Diagnostic Role of Inhibin a-Subunit and Inhibin/Activin B-Subunit in Adrenal Cortical and Medullary Tumors in Egyptian Patients
Hanan Mohammed Abd Elmoneim, PhD,* Rehab Monir Samaka, MD,t and Hanan Ali, MD#
Abstract: Inhibin and activins are dimeric glycoproteins that are structurally and functionally related to transforming growth factor-ß and are composed of 1 a-subunit and 1 of 2 B-subunits (BA or BB). In recent years, there has been controversy about their role in adrenal tumors and their suitability as a diagnostic/ predictive marker. Inhibin & and inhibin/activin ß protein ex- pression was assessed on 47 adrenal tissue specimens by means of immunohistochemistry. Positive immunoreactivity of inhibin- a was seen in all studied hyperplastic adrenal glands, 90.9% of cortical adenomas, and 83.3% of adrenal cortical carcinomas. In contrast, the adrenomedullary neoplasms had a statistically significantly different behavior (P = 0.001). We observed the negative expression of inhibin & in 85% and 80% of benign and malignant pheochromocytomas, respectively. As regards the immunoreactivity of inhibin/activin ß, 80% of adrenal hyper- plasias, 81.8% of cortical adenomas, and 83.3% of adrenal cortical carcinomas showed positivity. Strong-to-weak positive staining of inhibin/activin ß was observed in 70% of benign pheochromocytomas, whereas malignant pheochromocytomas showed positive immunohistochemical staining in 40% of cases with weak intensity. The scoring of inhibin/activin ß im- munoreactivity between adrenocortical and adrenomeullary neoplasia failed to reach the significant value (P = 0.1). Our results demonstrate that inhibin & had a diagnostic role, differ- entiating between the adrenocortical and adrenomedullary ne- oplasms. Moreover, inhibin/activin ß might play a predictive role for malignant potential in pheochromocytoma. Further studies are warranted to determine whether they play a diag- nostic/predictive role in adrenal tumors or are just surrogate markers for this group of neoplasia.
Key Words: adrenal tumors, inhibin a, inhibin/activin ß, im- munohistochemistry
(Appl Immunohistochem Mol Morphol 2012;20:462-469)
From the *Department of Pathology, Minia University, Minia; ¡Department of Pathology, Menoufiya University, Shebin Elkom; and ¿Department of Internal Medicine, Beni Sweif University, Bani Suef, Egypt.
The authors declare no conflict of interest.
Reprints: Hanan Mohammed Abd Elmoneim, PhD, Department of Pathology, Minia University, Minia 61111, Egypt (e-mail: annglasgow @hotmail.com).
Copyright @ 2012 by Lippincott Williams & Wilkins
T umors of the adrenal cortex have been identified in up to 9% of all autopsy cases. Most of them appear in hypertensive and diabetic patients.1 Masses found in 7% of patients older than 50 years are detected incidentally during assessment of patients by modern imaging modal- ities. Some adrenal tumors are hormone secreting and re- sponsible for endocrine pathology; however, the majority of them are nonfunctional.2,3 A patient’s physician, when faced with an incidental adrenal mass, must assess for the presence of subclinical disease and/or malignancy.4
Generally, adrenal tumors with a malignant poten- tial grow faster than those with a benign phenotype. However, it should be noted that even adrenal tumors smaller than 2 cm at detection might already be meta- static. The Adrenal Gland Scoring Scale (Pheochromo- cytoma of the Adrenal Gland Scaled Score) features intratumor necrosis, high cellularity, cellular monotony, tumor cell spindling, profound nuclear pleomorphism, nuclear hyperchromasia, capsular invasion, vascular in- vasion with irregular margins, and local metastasis as the initial characteristics suggestive of malignancy5,6; if these are not present regular imaging follow-up should be performed to help detect malignancy.7
Pheochromocytomas are rare tumors that produce significant amounts of catecholamines and give rise to the well-known clinical picture of pheochromocytoma.8-10 Autopsy series have revealed a much higher prevalence of pheochromocytomas,11 whereas they are relatively rare tumors found in about 4% of incidentally discovered adrenal masses.12
Adrenal cortical tumors sometimes resemble pheo- chromocytoma. Lipid degeneration indicates pheochro- mocytomas, whereas cytoplasmic lipids can develop in adrenal cortical tumors.13 Histologic evaluation, chro- maffin reaction, immunohistochemical analysis, and elec- tron microscopy can be helpful in the differential diagnosis between adrenal cortical tumors and pheochromocyto- ma.14 A distinction between adrenal cortical neoplasm and pheochromocytoma may be difficult to make, especially in small biopsies or cytology specimens. 15
