MHC CLASS II EXPRESSION- A NEW TOOL TO ASSESS DIGNITY IN ADRENOCORTICAL TUMOURS
CHRISTIAN MARX, GERNOT W.WOLKERSDÖRFER, 1JOHN W.BROWN, WERNER A. SCHERBAUM AND STEFAN R. BORNSTEIN
Department of Internal Medicine III, University of Leipzig, Ph .- Rosenthal-Strasse 27, 04103 Leipzig, Germany; 1 Veterans Affairs Medical Center, Miami, FL, U.S.A.
ABSTRACT. One in seventy randomly selected individuals is supposed to host an adrenal mass. The increasing number of incidentally detected adrenocortical adenomas requires the effective and reliable evaluation of dignity. So far this has been determined through a difficult multi-parametric analysis. Since MHC class II antigens are expressed in the normal adrenal cortex with a restriction to the zone reticularis, we examined 28 adrenocortical incidentalomas, 10 adenomas, 13 cortical carcinomas, 2 metastases, 10 controls as well as the adrenocortical carcinoma cell line NCI-H295 immunohistochemically for the expression of HLA class II antigens. We showed, that the majority of the adenomas still express class II ardigens, whereas the expression is abrogated in all carcinomas examined.
Our results indicate, that the detection of HLA class II positive tumour cells excludes malignancy. Therefore, MHC class II antigens may serve as a novel tumour marker in the evaluation of dignity in adrenocortical tumours. These findings could change the strategy for the assessment of adrenal masses.
I NCIDENTALLY detected adrenal masses have become a common clinical problem. Between 0.35 and 4.36 % of all CT scans reveal the presence of an adrenal tumour and the incidence is even higher in autopsy cases (1). The clinical relevance of these tumours often remains unclear. Only if the tumour is hormonally active or malignant (together less than 10 %) does the individual turn into a patient who needs to undergo surgery. Though a carcinoma is rare among the incidentalomas of cortical origin, the unfavourable prognosis requires safe diagnosis. However, the exact diagnosis is difficult and is still based on a multi-parametric analysis (2,3) which has to be performed postoperatively. Rather arbitrary criteria such as tumour size are commonly used to determine whether surgery is necessary or not. As yet there are no reliable histological markers justifying needle biopsy.
The expression of MHC class II expression has previously been examined in many other tumours and differing findings on the amount of HLA and its prognostic and diagnostic value have been reported (4). In the normal human adrenal, MHC class II expression is highly correlated with cellular differentiation, where it is expressed with restriction to the zona reticularis (5,6). Since pathologic
conditions like such as (adrenal) autoimmunity coincide with changes in MHC class II expression (7) we wondered, if changes in the expression of these antigens could have a diagnostic value in those adrenocortical tumours, which show a decrease of differentiation or undergo malignant transformation.
Materials and methods
Patients Specimens were examined from 28 individuals (21 females, 7 males; mean age 71.7 years) where an adrenocortical incidentaloma was detected on autopsy, 10 patients with cortical adenomas (6 females, 4 males; mean age 54.3 years), and from 13 patients with surgically removed carcinoma and 2 liver metastasis specimens (together 11 females, 4 males; mean age 50.8 years). The cortical origin of the tumours was confirmed by immunohistochemical staining against cytokeratin, vimentin, synaptophysin and D11 protein.
The available clinical data of the adenomas revealed 4 cases with clinical Cushing syndrome, 2 of them were virilizing, and 4 aldosteronomas. Among the carcinomas, 4 cases showed a Cushing syndrome, 1 of them androgen producing, and 3 tumours were functional inactive. Malignancy was diagnosed or excluded according to the criteria of Hough et al (2), Weiss et al (8) and Slooten et al (9).
The tumour size ranged between 1 and 5 cm in adenomas, the size of the carcinomas between 4 and 19 cm.
10 normal adrenals obtained from nephrectomized patients (6 females, 4 males; mean age 56.3 years) were used as controls. The patients had no history of autoimmune process. Cell culture The NCI-H295 carcinoma cells were maintained in RPMI 1640 medium (Gibco BRL, Eggenstein, Germany) containing Hydrocortison (3.625 µg/l), Insulin 5mg/ml, Transferrin (10 mg/ml), Estradiol (2.724 µg/1), Selenit (5 ug/ml), 2% fetal calf serum (all obtained from Sigma Aldrich, Deisenhofen, Germany) and antibiotics. The cells were grown at 37°℃ in a 5% CO2 humidified atmosphere under routinely changed media. Adherent cells were selected during the culture period and grown on culture slides for 72h prior to immunohistochemic staining.
