Different immunohistochemical patterns of TGF-61 expression in benign and malignant adrenocortical tumours
Adriana Boccuzzi*, Massimo Terzolo*, Susanna Cappiat, Paolo De Giulit, Carolina De Risit, Eugenio Leonardot, Silvia Bovio*, Mirella Borriero*, Piero Paccotti* and Alberto Angeli*
* Dipartimento di Scienze Cliniche e Biologiche, Cattedra di Medicina Interna, Azienda Ospedaliera S.Luigi, Università di Torino, Torino and + Servizio di Anatomia Patologica, Azienda Ospedaliera S. Luigi, Torino, Italy
(Received 7 July 1998; returned for revision 18 August 1998; finally revised 22 September 1999; accepted 21 November 1999)
Summary
OBJECTIVE Transforming growth-factor 81 (TGF-81) influences a number of specific functions of adreno- cortical cells in several animal species. The aim of our study was to evaluate by immunohistochemical analysis the presence and distribution of TGF-81 in normal adrenal tissue and in different adrenal tumours.
PATIENTS We analysed 8 functioning (5 adenomas and 3 carcinomas) and 15 non functioning (6 adeno- mas and 9 carcinomas) adrenal tumours and 6 normal adrenal glands.
RESULTS In normal adrenal glands, the glomerulosa and the reticularis zones displayed diffuse cytoplas- mic staining, while the fasciculata zone was almost completely negative. Functioning adenomas dis- played cytoplasmic staining restricted to compact cells while in nonfunctioning adenomas, prevalently composed by clear cells, no staining was observed. Overall, adrenal carcinomas were characterized by the lack of cytoplasmic positivity and by sporadic posi- tive cells around vessels both in functioning and in nonfunctioning tumours.
CONCLUSIONS TGF-81 expression is associated with active steroid secretion in normal adrenal tissue, as well as in benign cortical adenomas, while this rela- tionship is lost in primary adrenal malignancies.
Correspondence: Dr A. Boccuzzi, Clinica Medica Generale, Azienda Ospedaliera S. Luigi, Regione Gonzole, 10, 10043 Orbassano - Torino, Italy
These data provide indirect evidence for a regulatory role played by TGF-81 on steroid secretory pathways.
Transforming Growth Factor-ß (TGF-ß) belongs to a wide family of structurally related peptides which act as regulators at different steps of cell proliferation and cell differentiation. TGF-ßs are able to inhibit cell growth in epithelial tissues and also play an important role in the processes of embryonic development and tissue repair. Moreover, TGF-ßs are able to modulate hormonal activity in several types of endocrine cells (Adashi et al., 1989; Roberts & Sporn, 1990; Feige & Baird, 1991; Feige et al., 1991a; Benhamed et al., 1993).
To date, three isoforms, termed TGF-61, TGF-32 and TGF- B3, have been isolated in mammals. They are characterized by widespread expression in different mammals tissues. Each of the three forms is synthesized as a precursor that is cleaved intracellularly into two peptides, which further dimerize. These homodimers are non covalently bound to latency-associated peptides to form latent-TGF-6 which is secreted and activated outside the cell by proteolytic enzymes (Hering et al., 1995).
TGF-Bs induce their effects by binding to three different types of cell-surface receptors. These receptors have been identified by cross-linking experiments and are referred to as type I, type II (both are serine-threonine kinases) and type III, or beta-glycan. Only the type I and the type II receptors seem to be directly involved in signal transduction (Massagué, 1990; Lin et al., 1992; Wrana et al., 1992; Franzén et al., 1993; Bassing et al., 1994; Wrana et al., 1994).
The effects of TGF-61 on endocrine activity of the adrenal cortex have been extensively studied in different experimental models. Bovine adrenocortical cells (BAC) express TGF-6 receptors that are regulated by ACTH (Cochet et al., 1988). The presence of the three types of TGF-31 receptors has been demonstrated also in human adrenal cells (HAC), where type I receptor is the predominant form (Lebrethon et al., 1994), and in human fetal adrenal gland (Stankovic & Parker, 1995).
