A Case of Estrogen-secreting Adrenocortical Carcinoma with Subclinical Cushing’s Syndrome
NOZOMI FUKAI, YUKI HIRONO, TAKANOBU YOSHIMOTO, MASARU DOI, YUKIHIKO OHTSUKA*,
KEIKO HOMMA ** , HIROTAKA SHIBATA *** , HIRONOBU SASANO# AND YUKIO HIRATA
Department of Clinical and Molecular Endocrinology, Tokyo Medical and Dental University Graduate School, Tokyo 113-8513, Japan
*Department of Urology and Reproductive Medicine, Tokyo Medical and Dental University Graduate School, Tokyo 113-8513, Japan
** Department of Laboratory Medicine, Keio University, School of Medicine, Tokyo 160-8582, Japan
*** Health Center, Keio University, Tokyo 160-8582, Japan
Department of Pathology, Tohoku University Graduate School of Medicine, Sendai 980-8575, Japan
Abstract. A 25-year-old man was found to have a large right adrenal mass detected by abdominal echography and computed tomography, and presented with a mild gynecomastia. Endocrine study showed increased serum concentrations and urinary excretion of estrogens and dehydroepiandorosterone sulfate (DHEA-S). The patient had no Cushingoid features but autonomous cortisol secretion, compatible with the diagnosis of subclinical Cushing’s syndrome. Surgical removal of the adrenal tumor led to normalization of serum and urinary excretion of estrogens and DHEA-S. Histopathological examination revealed a high-grade adrenocortical carcinoma (ACC). The disorganized expression of all the steroidogenic enzymes in individual tumor cells was demonstrated by immunohistochemical analysis, and the abundant expression of both aromatase mRNA and insulin-like growth factor (IGF)-II mRNA was shown by RT-PCR. These data suggest the excessive secretion of estrogen as well as the ineffective steroidogenesis by the adrenal tumor. This is a very rare case of estrogen-secreting ACC associated with subclinical Cushing’s syndrome.
Key words: Adrenocortical carcinoma, Estrogen-secreting, Subclinical Cushing’s syndrome
(Endocrine Journal 53: 237-245, 2006)
ADRENOCORTICAL carcinoma (ACC) is a rare malignancy associated with poor prognosis [1, 2]. Clinical and pathological feature as well as its ap- proach to therapy is hampered by its low incidence (1-2/1,000,000/year) and heterogeneity [1, 2]. Tumors may be functional or nonfunctional depending on whether they produce biological active steroids, such as cortisol, aldosterone, androgens or estrogens. Dis- organized expression of steroidogenic enzymes in
individual carcinoma cells is considered to lead to ineffective corticosteroidogenesis, characteristic of altered steroid metabolism in ACC [3, 4]. We report herein a very rare case of estrogen-secreting ACC asso- ciated with subclinical Cushing’s syndrome in whom detailed analysis by endocrinological, biochemical, immunohistochemical, and pathological studies was conducted.
Methods
Immunohistochemistry
Immunohistochemical analysis of steroidogenic enzymes, including cholesterol side chain cleavage
Received: November 10, 2005
(P-450scc), 3ß-hydroxysteroid dehydrogenase (3ß-HSD), 21-hydroxylase (P-450c21), 11-hydroxylase (P-450116), 17a-hydroxylase (P-45017g), and dehydroepiandro- sterone sulfotransferase (DHEA-ST) was performed on routinely processed formalin-fixed, paraffin-embedded serial sections, using the biotin-streptavidin amplified method (Nichirei, Tokyo, Japan). Immunostaining procedure and characterization of primary antibodies used in the preset study have been described previously [5-7].