Immunohistochemical procedures Culture slides were fixed in 100% Acetone for 15 min and dried. Paraffin sections were deparaffinized in Xylene and hydrated in descending Ethanol row.
Prior to staining, the endogenous peroxidase was blocked in 1.5% H2O2 . All staining was performed using a standard avidin-biotin system (Dianova, Hamburg, Germany). Mouse anti-human HLA-DR (clone 3/43) and anti-HLA class II a- chain (TAL.1B5) antibodies were applied at dilutions of 1:50 and showed equal reactivity in all cases. Furthermore antibodies to CD45 (2B11+PD7/26) and CD68 (KP1) identifying infiltrating immune cells were used in consecutive sections. Adrenal medulla was detected by staining with chromogranin A (DAK-A3) antibodies (all Dako, Hamburg, Germany) and anti 17x-hydroxylase (kindly provided by Prof Waterman, Vanderbilt University, Nashville, TN) was employed to characterize zona reticularis cells.
Visualisation was achieved through the use of AEC (3- amino-9-ethylcarbazole) chromogen (Immunotech, Hamburg, Germany). After staining, the slides were counterstained with haematoxyline. Control staining was performed with mouse IgG instead of the primary antibodies and no unspecific staining was detectable. HLA class II reactivity of immune cells was used as an intrinsic positive control.
Quantification Two sections of each tissue were scored (0)- (3) according to the following scale: (1) positive cells only in areas of normal zonation; (2) scattered positive tumour cells in dispersed cords or islets of the section; (3) positive cells in about half of the areas of the section. There was no significant difference between these two sections.
Results
All normal adrenals demonstrated MHC class II expression within the zona reticularis (Fig.1). The distribution of the MHC class II expression in incidentalomas and adenomas is summarized in Table 1. The observed expression within the tumours varied from cords and islets of dispersed positive reticularis cells independent from infiltrating immune cells (Fig. 2a) to an abundant
pattern (when the tumour was composed of reticularis-like cells, Fig.2b). In some tumours, reactivity was only found in adjacent areas of normal zonation.
Two aldosteronomas and one adenoma of oncocytary phenotype (low differentiated), showed no expression of MHC class II antigens, whereas in tumours of fasciculata-like type only a few dispersed reticularis cells expressed MHC class II. The highest level of expression was seen within adenomas of reticularis-phenotype, independent from their hormonal activity.
None of the dedifferentiated adrenocortical carcinomas (Fig.3a,b) and liver metastases revealed class II positive tumour cells. The only sources of class II reactivity were of small extent and concerned areas of necrosis, mononuclear cells and connective tissue. Neither did the NCI H295 cell line, wich was shown to express 17a-hydroxylase, contain any MHC class II positive tumour cells (Fig.4).
| Score | Normal adrenals (n=10) | Inciden- talomas (n=28) | Adenomas (n=10) | Carci- nomas (n=13*) | Meta- stases (n=2) |
|---|---|---|---|---|---|
| 0 | n=3 | n=3 | n=13 | n=2 | |
| 1 | n=10 | n=8 | n=2 | ||
| 2 | n=15 | n=2 | |||
| 3 | n=2 | n=3 |
The table shows the score of MHC class II positive adrenocortical tissues: (0) no staining in tumour cells except infiltration, necrosis, connective tissue or endothel; (1) positive cells only in areas of normal zonation; (2) scattered positive tumour cells in dispersed cords or islets; (3) positive cells in about half of the areas of the section. *Including NCI H295 cell line
Figures
Fig.1: Normal adrenal stained for MHC class II antigens (x40). The inner zones of the cortex (c) are positive whereas the outer zones and the medulla (m) show no staining. Fig.2: Cortical adenoma. a) Cords of dispersed MHC class II positive reticularis cells (x100). b) Incidentaloma. Strong reactivity is found in tumours of reticularis-like phenotype independent from their hormonal activity (x40). Cortical carcinomas (Fig.3a,b) as well as cultured NCI H295 carcinoma cells (Fig.4) show complete loss of class II reactive tumour cells (x25/x40).
