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*Gene*GEO Profiles
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*Thymus Cancer
The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 5 2309-2317
Copyright © 2003 by The Endocrine Society

Sex Steroid Hormone Receptors in Human Thymoma

Hironori Ishibashi, Takashi Suzuki, Satoshi Suzuki, Takuya Moriya, Chika Kaneko, Touichirou Takizawa, Makoto Sunamori, Masashi Handa, Takashi Kondo and Hironobu Sasano

Department of Pathology, Tohoku University School of Medicine (H.I., T.S., T.M., C.K., H.S.); Department of Thoracic Surgery, Institute of Development, Aging, and Cancer, Tohoku University (S.S., T.K.); and Department of Surgery, Sendai Kousei Hospital (M.H.), Sendai 980-8575, Japan; and Departments of Pathology (T.T.) and Thoracic Cardiovascular Surgery (H.I., M.S.), Graduate School, Tokyo Medical and Dental University, Tokyo 113-0034, Japan

Address all correspondence and requests for reprints to: Hironori Ishibashi, M.D., Department of Pathology, Tohoku University School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi Prefecture, 980-8575, Japan. E-mail: hishiba{at}kf6.so-net.ne.jp.


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
In this study we examined the immunohistochemical localization of sex steroid receptors for estrogen {alpha} (ER{alpha}) and ERß, progesterone-A (PR-A) and PR-B, and androgen (AR) in human thymoma (n = 132) and correlated these findings with various clinicopathological parameters. We used RT-PCR and real-time PCR to further study the expression of these receptors in 20 thymoma cases. Immunoreactivity for all sex steroid receptors was detected in the nuclei of thymoma epithelial cells. The percentage of immunopositive cases and the H-score values for each receptor (mean ± SD) were: ER{alpha}, 66% and 85.8 ± 80.2; ERß, 7% and 7.2 ± 8.7; PR-A, 4% and 2.7 ± 4.9; PR-B, 49% and 55.8 ± 68.3; and AR, 15% and 14.1 ± 11.7, respectively. The results of real-time PCR were consistent with those of immunohistochemistry, especially results for ER{alpha}, PR-B, and AR. A significant positive correlation was detected between immunoreactivity for ER{alpha} and PR-B. ER{alpha} immunoreactivity was inversely correlated with tumor size, clinical stage, WHO classification, and Ki-67 labeling index. In addition, the status of ER{alpha} immunoreactivity was significantly associated with a better clinical outcome in thymoma patients. Results from our study suggest that estrogens may inhibit thymoma growth via ER{alpha}, and that ER{alpha} immunoreactivity may act as a prognostic factor in human thymoma.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
IT IS WELL known that sex steroids, such as estrogen, progesterone, and androgen, exert a wide variety of effects on target tissues in humans (1). The biological effects of sex steroid hormones are, in general, mediated through initial interactions with their native receptors. These receptors, including estrogen receptor {alpha} (ER{alpha}) (2), ERß (3), progesterone receptor-A (PR-A), PR-B (4), and androgen receptor (AR), belong to a family of nuclear hormone receptors (1, 5). Steroid receptors are generally known to function as dimers and to activate transcription in a ligand-dependent manner by binding to the responsive elements located in the promoter region of various target genes (5). A better understanding of steroid actions in normal and neoplastic human tissues can be obtained by analyzing the expression of specific steroid receptors in these tissues.

In a study by Anser-Ahmed et al. (6), sex steroid hormones were shown to have important roles in regulating the function of the rat thymus. Estrogen has also been reported to be involved in the processes of development and atrophy of the mouse thymus (7, 8, 9). In addition, ER was detected in the homogenates of mature human thymus (10, 11), and recent investigations using ER knockout mice have indicated that the growth and maturation of the thymus were at least in part under the regulation of estrogen (7, 8, 9). Considering that sex steroids regulate cell proliferation not only in normal tissues but also in various neoplasms derived from hormone- dependent tissues, such as breast and prostate (12, 13), it is reasonable to postulate that sex steroids may be involved in the development of human thymomas. However, the status of sex steroid receptors has not been examined in human thymomas, and thus, the biological significance of sex steroid actions remains unknown in human thymoma tissues. Therefore, in the present study we studied the immunolocalization of ER{alpha}, ERß, PR-A, PR-B, and AR in 132 cases of human thymoma tissues and correlated these findings with various clinicopathological parameters. Using RT-PCR and real-time PCR, the gene expression of these receptors was also analyzed in 20 cases of thymoma to further characterize these receptors in human thymoma.


