Prevalence of HLA-DRB1 Genotype and Altered Fas/Fas Ligand ...

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The Journal of Clinical Endocrinology & Metabolism 90(3):1768 –1774 Copyright © 2005 by The Endocrine Society doi: 10.1210/jc.2004-1406

Prevalence of HLA-DRB1 Genotype and Altered Fas/Fas Ligand Expression in Adrenocortical Carcinoma Gernot W. Wolkersdo¨rfer, Christian Marx, John Brown, Sabine Schro¨der, Monika Fu¨ssel, E. Peter Rieber, Eberhard Kuhlisch, Gerhard Ehninger, and Stefan R. Bornstein Departments of Internal Medicine I (G.W.W., C.M., G.E.), Internal Medicine III (S.R.B.), and Immunology (M.F., E.P.R.) and Institute of Medical Informatics and Biometry (E.K.), University of Technology Dresden, Dresden 01307, Germany; Veteran Affairs Medical Center (J.B.), Miami, Florida 33149; and Department of Immunology (S.S.), University of Leipzig, Leipzig 04129, Germany A distinctive feature of malignant adrenocortical neoplasms is decreased major histocompatibility complex (MHC) class II molecule expression. However, it is unknown whether there exists a restriction to certain MHC genotypes and whether this involves alterations of the Fas/Fas ligand system and thereby affects tissue homeostasis. Therefore, MHC class II phenotype and genotype and expression patterns of the Fas/Fas ligand system were investigated in 24 adrenocortical tumors (nAdenomas ⴝ 14, nCarcinomas ⴝ 10) and an adrenal cancer cell line (NCI-H295). No MHC class II antigen expression was detected in carcinomas. The DRB1*01 genotype was found in 54.5% of patients with carcinoma (P ⴝ 0.046). No prevalence of any genotype could be

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HE PROGNOSIS OF adrenocortical carcinomas is poor, because metastases have often developed by the time of initial diagnosis. The 5-yr survival in patients with stage III adrenocortical carcinoma (carcinoma extending beyond the adrenal gland or involvement of local lymph nodes) is less than 30% (1). Two age peaks around the 10th and between the 40th and 50th year of life have been reported (2, 3). Benign neoplasms of the adrenal gland are found at autopsy in 2–9% of cases, and incidentally discovered adrenal masses exceed 0.5–2% on computed tomography examinations (4, 5) and 9% in magnetic resonance imaging studies for renal artery stenosis evaluation (6). To date, no reliable test is available to exclude malignancy, and surgical therapy is recommended only when tumor size exceeds 5 cm or when circulating adrenal hormone levels increase. This does not provide a definitive diagnosis for the patient and as such leaves an element of doubt about the possible subsequent development of metastases. Thus, a recent National Institutes of Health State-of-the-Science conference on adrenal incidentaloma and an International Consensus Conference on Adrenal Cancer held in Ann Arbor (Michigan) in 2004 stressed the need for additional research including establishing tumor markers and prognostic factors for carcinomas. A distinctive feature of adrenocortical neoplasms is altered First Published Online December 7, 2004 Abbreviations: Fas-L, Fas ligand; MHC, major histocompatibility complex; NS, not significant; TCR, T cell receptor. JCEM is published monthly by The Endocrine Society (http://www. endo-society.org), the foremost professional society serving the endocrine community.

detected in patients with adenomas, which exhibited varying levels of antigen expression. Fas receptor expression was 75.0% in adenomas compared with 20.0% in carcinomas (P ⴝ 0.0196), whereas ligand expression was 37.7% in adenomas and reached almost 100% in the carcinomas investigated in this study (P ⴝ 0.0033). In summary, the DRB1*01 genotype may be correlated to a higher risk for malignancy. Additional studies on MHC class II genotype and phenotype and the altered Fas/Fas ligand system in adrenal neoplasms may help to identify mechanisms of immune escape and suggest new diagnostic approaches. (J Clin Endocrinol Metab 90: 1768 –1774, 2005)

