Maria Alexiadis

The FOXL2 C134W mutation is characteristic of adult granulosa cell tumors of the ovary.

Granulosa cell tumors of the ovary represent ∼5% of malignant ovarian cancers. It has recently been reported that 95–97% of adult granulosa cell tumors carry a unique somatic mutation in the FOXL2 gene. We undertook this study to verify the presence of the FOXL2 Cys134Trp mutation in two geographically independent cohorts of granulosa cell tumors and to examine the expression pattern of FOXL2 in these tumors. A total of 56 tumors with the histological diagnosis of adult granulosa cell tumor from two centers, Melbourne and Helsinki, were examined for the presence of the mutation using direct sequence analysis. Two granulosa cell tumor-derived cell lines, COV434 and KGN, three juvenile granulosa cell tumors and control tissues were also examined. The expression of the FOXL2 gene was determined using quantitative RT-PCR and/or immunohistochemistry. We found that 52 of the 56 adult granulosa cell tumors harbor the mutation, of which three were hemi/homozygous. Of the four cases with wild-type FOXL2 sequence, reappraisal suggests that three may have been misclassified at primary diagnosis. The KGN cells were heterozygous for the mutation, whereas the COV434 cells had a wild-type FOXL2 genotype. The expression levels of FOXL2 were similar across the adult granulosa cell tumors and the normal ovary controls; one mutation-negative granulosa cell tumor had high FOXL2 mRNA levels, whereas the COV434 cells and two of the three juvenile granulosa cell tumors lacked the expression of FOXL2. Our data provide confirmation of the frequent presence of the FOXL2 C134W mutation in adult granulosa cell tumors and demonstrate that the mutation is not associated with altered FOXL2 expression. The mutation analysis may be a useful tool to differentiate particularly between cell-rich diffuse granulosa cell tumors and mitotically active sex cord-stromal tumors. This unique FOXL2 mutation appears to be characteristic of adult granulosa cell tumors.

Nuclear Receptor Profiling of Ovarian Granulosa Cell Tumors

Granulosa cell tumors of the ovary (GCT) represent ~5% of malignant ovarian tumors. The adult form is defined by a mutation in the FOXL2 gene. GCT exhibit many of the features of normal proliferating granulosa cells. We have profiled the expression of the 48 human nuclear receptors (NR) by quantitative RT-PCR in a panel of GCT and in two GCT-derived cell lines, COV434 and KGN. The highest level of expression is seen for COUP-TF2 with abundant expression of PPARγ, SF-1, and TR-α. Estrogen receptor (ER)-β is the most abundant of the steroid receptors with relatively high expression also of AR, ER-α, and PR. The concordance of expression for each NR across the tumors is remarkably high with same discordance between the cell lines and the tumors, particularly the COV434 line. No significant differences were observed with respect to tumor stage for NR expression. These findings provide a full profile of NR expression in GCT which will enable full characterization of their roles and potential as therapeutic targets.

Expression status and mutational analysis of the PTEN and P13K subunit genes in ovarian granulosa cell tumors.

Granulosa cell tumors (GCT) are a unique subset of ovarian tumors which have a molecular phenotype resembling that of follicle stimulating hormone (FSH)-stimulated pre-ovulatory granulosa cells. FSH acts via its receptor to stimulate signaling pathways including the phosphatidylinositol 3-kinase (PI3K)-AKT pathway. Activation of this pathway occurs in solid tumors, including ovarian epithelial tumors, through mutation of the PI3K subunit genes or inactivation of the tumor suppressor, PTEN. Activation of this pathway would be predicted to be tumorigenic in granulosa cells. Expression of the 2 PI3K subunit genes, PIK3CA, which encodes the catalytic subunit, and PIK3R1, which encodes the regulatory subunit, together with the PTEN gene was determined in a panel of GCT, 2 human GCT-derived cell lines, COV434 and KGN, and normal ovary. Direct sequence analysis was used to screen for mutations. Expression of all 3genes was observed in the GCT without evidence of overexpression for the PI3K subunit genes or loss of expression for PTEN. Sequence analysis of amplicons spanning exons 9and 20, in which greater than 75% of mutations occur in the PIK3CA gene did not identify any missense mutations. Similarly, the previously reported deletions in exons 12 and 13 of the PIK3R1 were not found in the GCT. Three amplicons spanning the entire coding sequence of the PTEN gene were sequenced; neither deletions nor mutations were identified. These findings suggest that activation of PI3K signaling through PI3K/PTEN mutation or altered expression, in contrast to many other types of solid tumor, is not associated with GCT.

Seladin‐1/DHCR24 expression in normal ovary, ovarian epithelial and granulosa tumours

Objective  The human DIMINUTO/DWARF1 homolog seladin‐1/DHCR24 has been recently reported to be up‐regulated in adrenocortical adenomas. Seladin‐1 expression has been reported in the normal ovary. Granulosa cell tumours of the ovary (GCT) as with adrenocortical adenomas arise from a steroidogenic tissue, respond to pituitary hormone stimulation and synthesize steroid hormones.

