomas
Til OKT6 OKT3
T8 Anti-Leu-3a
Case 1
Case 2
Case 3
1.00 0.93 + 0.14 0.72 + 0.05 0.74 + 0.04 0.80 + 0.06
1.00 1.04 ±0.02 0.97 ±0.04 0.65 ± 0.02 0.93 ±0.02
1.00 0.94 ± 0.04 0.62 ± 0.01 0.48 ± 0.03 0.67 ± 0.01
Case 4 1.00
1.15 ±0.04 0.92 ± 0.09 0.75 ± 0.06 1.08 ±0.12
Results are expressed as a ratio of cells reacting with the panel of antibodies to Tl I positive cells. They represent the mean ± one standard error of the mean of the ratios obtained by three pathologists.
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FIG. 2. Frozen sections of thymomas stained with monoclonal antibodies to HLA-Dr and T-cell antigens: A. Anti-HLA-Dr; B. Anti-Tl 1; C OKT6; D. OKT3; E. anti-T8; F. anti-Leu-3a. The positive cells show a dark rim of reaction product. The proportion of cells positive for each antibody is listed in Table 2. Peroxidase-antiperoxidase (X250).
Discussion In our first three cases, the thymomas differed from each other in the proportions of epithelial cells and lymphocytes and the presence or absence of prominent perivascular spaces and microcystic change. The thymoma from case 4 had a distinctly different morphology. The diagnosis in all cases was confirmed by ultrastructural study. A number of investigators have shown that the lymphocytes infiltrating a thymoma are T-cells. 69 " The ability of the majority of these cells to form rosettes with sheep erythrocytes at 37°C and their binding of peanut agglutinin suggest that they are immature lymphocytes.9 The response of lymphocytes from thymoma to phytohemagglutinin (PHA) and concanavalin A (Con A) is generally much lower than that of mature peripheral blood lymphocytes. 2 ' 9 " Bhan and associates4 have shown that most of the cortical lymphocytes in normal thymus react strongly with the OKT6, OKT4 (anti-Leu-3a) and OKT8
(T8) antibodies and weakly with the OKT3 antibody, while the majority of the medullary lymphocytes, which are more mature cells, have lost their OKT6 reactivity and either OKT8 or OKT4 (anti-Leu 3a) reactivity. They also demonstrate a higher concentration of the OKT3 defined antigen. Reddick and Jenette 12 showed that the majority of lymphocytes in a cell suspension isolated from a thymoma reacted with the OKT6 antibody, suggesting that they have the cortical lymphocyte phenotype. We found that it is possible to quantitate the number of cells expressing a certain antigen in tissue sections with a fair degree of precision. Using this method in our immunohistochemical studies, we were able to demonstrate that almost all the lymphocytes expressed the OK.T6 antigen and TdT, regardless of the histologic appearance or the presence or absence of myasthenia gravis. Cells corresponding to mature thymic lymphocytes (OKT3+, OKT6—, TdT—) were almost completely absent. Many of the cells, however, showed a moderately strong reaction with OKT3 antibody, and about 25-50% of the cells
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reponsiveness of lymphocytes from thymomas of patients with and without associated myasthenia gravis. They found that lymphocytes from individuals with thymoma and myasthenia gravis gave higher responses. Since more mature lymphocytes show better response to mitogens than immature cells, this observation implies the presence of a higher percentage of more mature lymphocytes in myasthenic patients. Patient 2 in our series had mild localized myasthenia gravis, while patient 3 had a more severe generalized form. Like the other two cases, practically all the lymphocytes from the thymomas of these two patients bore the OKT6 defined antigen. The only marker that appeared to be significantly different in the thymomas of the myasthenic patients was the T8 defined antigen, which was found on a lower percentage of cells. If the lymphocytes in thymomas of myasthenic patients were functionally more mature than those of other thymomas, the loss of the T8 defined antigen might represent one of the first manifestations of maturation. More cases need to be studied before a firm conclusion can be drawn. In none of our cases did we see preferential localization of unique populations of cells, as defined by TdT or monoclonal antibodies, in particular regions of the tumor. This argues against differentiation of the thymoma into cortical and medullary areas.
FIG. 3. The anti-HLA antibody clearly is reacting with some of the epithelial cells. Many others are negative. A. Hematoxylin and eosin (X250). B. Peroxidase-antiperoxidase (X250).