or lost major histocompatibility complex (MHC) class II expression (7), whereas MHC class II molecules are restricted to the inner cortical zones in normal adrenals. Their expression coincides with the increase of androgen production during childhood (8). The function of MHC class II molecules within the immune system includes the processing and subsequent presentation of external antigens to immunocompetent T-helper cells. This antigen presentation can induce either stimulation of immunological response or its blunting via apoptosis induced by the Fas/Fas ligand (Fas-L) system (9, 10). Because the adrenal gland is not known to be involved in processing and presenting of external antigens, this unique expression of MHC class II molecules within an endocrine organ remains a matter of investigation and debate. Still, an interaction between the immune and endocrine systems is to be expected. Indeed, stimulation of hormone production can be demonstrated in experiments cocultivating adrenocortical cells with T cells (11); however, the involvement of MHC II molecules in this setting has yet to be proven. In addition, cytokine and angiogenic CXC chemokine expression by adrenocortical tumors has been shown to contribute to both clinical presentation and adrenal tumorigenesis. Other than the normal state of MHC class II molecule expression in the adrenal gland, two notable extremes, increased and general loss of MHC class II expression, have been described. First, a remarkable increase of MHC class II expression is found in autoimmune adrenalitis (Addison’s disease) (12). Programmed cell death is triggered or induced by invading lymphocytes in highly MHC class II-expressing glands of autoimmune adrenalitis, which progressively leads to the involution of the gland. The MHC class II type in

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Wolkersdo¨rfer et al. • HLA-DRB1 and Fas/Fas-L in Adrenocortical Carcinoma

patients with Addison’s disease shows a prevalence of HLADR3 and of HLA-B8 in class I (13–21). Second, reduced expression of MHC class II antigens seems to be linked to a low rate of apoptosis in adrenocortical neoplasms when compared with normal adrenals (22). MHC molecules are involved in Fas-L-dependent death of activated T cells via the Fas/Fas-L-system (23, 24). Fas-L is a transmembrane protein of the TNF superfamily, which binds to the Fas (APO1/CD 95) receptor and induces apoptosis. The Fas/Fas-L system assists immune balance and participates in T cell-mediated cytotoxicity (25). Fas-L is expressed in the testis (26), eye (27), brain (28), and placenta (29, 30), where it presumably contributes to the immune-privileged status of these organs, eliminating infiltrating lymphocytes. The discovery of Fas-L expression in tumors of the skin (31), brain (32–34), colon (21, 35–37), liver (38 – 40), breast (41), and lung (42) suggests that Fas-L-expressing tumor cells similarly use the cytotoxic effector Fas-L to kill activated, receptor-expressing lymphocytes. This has been referred to as the counterattack phenomenon. The presented study addresses the interaction of the MHC class II molecules with the Fas/Fas-L system in adrenocortical tumors. Immunodetection of MHC class II, Fas receptor, and Fas ligand has been used to investigate 10 adrenocortical carcinomas, 14 adenomas, and the adrenocortical carcinoma line NCI-H295. HLA-DR genotyping of the tumors was assessed to reveal a possible prevalence of particular HLA genotypes in adrenocortical tumors. Patients and Methods Patients Twenty-four patients (10 men and 14 women), 5– 84 yr of age, with adrenocortical carcinoma and adenoma surgically removed between 1990 and 1997, were included in this study. The tumors were analyzed according to the criteria of Weiss (43). Fourteen tumors without histological features of cancer were classified as benign. Ten tumors with either histological malignancy or documented metastasis were classified as malignant. The tumors were staged according to the MacFarlane classification (44). The human adrenal carcinoma cell line NCI-H295 (ATCC CRL-10296; American Type Culture Collection, Manassas, VA) (45) was included in this study. Patient characteristics, clinical presentation, and hormonal status are summarized in Tables 1 and 2. Informed consent for analysis of tumor DNA and access to the information col-

J Clin Endocrinol Metab, March 2005, 90(3):1768 –1774

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lected was obtained from all of the patients in accordance with national ethical rules. The study was approved by the ethical committee of the University of Leipzig (Germany) and the University of Technology Dresden (Germany).

Tissues Samples were collected from each tumor, fixed in 10% formalin (pH 7.4), and embedded in paraffin. Slices of 6 ␮m were used in all procedures. Tumor samples were also snap-frozen in liquid nitrogen and stored at ⫺80 C. Before staining, 8-␮m sections were fixed in 3% paraformaldehyde in 1⫻ PBS (10⫻ PBS: 1.3 m NaCl, 70 mm Na2HPO4, 30 mm NaH2PO4).