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
Patients and tissue specimens

One hundred thirty-two cases of thymoma were retrieved from surgical pathology files at Sendai Kosei Hospital (Sendai, Japan) and Tokyo Medical and Dental University (Tokyo, Japan). All specimens were fixed for 24 h in 10% formalin and embedded in paraffin wax. The clinical data, including patient age, sex, menopausal status, the presence or absence of myasthenia gravis, tumor size, clinical stage (14), and WHO classification (15), are summarized in Table 1AGo.


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  		Table 1A. The summary of clinical data in 132 patients with thymoma examined in this study

 
Freshly frozen specimens were also available for RT-PCR analysis and real-time PCR in 20 cases (Table 1BGo). Specimens for RNA isolation were immediately frozen in liquid nitrogen and stored at -80 C until used for RNA isolation. RNA was extracted within 2 wk after surgery.


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  		Table 1B. The summary of clinical data in 20 patients with thymoma for quantitative PCR

 
The research protocols used in this study were approved by the ethics committee at Tohoku University School of Medicine, Sendai Kosei Hospital, and Tokyo Medical and Dental University School of Medicine.

Antibodies

The antibodies used in this study are summarized in Table 2Go. Antibodies for the two isoforms of PR, PR-A (hPRa7) and PRB (hPRa2), were purchased from NeoMarkers Co. Ltd. (Fremont, CA). These monoclonal antibodies were raised in a mouse against PR isoforms obtained from a human endometrial carcinoma (EnCa 101). Clarke et al. (16) reported that the hPRa7 antibody recognizes both PR-A and PR-B in immunoblot analysis. However, using immunohistochemistry, Mote et al. (17) reported that hPRa7 did not recognize PR-B in fixed tissues even after antigen retrieval, as evidenced by the absence of immunostaining by this antibody in the PR-B-expressing MDA-MB-231/PR-B cell line. This is believed to be due to the inaccessibility of the epitope on PR-B recognized by hPRa7 in 10% formalin-fixed and paraffin-embedded tissue specimens (17).


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  		Table 2. Characteristics of primary antibodies used in immunohistochemistry

 
Immunohistochemistry

Immunostaining was performed by using the streptavidin-biotin amplification method using a Histofine Kit (Nichirei Co. Ltd., Tokyo, Japan) for ER{alpha}, PR-A, PR-B, AR, and Ki-67, and EnVision+ (DAKO Corp., Carpinteria, CA) for ERß. Sections were heated in an autoclave at 120 C for 5 min in citric acid buffer (2 mM citric and 9 mM trisodium citrate dehydrate, pH 6.0) after deparaffinization for antigen retrieval. The slides were incubated with primary antibodies for 12–18 h in a moist chamber at 4 C. The dilutions of primary antibodies used in our study are summarized in Table 2Go. The antigen-antibody complex was subsequently visualized with 3,3'-diaminobenzidine solution [1 mM 3,3'-diaminobenzidine, 50 mM Tris-HCl buffer (pH 7.6), and 0.006% H2O2] and counterstained with hematoxylin. Tissue sections used as positive controls in this study were as follows: breast cancer for ER{alpha}, nonpathological breast for ERß, endometrium for PR-A and PR-B, and prostate for AR. As a negative control for monoclonal antibody staining, normal mouse IgG was used instead of the primary antibodies. No specific immunoreactivity was detected in these sections. ERß (Upstate Biotechnology, Inc., Lake Placid, NY) is a polyclonal antibody raised in rabbits. An 18-amino acid peptide from the N terminus of ERß (amino acids 48–65) was made by solid phase synthesis on a multiple antigenic peptide backbone (Protein and Carbohydrate Structure Facilities, University of Michigan, Ann Arbor, MI). This antibody was characterized and reviewed for specificity using immunohistochemistry, including the preabsorption test and immunoblotting by Mitchner et al. (18). Because this antibody was unavailable, we used a normal rabbit IgG antibody instead of the primary antibody as a negative control for immunohistochemistry of ERß. No specific immunoreactivity was detected in these tissue sections.