TGF-61 is a potent inhibitor of basal and ACTH-induced cortisol synthesis in BAC and ovine adrenocortical cells (OAC). The cytokine accomplishes this effect through reducing the affinity of low density lipoprotein receptors (Feige et al., 1991b; Hotta & Baird, 1996), and restraining the synthesis of steroid 17a-hydroxylase (P-450 17a) mRNA and 33- hydro- xysteroid dehydrogenase (3-6-HSD) mRNA (Feige et al.,
1987; Rainey et al., 1990, 1991a,b; Naville et al., 1991; Perrin et al., 1991). Furthermore, TGF-61 reduces the number of angiotensin-II receptors in BAC cells (Feige et al., 1987) and ACTH-receptors in OAC cells (Rainey et al., 1989).
Similar evidence has been recently obtained for human fetal adrenocortical cells (Stankovic et al., 1994). On adult HAC, TGF-61 was found to reduce 17a-hydroxylase mRNA and increase 36-hydroxysteroid dehydrogenase mRNA (Lebrethon et al., 1994). However, the exposure of HAC to TGF-31 did not affect basal or ACTH-stimulated cortisol production (Lebre- thon et al., 1994).
The aim of the present study was to evaluate by immunohistochemical analysis the presence and distribution of TGF-31 in normal adrenal tissue and in functioning or nonfunctioning cortical tumours. We also tried to correlate TGF-31 expression with pathological characteristics of the tumours and their hormone secretion.
Materials and methods
Tissue specimens
Tissue specimens were obtained from 23 patients who underwent adrenalectomy because of an adrenal mass. In 8 cases the adrenal tumour was associated with ACTH- independent Cushing’s syndrome (5 adenomas and 3 carcino- mas), while 15 tumours were defined as nonfunctioning (6 adenomas and 9 carcinomas). Two additional patients were submitted to bilateral adrenalectomy because of occult ectopic ACTH syndrome. Pertinent clinical information is given in Table 1. Clinical presentation, hormonal and radiological studies, and follow-up data as well, were consistent with the histological diagnosis in all cases. Normal adrenal glands were obtained from 6 patients who underwent nephrectomy and adrenalectomy because of renal carcinoma (Stage I).
| Patient | Age | Sex | Histological diagnosis | Clinical presentation | Cortisol (%) | DHEAS (%) |
|---|---|---|---|---|---|---|
| 1 | 21 | F | Adenoma | CS | +40 | -95 |
| 2 | 15 | F | Adenoma | CS | -1 | - 94 |
| 3 | 62 | F | Adenoma | CS | - 18+ | -25+ |
| 4 | 21 | F | Adenoma | CS | + 50 | -85 |
| 5 | 52 | F | Adenoma | CS | + 32 | -88 |
| 6 | 57 | M | Adenoma | Incidentaloma | -30 | - 82 |
| 7 | 61 | F | Adenoma | Incidentaloma | -53 | -95 |
| 8 | 63 | F | Adenoma | Incidentaloma | -9 | - 80 |
| 9 | 54 | F | Adenoma | Incidentaloma | - 40 | -33 |
| 10 | 59 | M | Adenoma | Incidentaloma | -35 | -53 |
| 11 | 58 | F | Adenoma | Incidentaloma | -20 | - 87 |
| 12 | 62 | M | Carcinoma | CS | - 62* | und* |
| 13 | 23 | F | Carcinoma | CS, HA | +25 | + 100 |
| 14 | 18 | F | Carcinoma | CS, HA | + 28 | + 150 |
| 15 | 30 | F | Carcinoma | Abdominal pain | 0 | - 32 |
| 16 | 39 | M | Carcinoma | Abdominal pain, weight loss | -58 | - 79 |
| 17 | 38 | M | Carcinoma | Incidentaloma | - 34 | - 17 |
| 18 | 56 | M | Carcinoma | Incidentaloma | - 45 | -20 |
| 19 | 48 | M | Carcinoma | Incidentaloma | -51 | -38 |
| 20 | 27 | M | Carcinoma | Incidentaloma | - 12 | -36 |
| 21 | 20 | F | Carcinoma | Incidentaloma | - 43 | + 132 |
| 22 | 49 | F | Carcinoma | Abdominal pain | und* | und* |
| 23 | 28 | F | Carcinoma | Incidentaloma | - 18 | -48 |
| 24 | 45 | F | Bilateral adrenal hyperplasia | CS | + 300 | -51 |
| 25 | 34 | M | Bilateral adrenal hyperplasia | CS | +30 | -60 |
Hormonal data are expressed as percentage variation of the upper normal value of the reference laboratory [(observed value-upper normal value/upper normal value)× 100].
