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The Journal of Clinical Endocrinology & Metabolism Vol. 87, No. 1 49-56
Copyright © 2002 by The Endocrine Society

Special Features

Sertoli-Leydig Cell Tumor of the Ovary, a Rare Cause of Precocious Puberty in a 12-Month-Old Infant

Catherine S. Choong, Peter J. Fuller, Simon Chu, Yvette Jeske, Francis Bowling, Richard Brown, Peter Borzi, Nicholas D. Balazs, Ram Suppiah, Andrew M. Cotterill, Diane Payton, David M. Robertson and Henry G. Burger

Departments of Pediatric Endocrinology (C.S.C., Y.J., A.M.C.), Pediatrics (R.B., R.S.), Pediatric Surgery (P.B.), and Pathology (F.B., D.P.), Mater Misericordiae Hospitals, South Brisbane, Queensland, Australia 4104; Flinders Cancer Centre (C.S.C.), Flinders Medical Centre, Bedford Park, South Australia, Australia 5042; Southern Cross Pathology Australia (N.D.B.), Clayton, Victoria, Australia 3168; and Prince Henry’s Institute of Medical Research (P.J.F., S.C., D.M.R., H.G.B.), Clayton, Victoria, Australia 3168

Address all correspondence and requests for reprints to: Catherine Choong, Flinders Cancer Center, Flinders Medical Center, Bedford Park, South Australia, Australia 5042. E-mail: catherine.choong{at}flinders.edu.au


   Abstract
Top
 Abstract
Introduction
 Subject and Methods
 Results
 Discussion
 References
 
We report a 12-month-old infant who presented with a 4-month history of isosexual precocious puberty secondary to an estrogenizing Sertoli-Leydig cell tumor of the ovary. Total serum immunoreactive inhibin and subunits A and B were markedly elevated before surgical resection and subsequently decreased 7 wk later into the normal prepubertal range. Twenty weeks following surgical removal, the patient presented again with central precocious puberty; inhibin B levels were raised on this occasion, a luteinizing releasing hormone stimulation test confirmed central precocious puberty. This is the youngest reported occurrence of this rare sex cord stromal neoplasm. The prognosis of this extremely rare tumor presenting at this early juvenile stage is uncertain. This report illustrates the usefulness of serum inhibin as a tumor marker during therapeutic suppression with leuprorelin acetate for central precocious puberty. Analysis of genomic and tumor DNA revealed a normal nucleotide sequence for the LH receptor and the G{alpha}s gene. To understand the molecular pathogenesis of this tumor we analyzed mRNA levels for the inhibin A and B subunits, FSH receptor, LH receptor aromatase, steroidogenic factor-1 and the ER ß genes. Molecular characterization reveals the presence of genes specific for granulosa and Leydig cells; the relative expression of these genes, in addition to its histologic characteristics, suggests that this tumor may result from a dysdifferentiation of a primordial follicle.


   Introduction
Top
 Abstract
 Introduction
Subject and Methods
 Results
 Discussion
 References
 
SERTOLI-LEYDIG CELL TUMORS are rare sex cord-stromal neoplasms (1, 2) that account for less than 1% of ovarian tumors. Approximately 40% of these tumors are associated with evidence of androgen excess and more rarely estrogenization. Eighty percent of juvenile granulosa cell tumors of the ovary occurring in children younger than 10 yr of age are associated with isosexual pseudoprecocity. There are fewer reports of this phenotype resulting from Sertoli-Leydig cell tumors (2, 3). In isosexual pseudoprecocity, maturation is incomplete, with only one sexual characteristic developing early. This is distinct from isosexual precocity in which secondary sexual characteristics appropriate for the sex of the child occur. Isosexual precocity in girls resulting from disturbance of the hypothalamic-pituitary-axis may be secondary to neurogenic abnormality such as intracranial hamartoma. Other endocrine causes include tumors producing LH, human CG (HCG), or steroid excess from adrenal tumors or gonadoblastomas. Isosexual precocity may also occur following treatment of congenital adrenal hyperplasia. This results from maturation of the hypothalamic-pituitary-axis from the increased androgen levels present before adequate cortisol replacement.