Scoring of immunoreactivity

For semiquantitative analysis of immunoreactivity of steroid receptors, H-score (19) was used in this study. Briefly, more than 500 tumor cells were counted in each case, and the H-score was subsequently generated by adding the percentage of strongly stained nuclei (3x), the percentage of moderately stained nuclei (2x), and the percentage of weakly stained nuclei (1x), giving a possible range of 0–300. The score was independently obtained by two of the authors (H.I. and T.S.) after determining the areas of immunostained slides using a double-headed light microscope. In the present study interobserver differences were less than 5%, and the mean of the two values was obtained. Cases that were found to have an H-score more than 50 were noted as steroid receptor-positive thymomas, according to a report by Thike et al. (20).

Ki-67 immunoreactivity was scored in more than 500 tumor cells for each case, and the percentage of immunoreactivity regardless of the immunointensity, i.e. labeling index (LI), was determined in the manner described for steroid receptor immunoreactivity. Ki-67 LI indicates proliferative activity in various neoplastic tissues, including thymoma (21).

RT-PCR and real-time PCR

RT-RCR and real-time PCR analysis for ER{alpha}, ERß, PR-A, PR-B, or AR was performed in 20 cases of thymoma. Total RNA was extracted from frozen samples of thymoma with TRIzol reagent, according to the manufacturer’s instructions (Life Technologies, Inc.-BRL, Grand Island, NY). RNA concentrations were determined spectrophotometrically, and all RNA samples were stored at -80 C until used for cDNA synthesis. An RT kit (SuperScript Preamplification system, Life Technologies, Inc.-BRL) was used in the synthesis and amplification of cDNA.

Total RNA (5 µg) was denatured at 70 C for 10 min and then reverse transcribed in the presence of 25 ng/µl oligo(deoxythymidine) primer (Life Technologies, Inc., Tokyo, Japan), 2.5 mM MgCl2, 0.5 mM deoxy-NTPs, 10 mM dithiothreitol, and 10 U ribonuclease H- reverse transcriptase (SuperScript II RT, Life Technologies, Inc.) for 60 min at 42 C, 15 min at 50 C, and 15 min at 70 C. Subsequently, 1 µl of the resulting cDNA was used as a template for PCR. The resulting cDNA was used as a template for real-time PCR. Real-time PCR was carried out with the Light Cycler System (Roche, Mannheim, Germany) using the DNA binding dye Syber Green I (Roche) for the detection of PCR products. PCR was set up using 3 mM MgCl2, 10 pmol/liter of each primer (Table 3Go), and 2.5 U Taq DNA polymerase (Life Technologies, Inc.). An initial denaturing step of 95 C for 1 min was followed by 40 cycles, respectively, of 95 C for 0 sec; 15-sec annealing at 58 C (PR-AB, PR-B, and AR), 60 C [ERß and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)], and 62 C (ER{alpha}); and extension for 15 sec at 72 C. The fluorescence intensity of the double-strand specific Syber Green I, which reflects the amount of specific PCR products formed, was read by the LightCycler at 85 C after the end of each extension step. After PCR, the products were resolved on a 2% agarose ethidium bromide gel. Images were captured with Polaroid (Hertfordshire, UK) film under UV light. In initial experiments PCR products were purified and subjected to direct sequencing (ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit and ABI PRISM 310 Genetic Analyzer, PerkinElmer Corp., PE Applied Biosystems, Foster City, CA) to verify amplification of the correct sequences.


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  		Table 3. Oligonucleotide primer sequences used for RT-PCR

 
As a positive control, the MCF7 breast cancer cell line was used for ER{alpha} (23), and the T47D breast cancer cell line was used for ERß (23), PR-AB (24), and PR-B (25). Testis was used as a positive control for AR. Negative control experiments lacked the cDNA substrate to check for the possibility of exogenous contaminating DNA. No amplified products were observed under these conditions.

