Pathogenesis Of Granulosa Cell Tumors Research Articles

Transcriptomic Profiling of Gene Expression Associated with Granulosa Cell Tumor Development in a Mouse Model.

Simple SummaryOvarian granulosa cell tumors are rare ovarian tumors, with poorly understood etiology. The aim of this study is to define the molecular changes in ovarian granulosa cell tumors. We used a granulosa cell tumor mouse model that contains dysregulated transforming growth factor β signaling. Using RNA-sequencing technology, we identified differentially expressed genes between tumor tissues and normal controls. In addition, comparative analyses have been performed to reveal common genes altered in ovarian granulosa cell tumors between the mouse and the human. This study has revealed the molecular signature of ovarian granulosa cell tumors in a mouse model. The results are potentially important to understand cell signaling events that regulate the development of a class of rare ovarian tumors.Ovarian granulosa cell tumors (GCTs) are rare sex cord-stromal tumors, accounting for ~5% ovarian tumors. The etiology of GCTs remains poorly defined. Genetically engineered mouse models are potentially valuable for understanding the pathogenesis of GCTs. Mice harboring constitutively active TGFβ signaling (TGFBR1-CA) develop ovarian GCTs that phenocopy several hormonal and molecular characteristics of human GCTs. To determine molecular alterations in the ovary upon TGFβ signaling activation, we performed transcriptomic profiling of gene expression associated with GCT development using ovaries from 1-month-old TGFBR1-CA mice and age-matched controls. RNA-sequencing and bioinformatics analysis coupled with the validation of select target genes revealed dysregulations of multiple cellular events and signaling molecules/pathways. The differentially expressed genes are enriched not only for known GCT-related pathways and tumorigenic events but also for signaling events potentially mediated by neuroactive ligand-receptor interaction, relaxin signaling, insulin signaling, and complements in TGFBR1-CA ovaries. Additionally, a comparative analysis of our data in mice with genes dysregulated in human GCTs or granulosa cells overexpressing a mutant FOXL2, the genetic hallmark of adult GCTs, identified some common genes altered in both conditions. In summary, this study has revealed the molecular signature of ovarian GCTs in a mouse model that harbors the constitutive activation of TGFBR1. The findings may be further exploited to understand the pathogenesis of a class of poorly defined ovarian tumors.

Expression and mutation analyses of Fas, FLIP and Bcl-2 in granulosa cell tumor of ovary.

Mounting evidence indicates that evasion of apoptosis contributes to tumor pathogenesis. Although both Fas and Bcl-2 are crucial in apoptosis of normal ovarian cells, their roles in ovarian tumors, especially stromal tumors, are largely unknown. The aim of this study was to explore mutation of the Fas gene and expression of the apoptosis-related proteins Fas, FLICE-like inhibitory protein (FLIP) and Bcl-2 in granulosa cell tumor (GCT) of the ovary. We analyzed the expression of Fas, FLIP and Bcl-2 in 20 GCT tissues by immunohistochemistry. We also analyzed somatic mutations of the Fas gene in the same GCT tissues by polymerase chain reaction and single-strand conformation polymorphism assay. Expression of Fas was evident in 12 GCTs (60%), but the remaining 8 GCTs showed no or markedly decreased Fas immunostaining. Expression of FLIP was identified in 30% of the GCT samples and expression of Bcl-2 in 75%. All GCTs with positive Fas expression (n = 12) showed either FLIP or Bcl-2 expression. The GCTs were found to carry no somatic Fas mutations. Our data show that alterations of the apoptosis-related proteins Fas, FLIP and Bcl-2 are common in GCT, and suggest that expression of FLIP and Bcl-2 and loss of Fas expression might play role in the pathogenesis of GCT, possibly by inhibiting apoptosis.

SMAD3 Activation: A Converging Point of Dysregulated TGF-Beta Superfamily Signaling and Genetic Aberrations in Granulosa Cell Tumor Development?