Inhibin, a 32-kDa dimeric glycoprotein, is composed of an {alpha} subunit disulfide, linked to one of two ß subunits. Inhibin A and B contain the ßA subunit and ßB subunit, respectively; each subunit is the product of separate genes that are homologous to members of the transforming growth factor ß superfamily. The major known biological action of inhibin is as a gonadal regulator of FSH secretion. Studies in transgenic mice suggest a role for {alpha}-inhibin (4) as a tumor suppressor gene. Inhibin is normally secreted by the granulosa and Sertoli cells of the ovary; serum levels are elevated in epithelial ovarian neoplasms (4) including both adult ( 5) and juvenile granulosa cell tumors of the ovary (6) and Sertoli-Leydig cell tumors (7).

We report on a 12-month-old infant presenting with isosexual precocity secondary to a Sertoli-Leydig cell tumor and subsequent development of central precocious puberty following removal of the tumor. The serum inhibin concentration was the only identifiable tumor marker in this case. Analysis of mRNA expression for the inhibin subunits, FSH receptor, and ERß support a granulosa cell origin of this sex cord tumor; however, presence of the LH receptor expression suggest a dysdifferentiation during folliculogenesis.


   Subject and Methods
Top
 Abstract
 Introduction
 Subject and Methods
Results
 Discussion
 References
 
Patient history

The patient was born at term to nonconsanguineous parents following an in vitro fertilization pregnancy complicated by gestational diabetes. Delivery was by emergency lower uterine segment cesarean section for maternal preeclampsia. Birth weight was 2963 kg and length 44 cm. The perinatal course was uncomplicated. At 8 months of age, her parents first noted the appearance of pubic hair; this progressively darkened and coarsened and was associated with breast development at 9 months of age. Facial acne, downy axillary hair, and a viscous vaginal discharge but no bleeding was observed just before presentation at 1 yr of age. Informed consent for publication of this case study and deidentified photographs of the patient was obtained from her parents according to the guidelines of Mater Children’s Hospital Ethics Committee.

Clinical findings

At presentation, her height was 85.8 cm (97th percentile), weight 12.68 kg (97th percentile). Examination revealed an active, alert, irritable child with a nontender abdomen. Breast tissue was palpable and the contour of nipples and areolae was equivalent to Tanner stage 3 breast development (8). Pubic hair was coarse, pigmented, distributed over the labia majora, and equivalent to Tanner stage 3 (Fig. 1Go). Inspection of her external genitalia revealed estrogenized vulval mucosa with a clear viscous discharge, and the clitoris was slightly enlarged. Axillary hair was downy and acne was present on her forehead. A pale 5-cm ovoid café-au-lait macule was present on her left knee. No clear abdominal masses were palpable.



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  		Figure 1. Clinical photographs at presentation demonstrate Tanner stage 3 breast development (A) and pubic hair development (B) at 1 yr, 3 wk of age. C, Follow-up during suppression with leuprolide acetate 13 months after surgery demonstrates persistence of breast and loss of pubic hair.

 
X-ray of her left hand and wrist at a chronological age of 1 yr, 3 wk revealed a bone age of 2.5 yr according to female standards (9). Initial ultrasonographic investigation of the abdomen revealed an enlarged uterus with prominent endometrium and an 8.5 cm x 6 cm irregular, inhomogeneous mass lying above the uterus and crossing the midline. There was no evidence of cystic change within the mass; adrenals, liver, spleen, and kidneys were normal. Magnetic resonance imaging confirmed a left pelvic mass, with hypothalamic and pituitary regions showing a normal-sized pituitary and infundibulum with a normal posterior pituitary bright spot.

