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Origin of an Ovarian Steroid Cell Tumor Causing Isosexual Pseudoprecocious Puberty Demonstrated by the Expression of Adrenal Steroidogenic Enzymes and Adrenocorticotropin Receptor

Laboratório de Hormônios e Genetica Molecular LIM/42, Unidade de Endocrinologia do Desenvolvimento, Disciplina de Endocrinologia, Hospital das Clinicas (C.J.L., A.A.L.J., A.C.L., S.M., M.C.V.F., R.M.M., I.J.P.A., B.B.M.); and Departamento de Patologia (F.M.C.), Faculdade de Medicina, Universidade de Sao Paulo, CEP 01060–970 Sao Paulo, Brazil

Address all correspondence and requests for reprints to: Berenice B. Mendonca, M.D., Disciplina de Endocrinologia, Hospital das Clinicas, Caixa Postal 3671, CEP 01060–970 Sao Paulo, Brazil. E-mail: beremen{at}usp.br

	Abstract

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Ovarian steroid cell tumors are rare neoplasms composed of typical steroid hormone-secreting cells. Most ovarian steroid cell tumors, however, cannot be appropriately classified on a morphological basis, because the neoplastic cells closely resemble adrenal cortical cells. Nevertheless, the true adrenal origin of such tumors has been difficult to demonstrate. Here we report a 3-yr-old girl with isosexual pseudoprecocious puberty due to an ovarian steroid tumor whose adrenal cell origin was determined by the presence of messenger ribonucleic acid (mRNA) of adrenal-specific steroidogenic P450 enzymes (P450c11 and P450c21) and ACTH receptor (ACTHR). Her height was +2.3 SD, and she had Tanner stage III breast development, Tanner stage II pubic hair, and a normal clitoris. Bone age was 5 yr. Basal gonadotropin levels were undetectable (<0.6 U/L for LH and <1.0 U/L for FSH) and remained undetectable after stimulation with 100 µg GnRH, iv. Basal serum testosterone and 17-hydroxyprogesterone levels were slightly elevated, whereas basal serum androstenedione, estradiol, and dehydroepiandrosterone sulfate levels were clearly elevated. Pelvic ultrasound disclosed an enlarged uterus and an adnexal multicystic mass in the right ovary, and pathological studies disclosed an ovarian steroid cell tumor. To establish the cellular origin of the tumor we determined the presence of mRNA for P450c11, P450c21, and ACTHR in tumor tissue and normal adrenal and ovarian tissue. Detection of ACTHR, P450c21, and P450c11 mRNAs isoforms was achieved in tumoral and adrenal control tissue, but not in the ovary control tissue. The RT-PCR products of P450c11 from adrenal control tissue were composed by both BglI-sensitive and -resistant complementary DNAs, indicating the presence of both P450c11AS and P450c11ß, whereas RT-PCR product from the tumor was resistant to BglI digestion, indicating only the presence of P450c11ß.

We conclude that the histological origin of so-called adrenal rest tumor could be reliably determined by assessing the expression of specific genes in the tumor as P450c11ß and P450c21. The use of these molecular tools will allow a more precise classification of an important subset of the ovarian steroid cell tumors and can help to identify ectopic adrenal tissue in ovary and testis.

	Introduction

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OVARIAN STEROID cell tumors are rare neoplasms composed of cells that have typical morphological features of steroid hormone-secreting cells (1, 2, 3, 4, 5, 6, 7, 8). These neoplasms may secrete various steroids and may be uncommon causes of virilization (7, 8). Ovarian steroid cell tumors have different cellular origins and can be divided into different categories according to their originating cells (5). However, the cellular origin of a large subset of steroid cells tumors is uncertain, and they have been frequently designated not otherwise specified. In the literature, ovarian steroid cell tumors have also been called lipoid cell tumor, lipid cell tumor, adrenal-like tumor, masculinovoblastoma, luteoma, hypernephroid tumor, and adrenal rest tumor, demonstrating the difficulty in determining the cellular lineage of the neoplasm and its precise classification based solely on morphological criteria (9).

Here we report a 3-yr-old girl with isosexual pseudoprecocious puberty due to an ovarian steroid cell tumor whose adrenal origin was determined by the presence of messenger ribonucleic acid (mRNA) of adrenal-specific steroidogenic enzymes and ACTH receptor (ACTHR).

