CASE REPORT
Adrenocortical carcinoma manifesting pure primary aldosteronism: A case report and analysis of steroidogenic enzymes
T. Yoshimoto*, M. Naruse*, Y. Ito ** , K. Naruse*, T. Ueda *** , A. Tanabe*, S. Harada*, T. Nishikawa **** , H. Sasano ***** , T. Obara ** , and H. Demura
*Department of Medicine; ** Department of Endocrine Surgery, Institute of Clinical Endocrinology, **** Department of Surgical Pathology, Tokyo Women’s Medical, University, *** Yoshikawa Chuoh Hospital, ***** Departments of Pathology, Tohoku University School of Medicine, Japan
ABSTRACT. Adrenocortical carcinoma manifest- ing pure hyperaldosteronism is extremely rare. We report here a 61-year-old woman with bio- chemically proven primary aldosteronism due to right adrenocortical carcinoma. Computed to- mographic scan showed 4.5x5.3 cm lobulated mass with tiny calcification, while there was no significant uptake of 1311-iodomethyl norcholes- terol in the tumor. Immunohistochemical analysis demonstrated expression of steroidogenic en- zymes in the tumor tissue: P-450scc, P-450c21,
3ß-hydroxysteroid dehydrogenase, P45017%, and P-45011B. In addition, we could demonstrate mRNA expression of aldosterone synthase (P-450aldo: CYP11B2) in the tumor by specific ribonuclease protection assay. This is the first report of a case of primary aldosteronism due to adrenocortical carcinoma, in which expression of all sets of steroidogenic enzymes required for aldosterone synthesis was proven.
(J. Endocrinol. Invest. 23: 112-117, 2000) @2000, Editrice Kurtis
Although adrenocortical carcinoma (ACC) is a rare tu- mor, accounting only for 0.05-0.2% of all the malig- nancies (1), approximately half of these carcinomas are endocrinologically functioning. While most of the patients exhibit clinical manifestation of Cushing’s syndrome, virilism, or feminization, primary aldos- teronism is rarely caused by ACC: approximately 1-3% of the patients with primary aldosteronism (1). In addition, most of the cases of ACC manifesting pri- mary aldosteronism are accompanied by excess pro- duction of glucocorticoids and/or androgens in ad- dition to aldosterone reflecting potential ability of cancer cells to produce wide spectrum of adrenal steroids, albeit without clinical features of hypercor- tisolism and hyperandrogenism (1, 2). In this contex,
ACC that purely produces excessive amount of al- dosterone and fit Conn’s criteria for primary aldos- teronism is extremely rare (1, 2).
Adrenal steroidogenesis is achieved by a series of steroidogenic enzymes. The final step of aldos- terone synthesis in human adrenal cortex was me- diated by P-450aldo, which catalyzes 11ß-hydroxy- lation, 18-hydroxylation, and finally 18-oxidation of 11-deoxycorticosterone (3). It is therefore critical to demonstrate the expression of P-450aldo in addi- tion to other enzymes to prove aldosterone syn- thesis in the tissue. Recent advance in steroidogenic enzymology and immunohistochemistry could al- low us to investigate the expression of enzymes in- volved in the steroidogenesis in various adrenal dis- eases (4). However, since P-450aldo and P-45011B which catalyzes 11-deoxycortisol to cortisol through 11ß-hydroxylation share 93% of the amino acid se- quence (5), it has been difficult to distinguish these two enzymes. Consequently, studies on the mech- anism of aldosterone synthesis in the ACC mani- festing primary aldosteronism have been very lim- ited (6). In the present study, we report a rare case of ACC causing primary aldosteronism in which we
Key-words: Hyperaldosteronism, adrenocortical carcinoma, steroidogenic enzyme, aldosterone synthase, ribonuclease protection assay.
Correspondence: Takanobu Yoshimoto, M.D., Ph.D., Department of Medicine, Institute of Clinical Endocrinology, Tokyo Women’s Medical University, 8-1 Kawadacho, Shinjuku-ku, Tokyo 162-8666, Japan.
E-mail: RXK00525@niftyserve.or.p
could demonstrate expression of whole sets of steroidogenic enzymes for aldosterone production.
MATERIALS AND METHODS
All the experimental studies in the present report were approved by the ethical committee of Tokyo Women’s Medical University.
