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The FOXL2 C134W Mutation Is Characteristic of Adult Granulosa Cell Tumors of the Ovary

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The FOXL2 C134W Mutation Is Characteristic of Adult Granulosa Cell Tumors of the Ovary Modern Pathology (2010) 23, 1477–1485 & 2010 USCAP, Inc. All rights reserved 0893-3952/10 $32.00 1477 The FOXL2 C134W mutation is characteristic of adult granulosa cell tumors of the ovary Stacey Jamieson1,2, Ralf Butzow3,4, Noora Andersson3,6, Maria Alexiadis1, Leila Unkila-Kallio3,5, Markku Heikinheimo3,6, Peter J Fuller1,2 and Mikko Anttonen3,5 1Prince Henry’s Institute of Medical Research, Clayton, Victoria, Australia; 2Department of Medicine (MMC), Monash University, Clayton, Victoria, Australia; 3Women’s Health Research Program, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland; 4Department of Pathology, University of Helsinki, Helsinki, Finland; 5Department of Obstetrics and Gynecology, University of Helsinki, Helsinki, Finland and 6Children’s Hospital, University of Helsinki, Helsinki, Finland Granulosa cell tumors of the ovary represent B5% 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. Modern Pathology (2010) 23, 1477–1485; doi:10.1038/modpathol.2010.145; published online 6 August 2010 Keywords: FOXL2; granulosa cell tumor; ovarian cancer Granulosa cell tumors of the ovary represent B5% commonly presents during the perimenopausal or of malignant ovarian cancers.1,2 Two distinct sub- early postmenopausal period.3 Granulosa cell types have been described based on clinical pre- tumors are characterized by slow, indolent growth sentation and histological characteristics: the and a tendency to late recurrence, with intervals in juvenile and the adult form. The adult form excess of 10 or 20 years not being uncommon. accounts for 95% of granulosa cell tumors and most Although the prognosis is often favorable, those that recur and/or are at an advanced stage carry a poor prognosis with B80% of these patients succumbing Correspondence: Dr M Anttonen, MD, PhD, Women’s Health 3 Research Program and Department of Obstetrics and Gynecology, to their disease. Biomedicum 2U, University of Helsinki, PO Box 20, 00014 Although granulosa cell tumors seem to represent Helsinki, Finland, e-mail: [email protected] or Professor a relatively homogenous group of tumors, a mole- PJ Fuller, MBBS, PhD, FRACP, Prince Henry’s Institute of cular etiology has until recently eluded investiga- Medical Research, PO Box 5152, Clayton, VIC 3168, Australia, tors.4 The molecular changes that give rise to e-mail: [email protected] Received 15 April 2010; revised 13 May 2010; accepted 17 May granulosa cell tumors are likely to involve disruption 2010; published online 6 August 2010 of pathways that function during normal ovarian www.modernpathology.org FOXL2 in adult granulosa cell tumors 1478 S Jamieson et al development to regulate granulosa cell proliferation. PCR conditions used to amplify FOXL2 were those Indeed, granulosa cell tumors exhibit a gene expres- of Shah et al,8 using a VeritiTM 96-well Thermal sion profile consistent with that of normal prolifer- Cycler (Applied Biosystems, Foster City, CA, USA). ating granulosa cells of preantral follicles.5–7 A range In all cases, an amplicon of the predicted size of these genes, as well as genes commonly mutated (182 bp) when compared with the molecular weight in human malignancy, have been examined for standards was obtained. The PCR products were mutations and/or altered expression in granulosa purified using a QIAquick Gel Extraction Kit cell tumors without success.4 This situation abruptly (Qiagen, Hilden, Germany). For each sample, for- changed in 2009 with the report of a somatic ward and reverse sequences were determined mutation in the FOXL2 gene (C402G; Cys134Trp) using the primers described above as sequencing that was present in 97% of adult granulosa cell primers and a BigDyes Terminator Cycle Sequen- tumors.8 The mutation was found in only one cing Ready Reaction Kit, v3.1 (Applied Biosystems), juvenile granulosa cell tumor8 and not found in a according to the manufacturer’s protocol. The wide range of other tumors, both gonadal and analysis was carried out using ABI 3130xl Genetic non-gonadal.9,10 Curiously, diminished and/or loss Analyzer (Applied Biosystems) and