Inhibin and activin are dimeric glycoproteins that are structurally and functionally related to transforming growth factor-ß16,17 and are composed of 1 x-subunit and 1 of 2 B-subunits (BA or BB).18 Inhibins A (aßA) and B (aßB) inhibit the release of follicle-stimulating hormone from the pituitary gland, but activins (ABABA, B-BBBB,
AB-BAßB) increase the pituitary follicle-stimulating hor- mone secretion. 19-22
Inhibin a-subunit gene expression (which is needed for inhibin synthesis) has been detected in ovarian gran- ulosa cells and testicular sertoli cells, both of which are the main sources of circulating inhibin.23-25 Extragonadal inhibin expression has also been detected mainly in ste- roid-producing organs such as the placenta, pituitary gland, and adrenal gland.26,27 As ovarian granulose cell tumors and testicular sertoli cell tumors secrete inihibin, serum measurement can be used to detect recurrent or metastatic disease.24,28 Immunohistochemical staining for inhibin can be used to distinguish ovarian sex cord-stro- mal tumor from other neoplasms that mimic them.29-31
All zones of the human adult adrenal gland express both activin BA and BB subunits, suggesting that activins were synthesized in this organ.32 The physiological func- tion of activins in the adrenal gland is the inhibition of mitogenesis and enhancement of adrenocorticotropic hormone (ACTH)-stimulated cortisol secretion in human fetal zone cells and cultured bovine adrenocortical cells but not in adult adrenal cells.33
Tumor formation in the adrenal cortex has also been linked to inhibin and activin.32 Matzuk and col- leagues showed that inhibin x-subunit knockout mice developed adrenocortical tumors. Expression of the in- hibin a-subunit was investigated more widely, because the earlier data from animal models suggested that it was an adrenal tumor suppressor.34,35 Inhibin A had no mitogenic or steroidogenic effects and was suggested as a tumor suppressor for the adrenal gland in a gonadectomized an- imal model.17,34,36
Therefore, our study aimed to assess the im- munohistochemical staining of inhibin «-subunit and in- hibin/activin ß in adrenocortical and adrenomedullary neoplasia as well as in adrenal hyperplasia and determine whether differences exist that may serve to distinguish them from one another.
MATERIAL AND METHODS
This retrospective study investigated 47 adrenal tissue specimens. They were retrieved from the files of the departments of Pathology, El Minia University and Menoufyia University, and from the Internal Medicine Department, Bani Suef University, during the period between January 2000 and January 2010. These tissue specimens included 22 that were adrenocortical in origin and 25 that were adrenomedullary. The 22 adrenocortical tissue specimens comprised 5 adrenal cortical hyper- plasias, 11 adrenocortical adenomas, and 6 adrenocortical carcinomas. The 25 adrenonomedullary tissue specimens were composed of 20 benign pheochromocytomas and 5 malignant pheochromocytomas. Malignancies of adre- nocortical tumors were assessed according to the criteria of Pheochromocytoma of the Adrenal Gland Scaled Score, which was used for evaluation of the adrenome- dullary pheochromocytoma specimens.37,38 The histo- logically suspicious features suggestive of malignancy
were tumor weight, tumor size, presence or absence of tumor encapsulation, capsular invasion, vascular in- vasion, extension into the periadrenal adipose tissue, cell nests, necrosis, fibrosis, degenerative changes, calcifica- tions, cellularity, cytoplasmic quality, cellular pleomorphism, cellular monotony, nuclear hyperchromasia, pyknosis, nucleoli, intranuclear cytoplasmic inclusions, intracytoplasmic eosinophilic hyaline globules, giant cells, mitotic figures, atypical mitotic figures, and, if metastatic disease were present, whether it was histologically similar to the pri- mary tumor.5
Tumors were considered malignant if histologically or radiologically proven metastases were present or they were without metastases but having any of the histolog- ically suspicious features, that is > 5 mitoses/10 high- power fields, confluent tumor necrosis, or vascular or capsular invasion.