Immunohistochemistry for steroidogenic enzymes Immunohistochemical analysis of steroidogenic en- zymes, including cholesterol side chain cleavage (P-450scc), 3ß-hydroxysteroid dehydrogenase (3ß- HSD), 21-hydroxylase (P-450c11), 17a-hydroxylase (P-45017%), and 11ß-hydroxylase (P-45011B) was per- formed on routinely processed formaline-fixed, paraffin-embedded serial sections, using the biotin- streptavidin amplified method (Biogenex, Dublin, CA, USA). For negative control immunostaining, 0.01 mol/l phosphate-buffered saline or normal rab- bit or mouse IgG were used instead of primary an- tibodies. Immunostaining procedure and charac- teristics of primary antibodies used in the present study have been described previously (7).
Ribonuclease protection assay for aldosterone synthase mRNA
Aldosterone synthase (P-450aldo: CYP11B2 gene product), a variant of P-450118, is responsible for the fi- nal step of the formation of aldosterone from 11-de- oxycorticosterone. Since the specific antibody against P-450aldo for immunohistochemistry is not available, we constructed the ribonuclease protection assay spe- cific for P-450aldo in order to analyze the gene ex- pression of the enzyme in the tumor specimen, ac- cording to the method described previously (8) with some modifications. Briefly, 155 base pair of DNA fragment corresponding to entire sequence of exon 5 of CYP11B2 (5: Genbank Accession number J05140) was subcloned into pBluescript IISK (Stratagene, La Jolla, CA, USA). Antisense cRNA probe was synthe- sized by in vitro transcription with 100 uCi[32P]UTP (800 Ci/mmol) using RNA transcription kit (Stratagene). Total RNA extracts from the tumor specimen were ob- tained by the acid guanidinium-thiocyanate-phe- nolchloroform method. Ten ug RNA sample was hy- bridized with probe overright, digested with the com- bination of ribonucleases A and T1, and electropho- resed on 6% polyacrylamide gel containing 7M urea. The dried gel was exposed to X-ray films (Kodak XAR-5, Eastman Kodak, Rochester, NY, USA). Total RNAs extracted from the adrenal adenoma tissues of aldosterone-producing adenoma and Cushing’s syn- drome were simultaneously analyzed as positive con- trol and negative control, respectively.
CASE REPORT
The patient is a 61-year-old woman with a history of hypertension treated with a calcium channel blocker, amlodipine, since 1980. On October 1997, she be- gan to experience general fatigue, muscle weakness in both shoulder and arm, and admitted to the local hospital. The diagnosis of primary aldosteronism was suspected based on the combination of hypokalemia (1.4 mEq/l; normal: 3.4-4.9 mEq/l) and hypertension with metabolic alkalosis, suppressed plasma renin ac- tivity (PRA: 0.2 ng Ang I/ml/h; normal: 05-3.0 ng Ang I/ml/h), and increased plasma aldosterone concentra- tion (PAC: 22 ng/100 ml; normal: 2.2-15 ng/100 ml). She was referred to Tokyo Women’s Medical Uni- versity Hospital on December 1997. On admission, she showed muscle weakness, high blood pressure
A
B
Liver
L
R
(150/100 mmHg), and hypokalemia (3.3 mEq/l: even under 100 mEq/d of potassium supplementation). PAC was elevated (61.7 ng/100 ml) and PRA was sup- pressed (0.6 ngAng l/ml/h). The urinary excretion of 17-hydroxycorticosteroids (17-OHCS) and 17-ketos- teroids (17-KS) was 6.0 mg/day (normal: 1.6-8.8 mg/day) and 5.2 mg/day (normal: 2.4-11.3 mg/day), respectively. Plasma dehydroepiandrosterone sulfate (DHEA-S) concentration was 139 µg/100 ml (normal: 13-154 µg/100 ml). Plasma ACTH and cortisol (F) con- centration in the morning after overnight fasting were 52.8 pg/ml (normal: 10-60 pg/ml) and 17.8 µg/100 ml (normal: 4.5-24 µg/100 ml), respectively. All these endocrine data were compatible to the diagnosis of primary aldosteronism.
