0021-972X/98/$03.

00/0 Journal of Clinical Endocrinology and Metabolism Copyright 1998 by The Endocrine Society

Vol. 83, No. 1 Printed in U.S.A.

COMMENTS No Evidence of a Role for Mutations or Polymorphisms of the Follicle-Stimulating Hormone Receptor in Ovarian Granulosa Cell Tumors*
PETER J. FULLER, KAREN VERITY, YAN SHEN, PAMELA MAMERS, TOM JOBLING, AND HENRY G. BURGER
Prince Henrys Institute of Medical Research, Monash University, and Department of Obstetrics and Gynecology, Monash Medical Center, Clayton, Victoria 3168, Australia
ABSTRACT The molecular pathogenesis of granulosa cell tumors of the ovary is not understood, although recent studies have shown that immunoreactive inhibin secretion by these tumors may be used as a tumor marker. Granulosa cell tumors exhibit many features of normal granulosa cells, including a response to FSH and inhibin secretion. FSH levels are suppressed in patients with inhibin-secreting granulosa cell tumors, suggesting FSH-independent growth of these tumors. Activating mutations of the FSH receptor might, therefore, be involved in tumorigenesis. We sought to identify mutations in the FSH receptor genes of these tumors using PCR to amplify the exon encoding the transmembrane and cytoplasmic domains from the tumor DNA. Analysis of the amplicons for single strand conformational polymorphisms and direct sequencing confirmed a previously reported polymorphism in the C-terminal region of the receptor, but did not identify tumorspecific missense mutations and/or polymorphisms. In addition, ribonucleic acid from 3 granulosa cell tumors was used to confirm expression of the FSH receptor; expression was unexpectedly also observed in several ovarian mucinous cystadenocarcinomas used as controls. In conclusion, our failure to identify activating mutations of the FSH receptor in 15 granulosa cell tumors argues against a role for the FSH receptor in tumorigenesis and suggests that some subsequent component of this signal transduction pathway may be activated. (J Clin Endocrinol Metab 83: 274 279, 1998)

VARIAN SEX cord stromal tumors represent approximately 10% of all ovarian tumors. They are generally classified as granulosa cell tumors, thecomas, or luteomas; the former is the most common (1). Although the classification is primarily morphological, rather than functional or biological, these tumors exhibit many of the features of granulosa cells including FSH binding, a response to FSH, and the secretion of the peptide hormones inhibin (2 4) and Mu llerian inhibiting substance (5). Inhibin production by these tumors appears to suppress plasma FSH levels (3, 4), suggesting the FSH-independent growth of these tumors. Cyclin D2 is required for granulosa cell proliferation, and its expression is stimulated by FSH. High levels of cyclin D2 messenger ribonucleic acid (RNA) have recently been reported in granulosa cell tumors of the ovary (6), further arguing for constitutive activation of an FSH-dependent mitogenic signaling pathway in these tumors. Heterotrimeric G proteins couple cell surface receptors for

Received April 22, 1997. Revision received August 22, 1997. Accepted September 28, 1997. Address all correspondence and requests for reprints to: Dr. Peter J. Fuller, Prince Henrys Institute of Medical Research, P.O. Box 5152, Clayton, Victoria 3168, Australia. E-mail: peter.fuller@med.monash. edu.au. * This work was supported by a project grant from the Anti-Cancer Council of Victoria. Recipient of a Principal Research Fellowship from the National Health and Medical Research Council of Australia. Supported by a Monash University Graduate Research Scholarship.

hormones, including FSH, to intracellular second messenger systems. Activating mutations of both cell surface receptors and G proteins have been reported in various tumors (7, 8). Mutations of the G protein -subunit gene causing inhibition of the intrinsic guanosine triphosphatase activity and thereby yielding an activating oncogene, gsp, were first described in GH-secreting pituitary adenomas (9). The gsp oncogene is found in 40% of somatotroph adenomas as well as less frequently in nonfunctioning pituitary adenomas, corticotroph adenomas, thyroid adenomas, and the McCuneAlbright syndrome (7, 8, 10). In a retrospective study of archival material from many tumor types, Lyons et al. (10) reported the presence of analogous mutations in the Gi-2 subunit gene (gip2) in 3 of 10 sex cord stromal tumors and 3 of 11 adrenocortical tumors. In a recent study we were unable to find activating mutations at codon 179 of the Gi-2 gene in ovarian granulosa cell tumors (11), and Reincke et al. (12) were similarly unsuccessful when they examined 18 adrenocorticoid tumors. The FSH receptor is a member of the seven-transmembrane domain or G protein-coupled receptor (GPCR) superfamily of cell surface receptors. Activating mutations of these receptors have been described in a range of conditions (8). Activating mutations of the TSH receptor have been implicated in the pathogenesis of malignancy in hyperfunctioning thyroid adenomas (13, 14) and well differentiated thyroid carcinoma (15, 16). Recently, a virally encoded constitutively active GPCR of the chemokine subfamily has been impli274

