Neuroendocrine Differentiation of Adrenocortical Tumors
Harm R. Haak, M.D.,*t and Gert-Jan Fleuren, M.D.t
Background. The syndrome of multiple endocrine neoplasia type 1 frequently involves the adrenal cortex. The relationship between the diffuse neuroendocrine sys- tem and the adrenal cortex is not clear however, particu- larly as the adrenal cortex is not considered to be an in- trinsic part of the diffuse neuroendocrine system.
Methods. The possible relationship between the adrenal cortex and the diffuse neuroendocrine system was investigated in a study of the immunohistochemical characteristics of ten normal adrenal glands, four adre- nal adenomas, and 18 adrenocortical carcinomas using the indirect peroxidase method of staining intermediate filaments and neuroendocrine proteins.
Results. With synaptophysin, NSE, and vimentin there was focal staining in only few zona glomerulosa cells in the normal adrenal cortex, whereas adrenocorti- cal carcinomas and adenomas were extensively positive for these proteins. Keratin immunoreactivity, present in 100% of the normal cortices, was demonstrable in only half of the carcinomas and absent in all adenomas.
Conclusions. Adrenocortical tumors may originate from neuroendocrine foci. The findings illuminate the pathogenesis of adrenocortical carcinoma, and may carry significant implications about the choice of treatment of patients with this malignancy and other related tumors. Cancer 1995; 75:860-4.
Key words: adrenal cortex neoplasm, carcinoma, inter- mediate filament proteins, immunohistochemistry, neu- roendocrine differentiation.
Introduction
The adrenal gland is composed of two separate and dis- tinct types of endocrine tissue: the medulla and the cor- tex. The adrenal medulla and cortex differ in functional characteristics and originate from different embryologic tissues; the adrenal medulla originates from ectodermal
(neural-crest) tissue and the cortex from mesodermal tissue. The adrenal medulla belongs to the “diffuse neu- roendocrine system” (DNES), formerly called the amine precursor uptake and decarboxylation series. It was suggested by Pearse that all amine precursor uptake and decarboxylation cells originate from neuroendo- crine-programmed cells of the ectoblast. Amine precur- sor uptake and decarboxylation activity is also found, however, in endodermally and mesodermally derived tumors.1
Although the adrenal cortex is not considered to be an intrinsic part of the DNES,2 involvement of the adre- nal cortex in the multiple endocrine neoplasia type 1 (MEN-1) syndrome is very common.1 This syndrome is characterized by the combined occurrence of neuroen- docrine tumors of parathyroids, pituitary gland, and pancreatic islets. The pathologic picture of adrenocorti- cal involvement in MEN-1 is that of hyperplasia, ade- noma, or even carcinoma. Clinical evidence of hormone overproduction is only rarely observed.
The relationship between the diffuse neuroendo- crine system and the adrenal cortex is thus not clear. The role of neuroendocrine factors in the etiology of adrenocortical tumors has never been established. The question arises as to whether the presence of neuroen- docrine proteins in the adrenal cortex helps to explain neoplastic transformation, and whether this would have implications for the treatment of adrenal cortical carcinoma and other related tumors. For this reason, we investigated the possible relationship between the DNES and the adrenal cortex by studying the immuno- histochemical characteristics of neuroendocrine mark- ers and intermediate filaments in normal adrenal glands, in addition to pathologic adrenal glands having adenomas and carcinomas.
Patients and Methods
Patients
Eighteen patients with adrenocortical carcinoma, four with adrenocortical adenoma, and ten with normal adrenal glands were examined. The clinical data are
From the Departments of *Endocrinology and Pathology, Uni- versity Hospital Leiden, and the Department of tInternal Medicine, Diaconessenhuis Eindhoven, The Netherlands.
Address for reprints: H.R. Haak, M.D., Department of Internal Medicine, P.O. Box 90.052, 5600 PD Eindhoven, The Netherlands.
