Asian Cardiovasc Thorac Ann 2005;13:149-152
© 2005 Asia Publishing EXchange Ltd
Rearrangement of T-Cell Receptor Beta and Gamma Genes in Thymoma
Jun Nakajima, MD,
Jun Matsumoto, MD,
Eriho Takeuchi, MD,
Takeshi Fukami, MD,
Shinichi Takamoto, MD
Department of Cardiothoracic Surgery, Faculty of Medicine, University of Tokyo, Tokyo, Japan
For reprint information contact: Jun Nakajima, MD Tel: 81 3 3815 5411 Fax: 81 3 5684 3989 Email: nakajima-tho{at}h.u-tokyo.ac.jp, Department of Cardiothoracic Surgery, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan.
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ABSTRACT
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To investigate the differentiation stage of tumor-infiltrating lymphocytes in thymoma tissue, we performed Southern blot analysis of T-cell receptor beta and gamma genes in thymomas resected from 19 patients. At the same time, we conducted flow cytometric analysis of T-cell surface markers and examined the clinicopathological features of the thymomas. We found that the incidence of T-cell receptor gamma gene rearrangement was significantly higher in Masaoka stage I thymomas (11 of 12 cases) than in stage II or III invasive thymomas (3 of 7 cases). Moreover, gamma gene rearrangement was observed in all 10 type AB and B1 thymoma specimens and in 4 of 6 type B2 thymoma specimens. The 2 specimens of type B3 thymomas, which were classified as stage III, showed neither gamma nor beta gene arrangement and were single-positive for CD4 or CD8. Six thymoma specimens that showed beta gene rearrangement expressed both CD4 and CD8. In conclusion, thymomas have the capability of T-lineage cell differentiation, except for a subset of invasive thymomas with malignant characteristics.
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INTRODUCTION
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The tumor-infiltrating lymphocytes (TIL) in thymomas are immature T-lineage cells expressing both CD4 and CD8.1 We examined the rearrangement of T-cell receptor (TCR) beta and gamma genes in order to investigate the stage of differentiation of these TIL, and we also analyzed the relationship between the degree of malignancy and the extent of TCR gene rearrangement.
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PATIENTS AND METHODS
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Thymoma tissue specimens were obtained from 19 patients who had undergone extended thymectomy involving en bloc resection of the thymoma, the thymus, and the anterior mediastinal tissue around the thymus. Informed consent was given by the patients. To evaluate the degree of malignancy, the resected specimens were fixed in a 10% formaldehyde solution, embedded in paraffin, sectioned, and then stained with hematoxylin and eosin. They were then examined microscopically to determine their histological typing according to the World Health Organization classification.2 The thymomas were also subclassified according to Masaoka’s clinicopathological staging system.3
To determine the occurrence of TCR gene rearrangement, Southern blot analysis4 was performed as follows: DNA was extracted from lymphoid cells separated from the thymoma tissue, digested with restriction enzymes BamHI or HindIII, and separated by gel electrophoresis. After denaturation with sodium hydroxide and transfer to blotting paper, the blots were incubated with a labeled probe for the TCR beta gene C region or the gamma gene J region. If monoclonal bands were observed, besides germline bands, gene rearrangement was considered to have occurred in the DNA of the specimen.
To investigate T-cell differentiation, flow cytometric analysis of lymphoid cells in the thymoma tissue was performed as described elsewhere.5 Briefly, a neoplastic section of the resected tissue was minced in phosphate-buffered saline. The suspension was filtered through gauze to eliminate large particles, and lymphoid cells were separated by density-gradient centrifugation. The lymphoid cells were then stained with fluorescence-labeled monoclonal antibodies for CD4 and CD8. A FACSCalibur system was used for 2-color flow cytometry, and the 2- or 3-dimensional data generated were analyzed using CellQuest software. The monoclonal antibodies, flow cytometry system, and software were obtained from Becton, Dickinson and Co. (San Jose, CA, USA).
Differences in the incidence of TCR gamma gene rearrangement among the histologic subgroups were analyzed by the chi-squared test. Differences were considered significant when p < 0.05.
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RESULTS
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The thymomas from 12 of the 19 patients studied were classified as Masaoka stage I, 2 were stage II, and 5 stage III. One of the specimens was pathologically determined as thymoma type A, 4 as type AB, 6 as type B1, 6 as type B2, and 2 as type B3.
TCR gamma gene rearrangement was observed in 14 cases, shown by the monoclonal bands that appeared on the Southern blot. Among the 12 stage I thymomas, gamma gene rearrangement was found in 11 (92%). This incidence is much higher ( p = 0.02) than in the invasive stage II and III thymomas (3 of 7 cases, or 43%). In terms of histological classification, all 10 of the type AB and B1 thymomas and 4 of the 6 type B2 thymomas showed TCR gamma gene rearrangement. Rearrangement was not seen in the 1 type A thymoma and the 2 type B3 thymomas (types A, AB, and B1 vs. types B2 and B3, p < 0.05).