Immunohistochemistry Cryostat and paraffin sections were separately immunostained for Fas (anti-CD 95; mouse antihuman, clone DX2, Dianova, Hamburg, Germany), Fas-L [anti-Fas-L, mouse antihuman, clone G247-4, PharMingen, Hamburg, Germany, or anti-Fas-L(C-20)], rabbit antihuman, polyclonal (Santa Cruz Biotechnology, Santa Cruz, CA), and MHC class II (mouse antihuman, clone CR3/43, Dakopatts, Hamburg, Germany). The specimens were exposed to the specific antiserum against Fas, Fas-L, Fas-L(C-20), or HLA-DR for 12 h at 4 C at dilutions of 1:50. The reaction was visualized using the avidin-biotin staining method according to the catalyzed signal amplification system (Dako, Hamburg, Germany) with 3-amino-9-ethylcarbazole chromogen (Immunotech, Hamburg, Germany). Slides were counterstained with hematoxylin, rinsed in water, and mounted in glycerol gelatin (Sigma, Munich, Germany). As a control, the specific antiserum was replaced by an isotype-immune serum (Mouse IgG1, ␬ [anti-TNP], PharMingen).

Determination of HLA genotype Genomic DNA from snap-frozen tissues was prepared according to standard protocols. Low-resolution HLA genotyping of genomic DNA was carried out using the Amplicor HLA DRB test according to the supplier’s instructions (Dynal, Hamburg, Germany).

Statistical analyses Statistical analyses were performed with SPSS software using the two-tailed Mann-Whitney U test or two-sided ␹2/Fisher’s exact test to evaluate patient characteristics and immunohistochemistry data. The Zmax test (46) was used for comparison of MHC class II DR locus genotypes in adenoma and carcinoma. HLA gene frequencies of 406,503 normal bone marrow donors of the North American population as registered in the National Marrow Donor Program and as published by Mori et al. (47) served as controls. The frequencies were used to generate Z values for the different alleles. Results were considered statistically significant if P ⬍ 0.05.

TABLE 1. Comparison of carcinomas and adenomas with respect to clinical presentation

Age (mean ⫾ SD) Sex (female/male) Clinical presentation Endocrine symptoms Cushing’s syndrome Virilization Feminization Endocrine hypertension Incidentaloma Tumor syndrome Sex steroids Nonfunctional Tumor weight (g, mean ⫾

SD)

All patients (n ⫽ 25)

Adenoma (n ⫽ 14)

Carcinoma (n ⫽ 11)

P (carcinoma vs. adenoma)

49.7 ⫾ 21.6 15/10

39.5 ⫾ 16.5 8/6

62.5 ⫾ 21.0 7/4

0.004a NSb

4 (16%) 2 (8%) 1 (4%) 4 (16%) 6 (24%) 4 (16%) 4 (16%) 8 (32%) 149.7 ⫾ 264.5

3 (21.4%) 0 1 (7.1%) 3 (21.4%) 2 (14.3%) 0 3 (21.4%) 2 (14.3%) 18.6 ⫾ 30.7

1 (9.1%) 2 (18.2%) 0 1 (9.1%) 4 (36.4%) 4 (36.4%) 1 (9.1%) 6 (54.5%) 333.3 ⫾ 567.5

NSb NSb NSb NSb NSb 0.01b NSb 0.03a 0.0001a

Patients with carcinoma presented with older age and had more often nonfunctional tumors with a higher tumor weight. Patients with adenomas were younger, experienced more often endocrine symptoms, and their tumors were usually of lower weight. a Two-tailed Mann-Whitney U test. b Two-sided ␹2 test; NS, i.e. P ⱖ 0.05.

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TABLE 2. Features at presentation Activity and presentation

Carcinoma 1 Bilateral, metastases, steroids normal 2 Steroids normal, incidentaloma 3 Steroids normal, incidentaloma 4 Steroids normal, incidentaloma 5 Cortisone, steroids 6 Steroids normal, incidentaloma 7 Virilization; DHEA, testosterone, androsterone 8 Cortisone, invading the liver 9 Cortisone, Cushing, aortic metastasis 10 Aldosterone, hypertension H295 Hirsutism, edema, diarrhea; steroids, met Adenomas 1 Aldosterone 2 Cortisone, steroids; feminization 3 Infertility; progesterone, 17OH-progesterone 4 Steroids 5 Unknown 6 Aldosterone 7 Steroids 8 Cortisone 9 Cortisone, Cushing 10 Aldosterone 11 Cortisone, Cushing 12 Aldosterone 13 Unknown 14 Conn’s; aldosterone