Received August 11, 1994; revision received October 14, 1994; accepted October 14, 1994.
| Normal | Adenoma | Carcinoma | |
|---|---|---|---|
| n (male/female) | 10 (3/7) | 4 (1/3) | 18 (4/14) |
| Mean age (yr) (range) | 34 (8-59) | 32 (14-43) | 39 (8-69) |
| Cushing | - | 3 | 8 |
| Conn | - | 1 | 2 |
| Virilization | - | 2 | |
| Feminization | - | 1 | |
| No hormonal syndrome | - | 5 |
shown in Table 1. Two normal glands were harvested at autopsy. One normal adrenal gland was resected be- cause of mild hypercortisolism after two pituitary oper- ations for Cushing’s disease. One normal gland was re- sected during radical nephrectomy for renal cell carci- noma. Six normal adrenal cortices were obtained from glands resected for treatment of adjacent adrenal tu- mors (pheochromocytoma in four patients, Cushing’s adenoma in one and Conn’s adenoma in another pa- tient).
Tissues
Snap-frozen tissue was preserved in frozen tissue files (-70℃) of the Department of Pathology of the Leiden University Hospital (Leiden, the Netherlands). Frozen tissue was not available in five of the normal adre- nal glands: pheochromocytoma (n = 1), Cushing’s ade- noma (n = 1), Conn’s adenoma (n = 1), and glands ob- tained at autopsy (n = 2). In these patients, only paraffin embedded tissue could be evaluated.
Immunohistochemistry
Details of the antibodies used are shown in Table 2. The antibodies against the intermediate filament proteins
recognize keratin 8, 18, and 19, pankeratin, vimentin, neurofilament, and glial fibrillary acidic protein. The antibodies against neuroendocrine proteins recognize neuron-specific enolase (NSE), chromogranin, synapto- physin, “small cell carcinoma of lung MOC-1-related antigen” (MOC-1), and somatostatin. Staining with anti-NSE, antisomatostatin, and antichromogranin was performed in paraffin embedded material. The other antibodies were used on frozen tissues.
Indirect immunoperoxidase staining was per- formed on frozen and paraffin sections as described elsewhere.3 Rabbit antimouse Ig conjugated to horse- radish peroxidase was used as second antibody and 3- amino-9-ethylcarbazole was used as chromogen to vi- sualize the antigen/antibody reaction.
Experimental Design
Serial slides of tissue specimens were incubated with the antibodies (Table 2). PBS was used instead of the primary antibody as negative control. The presence of positive cells on the slides was scored semiquantita- tively as - (no positive tumor cells), + (1-24% positive cells), ++ (25-49% positive cells), +++ (50-74% posi- tive cells), and ++++ (75% or more positive cells).
Results
There was no difference in staining pattern between normal adrenal cortices and those with adjacent cortical pathology.
Intermediate Filaments
Keratin expression was present in 25-74% of cells in normal glands (see Table 3). Fifty percent of adrenocor- tical carcinomas were keratin positive. In the majority of these keratin-positive tumors less than 25% of the
| Antibody | Source | Dilution | Ag recognized | Reference no. |
|---|---|---|---|---|
| Clone M20 | Sanbio (NL) | 1:10 | Keratin 8 | 4 |
| Clone M9 | Sanbio | 1:10 | Keratin 18 | 5 |
| L2PK | Amersham (UK) | 1:10 | Keratin 19 | 6 |
| Clone 80 | Sanbio | 1:10 | Pankeratin | 7 |
| Clone V9 | Sanbio | 1:20 | Vimentin | 8 |
| Clone 2F11 | Sanbio | 1:10 | Neurofilament (NF) | 8 |
| M761 | DAKO (NL) | 1:4 | Glial filament (GFAP) | 9 |
| A589 | DAKO | 1:200 | Neuron-specific enolase (NSE) | 10 |
| A430 | DAKO | 1:40 | Chromogranin A | 11 |
| SY 38 | Boehringer (Ger) | 1:5 | Synaptophysin | 12 |
| MOC 1 | Eurodiagnostics (NL) | 1:10 | Small cell carcinoma of lung antigen | 13 |
| Clone 367 | Pathology Leiden (NL) | 1:100 | Somatostatin | 14 |
NL: The Netherlands; UK: United Kingdom; Ger: Germany.
| - | + | ++ | +++ | ++++ | Total + (%) | |
|---|---|---|---|---|---|---|
| Keratin 8 | ||||||
| Normal | 0 | 0 | 3 | 2 | 0 | 100 |
| Adenoma | 4 | 0 | 0 | 0 | 0 | 0 |
| Carcinoma | 9 | 5 | 1 | 0 | 3 | 50 |
| Vimentin | ||||||
| Normal | 0 | 3* | 2 | 0 | 0 | 100 |
| Adenoma | 0 | 1 | 0 | 2 | 1 | 100 |
| Carcinoma | 0 | 0 | 1 | 1 | 16 | 100 |
-: no positive tumor cells; +: 1-24% positive cells; ++: 25-49% positive cells; +++: 50-74% positive cells; ++++: ≥75% positive cells.