Real-time RT-PCR for aromatase (P-450 aroma) and insulin-growth factor (IGF)-II
P-450aroma and IGF-II mRNA levels were quantified by real-time quantitative RT-PCR using fluorescent SYBR Green technology (LightCycler; Roche Molecu- lar Biochemicals, Mannheim, Germany), as described previously [8]. Total RNA extraction from the surgi- cally resected tumor and first-strand cDNA synthesis was performed as described previously [9]. PCR prim- ers were designed as following; forward primer 5’- GTGGACGTGTTGACCCTTCT-3’ and reverse primer 5’-CACGATAGCACTTTCGTCCA-3’ for human P- 450 forward primer 5’-TGACCTCATTTCCCG ATACC-3’ and reverse primer 5’-GGGGAGAATT CGTCTGATTG-3’ for human IGF-II. The reaction produced 90 bp products for P-450aroma and 110 bp products for IGF-II. Total RNAs extracted from tumor tissues from each one aldosterone-producing adenoma and cortisol-producing adenoma were also analyzed for P-450aroma and IGF-II mRNA quantification as reference.
Urinary estrogen profile by gas chromatography/mass spectrometry (GC/MS)
Urinary estrogens (estrone, estradiol, estriol) con- centrations of 24 h-urine collection were determined by gas chromatography/mass spectrometry (GC/MS) method as previously reported [10, 11]. The reference values of urinary estrogens concentrations from 39 healthy men (age between 18 and 49 years old) were as follows: estrone (0-10 µg/g creatinine), estradiol (0- 4 µg/g creatinine), and estriol (0-10 µg/g creatinine), respectively.
Case Report
A 25-year-old man was well until June 2004, when he intermittently noticed a right flank pain. He visited Nakano General Hospital in December 2004, where he was found to have a large suprarenal mass by abdomi- nal ultrasound examination. He was referred to our University Hospital for further evaluation of the adre- nal mass in January 2005.
He was 166 cm tall and weighed 48 kg. Blood pres- sure was 106/74 mmHg. Physical examination re- vealed mild gynecomastia with tenderness and a solid mass palpable in the right upper quadrant. There were no Cushingoid features, such as moon facies, central obesity, or striae; the testes were normal in size. Com- plete blood cell counts, blood chemistry and urinalysis were almost within normal limits. Endocrine studies (Table 1) revealed that serum levels of dehydro- epiandrosterone sulfate (DHEA-S), estradiol and pro-
| Preoperative | Postoperative | |
|---|---|---|
| Plasma | ||
| ACTH (pg/ml) | <5 | 116 (7-56) |
| PRL (ng/ml) | 16.7 | 19.2 (3.58-12.78) |
| LH (mIU/ml) | 3.5 | 4.6 (1.22-7.05) |
| FSH (mIU/ml) | 2.3 | 5.5 (2.00-8.30) |
| Cortisol (µg/dl) | 19.1 | 6.0 (4.0-23.3) |
| DHEA-S (ng/ml) | 4567 | 599 (138-519) |
| Progesterone (ng/ml) | 0.63 | 0.25 (<0.7) |
| Teststerone (ng/ml) | 3.88 | 3.73 (2.01-7.50) |
| Estradiol (pg/ml) | 233.8 | N.D. (20-60) |
| Estriol (pg/ml) | 12 | 14 (<14) |
| Urine | ||
| Free Cortisol (ug/day) | 124 | N.D. (11.2-80.3) |
| 17-Total Ketosteroids | 20.3 | 8.1 (4.6-16.4) |
| (mg/day) | ||
| 17-Hydroxy Corticosteroids (mg/day) | 9.5 | 1.8 (2.9-11.6) |
| 17-Ketogenic Steroids (mg/day) | 2.11 | 5.1 (6.0-18.4) |
| 11-deoxyKGS (mg/day) | 0.44 | 2.6 (1.54-3.91) |
| 11-oxyKGS (mg/day) | 1.44 | 1.5 (3.86-13.8) |
| Pregnanediol (mg/day) | 1.85 | 0.15 (0.1-1.0) |
| Pregnanetriol (mg/day) | 3.63 | 0.86 (0.2-1.5) |
| Total Estrogen (ug/day) | 712 | 26.4 (2-20) |
| Estorone (ug/g creatinine)* | 462 | 5 (0-10) |
| Estradiol (µg/g creatinine)* | 18 | 1 (0-4) |
| Estriol (ug/g creatinine)* | 185 | 5 (0-10) |
N.D. (not detectable); Parentheses (reference value for healthy adult male); * determined by GS/MS (ref. 10, 11)
lactin were elevated, cortisol, testosterone, LH and FSH levels were normal, but ACTH and hCG levels were undetectable. 24 hour-urinary excretion of corti- sol, 17-KS, 17-OHCS, estrone, estradiol, estriol, preg- nandiol and pregnantriol were all increased.