Ovarian granulosa cell tumors (GCTs) are rare gynecologic tumors in women. Due to the rarity and limited research efforts invested, the etiology of GCTs remains poorly defined. A landmark study has discovered the mutation of forkhead box L2 (FOXL2) as a genetic hallmark of adult GCTs in the human. However, our understanding of the role of cell signaling in GCT development is far from complete. Increasing lines of evidence highlight the importance of TGF-beta (TGFB) superfamily signaling in the pathogenesis of GCTs. This review draws on findings using genetically modified mouse models and human patient specimens and cell lines to reveal SMAD3 activation as a potentially key converging point of dysregulated TGFB superfamily signaling and genetic aberrations in GCT development. It is anticipated that deciphering the role of TGFB superfamily signaling cascades in ovarian tumorigenesis will help develop new therapeutic approaches for GCTs by targeting core signaling elements essential for tumor initiation, growth, and progression.

Expression of H1.5 and PLZF in granulosa cell tumors and normal ovarian tissues: a short report.

Ovarian granulosa cell tumors (GCTs) typically exhibit an excellent prognosis, but their recurrences are associated with high mortality rates. In the past, immunohistochemistry (IHC)-based approaches have been used to facilitate the distinction between GCTs and other, more frequently occurring, primary or metastatic tumors. The purpose of this study was to assess the added value of H1.5 and PLZF protein expression in the correct delineation of GCTs. Consecutive 5-μm thick sections from routinely fixed and paraffin embedded tissues from 30 GCTs and 33 benign ovaries were processed for IHC using anti-PLZF and anti-H1.5 monoclonal antibodies. The respective protein staining intensities and distributions were quantified into reported scores for all tissue samples. Student's t-test and Fisher's exact test were used to compare the mean scores for each group. A p-value of <0.05 was considered statistically significant. Also, both the sensitivity and the specificity of the two antibodies were evaluated. A statistically significant difference in the expression of H1.5 between the GCT and normal ovary groups was observed (p < 0.0001). Normal ovarian tissues were found to strongly express H1.5, whereas GCTs were found to weakly express this protein. In contrast, PLZ expression was not found to be significantly different between both study groups. From our results we conclude that H1.5 is down-regulated in GCTs compared to normal ovarian tissues. Additional investigations on larger and more heterogeneous study populations, and on the molecular mechanism (s) underlying down-regulation of the H1.5 protein, may further substantiate the use of H1.5 as a diagnostic/prognostic marker and, in addition, provide insight into the pathogenesis of GCTs.

YAP regulates cell proliferation, migration, and steroidogenesis in adult granulosa cell tumors.

The Hippo signaling pathway has been implicated as a conserved regulator of organ size in both Drosophila and mammals. Yes-associated protein (YAP), the central component of the Hippo signaling cascade, functions as an oncogene in several malignancies. Ovarian granulosa cell tumors (GCT) are characterized by enlargement of the ovary, excess production of estrogen, a high frequency of recurrence, and the potential for malignancy and metastasis. Whether the Hippo pathway plays a role in the pathogenesis of GCT is unknown. This study was conducted to examine the expression of YAP in human adult GCTs and to determine the role of YAP in the proliferation and steroidogenesis of GCT cells. Compared with age-matched normal human ovaries, GCT tissues exhibited higher levels of YAP expression. YAP protein was predominantly expressed in the nucleus of tumor cells, whereas the non-tumor ovarian stromal cells expressed very low levels of YAP. YAP was also expressed in cultured primary human granulosa cells and in KGN and COV434 GCT cell lines. siRNA-mediated knockdown of YAP in KGN cells resulted in a significant reduction in cell proliferation (P<0.001). Conversely, overexpression of wild type YAP or a constitutively active YAP (YAP1) mutant resulted in a significant increase in KGN cell proliferation and migration. Moreover, YAP knockdown reduced FSH-induced aromatase (CYP19A1) protein expression and estrogen production in KGN cells. These results demonstrate that YAP plays an important role in the regulation of GCT cell proliferation, migration, and steroidogenesis. Targeting the Hippo/YAP pathway may provide a novel therapeutic approach for GCT.