At laparotomy, 1 wk following initial presentation, a solid tumor localized to the left ovary was identified. A left salphingo-oophorectomy was performed. The right ovary was normal on visual inspection and not removed. A short period of vaginal bleeding was noted a few days following surgery. Pubic hair was lost and her breasts became less prominent over the 8 wk following surgery. However, at 15 wk after surgery, her mother reported regrowth of pubic hair. Ultrasonographic imaging of her abdomen repeated at this time revealed a right ovary that measured 1 cm in length and contained one small follicle. Repeat magnetic resonance and computerized tomographic imaging of the head, chest, retroperitoneal abdomen, and pelvis and methylene diphosphonate whole-body bone scan did not reveal any evidence of recurrence or metastatic spread.

Endocrine investigations (Tables 1Go and 2Go) indicated development of central isosexual precocious puberty. Therapeutic suppression with leuprorelin acetate 7.5 mg/month im was initiated. Review at 12 months postoperatively revealed an active, well female child; weight and height continued on the 97th percentile, breasts were less prominent, and a few strands of pubic hair remained (Fig. 1CGo). Bone age had advanced to 4 yr, 2 months according female standards (9).


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  		Table 1. Preoperative endocrinology and tumor markers 1 wk before surgery

 

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  		Table 2. LHRH stimulation test

 
Endocrine investigations

The ACTH test was performed with an iv injection bolus of 250 µg 1–24ACTH (Synacthen, Ciba-Geigy, Basel, Switzerland) between 0800 and 0930 h. Blood samples were taken before and at 30 and 60 min after ACTH injection (Table 1Go). Intra- and interassay coefficients of variation for cortisol and 17OHP were 5.4% and 6.4%, respectively. The gonadotrophin-releasing hormone test required a slow iv injection of 100 µg gonadorelin (LHRH, Wyeth-Ayerst Laboratories, Inc., Baulkham Hills, Australia). Blood samples were taken 30 and 1 min before and following LHRH administration at 15-min intervals over 2 h. LH and FSH were measured by immunoenzymatic assay (Access, Beckman Coulter, Inc., Minneapolis, MN) referenced to World Health Organization 2nd International Reference Preparation for hLH (80/552) and hFSH (78/549). Intra- and interassay coefficients of variation were 3.6% and 4.4%, respectively.

Total serum inhibin was measured by heterologous RIA using 125I-labeled 31-kDa bovine inhibin as tracer, a rabbit anti-31-kDa bovine inhibin antiserum (reference no. 1989), and a serum standard of arbitrary unitage (10). The serum levels of inhibin A and B were determined by ELISA using reagents provided by Oxford-Bio-innovation Ltd. (Oxon, UK) based on published methods (11, 12). The tumor markers ßHCG, AFP, CEA, and CA-125 were assayed according to manufacturer’s protocols (AXYSYM, Abbott Laboratories, Abbott Park, IL). Plasma adrenal and ovarian steroids were measured by RIA, baseline samples taken before surgery and at follow-up. Müllerian inhibiting substance (MIS), assayed by the laboratory of Dr. John Hutson as described previously (13, 14), was not detected in the serum of our patient. The karyotype on analysis of peripheral lymphocytes from the patient was 46 XX.

Histological examination

Peritoneal fluid cytology showed mesothelial cells only.

Ovarian tissue was fixed in formaldehyde and paraffin sections were cut (4 µm) following routine techniques and stained with hematoxylin and eosin (Fig. 2Go).



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  		Figure 2. A, Resected fallopian tube and attached solid ovarian tumor. Paraffin sections stained with hematoxylin and eosin. B, Ovarian tumor showing cord-like and small tubule formation of tumor cells (magnification, x20). C, An ovarian follicle from the surrounding ovarian cortex showing thickened lamina propria filled and granulosa cells in a pattern suggestive of sex cord tubules (magnification, x40).