Examination of hypothalamicuitary-adrenal axis (Fig. 1) revealed a lack of circadian rhythm of cortisol, non-suppressibility to low- and high-doses (1, 2, 8 mg) dexamethasone, unresponsiveness of ACTH and/or cortisol to CRH stimulation, and no response of corti- sol to ACTH stimulation. These endocrine data are consistent with subclinical Cushing’s syndrome.
Computerized tomography (CT) and magnetic reso- nance imaging (MRI) of the abdomen revealed a huge (8.5 × 7.0 × 6.5 cm), heterogeneous right adrenal mass with possible central hemorrhagic necrosis (Fig. 2a-c); the mass displaced the liver and the kidney without an apparent evidence of infiltration or distant metastases. [13]]]-Adosterol adrenal scintigraphy showed mild in- crease of radioisotope uptake by the right adrenal gland without a distinct uptake by the left adrenal gland (Fig. 2d). These imaging data are compatible with ACC.
In February 2005, he underwent complete right adrenalectomy and partial resection of liver by a transperitoneal approach. Postoperative course was uneventful. Postoperative plasma estradiol level and urinary excretion of estrogens (estrone, estradiol, estri- ol) decreased to undetectable ranges. Low-dose (1 mg) dexamethasone suppression test showed normal corti- sol suppression, but low cortisol response to ACTH stimulation. Replacement therapy with hydrocortisone (20 mg) was substituted. After discharge, his gyneco- mastia and mild hyperprolactinemia gradually disap- peared about 3 months after surgery, and replacement of hydrocortisone was stopped. He received adjunctive chemotherapy (etoposide plus cisplatin) without mito- tane in combination with external radiation using Lineac.
Histopathological study
The resected adrenal tumor weighing 330 g, was 9.0 × 8.5 × 6.0 cm in size; it was brown-yellow, well- encapsulated, and unlobulated, accompanied by a com-
a. Diurnal Rhythm
b. CRH stimulation
20
Cortisol
20
Cortisol ( 1 g/dl)
20
20
ACTH(pg/ml)
ACTH(pg/ml)
Cortisol ( u g/dl)
Cortisol
10
10
10
10
ACTH
ACTH
0
0
0
0
8:00
16:00
23:00
0
30
60
Clock Time
90
120
Time
(min)
c. Dexamethasone
d. ACTH stimulation
20
20
Cortisol ( u g/dl)
Cortisol ( ut g/dl)
10
10
0
0
0
1
Dexamethasone
2
8
(mg)
0
30
60
Time
(min)
a
b
C
d
pressed atrophic non-neoplastic adrenal gland (Fig. 3). Histologically, the tumor tissue consisted of disorderly solid growth pattern of compact cells with pleomorphic nuclei. Mitotic figures were easily identifiable (1-2/ HPF) (Fig. 3b, c). Both Ki67 and p53 index were 13% and 11%, respectively (Fig. 4a, b). The histological findings fulfilled at least seven of the Weiss’s criteria for the histopathological diagnosis of ACC [11]: the presence of necrosis, diffuse architecture, high com- pact cells rate, high nuclear grade, high mitotic rate, sinusoidal invasion, and capsular invasion.
Immunohistochemical study
Immunohistochemical evaluation of steroidogenic enzymes revealed that immunoreactivities of all the steroidogenic enzymes (P-450scc, P-450C21, 30-HSD, P-45017g, P-45011B, DHEA-ST) examined were detected in the tumor cells to a varying degree (Fig. 5): diffuse
and weak immunostaining for P-450scc, P-450C21, and P-450116 (Fig. 5a-c); focally and intensely staining for P-45017a (Fig. 5d); diffuse and intense staining for DHEA-ST (Fig. 5e); intense and scattered staining for 3ß-HSD (Fig. 5f). These immunohistochemical fea- tures are consistent with the disorganized expression of steroidogenic enzymes in individual carcinoma cells, as described previously [3, 4]. Immunoreactivity of PRL was not detected in the tumor cells (data not shown).