* Subcapsular focal staining.
cells were stainable. Staining for keratin 8 was predom- inant over that for keratin 18; keratin 19 was negative in all cases. None of the adrenal adenomas stained for keratin.
Vimentin reactivity was found in all normal glands, but only focal positivity of subcapsular cells was found in three of the five glands. The other two normal glands showed vimentin immunoreactivity in 25 to 50% of the cells. All carcinomas showed extensive vimentin stain- ing. All four adenomas were positive also for vimentin (less than 25% of the cells in one, 50 to 74% in two, and greater than 75% in one).
Neurofilament and glial fibrillary acidic proteins were absent in all tissues studied.
Neuroendocrine Proteins
Synaptophysin was present subcapsularly, and only fo- cally in three normal adrenal cortices (Table 4). Two normal glands did not show synaptophysin immunore- activity. All adenomas, and 15 out of 18 carcinomas, showed mostly abundant, synaptophysin expression.
In carcinomas and adenomas, the staining pattern with the MOC-1 antibody was similar to that of synaptophy- sin, whereas 25 to 74% of the cells in normal glands were positive (Fig. 1).
Staining with anti-NSE showed positive cells fo- cally in the zona glomerulosa just inside the capsule in all ten normal adrenal glands. Adrenocortical carci- noma was more extensively positive for NSE (mean 50% of the cells). Three of the four adenomas were pos- itive for NSE.
All carcinomas were positive for at least two of the three above-mentioned neuroendocrine markers. Chro- mogranin and somatostatin immunoreactivity could not be detected in either the normal adrenal cortex, or in any of the adenomas or carcinomas.
Discussion
Immunohistochemical staining of intermediate fila- ments and tumor-associated proteins is useful to distin- guish different cell types, classify tumors, and improve the accuracy of the clinical diagnosis.15 In this respect, only few workers have studied immunohistochemical staining in the adrenal cortex. Our findings of staining patterns of intermediate filaments and neuroendocrine proteins may be of great importance not only in the di- agnostic, but also in the therapeutic aspects of adreno- cortical pathology.
Tumors derived from the adrenal cortex show ex- tensive vimentin positivity, as clearly illustrated in our study. In addition, expression of vimentin can be found in all normal adrenal glands. However, compared with that of adrenal tumors, vimentin expression is less in- tense in the majority of normal glands where it occurs only in a few subcapsular cells. These findings confirm results reported by others.16-19 As expected, keratin was present in all normal glands. However, keratin expres- sion could only be detected in 50% of the adrenal corti- cal carcinomas. Other carcinomas and all adenomas showed no keratin immunoreactivity.
| - | + | ++ | +++ | ++++ | Total + (%) | |
|---|---|---|---|---|---|---|
| Synaptophysin | ||||||
| Normal | 2 | 3 | 0 | 0 | 0 | 60 |
| Adenoma | 0 | 1 | 0 | 2 | 1 | 100 |
| Carcinoma | 3 | 2 | 1 | 5 | 7 | 83 |
| MOC-1-related antigen | ||||||
| Normal | 0 | 0 | 4 | 1 | 0 | 100 |
| Adenoma | 0 | 2 | 0 | 0 | 2 | 100 |
| Carcinoma | 1 | 2 | 3 | 3 | 9 | 94 |
| NSE | ||||||
| Normal | 0 | 10* | 0 | 0 | 0 | 100 |
| Adenoma | 1 | 2+ | 1 | 0 | 0 | 75 |
| Carcinoma | 1 | 5 | 9 | 3 | 0 | 94 |
-: no positive tumor cells; +: 1-24% positive; ++: 25-49% positive cells; +++: 50-74% positive cells; ++++: ≥75% positive cells.
NSE: neuron-specific enolase.
* Focal subcapsular staining.
t Adenoma with focal subcapsular staining.