assembled of FOXL2 expression has been reported in advanced using Sequencher 3.1.1 software. Sequencing was stage/aggressive juvenile granulosa cell tumors.11 performed by the Gandel Charitable Trust Sequen- In this study, we sought to confirm the presence cing Centre at the Monash Health Translation of this mutation in two geographically independent Precinct, Clayton. cohorts of granulosa cell tumors and to examine the expression pattern of the FOXL2 gene in Quantitative PCR analysis these tumors. Quantitative PCR was performed with the oligonu- cleotide primers described above using an Applied Materials and methods Biosystems 7900HT Fast Real-Time PCR System with all reactions performed in triplicate. Cycling Melbourne conditions comprised a 10 min initial denaturation step at 95 1C, then 40 cycles of denaturing (95 1C for Human tissue samples 30 s), annealing (55 1C for 30 s) and extension (72 1C Ovarian granulosa cell tumors and normal ovarian for 45 s). The b2-microglobulin (b2M) primers have tissue were obtained initially for a study of serum been described previously.15 Yields were converted inhibin levels in ovarian tumors12,13 and subse- to femtograms based on the standard curve for each quently for studies of the molecular pathogenesis PCR product, and the resultant mRNA levels were 5,14–19 of granulosa cell tumors. Normal ovarian tissue normalized to the b2M mRNA level per sample. The was obtained from premenopausal women who had data were calculated from the results of three undergone elective hysterectomy with oophorect- independent experiments. omy for a range of conditions not associated with ovarian malignancy (Supplementary Table 1). RNA from the tissue and the two granulosa cell tumor- Helsinki derived cell lines was extracted using the guanidine thiocyanate/cesium chloride method as described Human tissue samples previously.14 Tissue collection was approved by the The ethical committee of the Helsinki University research and ethics committee of the Monash Central Hospital and the National Supervisory Medical Centre, and all women gave written Authority for Welfare and Health in Finland informed consent before tissue collection. approved this study. A total of 37 adult granulosa cell tumor samples were collected in Helsinki Cell lines University Central Hospital between 1990 and The two human granulosa cell tumor-derived cell 2009 (Supplementary Table 2). Tissue samples were lines, COV434 and KGN, have been previously snap frozen in liquid nitrogen upon the initial described.20 COV434 cells were maintained in intraoperative diagnosis by a pathologist; the corre- DMEM and KGN cells in DMEM/F12 both supple- sponding paraffin-embedded tissue samples were mented with 10% FCS at 37 1C in a 95% air/5% CO2 utilized in immunostaining. The adult granulosa humidified incubator. cell tumor diagnosis was subsequently confirmed and tumor subtype, nuclear atypia rate and mitotic Mutation analysis index evaluated,7 before FOXL2 mutation analysis, Total RNA (1 mg) was reverse-transcribed for 60 min by an experienced pathologist (RB). Tissue samples at 50 1C in a total volume of 20 ml using SuperScript of three ovarian carcinomas (two endometrioid and III reverse transcriptase (Invitrogen, Carlsbad, CA, one serous) were analyzed for comparison. USA). First-strand synthesis was performed using 250 ng random hexamers. The oligonucleotide Mutation analysis primers (sense 50-CACAACCTCAACGAGTGC-30, DNA was extracted from frozen tissue samples with antisense 50-TTGCCGGGCTGGAAGTGCG-30) and a NucleoSpin Tissue kit (Machrey Nagel, Du¨ ren, Modern Pathology (2010) 23, 1477–1485 FOXL2 in adult granulosa cell tumors S Jamieson et al 1479 Germany) according to the manufacturer’s instruc- microscopy by two authors (NA and MAn) as to tions. The oligonucleotide PCR primers (forward the number of positive cells (nuclear positivity 50-CCAGTACATCATCGCGAAGTTCCCG-30, reverse for FOXL2) and intensity of staining: À negative, 50-CTCCGGCCCCGAAGAGCC-30) were those used þ /À weak, þ positive, and þþ highly positive. by Shah et al8 for direct sequencing. For PCR amplification, 50 ng of DNA sample was amplified with 10 pmol of each primer, and Biotools DNA Results polymerase and reaction mix (Biotools, B&M Labs, FOXL2 C143W Mutation Status Spain) in a final volume of 15 ml.
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