¡ Patient on ketoconazole treatment;
*patients on O,P’-DDD (Mitotane) treatment. CS, Cushing’s syndrome; HA, Hyperandrogenism; und: undetectable.
Histopathological evaluation
The tissues were fixed in 10% buffered neutral formalin and embedded in paraffin. Five micron sections were prepared and stained with haematoxylin-eosin and were reviewed by two expert pathologists. A part of tissue was placed in O.C.T. (Tissue Tek, Miles Laboratories) and frozen in liquid nitrogen; stored cryostatic sections (5 microns thick) were air dried and fixed in cold acetone for 10 minutes
In this study, adrenocortical adenomas were morphologically defined as adrenocortical masses well encapsulated, without foci of vascular or capsular invasion. Histopathological diagnoses were made following the criteria of adrenocortical malignancy developed by Weiss (1984).
Immunohistochemical analysis
Other five micron sections were prepared for immunohisto- chemical staining using the immunoperoxidase ABC method according to Hsu et al. (1981). Briefly, paraffin sections were dewaxed in xylene, rehydrated and washed in phosphate buffer saline 0.1 M pH 7-4 (PBS). Endogenous peroxidase was inhibited by 3% hydrogen peroxide in PBS. After washed in PBS the sections were incubated with normal horse serum and then with mouse antihuman TGF-1 (Clone TB21, Serotec, UK) for 60 minutes at room temperature (dilution 1 : 200). This antibody recognized either the dimeric (25 kD) or the mono- meric (12.5 kD) molecules of TGF-61, that means the form of TGF-31 not linked to the latency-associated peptide (LAP). Since accurate immunolocalization depends on preservation of the target protein during fixation we performed the immuno- histochemical analysis also on cryostatic sections. Cryostatic
sections of two normal glands, two adenomas associated with Cushing’s syndrome (cases 4 and 5), two nonfunctioning adenomas (cases 6 and 10) and two carcinomas (cases 12 and 15) were treated with PBS for 10 minutes, incubated with normal horse serum and then with mouse antihuman TGF-ß1 (Clone TB21, Serotec, UK) for 60 minutes at room temperature. After washes in PBS all the sections were incubated with horse antimouse immunoglobulin biotinyl antibody (Vector, Burlin- game) and then, after further washes, incubated with avidin- biotin-peroxidase complex (Vector, Burlingame). The site of the peroxidase reaction was visualized using a solution of 3-3’diaminobenzidine (DAB) with 0-3% Hydrogen peroxide. The evaluation of the staining was done by three different examiners. Negative controls were performed by omitting the primary antibody and by using unrelated antibodies of the same isotype as primary antibodies.
Results
Morphological findings
Different microscopic aspects have been observed among adrenal adenomas. The 5 adenomas associated with Cushing’s syndrome were composed by variable proportions of clear and compact cells. Compact cells were more numerous than clear cells in 4 of them while an inverse pattern was observed in 1 case. In the nonfunctioning adenomas clear cells always outnumbered compact cells which were detectable in only 3 out of 6 cases (Table 2).
In 8 out of 12 carcinomas compact cells outnumbered clear cells which were barely detectable in only two of these cases.
| Patient | Clinical presentation | Microscopic features | TGF-01 immunostaining | |||
|---|---|---|---|---|---|---|
| Compact cells | Clear cells | Compact cells | Clear cells | Pattern/intensity | ||
| 1 | CS | ++ | þ | 30% | Neg | Scattered/ + |
| 2 | CS | ++ | þ | 40% | Neg | Focal/+-+++ |
| 3 | CS | け | ++ | 10% | Neg | Scattered/ + |
| 4 | CS | ++ | þ | 80% | Neg | Diffuse/++ |
| 5 | CS | ++ | þ | 20% | Neg | Focal/++ |
| 6 | Incidentaloma | – | ++ + | – | Neg | – |
| 7 | Incidentaloma | – | +++ | – | Neg | – |
| 8 | Incidentaloma | ± | + + + | Neg | Neg | – |
| 9 | Incidentaloma | – | +++ | – | Neg | – |
| 10 | Incidentaloma | þ | ++ | Neg | Neg | – |
| 11 | Incidentaloma | ± | +++ | Neg | Neg | – |
CS, Cushing’s syndrome; Neg: negative.