Measurement of mRNA for P-450 aroma and IGF-II
Real-time quantitative RT-PCR for P-450aron and IGF-II revealed that the adrenal tumor tissue from the present case expressed far greater P-450aroma mRNA (more than 600 times) and IGF-II mRNA (more than 400 times) than those of other hormone-producing tumors (aldosterone-producing adenoma and cortisol- producing adenoma) used as references (Fig. 6).
a.
7
8
9
10
11
12
05B-1289
18 19 20 2
b.
C.
a. Ki67
b. p53
Discussion
ACC is a very rare tumor accounting only for 0.05- 0.2% of all malignancies, some of which are functional with regard to steroid hormone synthesis [1, 2]. Among 73 ACC cases previously reported from the Memorial Sloan-Kettering Clinical Center, 60% were proven to be functional in whom 73% are associated with Cushing’s syndrome and/or virilization, and only 2% with feminization [13]. Accordingly, Cushing’s syndrome with or without virilization due to excessive androgens is relatively common in functional ACC, however, estrogen-secreting ACC with feminization is quite rare. Although the rate of subclinical Cushing’s syndrome in ACC has not been well studied so far, the recent survey of 1004 cases of adrenal incidetaloma by
a. P450scc
b. P450c21
c. P450c11 B
d. P450c17
e. DHEA-ST
f. 3 B HSD
P450
IGF-II
a.
aroma
ACC
CS
PA
ACC CS PA
100bp
100bp
b.
700
700
mRNA levels (arbitrary unit)
mRNA levels (arbitrary unit)
500
500
aroma
P-450
300
IGF-II
300
100
100
1
1
0
0
ACC
PA
CS
ACC
PA
CS
the Italian Society of Endocrinology revealed 4 cases subclinical Cushing’s syndrome out of 47 ACC cases (8.5%) [14].
The criteria of Weiss have been extensively used to differentiate between benign and malignant adrenal tumors [12], and generally established as the only reli- able method of discerning malignancy of the adreno- cortical tumors. Immunohistochemical analyses of certain proliferation markers, such as p53 and Ki-67, have been demonstrated useful diagnostic markers for ACC: both proliferation indices are less than 10% in benign adrenal tumor, and correlates positively with malignant behavior in ACC [15, 16]. High index greater 10% for both Ki67 and p53 in the present tumor indicates malignancy with its highly proliferative character.
It has been shown that ACC is associated with IGF- II overexpression in the tumor tissue [17, 18], and the recent microarray analysis have revealed that gene expression profile of IGF-II cluster in adrenal tumor tissue showed strong predictive value for the diagnosis of ACC [19]. From the present quantitative RT-PCR of IGF-II mRNA expression in the adrenal tumors, we found that steady-state IGF-II mRNA levels in the
present adrenal tumor are far greater than those in two different functional (cortisol- and aldosterone- secreting) and benign adrenocortical tumors. These results strongly suggest that the expression of IGF-II mRNA by the tumor serves as a helpful diagnostic marker for ACC.
The present case with gynecomastia was shown to have increased serum and urinary excretion of estro- gens and mild hyperprolactinemia. Plasma PRL levels became normalized as gynecomastia gradually disap- peared postoperatively and the possible ectopic pro- duction of PRL by the tumor was excluded from the immunocytochemical study. Thus, his mild hyper- prolactinemia seems likely due to nonspecific nipple stimulation of his tender gynecomastia.