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cated in the pathogenesis of both Kaposis sarcoma and a rare -cell lymphoma (17). The possibility that activation of the FSH-dependent signaling pathways may play a role in the pathogenesis of granulosa cell tumors of the ovary, in a manner analogous to the role of the TSH receptor in the pathogenesis of thyroid tumors, encouraged us to analyze a series of granulosa cell tumors for activating mutations of the FSH receptor.
Materials and Methods Isolation of DNA from tissue specimens
Frozen granulosa cell tumor tissue (n 3) or paraffin-embedded tissue sections (n 12) were studied (11) as part of a much larger Melbourne-wide investigation of serum inhibin levels in ovarian tumors (3, 4). DNA extraction from fresh-frozen tissue was performed as previously described (11). Briefly, approximately 0.5 g tissue was homogenized in 5 mL extraction buffer (10 mmol/L Tris, 0.1 mol/L ethylenediamine tetraacetate, 20 g/mL ribonuclease, and 0.5% SDS), digested with 100 g/mL proteinase K at 37 C for 4 h, extracted twice with an equal volume of phenol and once with chloroform, precipitated with 0.2 vol 10 mol/L ammonium acetate and 2 vol 100% ethanol, and washed with 70% ethanol. DNA pellets were diluted in distilled water and stored at 4 C. For isolation of DNA from paraffin-embedded tissue, two or three 10-m sections were cut for each sample, and excess paraffin was removed using a disposable scalpel. Tissue sections were scraped into a 1.5-mL microcentrifuge tube. Paraffin was removed from the samples by two 20-min extractions with 1 mL xylene. Xylene was removed by two washes with 1 mL 100% ethanol and one wash with 70% ethanol. Tissue sections were vacuum-dried and digested overnight at 50 C with 400 ng/L proteinase K in 100 200 L 50 mmol/L Tris, pH 8.3, as previously described (11). Samples were then boiled for 8 min to inactivate proteinase K, cooled on ice, and spun briefly to pellet out debris. The supernatant was transferred into a fresh tube for storage at 20 C.

FIG. 1. Schematic representation of the structure of the FSH receptor. The amplicons (no. 15) that span the region of exon 10 encoding the transmembrane domains (TMD) and the cytoplasmic tail are indicated. The positions of the inactivating (Ala189 Val) (19) and activating (Asp567 Gly) (20) mutations are indicated, as is the position of the codon 680 polymorphism. Modified from Aittoma ki et al. (19).

Direct sequencing
To confirm the above findings, the PCR products were sequenced directly using a Sequenase 2.0 kit (U.S. Biochemical Corp., Cleveland, OH), [35S]deoxy-ATP, 15 pmol primer, and 500 ng of the PCR products for each sample.

Amplification of genomic DNA

PCR was used to amplify five overlapping amplicons covering exon 10 of the FSH receptor gene. The primer sequences are shown in Table 1, and the positions of the amplicons are shown schematically in Fig. 1. The amplicons ranged from 226 275 bp in length. The PCR reaction mixture consisted of 100 ng DNA extracted from frozen tissue or 5 L tissue section lysate, 30 pmol of each primer, 100 mol/L deoxy-NTPs, 1.8 mmol/L MgCl2, 2.5 U Taq polymerase (Boehringer Mannheim, Mannheim, Germany), and 1 PCR buffer [10 PCR buffer is 100 mmol/L Tris (pH 8.5), 500 mmol/L KCl, and 1% Triton X-100] in a total volume of 100 L. PCR was initiated at 94 C for 5 min, followed by 35 cycles of 94 C for 30 s, 55 C for 40 s, and 72 C for 60 s and a final extension at 72 C for 8 min. In all PCR experiments, reactions containing no DNA were included as negative controls.