Expression and Mutation Analyses of Fas, FLIP and Bcl-2 in Granulosa Cell Tumor of Ovary

Aims and background Mounting evidence indicates that evasion of apoptosis contributes to tumor pathogenesis. Although both Fas and Bcl-2 are crucial in apoptosis of normal ovarian cells, their roles in ovarian tumors, especially stromal tumors, are largely unknown. The aim of this study was to explore mutation of the Fas gene and expression of the apoptosis-related proteins Fas, FLICE-like inhibitory protein (FLIP) and Bcl-2 in granulosa cell tumor (GCT) of the ovary. Methods We analyzed the expression of Fas, FLIP and Bcl-2 in 20 GCT tissues by immunohistochemistry. We also analyzed somatic mutations of the Fas gene in the same GCT tissues by polymerase chain reaction and single-strand conformation polymorphism assay. Results Expression of Fas was evident in 12 GCTs (60%), but the remaining 8 GCTs showed no or markedly decreased Fas immunostaining. Expression of FLIP was identified in 30% of the GCT samples and expression of Bcl-2 in 75%. All GCTs with positive Fas expression (n = 12) showed either FLIP or Bcl-2 expression. The GCTs were found to carry no somatic Fas mutations. Conclusions Our data show that alterations of the apoptosis-related proteins Fas, FLIP and Bcl-2 are common in GCT, and suggest that expression of FLIP and Bcl-2 and loss of Fas expression might play role in the pathogenesis of GCT, possibly by inhibiting apoptosis.

Pharmacological targeting of mammalian target of rapamycin inhibits ovarian granulosa cell tumor growth

Few targeted therapies have been developed for ovarian granulosa cell tumor (GCT), even though it represents 5% of all malignant ovarian tumors in women. As misregulation of PI3K/AKT signaling has been implicated in GCT development, we hypothesized that the AKT signaling effector mammalian target of rapamycin (mTOR) may play a role in the pathogenesis of GCT and could represent a therapeutic target. Analyses of human GCT samples showed an increase in protein levels of mTOR and its downstream effectors RPS6KB1, RPS6, eIF4B and PPARG relative to normal granulosa cells, suggestive of an increase in mTOR pathway activity and increased translational activity and/or protein stability. We next sought to evaluate mTOR as a GCT therapeutic target using the Pten (tm1Hwu/tmiHwu);Ctnnb1 (tm1Mmt/+);Amhr2 (tm3(cre)Bhr/+) (PCA) mouse model, in which mTOR, RPS6KB1, eIF4B and PPARG are upregulated in tumor cells in a manner similar to human GCT. Treatment of PCA mice with the mTOR-specific inhibitor everolimus reduced tumor growth rate (1.5-fold; P < 0.05) and also reduced total tumor burden (4.7-fold; P < 0.05) and increased survival rate (78 versus 44% in the vehicle group) in a PCA surgical model of GCT peritoneal carcinomatosis. Everolimus decreased tumor cell proliferation and tumor cell volume relative to controls (P < 0.05), whereas apoptosis was unaffected. Phosphorylation of RPS6KB1 and RPS6 were decreased (P < 0.05) by everolimus, but RPS6KB1, RPS6, eIF4B and PPARG expressions were not affected. These results suggest that mTOR is a valid and clinically useful pharmacological target for the treatment of GCT, although its inhibition does not reverse all consequences of aberrant PI3K/AKT signaling in the PCA model.