@ 1999 Blackwell Science Ltd, Clinical Endocrinology, 50, 801-808
| Patient | Clinical presentation | Microscopic features | TGF-31 immunostaining | |||
|---|---|---|---|---|---|---|
| Compact cells | Clear cells | Compact cells | Clear cells | Pattern/intensity | ||
| 12 | CS | +++ | – | Neg | – | Sporadic positive cells around the vessels |
| 13 | CS, HA | け | +++ | Neg | Neg | Sporadic positive cells around the vessels |
| 14 | CS, HA | +++ | – | Neg | – | Sporadic positive cells around the vessels |
| 15 | Abdominal pain | +++ | ± | Neg | Neg | Positive cells around the vessels |
| 16 | Abdominal pain, weight loss | +++ | – | Neg | – | Sporadic positive cells around the vessels |
| 17 | Incidentaloma | け | +++ | Neg | Neg | Sporadic positive cells around the vessels |
| 18 | Incidentaloma | +++ | – | Neg | – | Sporadic positive cells. |
| 19 | Incidentaloma | +++ | – | Neg | – | Sporadic positive cells around the vessels. Higher density of vessels |
| 20 | Incidentaloma | +++ | – | Neg | – | Sporadic positive cells around the vessels |
| 21 | Incidentaloma | ± | +++ | Neg | Neg | Sporadic positive cells around the vessels |
| 22 | Abdominal pain | け | +++ | Neg | Neg | Sporadic positive cells around the vessels |
| 23 | Incidentaloma | +++ | ± | Neg | Neg | Sporadic positive cells around the vessels |
CS, Cushing’s syndrome; HA, hyperandrogenism; Neg, negative.
Conversely, clear cells predominated over compact cells in the remaining 4 carcinomas. The compact cell-only pattern was observed in 2 out of 3 carcinomas associated with Cushing’s syndrome (Table 3).
The 4 specimens of adrenal diffuse hyperplasia from the patients with ectopic Cushing’s syndrome (two for each case) showed a marked hyperplasia of the reticularis zone and a reduction of clear cells of the fasciculata zone; the glomerulosa zone was normally represented.
Immunohistochemical analysis in normal and hyperplastic adrenal glands
Immunohistochemical analysis of normal adrenal glands revealed a different staining in the three zones of the cortex. The glomerulosa displayed a diffuse cytoplasmic positivity. In the fasciculata the staining was restricted to a few cells and was of feeble intensity. In the reticularis the staining was present in the majority of the cells displaying a mild to strong intensity (Fig. 1 a, b). All the specimens examined shared this immunohistochemical pattern. No staining was observed when nonimmune mouse IgGs replaced the anti-TGF61 antibodies or when the first antibody was omitted.
In hyperplastic glands TGF-61 immunostaining showed a zone-specific pattern alike to that observed in normal glands.
TGF-31 immunostaining detected in cryostatic sections was highly comparable for distribution and intensity to that obtained in paraffin-embedded slices.
Immunohistochemical analysis in adrenal adenomas
In adrenal adenomas TGF-31 immunostaining varied in terms of intensity and distribution of the positive cells. The intensity ranged from weak to very marked while 3 patterns of distribution were observed: (a) scattered pattern which featured the presence of only a few positive cells in any one microscopic field; (b) focal pattern which was characterized by the clustering of positive cells in one or more groups; (c) diffuse pattern which featured staining of the majority of the cells (Table 2).
In the 5 adenomas associated with Cushing’s syndrome the staining was limited to the compact cells with a rate of positivity ranging from 10% to 80% (Fig. 2). Non-functioning adenomas did not display any staining because of the negativity of clear cells, the predominant cell type in such tumours (Fig. 3).
Cryostatic sections of cases 4 and 5 showed a cytoplasmic immunostaining with a distribution pattern similar to that observed in the corresponding paraffin-embedded tissues. The cryostatic slices of cases 6 and 10 did not stain at all for TGF-61 as was the case for the corresponding paraffin-embedded sections.
Overall, stromal and endothelial cells were always positive for TGF3-1.
Immunohistochemical analysis in adrenal carcinomas
In all adrenal carcinomas no cytoplasmic staining was observed, either in compact or in clear cells, while sporadic
@ 1999 Blackwell Science Ltd, Clinical Endocrinology, 50, 801-808
b
positive cells around the vessels were present (Table 3, Fig. 4). Stromal and endothelial cells were always positive for TGF6-1.