In 3 cases of stage I thymoma, 1 case of stage II, and 4 cases of stage III, we also examined TCR beta gene rearrangement and the expression of CD4 and CD8 on the cell surface. The results are shown in Table 1
. Beta gene rearrangement was observed in 6 of the 8 cases (Figure 1), 5 of whom also showed gamma gene rearrangement (Figure 2). The 2 cases of type B3 thymoma, both classified as stage III, showed neither beta nor gamma gene rearrangement, and their TIL were single-positive for either CD4 or CD8. The other 6 thymomas, which showed beta gene rearrangement, expressed both CD4 and CD8 on their TIL.
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Table 1. Patients’ Demographic Data, Thymoma Classification, and Gene Rearrangement and Expression of Surface Markers of T-Cell Receptors
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Figure 1. Southern blot analysis of germline DNA (Cont.) and DNA from tumor-infiltrating lymphocytes (Exp.) after digestion with BamHI and hybridization with a C beta 1 probe. Specimens 1 to 6 show a monoclonal band around 12 kb.
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Figure 2. Southern blot of germline DNA (Cont.) and DNA from tumor-infiltrating lymphocytes (Exp.) after digestion with HindIII (upper row) and BamHI (lower row) and hybridization with a J gamma probe. Specimens 1, 2, 3, 5 and 6 show monoclonal bands.
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DISCUSSION
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A thymoma is a neoplasm of the thymic epithelium with characteristic histological features. It exhibits varying degrees of malignancy, from a benign neoplasm characterized by a well-encapsulated tumor to a malignant one showing direct invasion of adjacent organs, dissemination in the pleural or pericardial cavity, and lymphogenous or hematogenous metastasis.6 The thymoma is relatively unique among tumors because its prognosis appears to be more closely related to invasive characteristics seen during surgery than to its histological appearance.7
The phenotypes of the lymphoid components in the thymoma have been reported to be immature and similar to those of normal thymocytes, which are mainly immature T-lineage cells. In contrast, the lymphoid cells infiltrating thymic carcinomas have been thought to be mature lymphocytes on the basis of immunohistochemical staining.1 Both encapsulated and invasive thymomas contain lymphoid cells in varying proportions. These lymphoid cells have been studied immunohistopathologically, and most of them are immature T-lineage cells characterized by the co-expression of CD4 and CD8, as in normal thymocytes.8 This is consistent with flow cytometry results which show that the majority of these cells express both CD4 and CD8. Thymomas also contain cell subsets expressing CD10 on their surface along with CD4 and CD38, which indicates that they are very immature pre-T cells.9
We previously reported that there was a subset of thymomas that did not contain CD4 and CD8 double-positive TIL but did contain CD4 or CD8 single-positive TIL.5 The neoplastic epithelium of the thymomas in this subset showed low-grade malignant features pathologically and clinically, suggesting the existence of types in between "thymoma" and "thymic cancer".
The TIL in thymoma tissue are derived from stem cells in the bone marrow. Thymoma tissue, like normal thymic tissue, may attract these cells and force them to differentiate to a certain extent. In the early stages of T-cell differentiation, rearrangement of TCR genes is a mandatory process that endows T-lineage cells with a diverse repertoire.10 T-cells must make productive rearrangements at the alpha- and beta-chain loci to produce alpha-beta T-cells, or at the gamma- and delta-chain loci to produce gamma-delta T-cells. According to the hierarchical theory of T-cell development,11 gene rearrangement starts with the delta and gamma chains followed by the beta and alpha chains. In the beta-chain locus, this process occurs via a hierarchical series of recombinations, the D (diversity) and J (joining) segments and then the V (variable) and D segments.
Pizer and colleagues12 reported that the monoclonal bands observed in the Southern blot analysis of TCR beta genes probably represent an intermediate deletion joining D beta 1 to a J beta 2 gene segment. They observed partial rearrangement of TCR beta genes, with linkage of the D and J segments, in thymoma. Linkage between the V and D segments was lacking, which indicates the accumulation of nonfunctioning T-lineage cells in thymus tissue.
In the present study, monoclonal bands were observed on Southern blot analysis with a gamma gene probe, suggesting that partial rearrangement of TCR gamma genes also occurred in thymoma. However, we did not find TCR gamma or beta gene rearrangement in stage III invasive thymomas that were histologically determined as type B3. Either CD4 or CD8 was expressed on the cell surface. These characteristics are identical to those of mature post-thymic T-cells.
In thymoma tissue, T-lineage cells are assumed to be on a normal differentiation path and should not be converted into neoplastic cells. According to the hierarchical sequence of TCR gene rearrangement, gamma gene rearrangement occurs at the immature stage of T-lineage cells before positive or negative selection in thymic tissue. The appearance of clonal bands on Southern blot analysis of TCR beta and gamma genes implies that the TIL in thymomas experience partial gene rearrangement just before the connection between the DJC complex and a locus of the V region. We conclude that most thymomas have the capability of making T-lineage cells differentiate, except the invasive stage III, type B3 thymoma, which contains only CD4 and CD8 single-positive cells.
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REFERENCES
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ABSTRACT
INTRODUCTION