Single strand conformational polymorphisms (SSCP)

The SSCP analyses were performed using minor modifications of the method described originally by Orita et al. (18). PCR reactions to be used for the SSCP analysis were performed in the presence of 0.5 Ci [-33P]deoxy-ATP [2000 Ci/mmol; DuPont (Australia), North Sydney, Australia] in a total volume of 50 L. The products were run on standard denaturing (7 mol/L urea) or nondenaturing 6% polyacrylamide sequencing gels at 28, 20, and 10 C with or without 10% glycerol in the gel. Constant temperature was maintained using a Strata Therm Temperature Controller (Stratagene, La Jolla, CA). The conditions of the PCR are described above.

Reverse transcriptase-PCR
RNA was extracted from the three frozen granulosa cell tumors (11), seven mucinous cystadenocarcinomas of the ovary, and normal human colon using the guanidine thiocyanate/cesium chloride method. One microgram of total RNA was reverse transcribed for 90 min at 42 C in a total volume of 20 L using AMV reverse transcriptase (Boehringer Mannheim) with the FSH receptor antisense primer at position 1769 (Table 1). Two microliters of this reaction were added to a PCR reaction as described above using the sense and antisense primers for amplicon 3 (Table 1). Ten microliters of each product were analyzed on ethidium bromide-stained 1% agarose gels. Reactions containing either no input RNA or no reverse transcriptase were included as negative controls.

TABLE 1. Primers used for PCR of exon 10 of the FSH receptor 1 Sense, 5-TCA TGG GGT ACA ACA TCC TCA-3 (1150) Antisense, 5-AAA AGC CAG CAG CAT CAC AGC-3 (1397) 2 Sense, 5-ATC ACA ACT ATG CCA TTG ACT-3 (1360) Antisense, 5-GTA GCT GCT GAT GCC AAA GAT-3 (1587) 3 Sense, 5-CTT TTG CAG CTG CCC TCT TTC-3 (1564) Antisense, 5-TTG GCG ATC CTG GTG TCA CTA-3 (1769) 4 Sense, 5-GCG GAA CCC CAA CAT CGT GTC-3 (1745) Antisense, 5-TGA AGA AAT CTC TGC GAA AGT-3 (1969) 5 Sense, 5-ATT CTG CTG GTT CTG TTT CAC-3 (1899) Antisense, 5-CAC ATT GTG TTT TAG TTT TGG-3 (2153) The primer positions, numbered according to the published sequence (36, 37), are shown in parentheses at the end of each line. The amplicons are numbered 15.

Results

The clinical details for 13 of the tumors analyzed have previously been reported (11). An additional 2 samples were obtained from paraffin blocks. Preoperative inhibin levels were not available for these 2 patients. In addition, DNA was

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obtained from normal human tissue for use as a control. All PCR amplicons obtained were of the expected size, and their identities were verified using direct DNA sequencing. All amplicons were subjected to SSCP analysis under the conditions described above. Several putative differences in the banding patterns were detected, but in none was this confirmed by direct sequencing. The only consistent difference in the pattern observed between samples for SSCP analysis was for the fifth amplicon, where 2 different alleles were clearly seen (Fig. 2). Direct sequencing revealed this to be a previously described (19) polymorphism where a G to A transition results in either a serine or an asparagine residue, respectively, at codon 680 (Fig. 3). We further examined DNA from 7 mucinous cystadenocarcinomas of the ovary and 22 randomly selected normal DNA samples for this polymorphism (Fig. 2). The distribution of the codon 680 alleles is tabulated in Table 2. No clear association of the codon 680 allele with the source of the DNA was observed. In their original description of this polymorphism, Aittoma ki et al. (19) found that neither allele was linked to hereditary hypergonadotropic ovarian failure, the phenotype being examined. In addition, direct sequencing was performed on amplicon 4 from 10 of the granulosa cell tumors, given that this region encompasses a previously reported activating mutation (20) of the FSH receptor and corresponds to a hot spot for activating mutations of the TSH receptor (1316) and has very recently been reported to contain an inactivatFIG. 3. Direct DNA sequencing of representative tumor samples either heterozygous or homozygous for the codon 680 polymorphism. TABLE 2. Distribution of codon 680 alleles
GCT MC N

Asn/Asn Ser/Asn Ser/Ser

4 8 3

6 1

5 9 8

GCT, Granulosa cell tumors; MC, mucinous cystadenocarcinomas; N, normal; Ser, serine; Asn, asparagine.