 
RNA analysis

RNA was extracted from the tumor tissue using the guanidine thiocyanate/cesium chloride method as described previously (15). As a control for the analysis of gene expression, RNA from normal premenopausal ovaries and from two adult granulosa cell tumors was used; the origin of these tissues has been described previously (16, 17). Expression of the inhibin {alpha}, ßA, and ßB subunit genes was examined using a coupled RT-PCR assay with gene-specific primers as described previously (17). Expression of the FSH receptor and ERß genes was also examined using previously described RT-PCR-based assays (15, 16). The RT-PCR assay for aromatase gene expression used primers described by Agarwal et al. (18). SF-1 gene expression was determined using primers derived from the published sequence (GenBank accession number HSU76388) at nucleotide positions 743 (5'-CGA TCT TGG GCT GCC TGC AG-3') and 973 (5'-CCT TGC CGT GCT GGA CCT GG-3'). An internal hybridization probe at position 851 was used for Southern blot analysis. Expression of the ß2-microglobulin gene was also determined as a control for RNA integrity (17). Five microliters of each product was analyzed on ethidium bromide-stained 2% agarose gels. Reactions containing no reverse transcriptase were included as a negative control.

The LH receptor was amplified using primers derived from the published sequence (GenBank accession number EO5678) at nucleotide position 1601 (5'-TCAATGTGGTGGCCTTCTTCATA-3') and 1834 (5'-TTGGCACAAGAATTGATGGGATA-3'). RT-PCR was used to amplify DNA fragments containing codon 201 and 227 of G{alpha}s (17) from tumor cDNA; genomic DNA was amplified using standard PCR conditions. Exons 8 and 9 of the G{alpha}s gene (GenBank accession number JO3647) were amplified from tumor cDNA using the forward primer (GSA535) 5'-CTGGACAAGATCGACGTGAT-3' and reverse primer (GSA803) 5'-TCCTGCAGGCGGTTGGTCT-3'. Purified PCR product was sequenced directly with the forward (GSA565) 5'-GACTATGTGCCGAGCGATC- 3' and reverse primers (GSA779) 5'-TCCTCCCGGATGACCATGTT-3' using the ABI PRISM Big Dye Terminator cycle sequencing ready reaction kit (Perkin-Elmer Corp., Boston, MA) according to manufacturer’s instructions. The resulting amplicons were subjected to direct dideoxy sequencing using an ABI377 automated DNA sequencer. PCR amplification of genomic DNA was carried out using GSA535 and GSA803 primers. Sequence analysis for genomic DNA was performed with following forward primers (GSA586–44) 5'-TCGGTTGGCTTTGGTCAGATCCAT-3', (GSA660–41) 5'-CCAGTCCCTCTGGAATAACCAGC-3' and reverse primers (GSA659+45) 5'-AGAAACCATGAT- CTCTGTTA-3', (GSA718+71) 5'-CAGCGACCCTGATCCCTAACA-3'.


   Results
Top
 Abstract
 Introduction
 Subject and Methods
 Results
Discussion
 References
 
Laboratory results

Preoperative endocrine investigations (Tables 1Go and 2Go) demonstrated that total serum inhibin and E2 levels were markedly elevated. Before removal of the tumor, cortisol levels were normal (Table 3Go). The LH level was just above the normal reference range for prepubertal females and did not alter with LHRH stimulation (Table 2Go). FSH levels were below the prepubertal limit. Serum progesterone and androstenedione were elevated (Table 1Go) and then fell into the normal range following removal of the tumor (Table 4Go). 17{alpha}-hydroxyprogesterone was initially above the reference for prepubertal females and in the range seen during the luteal phase of the menstrual cycle. Total serum inhibin was at the adult midluteal range before surgery and then remained elevated 12 h post surgery and subsequently fell into the normal prepubertal reference range when assayed 7 wk following removal of the tumor (Tables 1Go and 4Go).


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  		Table 3. Preoperative Synacthen stimulation test

 

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  		Table 4. Diagnostic endocrinology and tumor markers after surgery

 
Although T levels were not elevated above the reference range quoted for prepubertal females for the Mater Misericordiae Hospital laboratory, the level preoperatively was 1.9 nmol/liter, which was more than 10 times the levels obtained recurrently after removal of the tumor (Tables 1Go and 4Go). Therefore, it is very likely that at presentation the plasma T level was elevated relative to the normal levels for this child and was secondary to secretion from the malignant ovary. It is noteworthy that others (19) have reported a lower reference range of less than 0.5 mmol/liter for prepubertal children.