An immunohistochemical and molecular analysis of problematic and unclassified ovarian sex cord–stromal tumors

Most ovarian sex cord-stromal tumors (SCSTs) can be categorized on the basis of conventional histology, but approximately 10% of cases are unclassified because they present indeterminate or overlapping morphologic features. Immunohistochemical and molecular studies of unclassified ovarian SCST are very limited, but recently, it has been demonstrated that 2 major subgroups of SCST, adult-type granulosa cell tumor and Sertoli-Leydig cell tumor, are characterized by somatic mutations in FOXL2 and DICER1, respectively. In this study, 12 diagnostically problematic ovarian SCST, including 9 unclassified tumors, were investigated for FOXL2 and DICER1 mutations and for immunohistochemical expression of calretinin, CD56, CD99, estrogen receptor α, estrogen receptor β, FOXL2, inhibin, progesterone receptor, and steroidogenic factor-1. Four of 11 tumors with satisfactory analysis showed a FOXL2 mutation; 3 of these cases were reported initially as unclassified SCST and 1 as Sertoli-Leydig cell tumor. Conversely, 3 cases with an original diagnosis of granulosa cell tumor were FOXL2 mutation-negative, and none of 7 tumors with satisfactory analysis demonstrated a DICER1 mutation. All tumors expressed at least 4 of the immunomarkers examined, although staining was often focal and there was no consistent correlation with tumor morphology. In conclusion, molecular analysis is useful in the assessment of diagnostically challenging ovarian SCST. The absence of FOXL2 and DICER1 mutations in most unclassified SCST suggests that these could represent a distinct tumor subgroup with different molecular pathogenesis. Immunohistochemical profiles overlap with those of better categorized SCST, but staining may be focal or negative emphasizing the requirement for antibody panels in diagnostic assessment.

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.

Nuclear receptor expression in human differentiated thyroid tumors

BACKGROUND Nuclear receptors (NRs) play a key role in endocrine signaling and metabolism and are important therapeutic targets in a number of hormone-dependent malignancies. Studies on the role of NRs in thyroid cancer are limited. OBJECTIVE The objective of the study was to examine systematically the expression of the 48 human NRs in a series of benign and malignant thyroid tissues. Within the papillary carcinoma cohort, we sought to determine if NR expression differed significantly by BRAF mutation status. PATIENTS AND METHODS RNA was isolated from multinodular goiter (MNG; n=6), papillary carcinoma (PTC, n=14), follicular carcinoma (FC; n=5), and Hürthle cell carcinoma (HCC; n=7). The 48 human NRs were profiled in this panel by quantitative real time polymerase chain reaction. Protein expression for selected NRs (Rev-erbα and LXR-β) was examined by immunohistochemistry (IHC) on tissue microarrays comprising benign and malignant thyroid tissues. RESULTS Across all groups of benign and malignant thyroid tissue, there was prominent expression of LXR-β and ROR-γ. Key findings in PTC were marked overexpression of RXR-γ and Rev-erbα compared to MNG. Within the PTC cohort, when BRAF(V600E) tumors were compared with wild type BRAF, there was relative upregulation of RXR-γ and Rev-erbα and downregulation of AR, ERR-γ, and ROR-γ. In FC, EAR-2 was overexpressed, while PPAR-α and PPAR-δ were underexpressed compared to MNG. The NR expression profile of HCC was distinct, characterized by significant downregulation of a wide range of NRs. IHC for Rev-erbα and LXR-β localized protein expression to the tumor cells. Moderate to strong Rev-erbα immunostaining was seen in 22 out of 23 PTC, and, overall, staining was stronger than in the benign group. CONCLUSIONS These results represent the first systematic examination of NR expression in thyroid cancer. Our finding of tumor-specific patterns of NR expression, as well as significant differences in NR expression between BRAF(V600E) and wild type BRAF PTC, provides a basis for further mechanistic studies and highlights potential novel therapeutic targets for this malignancy.

Mutation profile of differentiated thyroid tumours in an Australian urban population.

The majority of differentiated thyroid cancers are characterised by one of several point mutations or gene rearrangements. Limited data are available on the prevalence and clinical correlations of these mutations in the Australian population.

Proteasome inhibition by bortezomib decreases proliferation and increases apoptosis in ovarian granulosa cell tumors.

Granulosa cell tumors of the ovary represent ∼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 (NFκB)-signaling pathway. The proteasomal inhibitor, bortezomib, has a complex mode of action which includes inhibition of NFκB 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 NFκB 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.

Molecular characterization of sarcomatous change in a granulosa cell tumor

Sarcomatous transformation of a granulosa cell tumor (GCT) is a rare event. We describe the development of a rapidly progressive sarcomatous change in a woman who had initially presented with a classical GCT. A first recurrence occurred 23 months after the initial diagnosis when she was treated with external beam radiotherapy to her pelvis. A second recurrence 76 months following her initial surgery was consistent with a GCT. At 92 months, she presented with a further recurrence, outside of the radiotherapy field. This last recurrence had a different histologic appearance with features of sarcomatous change. Molecular analysis, using both reverse transcription–polymerase chain reaction and complementary DNA microarrays, has been used to analyze tissue obtained before and after the observed change in the tumor. The data show that GCT-specific genes, such as inhibin α, estrogen receptor, and follicle-stimulating hormone receptor, have been downregulated in the sarcomatous change. Significant upregulation of genes associated with an inflammatory response was also noted in the sarcoma, and this was consistent with the presence of a marked inflammatory infiltrate seen on histopathology. This study represents the novel application of microarray technology and demonstrates the unexpected finding of expression of the fibroblast activation protein gene in normal ovary. Although tumors such as this may be targets for the novel fibroblast activating protein–directed chemotherapeutic monoclonal antibody sibrotuzumab, the finding of expression in the normal ovary suggests the need for caution