	Case Report

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A 3-yr-old girl had telarche at 2 yr and developed pubarche and accelerated growth velocity (9.0 cm/yr) 4 months later. A diagnosis of true precocious puberty was made in another hospital, and she was given cyproterone acetate therapy. Progression of pubertal development and growth velocity persisted despite the treatment. One episode of vaginal bleeding occurred after the discontinuation of medication, and she was sent to our hospital for reevaluation. Her familial and previous medical histories were unremarkable. Physical examination at 3 yr of age revealed a well nourished child, 106 cm height (+2.3 SD), 17 kg weight (+1.3 SD), and normal blood pressure. Bone age was 5 yr. Blood electrolytes and glucose were normal. She had Tanner stage III breast development, Tanner stage II pubic hair, and a 0.8-cm normal clitoris (10), and no abdominal mass was palpable. Serum TSH and T₄ levels were normal. Basal gonadotropin levels were undetectable (<0.6 U/L for LH and <1.0 U/L for FSH) and remained undetectable after stimulation with 100 µg GnRH, iv. Basal serum testosterone and 17-hydroxyprogesterone levels were slightly elevated, whereas basal serum androstenedione, estradiol, and dehydroepiandrosterone sulfate (DHEAS levels were clearly elevated. Basal serum cortisol, DHEA, 11-deoxycortisol, and aldosterone levels were normal (Table 1). Basal levels of hCGß, carcinoembryonic antigen, -fetoprotein, and CA-125 were normal. Pelvic ultrasound disclosed an enlarged uterus (6.2 x 3.3 x 2.2 cm) and a thick endometrium. An adnexal multicystic mass (4.4 x 4.1 x 3.2 cm) was visible on the right side of the pelvis. The left ovary was normal for chronological age. Computed tomography and magnetic resonance imaging confirmed the presence of a multicystic mass. The patient underwent surgical exploration, and resection of the right ovary was performed. Pathological studies disclosed an ovarian steroid cell tumor with morphological aspects similar to the adrenal glomerulosa zone. Two and 4 weeks after surgery the patient had normal hormonal levels followed by regression of breast and pubic hair development after 6 months. Two years after surgery she presented normal prepubertal levels of FSH and elevation of DHEA and DHEAS compatible with physiological adrenarche. During the 3 yr of follow-up she has maintained a normal growth velocity (6.4 cm/yr) and is free of the symptoms, with breast and pubic hair at Tanner stage I. At 6 yr and 6 months of age, the GnRH stimulation test was repeated, showing a normal prepubertal increase in LH from less than 0.6 to 3.5 U/L and in FSH from 1.6 to 19.3 U/L.

	Materials and Methods

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Tissue processing and RNA isolation

Tumor samples were obtained in the operating room soon after oophorectomy. After a macroscopic visual inspection, tumoral samples were collected from representative areas, avoiding those with necrosis or hemorrhage. Tissue samples were promptly frozen and stored in liquid nitrogen until used. Control ovarian tissue was obtained, after informed consent, from a 29-yr-old woman who underwent panhysterectomy due to endometrial adenocarcinoma. Histologically normal control adrenal tissue was obtained from the liquid nitrogen-stored tissue bank of our laboratory.

Total cellular RNA was extracted using a single step acid guanidinium thiocyanate-phenol-chloroform method and stored at -80 C. (11) The integrity of each sample was checked by the presence of bands corresponding to 28S and 18S of ribosomal RNA after electrophoresis on 1% agarose gel stained with ethidium bromide. The purity of the RNA samples was assessed using the 260/280 ratio and by the absence of bands corresponding to contaminating DNA in the agarose electrophoresis. The RNA concentration was estimated by spectrophotometric absorbance at 260 nm (Ultrospect III, Pharmacia LKB, Uppsala, Sweden).

RT-PCR

Four to 6 µg total RNA were reverse transcribed with 200 U Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc., Gaithersburg, MD). The RT was primed with 20 pmol oligo(deoxythymidine) primer and was carried out in 20 µL (total volume) complementary DNA (cDNA) buffer [50 mmol/L Tris-HCl (pH 8.3), 75 mmol/L KCl, and 3 mmol/L MgCl₂], 0.5 mmol/L of each deoxy (d)-NTP (dNTP mix, Pharmacia Biotech), 1 mmol/L dithiothreitol (Life Technologies, Inc.), and 10 U ribonuclease inhibitor (rRNasin Ribonuclease Inhibitor, Promega Corp., Madison, WI). The reaction mix was incubated at 37 C for 90 min. The products were frozen at -20 C until used. Ten microliters of each RT product were used in all PCR amplifications. The primer sequences used in the amplification of each specific transcript are available upon request.