During the second presentation with precocious puberty, LH, FSH, and E2 rose upon LHRH stimulation, indicating central gonadotrophin-dependent precocious puberty (Table 2Go). Serum inhibin B levels rose in association with symptoms of central precocious puberty at 22 wk (Table 4Go). ßHCG, AFP, and CEA were not elevated before surgery or following surgery; CA-125 was borderline but fell following surgery.

Ovarian histology

The resected specimen consisted of a fallopian tube and attached ovary weighing in total 95.3 g The ovary measured 75 x 48 x 42 cm with a smooth external surface that was cream gray in appearance with focal yellow areas. The cut section revealed a lobulated solid tumor with nodular yellow areas separated by grayish septa (Fig. 2AGo).

Microscopic examination showed that the majority of the ovary was replaced by a vaguely nodular tumor composed of hyalinized stroma containing a dual population of cells. Some of these cells were present in cords and nests, and these cells appeared slightly smaller than the second population of cells that formed small nodules or sheets of cells and were also present as scattered individual cells. The second population of somewhat larger cells had abundant slightly vacuolated cytoplasm, and the oil Red O stain showed that these cells contained lipid. Mitotic figures were present within these larger cells (Fig. 2BGo), and scattered apoptotic cells were present. Toward the periphery of the tumor the stroma was somewhat more edematous, and the tumor cells, which were arranged in cord-like structures, showed occasional areas strongly suggestive of poorly formed tubules. Within the surrounding ovarian tissue, there were follicles surrounded by thickened lamina propria. Some of these were filled with granulosa cells and others had sex-cord cells in solid tubule formation (Fig. 2CGo), suggesting that the Sertoli-Leydig cell tumor may have arisen from the granulosa cells (Scully R. E., personal communication). Normal oocytes were also present.

RNA analysis

RT-PCR with RNA from the tumor demonstrated clear expression of the three inhibin subunit genes (Fig. 3Go). When compared with RNA from two representative granulosa cell tumors (GCTs), the levels of both inhibin {alpha} and ßB gene expression were similar or higher in the patient than those in the GCT, although the comparison is clearly semiquantitative. Expression of the inhibin ßA subunit gene appeared to be less than that in the GCT. FSH receptor gene expression was also observed. The LH receptor was expressed in the tumor at levels that are much greater than in the GCT and comparable with levels seen in a normal ovarian sample that contains a corpus luteum (Fig. 3Go). The novel ERß isoform is predominantly expressed in the granulosa cells of the ovary and the epithelial cells of the prostate (20). We have recently reported high levels of ERß gene expression in GCT (16); in this tumor the levels were similar to those in the GCT. Aromatase gene expression was observed at levels that are similar to those observed in normal ovary and perhaps a little higher than those in the GCT. SF-1 gene expression paralleled that of aromatase (Fig. 3Go).



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  		Figure 3. Gene expression in the Sertoli-Leydig cell tumor. Representative ethidium bromide-stained agarose gels of RT-PCR products for the inhibin {alpha} (In{alpha}), inhibin ßA (InßA), inhibin ßB (InßB), ERß, FSH receptor (FSHR), LH receptor (LHR), aromatase (Arom), steroidogenic factor-1 (SF-1), and ß2-microglobulin genes. Molecular weight markers (M) are shown. RNA has been analyzed from the tumor, two adult granulosa cell tumors of the ovary and two normal premenopausal ovaries. In the case of the LHR, the RNA from a different normal ovary has been analyzed: This ovary contained a corpus luteum, thereby providing a positive control. A control of the tumor RT-PCR in which the RT has been omitted (no RT) is also shown.

 
DNA analysis

To determine the molecular pathogenesis of the tumor, we investigated the LH receptor and the G{alpha}s gene for activating mutations. Sequence analysis of the region encoding the transmembrane domain of the LH receptor revealed only wild-type sequence and in particular the Asp-His mutation at position 578 was not observed (21). Analysis of the G{alpha}s gene over 268 base pairs encompassing codons 201 and 227 did not reveal any mutations in the cDNA generated from tumor RNA or genomic DNA from peripheral white blood cells. Both tumor cDNA and genomic DNA from the patient were heterozygous for a conservative C-T transversion at nucleotide 555 (Ile185 Ile) in exon 7.