ing mutation in ovarian sex cord tumors (21). As with the SSCP analysis of this amplicon, direct sequencing failed to identify any nucleotide changes. In addition, we sought to establish that the FSH receptor was indeed expressed in granulosa cell tumors. RNA was able to be extracted only from the three frozen granulosa cell tumors and the seven mucinous cystadenocarcinomas whose DNA was analyzed above. Reverse transcriptase-PCR of the RNA from the granulosa cell tumors clearly confirmed expression of the FSH receptor gene (Fig. 4). Curiously, lower levels of expression than in the granulosa cell tumors were noted in most of the mucinous cystadenocarcinomas.
Discussion
FIG. 2. SSCP analysis of amplicon 5 of the FSH receptor. Analysis of DNA from 15 granulosa cell tumors and 7 mucinous cystadenocarcinomas of the ovary flanked by 2 normal DNA samples. The 33Plabeled PCR products have been run in a denaturing gel containing 10% glycerol at 28 C. The normal samples are both heterozygous for the polymorphism. The positions of the specific conformers for each allele are shown on the left (upper bands, the asparagine-containing allele) and right (lower bands, the serine-containing allele) of the figure.

Ovarian granulosa cell tumors are relatively rare, but exhibit an interesting phenotype with both unrestricted growth and ovarian peptide hormone hypersecretion. Granulosa cells are normally subject to hormonal regulation, particularly by FSH. FSH stimulates granulosa cells to secrete inhibin and Mu llerian inhibiting substance, both of which are members of the transforming growth factor- family of

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FIG. 4. Ethidium bromide-stained gels of reverse transcriptase-PCR products using the primer pair for the FSH receptor amplicon 3. A, The results from three granulosa cell tumors (GCT). Mol wt markers (M) and the amplicon from PCR of DNA (D) are also shown. B, The results from seven ovarian mucinous cystadenocarcinomas with no reverse transcriptase (T) and no RNA (R) negative controls. Colonic RNA (C) and jejunal RNA (J) negative controls are also shown. The FSH receptor band is indicated by an arrow.

dimeric growth factor molecules, as well as stimulates the secretion of estradiol. As part of the mitogenic response, FSH stimulates increased cyclin D2 expression (6). Recent clinical reports suggest that hyperstimulation of this signal transduction pathway during gonadotropin therapy for the treatment of infertility increases the incidence of granulosa cell tumors (22, 23); however, the validity of these studies is uncertain (24, 25). Granulosa cell tumors also arise in transgenic mice, in which the inhibin -subunit gene has been deleted. The pathogenesis of these tumors is unclear; however, the mice have markedly elevated levels of FSH and also activin (26). Up-regulation of the signal transduction pathways initiated by FSH might be postulated to play a role in the pathogenesis of these tumors. Lyons et al. (10) reported the presence of the gip2 oncogene in 3 of 10 ovarian sex cord stromal tumors. The relationship of Gi-2 to FSH-stimulated signal transduction is not clear; indeed, we were unable to confirm the presence of the gip2 oncogene in the granulosa cell tumors used in this current study (11).

In addition to mutations of G proteins, mutations of GPCR play a role in a range of inherited and acquired conditions (7, 8, 17). Inactivating mutations of these receptors have been extensively reported, including an alanine to valine substitution in the extracellular region of the FSH receptor first reported by Aittoma ki et al. (19) in hereditary hypergonadotropic ovarian failure. Activating mutations of GPCR were first identified in artificial constructs, but naturally occurring mutations have subsequently been identified in a range of receptors, including those for chemokines (17), calcium (8), PTH/PTH-related peptide (27), and gonadotropins. Activating mutations of the TSH receptor have been identified in both inherited and sporadic conditions. Inherited mutations of the TSH receptor cause familial nonautoimmune hyperthyroidism (28), whereas somatic mutations occur in both adenomas (13, 14) and well differentiated carcinomas (15, 16) of the thyroid. Activating mutations of the LH receptor have been reported in gonadotropin-independent male-limited precocious puberty (29, 30). A heterozygous A to G change at nucleotide 1700 in the human FSH receptor, causing an aspartate to glycine transition in codon 507, has been recently reported in a subject with pituitary-independent spermatogenesis (20). The substituted residue lies in the third intracytoplasmic loop. The majority of activating mutations of GPCR reported lie in this loop, the sixth transmembrane domain, or, less commonly, the second transmembrane domain. In the present study we sought to address the hypothesis that activation of some component of the FSH-stimulated signal transduction pathway in granulosa cells may contribute to the pathogenesis of granulosa cell tumors of the ovary. We examined the FSH receptor not only because it represents the most proximal component of the pathway, but also because of the reports of activating mutations in the three gonadotropin receptors. Our inability to identify any mutations in exon 10 of the FSH receptor in the DNA from 15 granulosa cell tumors would seem to argue strongly against a major role for FSH receptor mutations in the pathogenesis of these tumors. It does not exclude the possibility that mutations may occur in a subset of cells or a minority or tumors, and it remains possible, although unlikely, that our exhaustive search using SSCP analysis under a range of conditions has missed a specific mutation. It is reassuring that a previously reported polymorphism was clearly identified. Limited direct sequencing was also performed; although this should be 100% sensitive, it may still miss mutations of one allele where a degree of contamination of the tumor by normal tissue, such as stromal elements, dilutes the abnormal allele. Subsequent to the submission of this study, Kotlar et al. (21) reported a similar study in which they examined a smaller region of exon 10 of the FSH receptor gene in 13 ovarian sex cord tumors. They amplified the third intracytoplasmic loop together with parts of the fifth and sixth transmembrane domains. Using direct sequencing of PCR products from archival material amplified by nested PCR, they identified a missense mutation at nucleotide 1777 that converts codon 591 from phenylaline to serine (F5915) in 69% of the sex cord tumors (21). Functional studies suggest that this is an inactivating mutation (21), which runs counter to