Computed tomographic (CT) scan showed 4.5x5.5 cm of lobulated mass with tiny calcification in the right adrenal (Fig. 1A). While an adrenal scintigram with 131|-iodomethyl norcholesterol revealed a sig- nificant uptake of tracer in the left adrenal gland, there was no significant uptake of the tracer in the right adrenal (Fig. 1B). To confirm the site of the excess aldosterone secretion, adrenal venous sam- pling was performed. Figure 2 showed PAC and
plasma F concentration in blood samples obtained by adrenal vein catheterization. The PAC and PAC/F ratio in the right adrenal vein were two-fold greater than those in the left adrenal vein and in- ferior vena cava (Fig. 2). Taken together the results of endocrine tests and imaging techniques lead to the diagnosis of primary aldosteronism due to right adrenocortical carcinoma.
On February 1998, transabdominal right adrenalec- tomy was performed and retroperitoneal fat and regional lymph nodes were removed. The tumor was gray-yellow in its color, lobulated, and 6 x 5.5x 7 cm in size. Histologically, tumor tissue consisted of disorderly solid growth pattern of polygonal compact tumor cells with pleomorphic nuclei (Fig. 3A). The histological findings fulfilled at least five of the Weiss’s criteria for ACC: presence of sinu- soid invasion, capsular invasion, necrosis, diffuse architecture, paucity of clear cells, and high nucle- ar grade. All of the blood pressure, serum potassi- um level, PAC and PRA became normal after adrenal surgery. There has been no evidence for recurrence of the disease and long distance metas- tasis during the follow-up period for 7 months.
IVC
PAC
54.9
F
9.8
PAC/F 5.6
lt. adrenal vein
rt. adrenal vein
PAC 43.0
PAC 109
F
8.7
F
10.6
PAC/F 4.9
PAC/F 10.3
rt. Kidney
It. Kidney
IVC
PAC
68.7
F
11.1
PAC/F 6.2
A
B
C
D
E
F
RESULTS
Immunohistochemistry
All of P-450scc, P-450118, P-450c21, 30-HSD and P-45017g was immunohistochemically stained in the tumor cells. P-450scc and P-450118 were diffusely and intensely stained (Fig. 3B, 3F). P-450c21 was weakly but diffusely stained (Fig. 3C). 30-HSD was focally but intensely detected, especially in the large carcinoma cells with abundant cytoplasm (Fig. 3D). P-45017g was detected only in the scattered tumor cells (Fig. 3E).
Ribonuclease protection assay
Although the 155 bp of DNA sequence of exon 5 of P-450aldo is highly homologous to P-450118, there is a significant difference at the nucleotides levels. If the mRNA of P-450118 hybridizes to the cRNA probe for P-450aldo, the subsequent RNAase di- gestion occurs at the sequence of mismatch hy- bridization. Thus the presence of P-450aldo mRNA in the sample is detected as a protected fragment with 155 nucleotides in length. Figure 4 shows the
-Size marker
N CRNA probe
w t RNA
A ACC
0 APA
® Cushing
267 →
184 →
124 →
« 155 nt. protected fragment
result of ribonuclease protection assay for P-450al- do on polyacrylamide gel electrophoresis. The presence of 155 nucleotides of the protected frag- ment was clearly detected in the RNA sample from the present tumor specimen. The presence of P-450aldo mRNA was also detected in the RNA sample from an aldosterone-producing adenoma (positive control) (Fig. 4), while no protected frag- ment of P-450aldo mRNA was detected in the RNA sample from an adrenal adenoma of Cushing’s syn- drome (negative control).
DISCUSSION
In the present report, we demonstrated a quite rare case of pure primary aldosteronism due to ACC. Although ACC has been reported to frequently pro- duce glucocorticoids and/or androgenic steroids (1, 2), normal levels of plasma F, ACTH, DHEA-S, urinary 17-OHCS and 17-KS as well as normal up- take of 131|-iodomethyl norcholesterol in the con- tralateral adrenal gland suggested that the tumor produces mainly aldosterone.