Abstract A37: Differential response of LH and hCG on ovarian granulosa cells

Abstract LH hyperstimulation has been linked to conditions like PCOD and granulosa cell tumor. Chapekar had established a model of ovarian tumorigenesis by bilateral ovariectomy and splenic transplantation of ovaries, resulting in sustained LH levels because of absence of feedback inhibition. An in vitro model of the same phenomenon has resulted in goat ovarian granulosa cell line after exposure of primary granulosa cells to LH. In this study, we have exposed primary ovarian granulosa cells to either hCG or LH in a sustained manner (4 weeks). We observed a marked difference in the morphology and growth pattern between the LH treated, hCG treated and untreated cells. LH treated cells were transparent, spindle shaped with elongated cell extensions. hCG treated cells were distinctly polygonal with shorter cell extensions, while untreated cells were smaller with fewer extensions and lesser in number as compared to the hormone treated cells. Functional characterization of the granulosa cells was done by estimation of progesterone in supernatant by ELISA. Levels of progesterone were similar in all three subsets in primary culture. The untreated and hCG exposed cells showed markedly reduced cell proliferation after the fourth week of exposure while LH treated cells were proliferating rapidly even after 4 weeks. There were marked differences in downstream signaling molecule cAMP after short term (5 days) and long term stimulation (two weeks) with LH and hCG. Intacellular cAMP was assessed by ELISA. On prolonged exposure to LH and hCG, level of cAMP was significantly higher in hCG treated cultures as compared to LH treated cultures. Elevated cAMP levels on hCG stimulation is known to promote apoptosis in cells. This in vitro model system is suitable to study the effect of sustained hormonal stimulation on granulosa cells and can help in understanding the role of LH and hCG in etiology and pathogenesis of granulosa cell tumor which constitutes 1–2% of ovarian tumors. Citation Information: Cancer Prev Res 2010;3(1 Suppl):A37.

Alterations of the pituitary-ovarian axis in dogs with a functional granulosa cell tumor

Information on the pituitary-ovarian axis in dogs with a granulosa cell tumor (GCT) is lacking. Therefore, we investigated the plasma concentrations of luteinizing hormone (LH) and estradiol before and after gonadotropin-releasing hormone (GnRH) administration in seven bitches with a functional GCT (GCT-total), of which three were intact (GCT-intact) and four had remnant ovarian tissue (GCT-ROT). The results of the GnRH stimulation test were compared with those in six anestrous and six ovariectomized bitches. The most noteworthy results were as follows. The basal plasma LH concentrations of the GCT-ROT bitches were higher (P < 0.05) than those of the anestrous bitches. The increment in the plasma LH concentration after GnRH administration in the GCT-total bitches was lower (P < 0.001) than the increments in both the anestrous and ovariectomized bitches. The basal plasma estradiol concentrations in the GCT-total bitches were higher (P < 0.001) than those in the anestrous and ovariectomized bitches. In conclusion, the pituitary-ovarian axis is affected in bitches with a functional GCT and is characterized by relatively high plasma LH concentrations in GCT-ROT bitches and a subnormal LH response to GnRH stimulation in all GCT bitches compared with those in anestrous and ovariectomized bitches. The relatively high proportion of dogs with remnant ovarian tissue among the GCT bitches suggests a pathogenetic role for elevated gonadotropin secretion in the pathogenesis of GCT.

Pathogenesis of Granulosa-Cell Tumors of the Ovaries

The histopathologic diagnosis of granulosa-cell tumors (GCTs) — neoplasms that constitute <5% of ovarian cancers — is difficult even with

Proteasome Inhibition by Bortezomib Decreases Proliferation and Increases Apoptosis in Ovarian Granulosa Cell Tumors

Granulosa cell tumors of the ovary represent approximately 5% of malignant ovarian tumors. The molecular pathogenesis of granulosa cell tumors is not known but 2 human granulosa cell tumor-derived cell lines, COV434 and KGN, exhibit constitutive activation of the nuclear factor kappa-B (NFkappaB)-signaling pathway. The proteasomal inhibitor, bortezomib, has a complex mode of action which includes inhibition of NFkappaB signaling. We examined the effect of bortezomib on the COV434 and KGN cells. The COV434 and KGN cells both showed a dose-dependent inhibition of cell proliferation and viability in response to bortezomib together with an increase in apoptosis. This was achieved at concentrations within the range seen for clinically responsive tumors. The NFkappaB constitutive activity was not however decreased. Genes were identified that were regulated in both lines in response to bortezomib. This study suggests that advanced granulosa cell tumors, as represented by the cell lines, may respond to bortezomib either alone or in combination with other agents.