Estrogens are synthesized by P-450 through the conversion of androstenedione, testosterone, and 16a- hydroxyandorostenedione to estrone, estradiol, and estriol, respectively. This conversion occurs physio- logically at ovary and adipose tissue, while normal adrenal cortex possesses very low levels of P-450, activity [20]. We confirmed the extremely higher ex- pression of P-450aroma mRNA in the present tumor than that of benign functional adrenocortical tumors. Fur-
| Case | Age/Sex | Clinical manifestation | Endocrine data | Tumor weight (g) | Reference | |||||
|---|---|---|---|---|---|---|---|---|---|---|
| Gynecomatia | Vivilization | Cushing's syndrome | E1/E2 | A'dione/ DHEA-S | Cortisol | Aromatase | ||||
| 1 | 37yr/M | (+) | (+) | (-) | 1 / 1 | ND/ND | -> | ND | 800 | 27 |
| 2 | 19yr/M | (+) | (-) | (-) | 1 / 1 | Î /ND | ↑ | (+) | 1600 | 28 |
| 3 | 18mo/M | (+) | (+) | (-) | ND/ 1 | 1 / 1 | -> | ND | 300 | 29 |
| 4 | 2yr10mo/F | (+) | (+) | (-) | 1 / 1 | 1 / 1 | -> | (+) | 10 | 30 |
| 5 | 26yr/M | (+) | (-) | (-) | 1 / 1 | 1 / 1 | -> | (+) | 330 | Present case |
Abbrevations used: E, (estrone); E2 (estradiol); A’dione (androstenedione); DHEA-S (dehydroepiandrostenedione sulfate); yr (year); m (month); M (male); F (female); ND (not determined); 1 (increase); - > (normal)
thermore, the urinary analysis of estrogens excretion revealed that estrone is the major estrogens in the present case. Although possible conversion from adre- nal androgens to estrogens may take place in peripheral tissue, it is most likely that androstenedione produced by the tumor is directly converted to estrone by the tumor-derived P-450 aroma. By contrast, increased cir- culating estradiol may be derived from the conversion of estrone via peripheral 17ß-hydroxysteroid dehydro- genase type 1 [21], because plasma testosterone levels were within normal range.
Autonomous cortisol production without clinically overt Cushing’s syndrome, termed as subclinical Cush- ing’s syndrome, accounts for 5-12% of adrenal inci- dentaloma [14, 22-24]. However, ACC associated with subclinical Cushing’s syndrome has rarely been reported thus far [25, 26]. The diagnosis of subclinical Cushing’s syndrome in the present case was made by the lack of Cushingoid feature and the following endo- crine data; 1) suppression of plasma ACTH; 2) lack of circadian rhythm of cortisol; 3) no response of plasma ACTH and cortisol to CRH stimulation; 4) non- suppressibility of cortisol to low- and high-dose (1, 2, 8 mg) dexamethasone, and 5) postoperative adrenal insufficiency necessiating cortisol replacement [25]. Immunohistochemical study of steroidogenic enzymes demonstrated that the immunoreactivities of 3B-HSD was only detected in scattered tumor cells, while those of P-450118 was diffusely but weakly detected in most tumor cells, suggesting the ineffective corticosteroido- genesis by the tumor. This may in part explain the autonomous, but relatively low cortisol production, possibly due to the altered expression of steroidogenic
enzymes in individual tumor cells [3]. Alternatively, defective expression of cytoplasmic steroid transporter proteins, such as steroidogenic acute regulatory pro- tein, and/or altered membrane receptors, such as low- density lipoprotein receptor and ACTH receptor, resulting from malignant transformation may partly account for the ineffective corticosteroidogenesis by the tumor. Estrogen-secreting ACC is very rare world- wide [1, 2], and only four cases have been reported in Japan thus far [27-30]. Thus, our case adds to the liter- ature the fifth one. As summarized in Table 2, they are 4 male and 1 female, with the age ranging from 18- month-old to 37-year-old; all 5 cases presented with gynecomastia, 3 with virilization and none with Cush- ing’s syndrome.
In summary, we presented a rare case of estrogen- secreting ACC with subclinical Cushing’s syndrome. Data from endocrinological, pathological, immuno- histochemical and biochemical studies in this case sug- gest the excessive secretion of estrogens derived from conversion of adrenal androgens by the overexpressed P450 aroma as well as the low efficiency of cortisol pro- duction possibly due to ineffective steroidogenesis by the tumor.