   Discussion
Top
 Abstract
 Introduction
 Subject and Methods
 Results
 Discussion
References
 
Sertoli-Leydig cell tumors arise in young women, the majority present at an average age of 25 yr (1, 2). This patient appears to be the youngest reported patient with this tumor. The prognosis of Sertoli-Leydig cell tumors is usually good and correlates with the stage and degree of differentiation (1, 2, 22). This particular tumor would appear to be of intermediate differentiation (Meyer’s type II), which accounts for 54% of Sertoli-Leydig cell tumors. Of the intermediate differentiated tumors in one series of 207 Sertoli-Leydig cell tumors, 11 of 80 tumors behaved in a malignant fashion (2). Notably 2 of the 11 were children aged 2 and 4 yr. This tumor is totally confined to the ovary, indicating that it is stage 1 according to the staging classification of the International Federation of Gynaecology and Obstetrics. This is in keeping with reported series in which more than 90% of Sertoli-Leydig cell tumors are stage 1 at presentation. Because the tumor is stage 1 and the differentiation appears to be intermediate, the prognosis is expected to be good; however, the patient is being closely monitored.

The high levels of estrogen account for the breast development observed in this child. The development of pubic hair before removal of the tumor may be accounted for by the combination of elevated androstenedione and dehydroepiandrosterone and the elevated estrogen levels (23). Although the T levels were not elevated outside the normal range for our laboratory, the T levels were significantly higher preoperatively than those observed following removal of the tumor; therefore, T may also have contributed to the pubic hair growth. Elevated E2, dehydroepiandrosterone, androstenedione, and T observed preoperatively were most likely derived from the tumor. Androgenization is present in more than 50% of Sertoli-Leydig cell tumors (1, 2).

Serum inhibin concentration and estrogen levels were the primary tumor markers in our patient. Twelve hours post removal of the tumor, the serum inhibin level fell by 20%. Two weeks after surgery, total serum inhibin levels had fallen by 80% into the range associated with stage III-IV puberty (24) with concomitant decreases in the A and B forms. Assessment at 7 wk post surgery demonstrated serum inhibin levels in the prepubertal range (Tables 3Go and 4Go). Yamashita et al. (7) reported a reduction of inhibin A and B levels over a 1-wk period in a 16-yr-old patient with a poorly differentiated Sertoli-Leydig cell tumor with serum inhibin B levels decreasing at a slower rate than that of inhibin A. In two juvenile patients with granulosa cell tumors (6, 7), total inhibin levels reportedly decreased within 2 d. In our patient serum inhibin A were still elevated following resection of the tumor at 2 wk, whereas inhibin B levels had fallen into the prepubertal range. Persistent production of inhibin A from the remaining contralateral ovary or a prolonged half-life following marked elevation are two possible explanations for the elevated serum level of inhibin A at 2 wk after surgery in our case.

Several groups ( 25, 26) have reported that inhibin B is the major form expressed in serum during active malignant disease secondary to granulosa cell tumors, whereas both forms of inhibin were present in two patients with Sertoli-Leydig cell tumors (7). Additionally, others report elevation and subsequent reduction of inhibin B during treatment of central precocious puberty with a gonadotropin agonist (27); similarly in our patient, although serum inhibin A levels did not rise (Table 4Go), inhibin B levels rose above the prepubertal range with the appearance of central precocious puberty.

Endogenous inhibin is one of the nonsteroidal factors involved in the negative regulation of the FSH secretion in the female (28, 29). Therefore, it is noteworthy that we did not observe hypersecretion of FSH after cessation of suppression by the high inhibin and E2 levels associated with the tumor. FSH levels in our patient following removal of the tumor were not elevated above the normal prepubertal range at 2, 7, or 11 wk after surgery. At 22 wk the basal level of FSH rose from 2.6U/liter to a maximal level of 23.5 U/liter following stimulation with GnRH (Table 2Go), consistent with central precocious puberty. Primate studies suggest that inhibin inhibits both the synthesis and secretion of FSH from the pituitary (30); thus, the absence of FSH hypersecretion is not unexpected. Treatment of intact adult male Rhesus monkeys with an infusion of recombinant inhibin A to double normal circulating levels of immunoreactive inhibin within 12 h resulted in significant suppression of FSH levels by 54 h. Elevated inhibin levels were maintained for 4 d in this study; suppression of FSH persisted for the 6 h of sampling following termination of the inhibin infusion. When assessed 1 wk later, levels of FSH had rebounded to preinfusion levels in the primates. The authors did not report hypersecretion of FSH in this study (31).