Age at diagnosis (yr)

Sex

Size and weight of tumor

MacFarlane stage

DRB1 allele

74 65 68 74 68 84 5 64 73 65 48

m f m f m m f f f f f

260 g 81 g 118 g 119 g 270 g 750 g 350 g 1100 g 45 g 240 g 14 ⫻ 13 ⫻ 11 cm

IV I I I III III II III IV II IV

1,13 1, 7 13, – 4,11 11,13 15, – 1, 2 11, 4 1, 4 11, 4 1, –

31 74 36 21 16 39 22 48 47 57 28 61 39 35

f m f f m f m m f m f f f m

1.6 g 7.4 g 10.7 g 1.0 g 18.2 g 7.1 g 1.0 g 84.7 g 92 g 4.5 g 0.7 g 1.0 g 30.0 g 0.5 g

17, 7 11, 3 1,16 15,13 11,13 4,10 3, 7 4, 8 15,11 2, 3 3,12 2,11 3,11 1, 9

Summary of the age when surgery was performed, sex, hormonal activity, stage of the examined tumors, and summary of HLA-DRB1 genotype. DHEA, Dehydroepiandrosterone; met, metastases; f, female; m, male.

Results Patient characteristics

Clinical presentations of patients are summarized in Table 1, and individual patient characteristics together with the patient’s HLA-DRB1 allele are outlined in Table 2. No differences in sex or clinical presentation could be documented in the patients included in this study. Patients with adenomas presented with younger age. Adrenocortical carcinomas were significantly more often nonfunctional tumors and, consequently, were larger when the diagnosis was substantiated. As expected, patients with adrenocortical cancer suffered from weight loss, night sweats, anemia, or symptoms caused by metastases, which are further referred to as tumor syndrome. Immunohistochemistry

The data obtained in this study confirm our previous findings (7) that expression of MHC class II is altered in adrenal tumors. In adrenal adenomas, MHC class II was detectable in 27.7%. In adrenal carcinomas, none showed MHC II staining of adrenocortical epithelial cells (0%). The carcinoma cell line also failed to express MHC class II that reacted with MHC II antibodies. In the tumors, some infiltrating mononuclear blood cells expressed MHC class II. The MHC class II expression patterns of normal adrenal glands, adrenal adenomas, and carcinomas are demonstrated in Fig. 1 (A, normal adrenal; B, adenoma; C, carcinoma). Anti-Fas (CD 95) staining was weak in normal adrenals and seemed to increase toward the inner cortical zones with most intense staining in the cortico-medullar transition zone

FIG. 1. Expression of MHC class II antigens, Fas, and Fas-L in adrenocortical epithelial cells. MHC class II expression (A, normal adrenal; B, adenoma; C, carcinoma) is lacking in adrenocortical carcinoma. The expression of Fas (D, normal adrenal; E, adenoma; F, carcinoma) is weak in normal adrenals and adenomas but is absent in most of the adrenocortical carcinomas. Anti-Fas-L staining (G, normal adrenal; H adenoma; I, carcinoma) was strongest in adrenocortical carcinomas when compared with normal adrenal or adenoma.


(Fig. 1). In the adenoma group, 75% of tissues expressed the Fas receptor, compared with 20% in the carcinoma group (P ⫽ 0.0196; Fig. 1; D, normal adrenal; E, adenoma; F, carcinoma). Expression of Fas-L according to immunohistochemical staining with anti-Fas-L [anti-Fas-L(C-20), rabbit antihuman, polyclonal] (Fig. 1; G, normal adrenal; H, adenoma; I, carcinoma) occurred in all of the investigated tissue types. Adrenal adenomas exhibited a positive staining reaction in 37.7% of examined specimens, whereas expression could be documented in 100% of the carcinomas (P ⫽ 0.0033). The expression of Fas-L was further confirmed by a second antibody (anti-Fas-L, mouse antihuman, clone G247-4; data not shown). The data obtained using this antibody were similar to the data for anti-Fas-L(C-20). Occasionally, no immunoreaction was seen using the Fas-L (mouse antihuman, clone G247-4) antibody. Determination of HLA genotype