P-450aldo, which mediates 11ß-hydroxylation, 18-hydroxylation, and finally 18-oxidation of 11-de- oxycorticosterone, is known to be indispensable for the final step of aldosterone synthesis in human adrenal cortex (3). P-450118 and P450aldo share 93% similarity in amino acid sequence (5). Specific antibody against P-450aldo is not available. We therefore developed the ribonuclease protection assay for P-450aldo gene expression and could clearly demonstrate mRNA expression of P-450aldo in the tumor tissue. Although P-450aldo mRNA has been demonstrated in aldosterone-producing ade- noma and the adrenal cortex of idiopathic hyper- aldosteronism (9, 10), this is the first report on the gene expression of P450aldo in the ACC causing primary aldosteronism.
All the steroidogenic enzymes involved in the syn- thesis of mineralocorticoids were immunohisto- chemically detected in the tumor tissue of the present case. However, the intensity and distribu- tion of immunoreactivity of each enzyme varied from each other. Prominent staining of P-450scc and P-450116 and weak staining of P-450c21 were diffusely detected in tumor cells, while the im- munoreactivity of 30-HSD and P-45017% was de- tected focally. The expression pattern of both 3ß- HSD and P-45017g was clearly distinct from the homogeneous and diffuse pattern in functioning adrenocortical adenoma cells (4, 7) but was com- patible to the pattern in the ACC (4, 6). Although P-45017a is not required in aldosterone biosyn- thesis, expression of this enzyme has been re-
ported in a case of primary aldosteronism due to ACC (6). There was no sign of an excess produc- tion of androgens in the present case. Although details of the mechanism for this remain unknown, it may be attributed to the disorganized expres- sion of steroidogenic enzymes in individual car- cinoma cells: carcinoma cells positive for one steroidogenic enzyme do not necessarily express the other enzymes (6). It is also possible that num- ber of endocrinologically active cells is not suffi- cient to produce enough amount of androgens to raise plasma levels. Expression of 3-HSD was also shown in a cluster of cells of the tumor. Although the pattern of expression of the enzyme contrasts to the homogeneous pattern of expres- sion in the adenoma cells, it is compatible to that in the ACC (6). The findings of 3ß-HSD suggest that not all carcinoma cells are endocrinological- ly functioning and producing aldosterone.
Findings of the imaging techniques of the present case were significantly different from those of the usual aldosterone-producing adenoma. The size of aldosterone-producing adenoma is usually less than 3 cm in diameter. The size of the adrenal tumor of the present case was however larger than 3 cm in diameter and accompanied by calcification on CT scan. These findings suggested the diagnosis of ACC, since Farge et al. reported that aldos- terone-producing tumor above 3 cm in diameter with the internal calcification is very likely to be ma- lignant (1).
There was no significant uptake of 1311-iodomethyl norcholesterol in the tumor side on the adrenal scintigraphy. It has been reported that malignant lesion of the adrenals including even ACC mani- festing Cushing’s syndrome was not scintigraphi- cally visualized (11, 12). In the case of ACC causing primary aldosteronism, however, no or decreased uptake of the tracer has been reported in some cas- es (13-16) and an increased uptake of the tracer with (2) and without (17-19) pretreatment with dex- amethasone has been demonstrated in other cases. Therefore, the lack of significant uptake of the trac- er in the affected side was not necessarily the sign of malignancy in primary aldosteronism. Details of the mechanism for the lack of significant uptake in the tumor side despite of excess steroid produc- tion remain to be elucidated. It has been partly ex- plained by that the total amount of aldosterone production could be increased because of the large tumor size despite of even a small production in each tumor cell. It may also be attributed to an ac- celerated steroidogenesis after the first step of cholesterol uptake.
In conclusions, we reported a very rare case of ACC
manifesting pure primary aldosteronism. We de- monstrated all sets of enzymes needed for aldos- terone synthesis in the tumor tissue, especially P450aldo as a final and key enzyme by ribonuclease protection assay.
ACKNOWLEDGMENTS
We thank Ms. Chika Adachi and Ms. Yumi Tabei for their able secretarial and technical assistance. This work was supported in part by research grants from the Japanese Ministry of Health and Welfare “Disorders of Adrenal Hormones” Research Committee, the Japanese Ministry of Education, Science, and Culture, Tanabe Biomedical Conference (Tokyo, Japan), the Kidney Foundation (Tokyo, Japan) for the research of Kidney and NO, and the Smoking Research Foundation (Tokyo, Japan).
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