Acknowledgements
This study was supported in part by Grants-in-Aids from the Ministry of Welfare, Health and Labor, and the Ministry of Education, Culture, Sport, Science and Technology, Japan.
References
1. Schulick RD, Brennan MF (1999) Adrenocortical carcinoma. World J Urol 17: 26-34.
2. Dackiw AP, Lee JE, Gagel RF, Evans DB (2001) Adrenal cortical carcinoma. World J Surg 25: 914-926.
3. Sasano H, Suzuki T, Nagura H, Nishikawa T (1993) Steroidogenesis in human adrenocortical carcinoma: biochemical activities, immunohistochemistry, and in situ hybridization of steroidogenic enzymes and histo- pathologic study in nine cases. Hum Pathol 24: 397- 404.
4. Sasano H (1994) Localization of steroidgenic enzymes in adrenal cortex and its disorders. Endocr J 41: 471- 482.
5. Sasano H, Miyazaki S, Sawai T, Sasano N, Nagura H, Funahashi H, Aiba M, Demura H (1992) Primary pig- mented nodular adrenocortical disease (PPNAD): im- munohistochemical and in situ hybridization analysis of steroidogenic enzymes in eight cases. Mod Pathol 5: 23-29.
6. Sasano H, Sato F, Shizawa S, Nagura H, Coughtrie MW (1995) Immunolocalization of dehydroepiandro- sterone sulfotransferase in normal and pathologic hu- man adrenal gland. Mod Pathol 8: 891-896.
7. Nakamura Y, Suzuki T, Inoue T, Tazawa C, Moriya T, Saito H, Ishibashi T, Takahashi S, Yamada S, Sasano H (2005) 3ß-Hydroxysteroid dehydrogenase in human aorta. Endocr J 52: 111-115.
8. Yoshimoto T, Fukai N, Sato R, Sugiyama T, Ozawa N, Shichiri M, Hirata Y (2004) Antioxidant effect of adrenomedullin on angiotensin II-induced reactive oxygen species generation in vascular smooth muscle cells. Endocrinology 145: 3331-3337.
9. Sato R, Tsujino M, Nishida K, Otani Y, Minami T, Shichiri M, Hizuka N, Aiba M, Hirata Y (2004) High molecular weight form insulin-like growth factor II- producing mesenteric sarcoma causing hypoglycemia. Intern Med 43: 967-971.
10. Shackleton CH (1993) Mass spectrometry in the diag- nosis of steroid-related disorders and in hypertension research. J Steroid Biochem Mol Biol 45: 127-140.
11. Kikuchi E, Yanaihara H, Nakashima J, Homma K, Ohigashi T, Asakura H, Tachibana M, Shibata H, Saruta T, Murai M (2000) Urinary steroid profile in adrenocortical tumors. Biomed Pharmacother 54 (Sup- pl 1): 194s-197s.
12. Weiss LM (1984) Comparative histologic study of 43 metastasizing and nonmetastasizing adrenocortical tu- mors. Am J Surg Path 8: 163-169.
13. Pommier RF, Brennan MF (1992) An eleven-year experience with adrenocortical carcinoma. Surgery 28: 657-739.
14. Mantero F, Terzolo M, Arnaldi G, Osella G, Masini AM, Ali A, Giovagnetti M, Opocher G, Angeli A
(2000) A survey on adrenal incidentaloma in Italy. J Clin Endocrinol Metab 85: 637-644.
15. Sasano H, Imatani A, Shizawa S, Suzuki T, Nagura H (1995) Cell proliferation and apoptosis in normal and pathologic human adrenal. Mod Pathol 8: 11-17.
16. Arola J, Salmenkivi K, Liu J, Kahri AI, Heikkila P (2000) p53 and Ki67 in adrenocortical tumors. Endocr Res 26: 861-865.
7. Erickson LA, Jin L, Sebo TJ, Lohse C, Pankratz VS, Kendrick ML, van Heerden JA, Thompson GB, Grant CS, Lloyd RV (2001) Pathologic features and expres- sion of insulin-like growth factor-2 in adrenocortical neoplasms. Endocr Pathol 12: 429-435.