In low-resolution experiments, DRB1*01 occurred in six of 11 cases (54.5%), i.e. 27.3% of the observed alleles (vs. 10.2 ⫾ 0.041%; Zmax test, P ⫽ 0.0459) and DRB1*11 in four of 11 cases (36.4%), i.e. 18.2% of observed alleles [vs. 9.3 ⫾ 0.039%; not significant (NS)] of the carcinoma group. No prevalence of any genotype could be substantiated for adenomas. Two of 14 cases (14.2%) carried the DRB1*01 genotype, i.e. 7.14% of the observed alleles (vs. 10.2 ⫾ 0.041%; NS), and five of 14 cases (35.7%) carried the DR1*B11 genotype, i.e. 17.85% of the observed alleles (vs. 9.31 ⫾ 0.039%; NS) (47). Results are summarized in Table 3. Subgroup analysis of cancer patients with and without HLA-DRB1*01 allele did not reveal any significant difference, although patients with HLA-DRB1*01 allele appeared to be slightly younger, had a higher stage of disease, and a smaller tumor weight (Table 4). The difference did not prove to be statistically significant. Discussion

In the present study, we sought to compare results on phenotypic expression of MHC class II molecules with the MHC class II genotype in tumors of the adrenal cortex. In


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TABLE 4. Comparison of presentation of carcinomas with DRB1*01 allele and other alleles Carcinomas with

Age at diagnosis (mean) Sex (male/female) Weight of tumor (mean ⫾ MacFarlane stage

SD)

DRB1*01

Other DR1*allele

58.16 4/2 297.2 ⫾ 282.6 3.0 ⫾ 0.5

67.8 3/2 369.4 ⫾ 414.2 2.0 ⫾ 0.4

Carcinomas with DRB1*01 genotype presented with the lower age mean, lower tumor weight mean, and higher tumor stage. However, there was no statistical significance.

both adrenal adenomas and carcinomas, MHC class II was expressed only in a small portion of cases. Genotype analyses revealed a significant albeit small prevalence of DRB1*01, which was found in as many as 54.5% of patients in the carcinoma group (27.3% of alleles vs. 10.2 ⫾ 0.041%) (47). When subgroup analysis was performed, at least lower tumor weight but higher stage of disease have been suggestive for a more aggressive tumor with a probable tendency toward early development of metastases for patients with the HLA-DRB1*01 allele. This is the first report to show a prevalence of an HLA genotype in adrenocortical tumors. So far, HLA prevalence has been documented in congenital adrenocortical hyperplasia due to 21-hydroxylase deficiency, in which HLA-B5 and HLA-Bw45 have been found. The 21-hydroxylase gene and its pseudogene are located on chromosome 6 between the loci of MHC classes II and III, respectively; therefore, a link between adrenally expressed genes and the HLA system might exist. The normal expression of MHC class II in the human adrenal gland is restricted to the inner cortical zones, where cortical androgens are produced and secreted. Cortical androgens have been shown to counterbalance glucocorticoid effects on T lymphocytes (48, 49). The natural ligand of the MHC class II-complex is the T cell receptor (TCR) complex on CD 4-positive T-helper lymphocytes. T lymphocytes, in turn, have been shown to express the Fas ligand upon stimulation. Hence, a bidirectional, regulatory circuit between adrenocortical cells and subgroups of lymphocytes is con-

TABLE 3. HLA DRB1* allele frequencies of the North American population and observed allele frequencies in adrenocortical tumors DRB1*

DRa

Population frequencya,b

01 15/16 03 04 11/12 13/14 07 08 09 10 Other Total

1 2 3 4 5 6 7 8 9 10 Other

10.23 15.24 10.87 16.76 11.22 14.39 13.28 2.88 1.06 1.48 2.58 100.00

Adenoma n

Frequency

2 5 6 2 5/1 2/0 2 1 1 1 0 28

7.14 17.85 21.42 7.14 21.42 7.14 7.14 3.57 3.57 3.57 0.0 100.00

Carcinoma b

n

Frequency

6 3/0 0 4 4/0 4/0 1 0 0 0 0 22

27.27 13.63 0 18.18 18.18 18.18 4.54 0.0 0.0 0.0 0.0 100.00

All patients b

n

Frequencyb

8 8 6 6 10 6 3 1 1 1 0 50

16.0 16.0 12.0 12.0 0.2 12.0 6.0 2.0 2.0 2.0 0.0 100.00

Due to revision of the nomenclature of the HLA system, marker DR2 was split into DRB1*15 and DRB1*16, DR5 into DRB1*11 and DRB1*12, and DR6 into DRB1*13 and DRB1*14. a Population frequency depicted without SE (47); other, DRX, DR5 unsplit. b Frequency in percentage.