The mRNA levels for ER{alpha}, ERß, PR-AB, PR-B, and AR in each case are summarized as a ratio of GAPDH and evaluated as a ratio (percentage) compared with that of each positive control. Conventional quantitative PCR requires utilization of a purified plasma cDNA in the construction of a standard curve, but PCR products could be semiquantitated with the LightCycler using purified cDNA of known concentrations (26, 27).

Statistical analysis

Statistical analyses among patient age, tumor size, Ki-67 LI, H-scores, and steroid receptor mRNA levels were performed using a correlation coefficient (r) and a regression equation. Statistical differences between the H-score for steroid receptors and sex, status of myasthenia gravis, clinical stage, or WHO classification were evaluated using Mann-Whitney and Kruskal-Wallis tests (28). Overall survival curves were generated according to the Kaplan-Meier method (29), and statistical significance was calculated using the log-rank test (30). Univariate and multivariate analyses were evaluated by a proportional hazard model (COX) using PROC PHREG in our SAS software. P value less than 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
Immunohistochemical expression of sex steroid receptors

ER{alpha}, ERß, PR-A, PR-B, and AR immunoreactivity was detected predominantly in the nuclei of epithelial cells of thymoma (Fig. 1Go, A and B), but not in lymphocytes, except for ERß (Fig. 1Go, C and D). The number of positive cases and the H-score value (mean ± SD) for each receptor in 132 cases of thymoma were as follows: ER{alpha}, 87 (66%) and 85.8 ± 80.2; ERß, 9 (7%) and 7.2 ± 8.7; PR-A, 5 (4%) and 2.7 ± 4.9; PR-B, 65 (49%) and 55.8 ± 68.3; and AR, 20 (15%) and 14.1 ± 11.7, respectively (Fig. 2AGo). There was a significant positive correlation between the immunoreactivity for ER{alpha} and PR-B (r = 0.50; P < 0.0001; Fig. 2BGo), but no other significant correlations were detected among the findings for sex steroid receptor immunoreactivity in this study.



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  		Figure 1. Immunohistochemistry for ER{alpha} (A and C) and PR-B (B and D) in tissue specimens of human thymoma. Immunoreactivity for ER{alpha} and PR-B was detected in the nuclei of thymoma epithelial cells, but not in lymphocytes. A and B, Epithelial cell dominant type, which is type A by WHO classification (same field); C and D, lymphocyte dominant type, which is type B1 by WHO classification (same field). Original magnification, x400. Bar, 25 µm.

 


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  		Figure 2. A, Scattered plots showing variation in the H-score for ER{alpha}, ERß, PR-A, PR-B, and AR in 132 human thymomas. The average H-score for each receptor and the percentage of positive cases are also shown. B, Correlation between the H-score for ER{alpha} and PR-B in 132 cases of thymoma. A significant correlation (r = 0.50; P < 0.0001) was detected. y = 19.45 + 0.42x; r2 = 0.25.

 
Real-time PCR analysis

In the present study mRNA expression for ER{alpha}, ERß, PR-AB, PR-B, AR, and GAPDH was detected as a specific single band at 168, 225, 196, 196, 195, and 307 bp, respectively (data not shown). The clinical data for the 20 patients included in our PCR analyses, including sex, age, tumor size, the presence or absence of myasthenia gravis, WHO classification, and clinical stage, are summarized in Table 1BGo.

A significant positive correlation was detected between the immunoreactivity (ir) for ER{alpha} and ER{alpha} mRNA levels (r = 0.83; P < 0.0001; Fig. 3AGo), irPR-B and PR-B mRNA levels (r = 0.78; P < 0.0001; Fig. 3BGo), or irAR and AR mRNA levels (r = 0.72; P = 0.00030). There were no significant positive correlations between the immunoreactivity for ERß and ERß mRNA levels (r = 0.032; P = 0.21), irPR-A and PR-AB mRNA levels (r = 0.28; P = 0.13), or irPR-B and PR-AB mRNA levels (r = 0.33; P = 0.091).