Amplifications of ACTHR cDNAs were carried out in 100 µL reaction mixture [50 mmol/L KCl, 1.5 mmol/L MgCl₂, 10 mmol/L Tris-HCl (pH 9.0), 100 µmol/L dNTPs, 10 pmol of each primer, and 5 U Taq polymerase]. ACTHR mRNAs were amplified using the same protocol. A 3-temperature pre-PCR incubation (5 min at 98 C, 5 min at 55 C, and 5 min at 72 C) was followed by 30 denaturation cycles at 98 C for 30 s, annealing at 55 C for 30 s, and a 3-min extension at 72 C. A 3-temperature post-PCR incubation completed the reaction (98 C for 30 s, 55 C for 30 s, and 72 C for 15 min). P450c21 cDNA was amplified in 50 µL PCR buffer (50 mmol/L KCl, 1.5 mmol/L MgCl₂, and 10 mmol/L Tris-HCl, pH 9.0), 2 U Taq polymerase, 15 pmol of each primer, and 200 µmol/L dNTPs. The RT products were amplified for 32 cycles of denaturation at 94 C for 1 min, annealing at 63 C for 1 min, and extension at 72 C for 2 min. An additional extension step at 72 C completed the amplification protocol.

The fragment of P450c11 cDNA corresponding to exons 1 and 2 was PCR amplified using primers that do not distinguish between P450 c11ß and P450 c11AS. The amplification was performed in 100 µL reaction buffer (50 mmol/L KCl, 2.5 mmol/L MgCl₂, and 10 mmol/L Tris-HCl, pH 9.0), 100 µmol/L dNTPs, 25 pmol forward and reverse primers, and 5 U Taq polymerase (Taq DNA polymerase, Pharmacia Biotech). The amplification protocol consisted of initial denaturation at 94 C for 4 min, followed by 35 cycles of denaturation at 94 C for 1 min, annealing at 63 C for 30 s, and extension at 73 C. The amplification cycles were completed with 10 min of extension at 72 C. PCRs were carried out in a sample-sensing thermocycler (GeneAmp PCR System 9600, Perkin-Elmer Corp./Cetus, Palo Alto, CA).

Digestion with BglI

One microliter of P450c11 RT-PCR product was digested with 10 U BglI (Life Technologies, Inc.) at 37 C for 1 h. As P450c11ß and P450c11AS share 93% sequence identity, this is the approach of choice to assess quickly and correctly the expression of P450c11ß and P450c11AS. The P450c11AS carries a BglI site at the position corresponding to amino acid 29. There is no BglI site at the same position in P450 c11ß cDNA (12). Thus, after BglI digestion, P450 c11AS cDNA should be cleaved into a fragment of 307 bp and a fragment of 85 bp. On the other hand, the 392-bp-long P450c11ß cDNA should be resistant to BglI digestion. The digestion products were resolved in a 2% agarose gel.

	Results

Top Abstract Introduction Case Report Materials and Methods Results Discussion References

 
We amplified P450c21 and ACTHR cDNAs in the adrenal control tissue, but not in the ovarian control tissue (Fig. 1). Detection of ACTHR, P450c21, and P450c11 mRNAs isoforms was achieved in tumoral and adrenal control tissue, but not in the ovary. The RT-PCR products of P450c11 from adrenal control tissue were composed by both BglI-sensitive and -resistant cDNAs, indicating the presence of both P450c11AS and P450c11ß. The RT-PCR product from the tumor was resistant to BglI digestion, indicating only the presence of P450c11ß (Fig. 2). This resistance to BglI digestion was further confirmed by longer incubation with a higher enzyme concentration (data not shown).

View larger version (51K): [in this window] [in a new window]   Figure 1. Detection of mRNA for P450c21 and ACTHR through RT-PCR. The PCR products were resolved on a 1% agarose gel electrophoresis. The results of the experiment for detection of P450c21 mRNA is grouped on the left side of the gel. PCR amplification of genomic DNA was used as positive control for the PCR reaction. , Molecular weight marker. B, Negative control (RT and PCR).

View larger version (73K): [in this window] [in a new window]   Figure 2. Detection of P450c11 and P450c11ß in tumoral and control tissue through RT-PCR. The PCR products were separated on a 2% agarose gel electrophoresis. Digestion with BglI identified the expression of P450c11ß in tumor tissue, whereas normal adrenal tissue expressed both P450c11AS and P450c11ß. PCR amplification of genomic DNA served as a control for both PCR and BglI digestion. , Molecular weight marker. B, Negative control (RT and PCR).

	Discussion

Top Abstract Introduction Case Report Materials and Methods Results Discussion References

Ovarian steroid cell tumors present a variety of gross appearances, ranging from small solid masses to large multicystic masses as seen in our patient (15). These rare ovarian tumors are composed of steroid hormone-secreting cells and have been divided into three subgroups: stromal luteoma, Leydig cell tumor, and steroid cell tumor "not otherwise specified" (9). The last group is the largest one, because most ovarian steroid cell tumors cannot be appropriately classified on a morphological basis. Due to their cytological composition, similar to adrenocortical cells, these unclassified ovarian steroid cell tumors are frequently designated adrenal rest tumors. Nevertheless, the true adrenal origin of such tumors has been difficult to demonstrate because of the absence of reliable classification criteria and technology. Previous studies in adrenal tissue based on immunohistochemical analysis for adrenal enzymes (16) or steroidogenic mRNAs quantification (17) suggested the adrenal origin for a testicular adrenal rest tumor and Leydig cell testicular tumor, respectively.