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their and our initial hypothesis. We have been unable to identify this mutation in our tumors from the SSCP analysis, by direct sequencing of both strands of this amplicon from 10 of our tumors, or by allele-specific hybridization with the primers described by Kotlar et al. (21). It is difficult to explain the discrepancy between our study and that of Kotlar et al. (21); the methodology is fundamentally similar, except, perhaps, that we did not need to use nested PCR. The main difference, therefore, lies in the tumor populations studied. Our tumor sample represents essentially consecutive granulosa cell tumors identified in Melbourne as part of a larger study of inhibin levels in ovarian tumors (3, 4), whereas Kotlar et al. (21) examined archival material accumulated by referral of specific blocks of tumors over several decades for confirmation of difficult histological diagnosis. They include a large number of juvenile granulosa cell tumors. The mutation was also observed in a curious subgroup of ovarian tumors, small cell carcinomas of the hypercalcemia type. In their discussion, Kotlar et al. (21) note that in two additional, presumably relatively unselected, cases, the mutation was not observed. An exciting conclusion that might be drawn from this dichotomy is that distinct subgroups of sex cord tumors might be defined on the basis of the F591S mutation in the FSH receptor gene. The polymorphism at codon 680 was first recognized as such by Aittoma ki et al. (19). Although the polymorphism would clearly seem to have no correlation to ovarian cancer and to be fairly evenly distributed, it represents a nonconservative substitution. This region of the receptor is not well conserved compared to the TSH or LH receptor, but is reasonably well conserved across species (31). Asparagine is present at this position in seven other species of FSH receptor (31). We know of no studies that examine the significance of this substitution. The cytoplasmic tail is known to be important in G protein coupling (7, 8), and thus changes in this region might alter the strength or specificity of such interactions. A serine at this position also creates a potential phosphorylation site (32). FSH binding and stimulation of adenylyl cyclase activity in granulosa cell tumors have previously been reported (33, 34), and our demonstration of FSH receptor gene expression confirms this finding. The presence of FSH receptor messenger RNA, albeit at low levels, in mucinous cystadenocarcinomas is of interest given that these tumors also secrete inhibin (3). The lack of activating mutations of the FSH receptor in granulosa cell tumors in this study, although in contrast to the situation with the TSH receptor, parallels the findings of studies of adrenal tumors in which activating mutations of the ACTH receptor were not identified (35). In conclusion, it would seem that the FSH receptor, like the gip2 oncogene (11) or the gsp oncogene (10), is unlikely to play a major role in the pathogenesis of granulosa cell tumors of the ovary, although an inactivating mutation may play a role in the pathogenesis of a subset of sex cord tumors (21). The pathogenesis of ovarian sex cord stromal tumors, therefore, remains largely unexplained, and perhaps because of their relative rarity, few studies have specifically examined the molecular genetics of these tumors. The evidence, however, that upregulation of this signaling pathway occurs remains com-

pelling, and future studies will need to examine molecules distal to the FSH receptor but proximal to cyclin D2 up-regulation.
Acknowledgments
We thank Sue Panckridge and Claudette Thiedeman for preparation of the manuscript. and Simon Chu for performing the allele-specific hybridization. We also acknowledge the contribution of our pathologist sto colleagues, Drs. Beatrice Susil, Virginia Bilson, Andrew O r, and James Scurry, for making available the tissue blocks used.

References
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