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  		Figure 3. Correlation between the H-score and the mRNA level of ER{alpha} or PR-B. Real-time PCR analysis was performed for ER{alpha}, ERß, PR-A, PR-B, and AR in 20 cases of thymoma. A, Correlation between the H-score of ER{alpha} and mRNA level of ER{alpha} (r = 0.83; P < 0.0001; y = 0.14 + 0.005x; r2 = 0.70). B, Correlation between the H-score of PR-B and the mRNA level of PR-B (r = 0.78; P < 0.0001; y = 0.022 + 0.001x; r2 = 0.61).

 
Correlation between immunoreactivity for sex steroid receptors and clinicopathological parameters

The association between ER{alpha} immunoreactivity and clinicopathological factors for thymomas is summarized in Table 4Go. There was an inverse correlation between ER{alpha} immunoreactivity and tumor size (r = 0.28; P = 0.0010), clinical stage (P < 0.0001), WHO classification (P = 0.015), and Ki-67 LI (r = 0.43; P < 0.0001; Fig. 4AGo). However, in this study there was no significant relationship between ER{alpha} immunoreactivity and sex, patient age, menopausal status, and/or status of myasthenia gravis.


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  		Table 4A. Correlation between ER{alpha} immunoreactivity and pathological parameters in 132 human thymomas

 


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  		Figure 4. Correlation between the H-score for ER{alpha} (A) or PR-B (B) and Ki-67 LI in 132 cases of thymoma. ER{alpha} and PR-B immunoreactivity was inversely correlated with Ki-67 LI (for ER{alpha}: r = 0.43; P < 0.0001; for PR-B: r = 0.38; P < 0.0001; for ER{alpha}: y = 2.21 + 0.005x; r2 = 0.18; for PR-B: y = 2.07 + 0.005x; r2 = 0.14).

 
The association between PR-B immunoreactivity and clinicopathological factors for thymomas is summarized in Table 4BGo. PR-B immunoreactivity was inversely correlated with clinical stage (P = 0.00030), WHO classification (P = 0.028), and Ki-67 LI (r = 0.38; P < 0.0001; Fig. 4BGo).


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  		Table 4B. Correlation between PR-B immunoreactivity and pathological parameters in 132 human thymomas

 
In the present study there was no significant correlations between immunoreactivity for other steroid receptors and clinicopathological parameters.

Correlation between immunoreactivity for sex steroid receptors and overall survival of patients with thymoma

There was a significant positive correlation between ER{alpha} immunoreactivity and clinical outcome (P = 0.00010; Fig. 5AGo). No significant correlations were detected between the immunoreactivity for other steroid receptors and clinical outcome in patients with thymoma (Fig. 5BGo). In our female patients there was a significant positive correlation between ER{alpha} immunoreactivity and clinical outcome (P = 0.00060; Fig. 5DGo) and between PR-B immunoreactivity and clinical outcome (P = 0.048; Fig. 5FGo), but there were no significant correlations between ER{alpha} immunoreactivity (P = 0.13; Fig. 5CGo) or PR-B immunoreactivity (P = 0.21; Fig. 5EGo) and clinical outcome in male patients with thymoma. After univariate analysis (Table 5Go), clinical stage (P = 0.0017), ER{alpha} immunoreactivity (P = 0.0021), and tumor size (P = 0.0024) were demonstrated to be significant prognostic factors for overall survival in 132 thymoma patients. A multivariate analysis revealed that only clinical stage (P = 0.024) and ER{alpha} immunoreactivity (P = 0.036) were independent prognostic factors with relative risks over 1.0 in our series.



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  		Figure 5. Overall survival of 132 patients with thymoma with respect to ER{alpha} (A) or PR-B (B) immunoreactivity (Kaplan-Meier method). ER{alpha} immunoreactivity was significantly associated with an improved overall survival (P = 0.00010); however, PR-B immunoreactivity was not significantly associated with overall survival (P = 0.47). There was a significant positive correlation between ER{alpha} immunoreactivity and clinical outcome (P = 0.00060; Fig. 5DGo) in female patients, but no significant correlations in male patients (P = 0.13; Fig. 5CGo). Furthermore, a significant positive correlation between PR-B immunoreactivity and clinical outcome (P = 0.048; Fig. 5FGo) was found in female patients, but no significant correlations were identified in male patients (P = 0.21; Fig. 5EGo).