Loss of Betaglycan Contributes to the Malignant Properties of Human Granulosa Tumor Cells

Betaglycan is a type III TGFbeta receptor that modulates cellular sensitivity to inhibins and TGFbeta. Previous studies have suggested that betaglycan acts as a tumor suppressor in certain human epithelial cancers. However, the roles of betaglycan in ovarian granulosa cell tumors (GCTs) are poorly understood. The objective of this study was to determine whether human GCTs exhibit betaglycan expression and, if so, what impact this receptor has on tumor biology. Real-time PCR was used to quantify betaglycan transcripts in human GCTs (n = 17) and normal premenopausal ovaries (n = 11). This analysis established that GCTs exhibited a significant 2-fold lower mean betaglycan mRNA level as compared with the normal ovary (P < 0.05). Similarly, two human GCT cell lines, KGN and COV434, exhibited low betaglycan expression and poor responsiveness to TGFbeta and inhibin A in luciferase reporter assays, which was restored by stable transfection of wild-type betaglycan. Betaglycan significantly increased the adhesion of COV434 (P < 0.05) and KGN (P < 0.0001) cells, decreased cellular invasion through Matrigel, and inhibited wound healing. Expression of mutant forms of betaglycan that are defective in TGFbeta and/or inhibin binding in each GCT cell line revealed that the inhibitory effects of betaglycan on wound healing were most strongly linked to the inhibin-binding region of betaglycan. Furthermore, knockdown of INHA mRNA expression abrogated the betaglycan-mediated inhibition of wound healing and invasion, whereas both INHA silencing and TGFbeta neutralization abolished the betaglycan-mediated increase in adhesion to substrate. These data suggest that loss of betaglycan contributes to the pathogenesis of GCTs.

Insulin-like growth factor, insulin-like growth factor–binding protein-4, and pregnancy-associated plasma protein-A gene expression in human granulosa cell tumors

The insulin-like growth factor (IGF) system plays an important role in folliculogenesis. It is also thought to contribute to the pathogenesis of many cancers, including those of the ovarian epithelium. In the human follicle, the predominant IGF is IGF-II and its actions are modulated by insulin-like growth factor-binding protein-4 (IGFBP-4), the IGFBP-4 protease, and the pregnancy-associated plasma protein-A (PAPP-A). These peptide components are synthesized by the granulosa cells of the developing follicle. The aim of this study was to characterize the expression of these components of the IGF system in granulosa cell tumors (GCT) of the ovary. IGF-I, IGF-II, IGFBP-4, and PAPP-A gene expression was determined in a panel of GCT and compared to the levels in normal ovary and in epithelial ovarian tumors. Although both the IGF-I and IGF-II genes were expressed in the GCT, the levels were lower than in the other tissue groups. IGFBP-4 expression was also low in the GCT, whereas PAPP-A gene expression was highest in the GCT. These findings were unexpected given the prominent role this signaling system plays in normal granulosa cells. In conclusion, these observations suggest that the IGF system may have a limited role in the pathogenesis of GCT with PAPP-A subserving a function other than IGFBP-4 proteolysis.

Expression status and mutational analysis of the ras and B-raf genes in ovarian granulosa cell and epithelial tumors

Objectives. The molecular pathogenesis of granulosa cell tumors of the ovary (GCT) is not understood. Activating mutations in the K-, N-, and H-ras protooncogenes have been identified in a wide range of human cancers, including ovarian epithelial tumors. Furthermore, an apparent association has recently been reported between the presence of ras and B-raf mutations in the same cancer types. Activation of the ras/raf pathway would be predicted to be tumorigenic in granulosa cells. Methods. Gene expression patterns of the three ras and B-raf genes were determined in a panel of GCT and ovarian epithelial tumors, and in normal premenopausal ovaries. Expression was determined by RT-PCR using gene-specific primers combined with Southern blot analysis of the PCR products using gene-specific 32P-labeled probes. Direct sequence analysis was used to screen for known activating mutations. Results. Widespread expression of the four genes was observed in all tumor types examined. Compared to the normal ovaries, none of the genes was expressed at significantly higher levels in any of the tumor types examined. A heterozygous point mutation in codon 12 of the K-ras gene was found in five of the 10 mucinous tumors. No B-raf mutations were detected in the mucinous tumors. No mutations were detected in any of the genes in the cohort of GCT. Conclusion. These results suggest that neither overexpression nor activating mutations of the ras or B-raf genes are associated with the development of GCT.

Transrepression of estrogen receptor beta signaling by nuclear factor-kappab in ovarian granulosa cells.

