HORMONE RESEARCH
Horm Res 2007;67:7-11 DOI: 10.1159/000095806
Received: February 1, 2006 Accepted: June 20, 2006 Published online: September 15, 2006
An Inhibin B and Estrogen-Secreting Adrenocortical Carcinoma Leading to Selective FSH Suppression
Maria Candida Barisson Villares Fragosoª Maria Beatriz Fonte Kohekb Regina Matsunaga Martina Ana Claudia Latronicoª Antonio Marmo Luconª Maria Claudia Zerbinid Carlos Alberto Longuie Berenice Bilharinho Mendoncaª Sorahia Domeniceª
ªUnidade de Endocrinologia do Desenvolvimento, Laboratório de Hormônios e Genética Molecular LIM/42, Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, São Paulo, bDepartamento de Ciências Fisiológicas da Fundação Faculdade Federal de Ciências Médicas de Porto Alegre, Porto Alegre, ‘Departamento de Urologia e dDepartamento de Anatomia Patológica do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, eFaculdade de Ciências Médicas da Santa Casa de São Paulo, São Paulo, Brazil
Established Facts
· Hormone-secreting adrenocortical carcinomas are usually associated with endocrine syndromes resulting from secretion of cortisol, adrenal androgens, mineralocorticoids and their precursors and rarely estrogens.
· Inhibin was first identified as a gonadal-derived regulator of pituitary FSH.
· It has been demonstrated that testicular and adrenal tumors can synthesize inhibin subunits.
· Human fetal, adult and neoplastic adrenocortical cells may produce both inhibins A and B.
· A single adrenal adenoma co-secreting E2 and inhibin B was described as being associated with blunted FSH in both basal and GnRH-stimulated conditions.
Novel Insights
· This is the first report of inhibin and estradiol-secreting adrenocortical carcinoma.
· The selective FSH inhibition, blunted in both basal and GnRH-stimulated conditions, suggested the secretion of inhibin B by the adrenocortical tumor in this case.
· This report illustrates, in vivo, the critical role of inhibin B in the physiological mechanisms involved in gonadotropin regulation, particularly FSH release.
· Elevated serum inhibin B and undetectable FSH levels may be a useful marker of an inhibin B-secreting adrenocortical carcinoma recurrence.
Key Words
Inhibin B · Estrogen · FSH · Adrenocortical carcinoma
Abstract
Objective: Hormone-secreting adrenocortical tumors are frequently associated with endocrine syndromes. We de-
scribe a 30-year-old man who had abdominal pain, a nodule in the right breast and loss of libido. Abdominal magnetic resonance imaging revealed a very large tumor in the right adrenal gland. Methods: Hormonal profile disclosed in- creased levels of estradiol and slightly low testosterone lev- els. The basal and stimulated LH levels were normal, where- as basal and stimulated FSH levels were totally suppressed.
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Cortisol and adrenal androgen levels were normal. The un- usual finding of selective FSH suppression suggested secre- tion of inhibin B by the adrenocortical tumor. A very high level of serum inhibin B (405 pg/ml) was demonstrated by ELISA assay. Right adrenalectomy and nephrectomy were performed and the tumor was classified as a malignant tu- mor (Weiss score: 7.0) and unilateral mastectomy disclosed a lipoma. Results: One week after surgery, a GnRH-stimula- tion test disclosed normal basal and stimulated FSH levels and low levels of inhibin B and estradiol. Immunohistochem- ical analysis with anti-B-inhibin antibody revealed intense staining in the adrenocortical tumor cells. One month after surgery, an abdominal magnetic resonance imaging re- vealed a local recurrence of the tumor and a second surgery was performed with partial resection of the tumor and the patient died 1 year after the first surgery. Conclusion: We herein report the first inhibin B and estradiol-secreting ad- renocortical carcinoma. The unusual selective inhibition of FSH secretion should be considered a valuable hormonal finding for the diagnosis of inhibin B-secreting adrenocorti- cal tumors. Copyright @ 2007 S. Karger AG, Basel
Introduction
Adrenocortical carcinomas are rare malignant neo- plasms [1]. Hormone-secreting adrenocortical tumors are usually associated with endocrine syndromes that re- sult from secretion of cortisol and its precursors, adrenal androgens and their precursors, or rarely, estrogen or mineralocorticoids [2-5]. The most common syndrome associated with adrenal tumors in adults is Cushing’s syndrome [3, 4], which is present in 30-40% of patients with adrenocortical carcinoma. Virilization occurs in 20-30% of adults with functional adrenal neoplasms, whereas it is the most common hormonal syndrome in children with adrenocortical tumors [6-8]. Feminiza- tion, as a pure hormonal syndrome, is a quite rare mani- festation of adrenocortical neoplasms [5, 9-10]. In this report, we describe the first inhibin B and estrogen-se- creting adrenocortical carcinoma leading to a selective FSH suppression in a male.