The original intermediate filament protein pattern is usually maintained during neoplastic transforma- tion.15 Because the adrenal cortex is of mesodermal ori- gin, the extensive presence of vimentin in tumors of the adrenal cortex is to be expected, despite the often only sparse vimentin reactivity of normal glands. A remark- able loss of keratin expression was observed in adreno- cortical tumors. The loss of keratin reactivity may be incomplete in some adrenocortical tumors, due to prob- lems associated with the detection of very small amounts of keratins using the immunohistochemical technique. Nevertheless, the observed loss of keratin and the appearance of extensive vimentin reactivity in- dicate a change in differentiation of the adrenal cortex upon neoplastic transformation.20
The neuroendocrine proteins NSE, synaptophysin, and MOC-1-related antigen were present in normal glands. We were unable to demonstrate synaptophysin in two normal adrenal glands. It may be possible that in those two normal glands, synaptophysin was truly not expressed. However, the apparent negativity might re- flect the limit of detection of small amounts of this pro- tein by the method applied. Similar to vimentin, the neuroendocrine proteins were present in small foci in the zona glomerulosa of normal glands, just within the outer capsule. In contrast, the neuroendocrine proteins were clearly present in all adrenocortical carcinomas and in most of the adenomas.
Given the concept of the DNES, of which the adre- nal cortex is not considered to be an intrinsic part,2 the presence of these neuroendocrine markers in the adre- nal cortex is an unexpected but clear finding. Synapto- physin is believed to be one of the best markers for neu- roendocrine tissues.21 The function of synaptophysin in
neuroendocrine tissues is still unknown, but it probably participates in transmitter transport. The presence of synaptophysin outside the DNES to our knowledge has not been described.
The presence of NSE in nonneuroendocrine tissues and various tumors, however, has been reported.22 NSE is an enzyme that catalyses the interconversion of 2-phos- phoglycerate and phosphoenolpyruvate. Its presence in neoplastic cells is ascribed to the need for the opening up of metabolic pathways that under normal conditions are operable only in neuroendocrine cells. The MOC-1-related antigen is present outside the lung in a subset of normal endocrine and neural cells. The zona glomerulosa and fas- ciculata of the adrenal cortex previously have been found to be positive for this protein.13
The focal positivity of NSE, synaptophysin (and vi- mentin), and the more abundant presence of the MOC- 1-related antigen in the normal cortex, combined with extensive positivity of these proteins in adrenal tumors, all support considering the adrenal cortex as part of the DNES. The neuroendocrine marker-positive foci in the normal cortex could be remnants of foetal adrenal glands, which constitute 80% of the cortex at term but involute very soon after birth.23,24 The cells in these foci thus are to be considered as progenitor (stem) cells, from which tumors may arise after clonal expansion. This speculation supports oncogenesis of adrenocortical tu- mors as part of the concept of “blocked ontogeny.” 25,26 Conversely, the tumor may also emerge as a result of retro-differentiation or differentiation of mature, spe- cialized cells.26 The latter view supports de-depression of gene regulation as a cause for the highly specialized cell to revert to a multipotent state with possible expres- sion of proteins of the progenitor cells.
The similarities between adrenocortical carcinomas and neuroendocrine tumors may have implications, not only for understanding of gene transcription, but also to form a rationale for the treatment of these tumors. It may explain at least partially the disappointing results of various treatment modalities. Thus, in neuroendocrine tumors of the pancreas, streptozotocin therapy resulted in tumor re- gression in only 14 out of 38 patients treated.27 Streptozo- tocin is also active in adrenocortical carcinoma,28 but the use of this drug in patients with neuroendocrine tumors and in patients with adrenocortical tumors in general has yielded only modest clinical results.
From the above findings, it can be concluded that there is a relationship between adrenocortical carcino- mas and neuroendocrine tumors. Therefore, any agent that would prove to be successful in the management of one of these tumors may carry the same potential in the management of the others.
These speculations based on our immunohistochem- ical results of course remain to be proven. Results from the present study lead us to conclude, however, that after neoplastic transformation, adrenal cortical cells show (fur- ther) neuroendocrine differentiation with a mesenchymal phenotype including vimentin expression and loss of ker- atin reactivity. These immunohistochemical findings may prove to be useful in tumor diagnosis, especially in cases where metastatic abdominal tumors cannot be readily classified on the basis of conventional techniques. These findings may also be useful in the development of new therapeutic regimens in the, so far, disappointing chemo- therapeutic management of adrenal cortex carcinoma, and other neuroendocrine tumors.
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