 

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  		Table 5. Univariate and multivariate analyses of overall survival in 132 thymoma patients examined

 

   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
This is the first study demonstrating a correlation between the expression of sex steroid receptors and clinicopathological parameters in human thymoma. Various sex steroid receptors were found to be expressed predominantly in the epithelial cells of human thymoma. ER{alpha} immunoreactivity was detected in the great majority (66%) of human thymoma epithelial cells examined in our study, whereas ERß immunoreactivity was detected in only 7% of the cases examined. Kawashima et al. (31) described the immunolocalization of ER and PR in epithelial cells of the rat thymus. A study using ER{alpha} knockout mice demonstrated that estrogen-ER{alpha} interaction was essential for the normal development and maturation of the thymus (9). In addition, estrogens have been suggested to regulate the production of thymosine {alpha}, a hormonal factor released from thymic epithelial cells (32). The relatively wide distribution of ER{alpha} immunoreactivity in human thymoma described in the present report appears to suggest the importance of estrogenic actions in human thymoma via ER{alpha}. Seiki and colleagues (33) demonstrated that estrogens could influence the T lymphocyte population in the thymus of ovariectomized female rats. However, in our study ER{alpha} immunoreactivity was not detected in lymphocytes, although ERß was detected in lymphocytes in 10 cases (8%). Thymosine {alpha}, which is in part regulated by estrogen, is involved in immune modulations, including T lymphocyte differentiation (34). Recently, Tornwall et al. (35) reported that ER{alpha} was detected in all lymphocytes in the mouse thymus. Moreover, ERß was shown to be expressed at low levels in thymic CD4-/CD8- T cells in the mouse thymus (35). Carbone et al. (36) examined the estrogen binding capacity of peripheral blood mononuclear cells and lymphocytes from two young female patients with myasthenia gravis and reported high levels of estrogen-binding sites in both prethymectomy peripheral blood mononuclear cells and lymphocytes from the hyperplastic thymus of myasthenia gravis patients. Therefore, differences from the results of our present study may represent differences between human and mouse species or between the neoplastic and nonneoplastic thymus. It is not clear at present what causes these changes; therefore, further investigations are required to clarify these discrepancies.

In the present study ER{alpha} immunoreactivity was inversely correlated with Ki-67 LI (P < 0.0001) and tumor size (P = 0.0010). Using the WHO classification, ER{alpha}-positive thymomas were found to be expressed at earlier clinical stages (P < 0.0001) with lower histological grades (P = 0.015). Moreover, patients with ER{alpha}-positive thymoma showed significantly better clinical outcome than those with ER{alpha}-negative thymoma (P = 0.00010). In addition to clinical stage, a well established diagnostic modality in human thymoma (14), the results of both univariate and multivariate analysis in our study demonstrated that ER{alpha} immunoreactivity is an independent prognostic factor (P = 0.0021 and P = 0.036, respectively) for human thymoma. Estrogen is well known to exert a wide variety of effects in target organs, including cell proliferation in breast cancer (12) and inhibition of cell growth in prostatic cancer (37) or vascular smooth muscle cells (38). In a study by Ezaki and co-workers (39), thymomas in males were reported to be larger than those of females in age-matched BUF-Mna rats. Furthermore, Ezaki’s study (39) indicated that estrogen inhibited or retarded the development of spontaneous thymomas in BUF/Mna rats. The results from our present study are in good agreement with these published findings. These results all appear to suggest that estrogen may inhibit cell proliferation in human thymoma. ER{alpha}-positive thymoma may also be associated with increased estrogenic actions resulting in an improved prognosis.