1
2à
3a
b
4
Discussion
MHC class II was demonstrated in the reticularis cells of all normal human adrenals examined. The class II expression in the incidentalomas and active adenomas seems to reflect the functional conversion of some tumour cells to MHC class II positive cells, as well as the displacement of cords of originally positive normal reticularis cells caused by the proliferative activity of the tumour. This is likely due to the circular arrangement of positive cells around nodular tumour proliferation zones. In some tumours, positive staining for HLA class II also involves fasciculata or glomerulosa cells. In these cases, the strong staining of HLA class II could in part be due to the influence of immunocompetent cells. In some incidentalomas and adenomas class II reactivity occurs exclusively in areas of normal zonation but not in adenomatous areas. The reasons for these expression patterns may be heterogenous:
Some tumours consist of a priori negative glomerulosa or fasciculata cells- in these cases, class II positivity is found only within the adjacent normal cortex. This underlines the close dependence of the HLA class II expression on the cellular consistence of the tumour. In incidentalomas of “intermingling” phenotype, class II may be absent in reticularis-like tumour cells but areas of normal zonation remain positive. The reasons for the absent reactivity in these cases and the complete absence of MHC class II antigens in three further cases remain unclear. The level of MHC class II in the normal adrenal cortex seems to be linked to the level of dehydroepiandrosterone secretion which is maximal in younger people and declines with increasing age (6). We were not able to observe a correlation between the expression and age in our tumours, but age dependent changes should certainly have individual influence on the HLA class II expression.
The function of MHC class II expressed by adrenocortical cells is not clear. Interestingly, TNF- a, which is known to induce MHC class II antigens, is produced by class II positive steroid cells. The steroid enzyme 17a-hydroxylase, involved in the production of dehydroepiandrosterone, is equally found to be expressed by these MHC class II positive reticularis cells (10). Nevertheless we could not detect the expression of MHC class II antigens in unstimulated NCI-H295 cells which show the expression of 17a-hydroxylase.
The expression of MHC class II is abrogated in all adrenocortical carcinomas, examined. The malignant transformation of cortical tumour cells coincides with a decline in cellular differentiation and loss of normal zonation. We believe that the malignant transformation of an originally MHC class II positive adrenocortical tumour coincides with the loss of these antigens. This finding reflects the aggressive behaviour of these carcinomas.
Interestingly, one tumour, initially considered to be potentially malignant, showed MHC class II expression. According to this finding, we assumed a benign behaviour of this tumour and this was confirmed by the results of the final pathological examination. Reversely, one dedifferentiated oncocytary adenoma showed no MHC class II expression. This supports the assumed potential malignant behaviour.
In summary, we conclude: i) MHC class II is found in normal adrenals as well as approximately 80 % of benign cortical tumours. ii) In the majority of cases MHC class II is also present in the adenomatous tissue. iii) The degree of expression seems to be dependent on the cellular differentiation of the tumour (glomerulosa vs. fasciculata vs. reticularis cells or dedifferentiation), the inductive influence of immune cells as well as age dependent changes. iv) Most importantly, our results indicate, that the detection of adrenocortical tumour cells expressing HLA class II antigens independent from infiltration, excludes malignancy at the time of examination.
This observation provides a simple but straightforward tool to separate malignant from benign adrenocortical tumours. The antibodies we used are commercially available and the immunohistochemical detection system may be routinely used in the pathological examination of tissues. In a high number of cases, the
administration of this new tumour marker could accelerate and confirm the correct diagnosis which is important for rational treatment.
It will be of great clinical interest to test biopsy specimens of these tumours for HLA class II expressing tumour cells. We hope that, if these results are confirmed, the use of this marker may lead to a better defined rationale for surgery in patients with adrenocortical tumours.
Acknowledgements
We thank Prof S.Schröder, Hamburg, and Dr Ziegan, Leipzig, for providing tumour specimens and Mrs. S.Brauer and Mrs. R.Blaschke for technical assistance.
This work was supported by The Deutsche Forschungsge- meinschaft (DFG Bo 1141/ 2-3) and a Heisenberg grant to S.B.
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