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ceivable. The possible benefit for the organism may be control and regulation of tissue homeostasis in both adrenocortical cells and lymphocytes. In adrenocortical tumors, this circuit might exist in a similar fashion and seems to be interrupted because they exhibit an altered MHC class II phenotype. In the study presented, we found only 27.7% of the adrenal adenomas and none of the carcinomas with positive immunostaining for MHC class II in epithelial cells. However, the expression of MHC molecules in adenoma was normal or even enhanced in tissue adjacent to benign adenomas. The involvement of MHC class II, the TCR complex, and CD 4 molecules in control and regulation of T-helper lymphocytes has been described (24, 50 –52). Recent data have been reported on MHC class II-mediated interaction between CD 4-positive T cells, which induced Fas/Fas-L-triggered programmed cell death (24). Normal adrenal glands express both MHC class II molecules and the Fas receptor (53). However, the question arises as to how decreased or lost MHC class II expression as observed in adrenal carcinomas could result in counterattacking and escape from immune surveillance, when activation-induced cell death cannot be induced via CD 4-MHC II-TCR complex interaction. First, lost MHC class II expression will impair such interaction of adrenocortical tumor cells with resting T cells. This altered or abolished interaction has been shown to inhibit positive selection of peripheral CD 4-expressing T cells (54) and, moreover, to induce Fas-mediated apoptosis (23, 24, 50). Hence, negative selection of as yet unactivated T cells could be considered. Second, the data obtained demonstrate a reduced Fas receptor expression pattern in carcinomas, whereas the expression of Fas ligand is increased. Indeed, the capacity to undergo apoptosis is diminished in adrenocortical carcinomas (22). Although the list of tumors that demonstrate Fas-L expression is growing, involvement of Fas-L in immune escape has been questioned by others (55). Fas-L-expressing tumors include such histologically diverse tumors as hematopoietic neoplasms, melanoma, carcinomas, and glioma. The expression of Fas-L by cancer cells could provide them with a way to escape immune surveillance (56 – 61). To effectively launch a counterattack, tumor cells must be resistant to their own Fas-L. This can be accomplished by mutations in the Fas receptor gene (62– 64), abrogation or blocking of the downstream apoptotic pathway (65, 66), or down-regulation of Fas expression, as seen in liver and cholangiocarcinomas (40, 67). In this study, we show that the latter mechanism is most likely in adrenocortical carcinomas, because Fas immunoreactivity was lower in adrenal carcinomas than in normal adrenal cortex tissues. However, the phenomenon of immune escape in adrenocortical tumors may be unique and differs from other tumors in that the natural interaction between adrenocortical cells and the immune system via MHC class II molecules is disrupted. High expression of MHC class II correlates with a more favorable prognosis in some tumors (68), whereas its downregulation or lack of expression has been suggested to render prognosis poor in other malignancies (69, 70). The phenotypic loss of MHC class II expression in malignant adrenocortical tumors seems to be related to the DR1*B01 allele. Additional studies are needed to confirm these associations


in a higher number of patients with different ethnic backgrounds and to investigate whether this association is due to causal involvement or linkage disequilibrium. The immunoadrenal interaction may be of particular relevance for both the endocrine and the immune systems in regulating hormonal activity and functional immunological properties as well as tissue homeostasis of the adrenal gland (32). In summary, we demonstrate a possible mechanism of immune escape in adrenocortical carcinomas because of the lost MHC class II expression and the altered Fas/Fas-L expression. Second, the phenotypic loss of MHC class II expression shows a relation to the DRB1*01 allele. Third, in incidentally discovered adrenal masses, the determination of the HLA type may serve in the future as a prognostic factor. Together with imaging criteria and gene profiling of adrenal biopsies it may enable us to define new strategies for the management of adrenal lesions not solely based on size criteria and hormone secretion. Acknowledgments We thank Dr. M. Meredyth-Stewart for her assistance during the preparation of the manuscript. Received July 23, 2004. Accepted December 1, 2004. Address all correspondence and requests for reprints to: Gernot W. Wolkersdo¨rfer, Medical Department I, Faculty of Medicine Carl Gustav Carus, University of Technology Dresden, Fetscherstrasse 74, Dresden 01307, Germany. E-mail: [email protected].

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