At presentation LH levels were slightly higher than expected; interestingly, an alternative assay for LH (Abbott Diagnostics, Abbott Park, IL) did not show this mild elevation (Table 2Go). It is possible that the difference in the LH levels measured between the two assays may reflect differing sensitivity to LH isoforms. It has been noted that changes in gonadotropin glycoforms occur through the menstrual cycle, which are related to changes in the prevailing steroid environment (32). Elevated levels of gonadotropins have been previously reported in isosexual precocity associated with juvenile granulosa cell tumors (3), and differing isoforms may account for this apparent contradiction because gonadotrophins would be expected to be depressed when estrogen secretion from a peripheral source is responsible for sexual precocity. Elevated levels of SHBG and PRL preoperatively may have resulted from the high E2 levels. Following representation, LH and FSH responses to GnRH stimulation were characteristic of central precocious puberty.

Although it is possible, given that estrogen and inhibin suppresses FSH production, it would seem unlikely that the patient had developed central isosexual precocity before surgery because one would have anticipated initial LH levels to rise with GnRH stimulation as they did in the second test (Table 2Go). Secondary central precocious puberty developed within 22 wk of removal of the tumor. Peripherally mediated precocious puberty (e.g. congenital adrenal hyperplasia) (33) may precipitate maturation of hypothalamic centers controlling pubertal progression. An ultrasensitive bioassay indicates that E2 levels are 8-fold higher in normal prepubertal girls than boys and are therefore likely to promote epiphyseal maturation and earlier onset of puberty in girls (34). Thus, the extremely high E2 levels that may have been present for at least 4 months before treatment may account for the unusually rapid onset of central precocious puberty in our patient. Advanced bone age and the risk of compromising adult height along with psychosocial considerations warranted initiation of treatment with the gonadotrophin agonist leuprolide acetate. Careful monitoring is needed because the long-term safety of leuprolide acetate remains to be established (35).

Total inhibin is a sensitive immunohistochemical marker of ovarian sex cord-stromal tumors (36, 37). Studies in testicular-derived sex cord tumors indicate that inhibin is also a sensitive immunohistochemical marker for Sertoli and Leydig tumors (38, 39) and differentiate these malignancies from seminoma and embryonal carcinoma and possibly from the rare occurrence of metastases to the testes (40).

Immunohistochemical studies indicate that MIS staining in testes is limited to Sertoli cells and absent in Leydig cells; in a normal ovary, granulosa cells are positive for MIS, but thecal cells are only focally reactive. In testicular Sertoli-Leydig cell tumors, MIS staining is limited to the Sertoli cells (37). The absence of MIS expression in serum from our patient is of interest and suggests that, in keeping with its histological appearance, the tumor reported here may have thecal/Leydig cell differentiation or alternatively, absent MIS expression has occurred as a result of malignant transformation of granulosa cells. The second subtype of estrogen receptor, ERß, is the predominant isoform expressed in granulosa cells in normal ovaries. In normal human testes, ERß mRNA was not detected by in situ hybridization in Leydig cells; however, it is present in germ cells and Sertoli cells at later stages of differentiation (20). Others have identified ERß-specific staining by immunohistochemistry in both rat Sertoli and Leydig cells (41). The abundance of ERß expression in this Sertoli-Leydig cell tumor is similar to levels we have reported in granulosa tumors (16). The FSH receptor normally specifically expressed in the Sertoli cells of the testis and granulosa cells of the ovary (42) is present in this tumor. The aromatase/SF-1 expression is predictable given the estrogen synthesis and is consistent with a granulosa cell origin. The LH receptor expression in clearly more consistent with a Leydig cell origin. These data in combination may reflect a "dysdifferentiation" during early folliculogenesis allowing a mixed cytopathological and molecular phenotype.