showed no T8 defined antigen. A lesser percentage had lost its Leu-3-defined antigen (Table 2). These observations suggest that some of the cells had matured slightly beyond the normal cortical thymic lymphocyte stage. The almost constant association of neoplastic epithelium with cortical thymic lymphocytes suggests that the epithelial cells still retain certain functional characteristics and provide a microenvironment that can retain or perhaps attract immature T-cells. Baroni and associates2 studied the PHA and Con A
HLA-Dr antigens have been demonstrated on normal human thymic epithelium. In the thymomas we studied, it appeared that the number of cells bearing HLA-Dr antigen was smaller than the number of epithelial cells seen in the tissue sections stained with hematoxylin and eosin. This was especially evident in case 4, where many areas contained predominantly epithelial cells but only some of the cells reacted with anti-HLA-Dr antibody. This indicates heterogeneity in surface antigen expression. In the normal thymus, only the periphery of the lobules showed a positive reaction with anti-Leu-7. In our four thymomas, the staining pattern was highly variable. One of the cases (case 3) showed an extensive network of positivity. Case 2 showed a similar pattern of reaction but with fewer positive cells and less intense reaction. In both cases, the lining of the perivascular spaces showed a positive reaction. It has been shown ultrastructurally that the perivascular space in thymomas and the lobular margin in the normal thymus both are lined by epithelial cells and their processes.15 It is possible that this lining is the target for the anti-Leu-7 antibody. To confirm this hypothesis and to exclude the possibility that the reactivity was directed against basement membrane at these sites will require ultrastructural immunohistochemical studies. The general pattern of staining in cases 2 and 3, however, were also similar to the pattern of anti-HLA-Dr staining described in normal thymus, supporting the possibility
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FlG. 4. The reactivity of anti-Leu-7 against: A. normal thymus, note staining of the periphery of the lobule; B. case 1, note focal reactivity; C. case 3, note the diffuse linear-reticular pattern and prominent staining of the lining of the perivascular space; and D. case 4, note the focal staining and the accentuation at the margin of cellular nodules. Peroxidase-antiperoxidase (X250).
that anti-Leu-7 was directed against the epithelial cell component. The fact that only the lobular margin of the normal thymus was reactive suggests that anti-Leu-7 reacted only with a type of specialized epithelial cell, i.e., those cells forming a boundary between the thymic parenchyma and its surrounding fibrous tissue. This type of cell also may line the perivascular spaces in thymomas and form the boundary between large fibroepithelial bundles and more cellular tissue as seen in case 4. The fact that no Leu-7 positive epithelial cells were detected outside of the periphery of the lobules in normal thymus but could be demonstrated diffusely or focally in thymomas
indicates varying degrees of disorganization of epithelial cell architecture or disorderly differentiation. Many cases of pure red blood cell aplasia and primary acquired hypogammaglobulinemia are associated with thymoma. 15 It would be of interest to determine if the lymphocytes and epithelial cells in such instances show any unique characteristics. Immunohistochemical studies should help to provide some of this information. Acknowledgment. The authors thank Dr. Geraldine Pinkus for her advice in immunohistochemistry, Ms. Sherry Jacobs for typing the manuscript, Mr. Robert Santorianni, Mr. Willie Thomas, Ms. Dabo Brantley, and Ms. Donna Bassinger for technical assistance.
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1. Abo T, Balch CM: A differentiation antigen of human NK. and K cells identified by a monoclonal antibody (HNK-1). J Immunol 1981; 127:1024-1029 2. Baroni CD, Rigato P, Ruco L, Uccini S, Mineo T, Ricci C: PHA and Con A lymphocyte response in normal, hyperplastic and neoplastic human thymus: Morphologic and functional correlations. Cancer 1980; 46:2055-2061 3. Battifora H, Sun TT, Bahu RM, Rao S: The use of antikeratin antiserum as a diagnostic tool: Thymoma versus lymphoma. Hum Pathol 1980; 11:635-641 4. Bhan AK, Reinherz EL, Poppema S, McCluskey RT, Schlossman SF: Location of T-cell and major histocompatability complex antigens in the human thymus. J Exp Med 1980; 152:771-782 5. Evans RL, Wall DW, Platsoucas CD, et al: Thymus-dependant membrane antigens in man: Inhibition of cell-mediated lympholysis by monoclonal antibodies to TH2 antigen. Proc Natl Acad Sci (USA) 1981; 78:544-548 6. Griffin JD, Aisenberg AC, Long JC: Lymphocytic thymoma associated with T-cell lymphocytosis. Am J Med 1978; 64:10751079 7. Janossy G, Tidman N, Papageorgiou ES, Kung PC, Goldstein G:
8. 9. 10. 11. 12. 13. 14. 15.
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Distribution of T-lymphocyte subsets in the human bone marrow and thymus: An analysis with monoclonal antibodies. J Immunol 1981; 126:1608-1613 Lampson LA, Levy R: Two populations of la-like molecules on a human B cell line. J Immunol 1980; 125:293-299 Lauriola L, Maggiano N, Marino M, Carbone A, Piantelli M, Musiani P: Human thymoma: Immunologic characteristics of the lymphocytic component. Cancer 1981; 48:1992-1995 Loning Th, Caselitz J, Otto HF: The epithelial framework of the thymus in normal and pathological conditions. Virchows Arch [Pathol Anat] 1981;392:7-20 Pedraza MA: Thymoma immunological and ultrastructural characterization. Cancer 1977; 39:1455-1461 Reddick RL, Jennette JC: Immunologic and ultrastructural characterization of the small cell population in malignant thymoma. Hum Pathol 1983; 14:377-380 Reinherz EL, Schlossman SF: The differentiation and function of human T-lymphocytes. Cell 1980; 19:821-827 Reinherz EL, Schlossman SF: Human T-lymphocyte differentiation. Immunology Today 1982; 3:237a Rosai J, Levine GD: Tumors of the thymus. Second Series, Fasicle 13, Armed Forces Institute of Pathology. Washington, D.C., 1976