Case Report
A 30-year-old man with abdominal pain and loss of libido in the previous 2 years was referred to Endocrinology Unit of Hos- pital das Clínicas, São Paulo, Brazil, for endocrine evaluation. At physical examination, only a hard 2-cm nodule in the right breast
was observed. He had normal blood pressure and his body mass index was 27 kg/m2. Magnetic resonance image of the abdomen revealed a right adrenal heterogenic mass of 9.0 × 7.0 × 6.2 cm. Hormonal evaluation disclosed elevated E2 (661 pmol/l) and slightly low testosterone (T) levels (892 pmol/l). Basal and GnRH- stimulated LH levels were normal, whereas FSH levels were sup- pressed (table 1). Serum inhibin B levels were 405 pg/ml (normal values: 190-266 pg/ml) (table 1). The patient underwent a right adrenalectomy, nephrectomy and breast nodulectomy. A large tumor, weighting 330 g and measuring 110 × 55 × 50 mm was removed. The histopathological analysis showed adrenocarcino- ma with Weiss score 7 [11]. The kidney was free of tumor cells. The breast nodule was diagnosed as a lipoma. One week after surgery, a GnRH stimulation test disclosed normal basal and stimulated FSH levels (10.4 and 15.7 U/1, respectively). The in- hibin B and estradiol levels were significantly low at that time (table 1). One month after surgery, the patient developed ascites. Abdominal magnetic resonance imaging revealed a local recur- rence of the tumor and a neoplasic thrombus, which extended from the vena cava to the right cardiac atrium. Estradiol and in- hibin B levels had increased again (209 pmol/l and 408 pg/ml, respectively) at that time. A second surgery was performed with a partial resection of the tumor mass. He did not respond to mi- totane treatment (6 g/day) and died 1 year after the first sur- gery.
Materials and Methods
The patient gave his informed consent for all endocrine testing as well as for adrenal tumor tissue studies. The study was ap- proved by the Ethics Committee of Hospital das Clínicas, São Paulo, Brazil.
Hormone Analysis
Hormonal evaluation consisted of basal measurements and dynamic endocrine tests, including gonadotropin responses to an acute i.v. administration of 100 µg GnRH, before and after sur- gery (table 1) and cortisol levels after dexamethasone (1.0 mg overnight). Serum LH, FSH, testosterone, cortisol and estradiol levels, and 24-hour urinary cortisol were measured by automated immunofluorometric or fluorometric assays kits (AutoDelfia, Wallac, Finland), whereas DHEAS and androstenedione were measured by iodide radioimmunoassay kits (DSL, Webster, Tex., USA).
Inhibin B Analysis
Serum inhibin B levels were measured with a solid phase sand- wich ELISA (inhibin B dimmer assay, Serotec, Oxford Bio-Inno- vation, UK).
To study the levels of inhibin B in tumor tissue, the protein isolation was performed by TRI Reagent (Sigma Chemical CO, St. Louis, Mo., USA) from frozen adrenal tumor tissue and from nor- mal adrenal and testicular tissues. In these tissues, inhibin B lev- els were also measured by ELISA assay (table 1).