The adrenal tissue specimens were subjected to the immunohistochemical staining of inhibin a-subunit and inhibin/activin ß-subunit. The expression of the a-sub- unit, and thus of inhibins, seems to be more restricted than that of the B-subunit and therefore of activins. However, when both are expressed, inhibins seem to op- pose the actions of activins.32 Clinical findings in all cases confirmed the histologic diagnoses. Diagnosis of Cushing syndrome was based on clinical findings of hypertension, oligomenorrhea, a moon face, a buffalo hump, striae over the patient’s abdomen and thighs, trunkal obesity, and hirsutism. Laboratory investigations included the fol- lowing: urinary-free cortisol levels on 2 separate collec- tions (serum cortisol and cortisol levels after being given dexamethasone suppression of 1 and 8 mg overnight), ACTH level, and dehydroepiandrosterone sulfate level. The presence of malignant tumors was clinically sus- pected in patients with weight loss, high serum concen- tration of dehydroepiandrosterone sulfate, and the presence of an irregular, large (usually with a diameter >6 cm) mass found by computed tomography imaging. The diagnosis of pheochromocytoma was suspected in patients with poorly controlled hypertension associated with bouts of sweating, headache, palpitations, and a high level of metanephrins in urine. In patients with non- functioning adenomas, the tumors were incidentally found during computed tomography imaging.
Immunohistochemical Staining
Sections of 5-um thickness were cut from the par- affin-embedded blocks with subsequent steps of depar- affinization and dehydration in xylene and a decreasing graded series of alcohol. Antigen retrieval was performed by pretreatment of sections in a microwave oven in 10-mmol/L citrate buffer, pH 6.0, at 600 W for 20 mi- nutes. The slides were incubated overnight at room tem- perature with the primary monoclonal antibodies for inhibin a-subunit (dilution 1:40; Serotec Ltd, Kidlington, Oxford, UK) and inhibin/activin ß-subunits (dilution 1:50 both from Serotec Ltd). The Envision 1 (Dako, Glostrup, Denmark) method was used for detection of the used antibodies. The reaction was visualized by an appropriate
substrate/chromogen (diaminobenzidine) reagent with Mayer hematoxylin as a counter stain. Normal ovarian tissue was used as a positive control. The negative control obtained by substitution of primary antibodies with blocking buffer was included in the staining procedure. Negative controls also included the kidney cortex, which was expected to be negative.
Interpretation of Immunostaining Results
Granular brown cytoplasmic staining was consid- ered positive. The percentage of positively stained cells was analyzed from 1 slide per tumor. Five representative high-power fields were chosen, and the percentage of positive staining cells was calculated. The final result was estimated from the whole slide and was categorized as follows: negative (no staining), weak (1% to 10%), moderate (11% to 50%), and strong (over 51%).39,40 Unintentional bias was prevented by coding patient tissue samples so that immunohistochemistry could be studied without knowledge of the patient outcome and tumor characteristics. Regarding the reproducibility of the im- munostaining results, 2 authors (R.M.S. and H.M.A.E.) evaluated the slides separately. Minor differences were neglected, but in cases with major differences they reevaluated the cases together to reach a consensus score.
Statistical Analysis
Data were collected, tabulated, and statistically analyzed using a personal computer with the statistical package for the social sciences version 16 for Windows. The x2 test was applied for comparison between groups. Correlation between markers in different lesions was as- certained using the Spearman correlation test. Differences were considered statistically significant with P≤0.05.
RESULTS
Twenty-three (48.9%) patients were male and 24 (51.1%) were female. Patients’ ages ranged from 38 to 67 years, with mean ± SD of 52.91 ± 7.31 years. As regards the adrenocortical tissue specimens, all 5 (100%) adrenal hyperplasias (Fig. 1A), 10/11 (90.9%) cortical adenomas (Fig. 1B), and 5/6 (83.3%) adrenal cortical carcinomas (Fig. 1C) were positive for inhibin a. The staining in- tensity was distributed, as 11 were strongly positive, 6 were moderately positive, and 3 were weakly positive. The differential distribution of inhibin & immunoreactivity in the adrenocortical tissue specimens was more intense in relatively small polygonal cells with eosinophilic cyto- plasm resembling normal adrenal cortical cells of zona reticularis. The large cells with clear cytoplasm in the neoplasms that resembled adrenal cortical cells of zona fasciculata were negative or weakly positive for inhibin a.