Estrogen receptor (ER) beta is the predominant ER in granulosa cells of the ovary. ERbeta is expressed at high levels in granulosa cell tumors (GCT) and in the human GCT-derived cell lines, COV434 and KGN. To gain insight into ERbeta function in granulosa cells and in GCT, we have used the COV434 and KGN cell lines. Although the cells bind estradiol (E2), transcriptional activation of a transfected estrogen-responsive reporter vector construct (ERE2-luc) by E2 was not observed. Transactivation was also not observed with cotransfected ERalpha or beta. This transcriptional resistance is specific to steroid receptor transactivation; reporter plasmids that are activated by the transcription factors activator protein 1 (AP-1) and nuclear factor kappaB (NF-kappaB) demonstrate both constitutive and inducible transactivation. AP-1 and NF-kappaB are known to cause transrepression of both ERalpha- and glucocorticoid receptor-mediated transcription. We therefore examined the possibility that activation of these pathways was responsible for the lack of a response to estrogen by using inhibitors of AP-1 or NF-kappaB. The AP-1 inhibitors alone had no effect, whereas inhibition of NF-kappaB signaling allowed a 3- to 4-fold E2-mediated induction of ERE2-luc. This response was both ligand and ER dependent. Repression of ERbeta signaling by NF-kappaB has not previously been reported. Recent evidence suggests that ERbeta may function to promote differentiation. The inhibition of ERbeta in combination with the antiapoptotic properties of NF-kappaB may therefore contribute to pathogenesis of GCT.

Differential expression of GRK isoforms in nonmalignant and malignant human granulosa cells.

Granulosa cell tumors are serious ovarian neoplasms that can occur in women of all ages. While there have been numerous attempts to understand the cause of these malignancies, the pathogenesis of granulosa cell tumors (GCTs) still remains largely unknown. G-protein coupled receptor kinases (GRKs) are important regulators of signal transduction through the process of receptor desensitization and internalization. Receptors that are regulated by GRKs are members of the large family of seven-transmembrane receptors and include the follicle stimulating hormone receptor (FSHR). In granulosa cells, the FSH signaling system is responsible for cell proliferation, differentiation, and steroidogenesis. In the studies presented, we examined GRK mRNA and protein expression in nonmalignant human granulosa cells, in KGN cells, a human GCT cell line, and in a panel of human GCT samples. The KGN tumor cells express significantly less GRK4 alpha/beta protein and higher levels of GRK2 and GRK4 gamma/delta protein as compared to nonmalignant human granulosa cells. In human GCT samples, GRK4 alpha/beta protein was detected in 3 of the 13 tumor samples, whereas gamma/delta proteins expression was detected in all samples. These findings suggest that GRK protein expression is altered in GCTs and may be involved in the pathogenesis of these tumors.

No Evidence of a Role for Mutations or Polymorphisms of the Follicle-Stimulating Hormone Receptor in Ovarian Granulosa Cell Tumors1

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 tumor-specific 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.

Absence of the previously reported G protein oncogene (gip2) in ovarian granulosa cell tumors.

The molecular pathogenesis of granulosa cell tumors of the ovary is not understood, although recent studies have shown that authentic inhibin secretion by these tumors may be used as a tumor marker. G proteins are heterotrimeric membrane-based polypeptides that mediate signal transduction. Activating mutations of the alpha-subunit have been reported in several tumors in which the resulting constitutively activated signal transduction pathway may be involved in tumorigenesis. Activating mutations of the G protein, G alpha I-2, have been reported to be present in 30% of ovarian sex cord tumors and in adrenocortical tumors; this activated G alpha I-2 has been designated the gip2 oncogene. We sought to explore the frequency of this oncogene in granulosa cell tumors, the most common of the sex cord tumors. Genomic DNA was obtained from either fresh-frozen tumor tissue or paraffin-embedded sections. Using both allele-specific oligonucleotide hybridization and direct sequencing of a PCR-amplified region of the G alpha I-2 gene, we were unable to confirm the presence of the previously reported mutation in any of the 13 tumors examined. The gip2 oncogene does not appear to be present at high frequency in ovarian granulosa cell tumors.