The normal adrenal tissue was obtained from a male patient, submitted to surgical treatment of renal tumor. The gonadal tis- sues (testis and ovary) were obtained from two patients with di- agnosis of transexualism, who were submitted to gonadectomy.
A
B
| FSH, IU/l | LH, IU/l | Aldo pmol/l | E2 pmol/l | Testo- sterone pmol/l | Androste- nedione pmol/l | 11-deoxy- cortisol pmol/l | Inhibin B pg/ml | DHEAS nmol/l | F, nmol/l | Fu | |||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| basal | peak | basal | peak | basal | after dexa | nmol/ day | |||||||
| Before surgery | |||||||||||||
| <1.0 | <1.0 | 1.5 | 57.4 | 360 | 661 | 892 | 8.7 | 10.4 | 405 | 3,676 | 440 | 55 | 692 |
| 10th day after surgery | |||||||||||||
| 10.4 | 15.7 | 9.9 | 32.4 | <69 | 95 | 718 | 5.6 | 14 | 40 | 6,334 | 495 | – | – |
| 120th day after surgery | |||||||||||||
| <1.0 | – | 0.8 | – | – | 209 | 187 | 8.0 | 9.2 | 408 | – | – | – | – |
| Normal values | |||||||||||||
| 1.0-1.2 | 1.4-9.2 | 250-499 | <128 | 694-3,298 | 2.8-9.7 | 6.3-10.5 | 190-266 | 1,842-11,214 | 192-853 | <49 | 82-827 | ||
peak = Peak after acute GnRH test; after dexa = cortisol (F) after 1 mg suppression dexametasona test; Fu = 24 h urinary cortisol; E2 = estradiol.
All donors had normal LH, FSH, testosterone, estradiol and adre- nal hormone levels. Their age ranged from 28 to 50 years.
Immunohistochemistry Studies
Sections of 3 mm paraffin were submitted to classical immu- nostaining procedures using the streptavidin-biotin-peroxidase complex and heat antigen retrieval. Briefly, endogenous peroxi- dase was blocked with hydrogen-peroxide, followed by antigen retrieval in citrate buffer (0.001 M/1, pH 6.0). Monoclonal mouse anti-human inhibin & (clone R1, Dako, USA, 1:50) was used as primary antibody and incubated overnight at 4℃ in a humidified chamber. Reactivity was detected through incubation with rabbit anti-mouse secondary anti-inhibin & antibody, streptavidin-bio-
tin-peroxidase complex (LSAB plus, DAKO, USA), using 3,3 di- aminobenzidine as the chromogen. Slides were counterstained with Harris hematoxylin solution, dehydrated, mounted in xy- lene, and examined with a light microscope.
Results
Immunohistochemistry. Labeling of adrenocortical carcinoma sections with anti-inhibin & antibody revealed the presence of immunoreactive cell clusters, diffusely distributed in the neoplastic cells (fig. 1).
| Tissue | Inhibin B pg/ml |
|---|---|
| Adrenal tumor | 433 |
| Normal adrenal control | <15.6 |
| Normal ovary control | 697 |
| Normal testis control | 236 |
In vitro Studies. The levels of inhibin B protein ex- tracted from the adrenal tumor were 27 and 1.8 times higher than those from the adrenal normal control and normal testis control, respectively (table 2).
Discussion
Inhibin was first identified as gonadal-derived regula- tor of pituitary FSH and, subsequently multiple actions were assigned to a wide range of tissues [12]. It is a di- meric glycoprotein hormone, member of TGF-ß super- family of growth and differentiation factors and consists of either of the ß-subunits dimerized with a common a- subunit [13]. Inhibin production is restricted mainly to the steroidogenic tissues and to the pituitary [14]. Human fetal, adult, and neoplastic adrenocortical cells may pro- duce both inhibins A and B [15-16].