On the other hand, there was a significant positive correlation between the immunoreactivity for ER{alpha} and PR-B (r = 0.50; P < 0.0001). PR, which is coexpressed with ER, has been regarded as one of the markers of functional estrogen pathways, i.e. transcription of the PR-B gene is enhanced and maintained by estrogens (12, 40). Therefore, PR-B status in human thymoma may also reflect estrogenic actions in human thymoma tissues, but other possibilities also exist concerning the biological significance of PR-B status in human thymoma. We examined survival data from patients in the present study according to the status of ER{alpha} and PR-B, separately in male and female patients diagnosed with thymoma. There was a significant positive correlation between ER{alpha} immunoreactivity and clinical outcome (P = 0.0018; Fig. 5DGo) and between PR-B immunoreactivity and clinical outcome (P = 0.048; Fig. 5FGo) in female patients, but no such significant correlations were detected in male patients with thymoma. These findings also suggest that the expression of ER{alpha} may represent a marker associated with better clinical outcome, especially in female patients with thymoma. PR-B status in human thymoma may also reflect estrogenic actions in thymoma tissues, but further investigations are required to demonstrate the presence of direct estrogenic actions in human thymoma.

The combination ER{alpha} analysis in resected surgical pathology specimens by immunohistochemistry and recently developed selective ER modulators has been considered for the diagnosis and treatment, respectively, of patients with advanced thymoma, especially females. However, further investigations are required to clarify the role of ER in the diagnosis, prevention of disease progression, and/or pathogenesis.

In this study immunoreactivity for PR-B was dominant in human thymomas (49%) compared with that for PR-A (4%). To date, two different isoforms of PR, PR-A (81–83 kDa) and PR-B (116–120 kDa), have been identified (4). The exact roles and functions of each receptor isoform remain unclear, but PR-B is believed to be necessary for the activation of progesterone target genes (4), whereas PR-A functions as a repressor of PR-B function (41). Transcription of the PR-B gene is enhanced and maintained by estrogen. PR, which is coexpressed with ER, has been regarded as one of the markers of functional estrogen pathways (12, 40). Therefore, PR-B status in human thymoma may also reflect estrogenic actions in human thymoma tissues. However, further investigations are required to clarify the role of PR-B in human thymoma.

In this study AR immunoreactivity was detected in only 15% of human thymomas, and the status of AR was by no means significantly correlated with any clinicopathological parameters examined in this study. Androgenic action in the thymus is still very controversial. Castration of normal male rodents results in significant enlargement of the thymus (42). Furthermore, a study by Nancy et al. (42) reported that AR immunolocalization in the thymic epithelium and androgens modulated the size of the thymus and thymocyte development in the male mouse. Dulos et al. (43) found that indirect actions of androgen could accelerate the apoptosis of thymocytes in the mouse model. However, Ezaki et al. (39) reported that androgens were not involved in spontaneous thymoma tumor development in BUF/Mna rats. In this study we did not investigate the role of androgens in human thymoma tissues. Further studies are required to clarify the role that androgens play in the pathogenesis of human thymoma.

In summary, we have characterized the expression of various sex steroid receptors in human thymoma. We found ER{alpha} and PR-B immunoreactivity to be widely distributed in thymoma epithelial cells. ER{alpha} and PR-B immunoreactivity was inversely associated with tumor size, clinical stage, WHO classification, and Ki-67 LI; moreover, ER{alpha} was found to be an independent prognostic variable in multivariate analysis. Furthermore, the results from our present study suggest that estrogen inhibits the growth of thymoma via ER{alpha}, and that ER{alpha} immunoreactivity is a potent prognostic factor in human thymoma.


   Acknowledgments
 
We thank Prof. K. Hirokawa (Department of Pathology and Immunology, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan) for his invaluable advice, and Dr. T. Akashi (Department of Pathology, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan) and Dr. H. Akamatsu (Department of Thoracic Cardiovascular Surgery, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan) for their cooperation in retrieving the specimens of thymoma. We also appreciate the skillful technical assistance of Ms. K. Hidaka (Department of Pathology, Tohoku University School of Medicine, Sendai, Japan).


   Footnotes
 
Abbreviations: AR, Androgen receptor; ER, estrogen receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ir, immunoreactivity; LI, labeling index; PR, progesterone receptor.

Received August 26, 2002.

Accepted February 10, 2003.


   References
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

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