However, 17/20 (85%) benign pheochromocytomas (Fig. 1D) and 4/5 (80%) malignant pheochromocytomas (Fig. 1E) were negative for inhibin a. Immunoreactivity of the positive cases was weak. A statistically significant dif- ference was found between the studied groups with regard to inhibin o-subunit expression (P < 0.001) (Table 1). In addition, inhibin a-subunit expression showed a statisti-
cally significant difference between adrenocortical and adrenomedullary tumors (P = 0.001) (Table 2).
As regards the immunoreactivity of inhibin/activin B, 4/5 (80%) adrenal hyperplasias (Fig. 2A), 9/11 (81.8%) cortical adenomas (Fig. 2B), and 5/6 (83.3%) adrenal cortical carcinomas (Fig. 2C) showed positive granular cy- toplasmic staining. Six adrenocortical tissue specimens were strongly positive, 5 were moderately positive, and 7 were weakly positive. Most of the benign pheochromocytomas [14/20 (70%)] expressed positive immunohistochemical staining of inhibin/activin ß (Fig. 2D). Nine tumors were strongly positive and 5 were moderately or weakly positive. Forty percent of the studied malignant pheochromocytomas showed weak focal positive immunohistochemical staining for the inhibin/activin ß-subunit (Fig. 2E). No statistically significant differences were noticed in immunohistochemical expression of inhibin/activin ß between the studied groups (P = 0.5) (Table 1), nor between adrenocortical and adrenomedullary tumors (P = 0.1). However, a statistically significant difference was noticed between benign and malignant pheochrocytoma (P = 0.05).
The density of sustentacular cells may vary markedly within a single tumor, with some areas containing very few cells. Immunostaining for inhibin «-subunit and inhibin/ activin ß-subunit may be difficult to interpret in some cases because of positivity in tumor cells with the absence of inhibin «-subunit-positive and inhibin/activin ß-subunit- positive sustentacular cells in malignant tumors.
No statistically significant correlation was found between inhibin a-subunit and inhibin/activin ß-subunit immunoreactivity in adrenocortical and adrenomedullary tumors (r = 0.863, P = 0.2; data not shown in Table).
DISCUSSION
Although gonads are the main source of circulating inhibins, adrenal venous blood has a higher concentration of inhibins than does peripheral blood, suggesting some contribution of adrenals as well.41 A negative auto- regulation of inhibin o-subunit expression was suggested in a transgenic mouse study, where gonadal inhibins were shown to downregulate the expression of inhibin o-subunit gene in the adrenal gland.35 In the meantime, inhibins and activins are endocrine and paracrine hormonal regulators of the hypothalamic-pituitary-gonadal axis, and they are expressed in many tissues such as pituitary, bone marrow, central nervous system, and placenta.19,42,43 They may exert a pleiotropic effect and seem to inhibit hepatocyte growth in an autocrine manner by influencing apoptosis.44 They may play a role in inflammatory processes45 and a significant role in embryogenesis.46
In the present study, we found a positive im- munoreactivity with the anti-inhibin o-subunit mono- clonal antibody in hyperplastic adrenal glands (100%), in adrenal cortical adenomas (90.9%), and in adrenal cort- ical carcinomas (83.3%). Similar results were obtained from other studies that included both cortisol-producing and aldosterone-producing and hormonally nonfunc- tional lesions. Production and secretion of inhibin into
A
B
C
0
D
E
the circulation has previously been demonstrated in hu- man adrenal tumors.41,47 An early study detected inhibin a-subunit immunoreactivity in hyperplastic tissues and adrenocortical carcinoma.48 This observation was sup- ported by data from larger, subsequent studies of tissues from patients with adrenal cortical neoplasia.32,49-51 In general, inhibin & immunoreactivity was detected in adrenal cortical adenomas and carcinomas. Inhibin a immunoreactivity provides a diagnostic marker that can be used to differentiate adrenal cortical tumors from