The interesting histological feature of this tumor and the surrounding ovary is the presence of the abnormal follicle in the ovarian tissue surrounding the tumor. Similar histologic features have been described as infant ovaries with annular tubule-like and gonadoblastoma like structures. These have been regarded as normal regressive changes in primordial follicles. Some authors (43) have suggested that these may constitute a first-hit phenomena on the path to neoplastic transformation. Because the structures appear to be present in a high proportion of pediatric autopsies with a peak in preterm infants and infants through the first 6 months of life, it is possible that these tumor-like structures are examples of abnormal folliculogenesis that progressively disappear through the first year of life.

Recently, Chin et al. (44) reported identification of expression of the genes for the adrenal-specific P450c21 and P450c11steroid enzymes and the ACTH receptor in an ovarian tumor from a 3-yr-old child with isosexual pseudoprecocious puberty supporting an adrenal origin. The biochemical features of this adrenal rest tumor included a 7-fold elevation of dehydroepiandrosterone sulfate levels, and an 8-fold elevation of E2 whereas in our patient there was a slight elevation in the level of dehydroepiandrosterone sulfate, with a greater that 30 fold elevation of serum E2 (Table 3Go). We did not test the tumor presented here for the expression of adrenal cortical specific genes, however the presence of the FSH receptor supports a gonadal origin.

The G proteins are involved in the transduction of signals from specific cell surface receptors to intracellular second messengers. These heterotrimeric proteins are composed of {alpha}, ß, and {gamma} subunits. Somatic mutations at codons 201 and 227 of the {alpha} subunit (gsp) have been identified in human GH-secreting adenomas and thyroid nodules and are associated with the McCune-Albright syndrome. The gsp R201C mutation has been reported in four Leydig cell tumors of a mixed cohort of 12 ovarian sex chord stromal tumors (45). A mutation, Asp578His, in the sixth transmembrane domain of the LH receptor was reported in three Leydig cell tumors (21). This led us to examine the tumor specifically for activating mutations at gsp codons 201 and 227 and the LH receptor codon 578. We did not identify any mutations in either of these genes by direct sequence analysis. It remains possible that the molecular pathogenesis of the tumor reported here may result from mutations of other genes involved in the G protein-coupled FSH receptor pathway given FSH (46) and cAMP ( 47) stimulate inhibin expression and secretion. Paracrine actions of high inhibin levels at theca cells may enhance androgen production (48) and subsequently lead to the extremely high E2 levels observed in this case. Alternatively, dysregulated expression of cell cycle activators such as the cyclin-dependent kinase activator cyclin D2 and the cell cycle inhibitor p27 kip1, which are induced during follicular proliferation and differentiation by FSH and LH may account for abnormal folliculogenesis observed in this sex cord tumor (49).

In summary, we have reported a rare case of Sertoli-Leydig cell tumor presenting with isosexual pseudoprecocious puberty during infancy. This tumor was associated with elevated serum inhibin A and B levels. Molecular characterization reveals the expression of genes specific for granulosa and Leydig cells, suggesting that this tumor may result from a dysdifferentiation of the primordial follicle.


   Acknowledgments
 
We acknowledge the expert opinion and comments of Dr. Robert E. Scully, Harvard Medical School. We thank Marija Luketic and Wah Chin Boon for kindly providing the aromatase and SF-1 primers and Mitzi Dyson and Sandra Johnson for excellent technical assistance in this work.


   Footnotes
 
This work was supported by the National Health and Medical Research Council of Australia and the Mater Hospital Trust.

Abbreviations: GCT, Granulosa cell tumor; HCG, human CG; MIS, Müllerian inhibiting substance.

Received February 15, 2001.

Accepted October 5, 2001.


   References
Top
 Abstract
 Introduction
 Subject and Methods
 Results
 Discussion
 References
 

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