ACTH stimulates the production of inhibins A and B via the PKA signal transduction pathway and decreases activin/inhibin secretion ratio in cultured adrenal cells [17]. Moreover, human adrenocortical cells express mRNAs for inhibin receptors, suggesting the presence of specific receptors for these peptides in the adrenal gland [17]. Gonadotropin secretion may be modified in some adrenocortical carcinoma cases with functioning lesions, in the presence of excessive tumor testosterone or estro- gen production. In the rare adrenocortical tumor cases described with excessive tumor estrogen production, go- nadotropin levels were reported as being suppressed [9, 10, 18]. Bouraïma et al. [18] described a patient with a feminizing adrenocortical adenoma, wherein LH and FSH pulsatility was completely abolished, indicating the fundamental participation of E2 in the control of gonado- tropin secretion, although the inhibin was not investi- gated in this case.
To our knowledge, there is a single adrenal adenoma described in the literature in which E2 and inhibin B co- secretion was determined [10]. The patient described in that study presented a feminizing adrenocortical tumor and the pathological examination confirmed an adeno- ma. This unusual secretory pattern appeared to be due to a rise of aromatase activity and increased inhibin B synthesis in the adrenal adenoma. Similarly to our case, gonadotropin secretion was inhibited, with a more pronounced FSH suppression compared to LH secre- tion.
In our case, the inhibin production by adrenal tumor cells was determined using different strategies. First of all, very high levels of serum inhibin B were identified in the pre-surgery condition. The reduction of serum in- hibin B and E2 levels after the excision of the adrenal mass confirmed their production and secretion by the adreno- cortical carcinoma. Later, we observed the increment in basal FSH secretion and its release after GnRH-stimula- tion, suggesting the discontinuation of the blunted effect. The presence of inhibin in the tumor tissue was revealed by the immnohistochemistry studies as well as by inhib- in B measurements in the adrenal tumor tissue, indicat- ing that its production source was the tumor adrenal tis- sue (table 2).
The high inhibin B associated with selective FSH sup- pression represents a very unusual and interesting model for understanding the relationship between the gonado- tropin axis and the adrenal secretion. The negative feed- back inhibition of gonadal hormones on FSH secretion involves both gonadal peptides (inhibin, activin) and ste- roid hormones (testosterone and estradiol). On the other hand, FSH and androgen are necessary for normal in- hibin production by gonadal tissues [19].
The presence of normal LH levels and totally blunted FSH secretion in both basal and GnRH-stimulated con- ditions of our patient, led us to hypothesize that a tumor producing inhibin B may be more significant than estro- gen, regarding the mechanisms involved in the regula- tion of pituitary FSH control. However, the combined ef- fects of inhibin and elevated estradiol should also be con- sidered.
The particular hormonal condition described in this patient confirmed the ability of adrenocortical carcino- ma to produce and secrete inhibin B in addition to steroid hormones. Moreover, this study allowed us to illustrate, in vivo, the critical role of inhibin B in the physiological mechanisms involved in FSH release and to add further elements to help elucidate the mechanisms of gonadotro- pin regulation.
Conclusion
We conclude that the measurement of inhibin B levels as well as estradiol can be performed on a regular basis, in adrenal tumors that present suppressed levels of go- nadotropins. In fact, plasma inhibin B levels may offer an additional clinically useful serum marker of adrenal tu- mor recurrence, particularly when the hormonal pattern includes undetectable plasma FSH levels.
Acknowledgements
The authors thank the staff of the Laboratório de Hormônios e Genética Molecular LIM/42 particularly Dr. Mirian Nishi, Ma- ria Aparecida Medeiros for providing excellent technical support. We also thank Valeria Q. Passos, Katia Seidemberg for patient’s care and Dr. Sheila Aparecida Siqueira for providing immunohis- tochemistry studies.
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Copyright: S. Karger AG, Basel 2007. Reproduced with the permission of 5. Karger AG, Basel. Further reproduction or distribution (electronic or otherwise) is prohibited without permission from the copyright holder.
Copyright: S. Karger AG, Basel 2007. Reproduced with the permission of 5. Karger AG, Basel. Further reproduction or distribution (electronic or otherwise) is prohibited without permission from the copyright holder.