In this study, we demonstrated the adrenal origin of an ovarian steroid cell tumor tissue through the expression of both P450c11ß and P450c21. These steroidogenic P450s are normally expressed only in the zona fasciculata and reticularis of adrenal glands. No other human cell line, except those of adrenal origin, has been demonstrated to express P450c11 (12).

We conclude that the histological origin of the so-called adrenal rest tumor could be reliably determined by assessing the expression of specific genes in the tumor as P450c11ß and P450c21. The molecular biology tools employed are simple, quick, and affordable. The use of such tools will allow more precise classification of an important subset of ovarian steroid cell tumors and can help to identify ectopic adrenal tissue in ovary and testis.

	Acknowledgments

 
We thank the staff of Laboratorio de Hormonios e Genetica Molecular for their technical assistance, and Prof. Walter Miller for his useful suggestions.

Received September 27, 1999.

Revised November 23, 1999.

Accepted December 2, 1999.

	References

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1. Taylor HB, Norris HJ. 1967 Lipid cell tumors of the ovary. Cancer. 20:1953–1962.[CrossRef][Medline]
2. Hughesdon P. 1966 Ovarian lipoid and thecal cell tumors: their origins and inter-relationships. Obstet Gynecol Surv. 21:245.[Medline]
3. Pedowitz P, Pomerance W. 1962 Adrenal-like tumors of the ovary: review of the literature and report of two new cases. Obstet Gynecol. 19:183.[Free Full Text]
4. Hayes MC, Scully RE. 1987 Ovarian steroid cell tumors (not otherwise specified)–a clinicopathological analysis of 63 cases. Am J Surg Pathol. 11:835–845.[CrossRef][Medline]
5. Scully RE, Young RH, Clement PB. 1998 Tumor of ovary maldeveloped gonads, fallopian tube and broad ligaments. In: Atlas of tumor pathology, series 3, fascicule 23. Washington, DC: Armed Forces Institute of Pathology; 27–31.
6. Campbell PE, Danks DM. 1963 Pseudoprecocity in an infant due to a luteoma of the ovary. Arch Dis Child. 38:519–523.
7. Imperato-McGuinley J, Peterson RE, Dawood MY, et al. 1981 Steroid hormone secretion from a virilizing lipoid cell tumor of the ovary. Obstet Gynecol. 54:525–553.
8. Harris AC, Wakely PE, Kaplowitz PB, Lovinger RD. 1991 Steroid cell tumor of the ovary in a child. Arch Pathol Lab Med. 115:150–154.[Medline]
9. Saigo PE. 1993 The histology of malignant ovarian tumors. In: Markman M, Hoskin WJ, eds Cancer of ovary. New York: Raven Press; 21–46.
10. Oberfield SE, Mondok A, Shahrivar F, Klein JF, Levine LS. 1989 Clitoral size in full-term infants. Am J Perinatol. 6:453–454.[Medline]
11. Chomczynskii P, Sacchi N. 1987 Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 162:156–159.[Medline]
12. Staels B, Hum DW, Miller WL. 1993 Regulation of steroidogenesis in NCI-H295 cells: a cellular model for the human fetal adrenal. Mol Endocrinol. 7:423–433.[Abstract]
13. Grumbach MM, Styne DM. 1998 Puberty: ontogeny, neuroendocrinology, physiology and disorders. In: Wilson J, Foster DW, Kronenberg HM, Reed Larsen P, eds. Williams textbook of endocrinology, 9th Ed. Philadelphia: Saunders; 1584.
14. Sklar CA, Kaplan SL, Grumbach MM. 1981 Lack of effect of oestrogen on adrenal androgen secretion in children and adolescents with a comment on oestrogens and pubic growth. Clin Endocrinol (Oxf). 14 93):311–320.
15. Outwater EK, Wagner BJ, Mannion C, McLarney JK, Kim B. 1998 Sex cord-stromal and steroid cell tumors of the ovary. Radiographics. 18:1523–1546.[Abstract]
16. Clark RV, Albertson BD, Munabi A, et al. 1990 Steroidogenic enzyme activities, morphology, and receptor studies of a testicular adrenal rest in a patient with congenital adrenal hyperplasia. J Clin Endocrinol Metab. 70:1408–1413.[Abstract]
17. Solish SB, Goldsmith MA, Voutilainen R, Miller WL. 1989 Molecular characterization of a Leydig cell tumor presenting as congenital adrenal hyperplasia. J Clin Endocrinol Metab. 69:1148–1152.[Abstract]

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