histologically similar tumors, including phechromocyto- mas and hepatocellular and renal cell carcinomas.52,53
Our study showed intense immunoreactivity in compact cells with eosinophilic cytoplasm that histologi- cally resembled those of the zona reticularis. The zona fasciculata cells of the normal adrenal gland showed an occasional weak immunoreaction for inhibin a. These findings indicate a zonal specificity in the degree of im- munoreactivity for inhibin a-subunit in the adrenal cor- tex.17,52 Adrenocortical tumor cells are derived from
| *Inhibin a | ** Inhibin/Activin ₿ | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Negative N | Weak N | Moderate N | Strong N | Negative N | Weak N | Moderate N | Strong N | |||
| (%) | (%) | (%) | (%) | P | (%) | (%) | (%) | (%) | P | |
| Hyperplasia (n = 5) | 0 (0) | 1 (20) | 1 (20) | 3 (60) | < 0.001 | 1 (20) | 2 (40) | 1 (20) | 1 (20) | 0.5 |
| Adenoma (n = 11) | 1 (9.1) | 1 (9.1) | 2 (18.2) | 7 (63.6) | 2 (18.2) | 3 (27.3) | 2 (18.2) | 4 (36.4) | ||
| Carcinoma (n = 6) | 1 (16.7) | 1 (16.7) | 3 (50) | 1 (16.7) | 1 (16.7) | 2 (33.3) | 2 (33.3) | 1 (16.7) | ||
| Benign pheochromocytoma (n = 20) | 17 (85) | 2 (10) | 1 (5) | 0 (0) | 6 (30) | 1 (5) | 4 (20) | 9 (45) | ||
| Malignant pheochromocytoma (n = 5) | 4 (80) | 1 (20) | 0 (0) | 0 (0) | 3 (60) | 1 (20) | 1 (20) | 0 (0) | ||
Test of significance: x2 test.
*Hyperplasia versus benign pheochromocytoma, P < 0.001; hyperplasia versus malignant pheochromocytoma, P < 0.001; adenoma versus benign pheochromocytoma, P <0.001; adenoma versus malignant pheochromocytoma, P < 0.001; carcinoma versus benign pheochromocytoma, P <0.001; carcinoma versus malignant pheochro- mocytoma, P = 0.003.
** Benign pheochromocytoma versus malignant pheochromocytoma, P = 0.05.
pluripotent adrenocortical progenitor cells that adopt a gonadal fate because of the convergent loss of inhibin and chronic exposure to elevated gonadotropins.54
In our study, a decrease in expression of inhibin & was noticed in adrenocortical carcinoma than in hyper- plasia and adenoma. Histologic recognition of an adrenal cortical adenoma or carcinoma is generally straightfor- ward, especially when there is a history of hormone production. However, adrenal cortical carcinoma is often hormonally inactive.47 Adrenal cortical carcinoma is a rare and heterogenous malignancy with incompletely understood pathogenesis and poor prognosis.55 It can be functional or nonfunctional with regard to hormone synthesis and clinical features.56 Inhibin & was not de- tected in carcinoma samples, but inhibin ß-subunit mRNA was found to be slightly decreased in carcinomas, suggesting that tumor formation in carcinomas might have been caused by loss of expression of the a-subunit or decreased ß-subunit expression. These findings suggest that inhibin and activin signaling is dependent on tumor status of the adrenal cortex and may itself play a role in tumor formation.52
Inhibin, however, has been identified in several organs, including the gonads and adrenal gland.48,57 Its
diagnostic use in the differential diagnosis of gonadal sex cord-stromal tumors and in adrenocortical neoplasms has been proven.47,58 In the current study, adrenocortical neoplasms showed positive immunoreactivity for inhibin a, whereas benign and malignant pheochromocytomas were negative (85% and 80%, respectively). Immuno- reactivity for inhibin a-subunit was reported in a few cases of pheochromocytomas with weak focal to moder- ate immunoreactivity.49,52 This finding is different from that of previous reports that showed complete negativity of inhibin a in pheochromocytoma.5,47 The difference in embryologic origins and function of these 2 distinct adrenal endocrine tissues might explain this observation. Although the cortex is of mesodermal origin and secretes steroid hormones, the medulla is of neuroectodermal origin and is rich in catecholamines.59