18. Fottner Ch, Hoeflich A, Wolf E, Weber MM (2004) Role of the insulin-like growth factor system in adre- nocortical growth control and carcinogenesis. Horm Metab Res 36: 397-405.
19. De Fraipont F, El Atifi M, Cherradi N, Le Moigne G, Defaye G, Houlgatte R, Bertherat J, Bertagna X, Plouin PF, Baudin E, Berger F, Gicquel C, Chabre O, Feige JJ (2005) Gene expression profiling of human adrenocor- tical tumors using complementary deoxyribonucleic acid microarrays identifies several candidate genes as markers of malignancy. J Clin Endocrinol Metab 90: 1819-1829.
20. Bulun SE, Noble LS, Takayama K, Michael MD, Agarwal V, Fisher C, Zhao Y, Hinshelwood MM, Ito Y, Simpson ER (1997) Endocrine disorders associated with inappropriately high aromatase expression. J Steroid Biochem Mol Biol 61: 133-139.
21. Martel C, Rheaume E, Takahashi M, Trudel C, Couet J, Luu-The V, Simard J, Labrie F (1992) Distribution of 17 beta-hydroxysteroid dehydrogenase gene expression and activity in rat and human tissues. J Steroid Bio- chem Mol Biol 41: 597-603.
22. McLeod MK, Thompson NW, Gross MD, Bondeson AG, Bondeson L (1990) Subclinical Cushing’s syn- drome in patients with adrenal gland incidentalomas. Am Surg 56: 398-403.
23. Reincke M, Nieke J, Krestin GP, Saeger W, Allolio B, Winkelmann W (1992) Preclinical Cushing’s syn- drome in adrenal “incidentalomas”: comparison with adrenal Cushing’s syndrome. J Clin Endocrinol Metab 75: 826-832.
24. Nawata H, Demura H, Suda T, Takayanagi R (1996) Preclinical Cushing’s syndrome. In: Nawata H (ed) Annual Report of the Ministry of Health and Welfare ‘Disorders of Adrenal Hormones’ Research Commit- tee, Japan. 1995. Fukuoka, 1996: 223-226 (in Japa- nese).
25. Midorikawa S, Hashimoto S, Kuriki M, Katoh K, Watanabe T, Sasano H, Nishikawa T (1999) A patient with preclinical Cushing’s syndrome and excessive
DHEA-S secretion having unilateral adrenal carcinoma and contralateral adenoma. Endocr J 46: 59-66.
26. Hofle G, Gasser RW, Lhotta K, Janetschek G, Kreczy A, Finkenstedt G (1998) Adrenocortical carcinoma evolving after diagnosis of preclinical Cushing’s syn- drome in an adrenal incidentaloma. A case report. Horm Res 50: 237-242.
27. Nishiki M, Amano K, Itoh H, Ezaki H, Miyachi Y (1980) Feminizing adrenocortical carcinoma in man. Jpn J Surg 10: 159-163.
28. Kimura M, Itoh N, Tsukamoto T, Kumamoto Y, Takagi Y, Mori Y (1995) Aromatase activity in an estrogen- producing adrenocortical carcinoma in a young man.
J Urol 153: 1039-1040.
29. Watanabe T, Yasuda T, Noda H, Wada K, Kazukawa I, Someya T, Ohnuma S, Kohno Y, Harada S (2000) Estrogen secreting adrenal adenocarcinoma in an 18- month-old boy: aromatase activity, protein expression, mRNA and utilization of gonadal type promoter. Endocr J 47: 723-730.
30. Hirai K, Mori T, Murata K, Hashimoto R, Takii Y, Honda M, Ikeda I, Hara M, Osano M, Endo M, Katsumata K, Fujimoto M, Kato K (1989) Virilizing adrenocortical carcinoma weighing 10 gm with eleva- tion of serum estrogen in a girl. J Jpn Pediatr Soc 93: 2013-2016 (in Japanese).