In cryostatic sections of cases 12 and 15 no intracytoplasmic immunostaining was detected but stromal and endothelial cells were positive.
Discussion
TGF-31 has been viewed as a candidate for the role of auto/ paracrine regulator of the endocrine activity of the adrenal cortex. TGF-31 has been found to induce variable biochemical responses in different species (Flanders et al., 1989; Thompson et al., 1989), and it has been shown to be a growth regulator of
both definitive zone and fetal zone cells in human fetal adrenal gland (Parker et al., 1992). Therefore, locally produced TGF-61 may indeed participate in the regulation of different aspects of human adrenocortical physiology and pathophysiology.
We therefore thought it worth evaluating the distribution of TGF-31 in normal human adrenal gland and in different adrenal tumours. We tried to correlate the different immunohisto- chemical patterns with the benign or malignant behaviour of the tumours and their endocrine activity.
TGF-61 staining was present at variable degrees of intensity in the different layers of the normal adrenal gland. The maximal intracellular staining was evident in the zona reticularis, that is mainly composed by cells with ‘compact’ characteristics,
because of their scant lipid content. The staining progressively decreased towards the zona fasciculata, which was almost completely negative, being composed only by ‘clear’ cells. A diffuse staining was present in the zona glomerulosa.
Such a clear zonal distribution of the TGF-61 immunostain- ing suggests a relationship between TGF-31 expression and steroidogenic activity in the normal adrenal cortex. TGF-61 may regulate steroid synthesis in compact cells or their proliferation rate, or both. Since the zona reticularis is admittedly the site of synthesis of dehydroepiandrosterone (DHEA) and its sulphate form (DHEA-S), our findings provide indirect evidence that TGF-31 may be physiologically involved
in regulating the production of these steroids. Previous studies have provided data consistent with an inhibitory role of TGF-31 upon DHEA-S secretion. (Lebrethon et al., 1994; Parker et al., 1995)
The outcome of our study on TGF-01 expression in adrenocortical adenomas suggests that this cytokine may play other actions. Only the adenomas associated with Cushing’s syndrome showed significant TGF-61 immunostaining. They were mainly composed by compact cells which were found to express higher levels of steroidogenic enzymes in comparison to clear cells (Suzuki et al., 1992a, b; Sasano et al., 1993). Therefore, it is possible to speculate that TGF-61 may modulate
cortisol secretion and not only restrain DHEA-S synthesis, at least in states of cortisol hypersecretion. In fact, DHEA-S levels were in the low-normal range in all the adenomas evaluated, while TGF-61 staining was a unique characteristic of glucocorticoid-producing tumours. This hypothesis fits well also with the significant TGF-31 immunostaining observed in ACTH-dependent adrenal hyperplasia.
Overall, adrenal carcinomas displayed no significant expres- sion of TGF-61 even in tumours with active glucocorticoid secretion. Therefore, the coupling between TGF-61 expression and cortisol hypersecretion seems to be disturbed by cellular atypia and dedifferentiation. However, in all carcinoma speci- mens some TGF-61 positive cells were detected nearby the vessels. This finding recalls the well-described angiogenetic activity of TGF-31, which was found able to induce vessel formation in vivo and to stimulate endothelial cell arrangement in tubular structures in vitro (Benzakour, 1994; Roberts et al., 1986; Madari et al., 1988; Pellerin et al., 1993). Comparable results were obtained in formalin-fixed paraffin embedded tissues as in frozen preparations, thus excluding that the staining patterns were induced by the method of tissue preparation.
Our data agree with preliminary results of Arnaldi et al. (1995) who reported a significant reduction of TGF-31 mRNA content in adrenal carcinomas (either functioning or nonfunc- tioning) in comparison to normal adrenal glands and cortisol- producing adenomas. The analysis of TGF-81 immunostaining seems of limited utility in the differential diagnosis between adrenal adenomas and carcinomas when the tumour is devoid of endocrine activity. Conversely, the study of TGF-61 immuno- localization may be of help in differentiating benign from malignant adrenocortical neoplasms associated with Cushing’s syndrome.
In conclusion, the present data raise interesting speculations on TGF-31 as a modulator of human adrenal function either in normal physiology or in disease states. Further studies are warranted to clarify the relationship between TGF-61 and adrenal steroidogenesis.
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