Human fetal, adult, and neoplastic adrenocortical cells are capable of producing dimeric activin A, inhibin A, and inhibin B peptides. ACTH stimulates the production of inhibins A and B through the protein kinase A signal transduction pathway and decreases the activin/inhibin secretion ratio in cultured adrenal cells. Human adrenocortical cells express mRNAs for activin/ inhibin receptors, suggesting the presence of specific
| Inhibin a | P | Inhibin/Activin B | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Negative N | Weak N | Moderate N (%) | Strong N | Negative N | Weak N | Moderate N | Strong N | |||
| (%) | (%) | (%) | (%) | (%) | (%) | (%) | P | |||
| Adrenocortical neoplasia (n = 22) | 2 (9.1) | 3 (13.6) | 6 (27.3) | 11 (50) | 0.001 | 4 (18.2) | 7 (31.8) | 5 (22.7) | 6 (27.3) | 0.1 |
| Adrenomedullary neoplasia (n = 25) | 21 (82) | 3 (12) | 1 (4) | 0 (0) | 9 (36) | 2 (8) | 5 (20) | 9 (36) | ||
Test of significance: The x2 test was conducted between the total number of studied groups of adrenocortical and adrenomedullary origin. For a medullary tumors a statistical significant difference was noticed between benign and malignant pheochromocytoma (P = 0.05).
A
B
C
D
E
receptors for these peptides. Activin/inhibin system is a paracrine and autocrine mediator of ACTH in adreno- cortical cells.60
In the current study, we detected the expression of inhibin/activin ß-subunits in 80% of adrenal hyper- plasias, 81.8% of cortical adenomas, and 83.3% of adrenocortical carcinomas. The presence of inhibin/acti- vin ß-subunits has been documented in the adrenal gland. Immunoreactivities for BA-subunit and BB-subunit were
reported in the adrenal cortex.32 However, benign pheo- chromocytomas expressed inhibin/activin ß-subunit immuno- histochemically in 70% of cases with moderate-to-strong intensity, whereas 40% of malignant pheochromocyto- mas showed weak focal immunoreactivity for the inhibin/ activin ß-subunit. Inhibin/activin ß-subunit that is ex- pressed in the normal adrenal medulla has been found to be high in benign pheochromocytomas and near negative in malignant tumors. 33,40
The clinical presentation of pheochromocytoma may mimic other disease conditions such as hyperthyroidism and panic disorders, consequently causing a delay in diagnosis. Biochemical tests remain the hallmark of diagnosis. This in- cludes the determination of urinary metabolites of catechol- amine and urinary vanillyl mandelic acid.61-63 No definite immunohistochemical markers have been found for malig- nant pheochromocytomas, even though p53 mutations are believed to have a role in the development of multiple and malignant pheochromocytomas.64 In our series, the 2 positive cases of malignant pheochromocytomas had weak focal im- munoreactivity for the BB-subunit. In contrast, most benign tumors showed moderate-to-strong immunopositivity. It seems that pheochromocytomas arising from chromaffin cells lose their potential to express inhibin/activin BB-subunit when they become malignant.40 Although the diagnosis of a malignant pheochromocytoma requires evidence of meta- stases, Linnoila et al65 reported that malignancy often cor- relates with extra-adrenal location and with histologic features, including coarse nodularity, confluent necrosis, and absence of hyaline globules. Benign adrenal pheochromo- cytomas were mostly positive for the ß-subunit, whereas malignant ones had weak or no expression of it. A clear ß-subunit expression in pheochromocytomas favors a benign nature.40
CONCLUSIONS
The results of this study show that inhibin a is dif- ferentially expressed in cortical and medullary lesions of the adrenal and can serve as an adjunct in distinguishing cort- ical from medullary lesions in small biopsies and cytopa- thology samples. The loss of inhibin & expression in some cortical carcinomas suggests a role in tumor progression. Similarly, the loss of inhibin/activin ß in malignant pheo- chromocytomas also suggests a role in progression for these tumors.
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