Mutational analysis of FOXL2 codon 134 in granulosa cell tumour of ovary and other human cancers

A missense somatic mutation in the FOXL2 gene affecting codon 134 has recently been reported in granulosa cell tumour (GCT) and thecoma of the ovary. Such a recurrent nature of the mutation strongly suggests that the FOXL2 mutation may play an important role in the development of ovarian sex cord‐stromal tumours. The aim of this study was to characterize the FOXL2 mutation in human tumour tissues. We analysed 1353 tumour tissues from various origins, including ovarian tumours and other common cancers, by single‐strand conformation polymorphism analysis. We found the FOXL2 codon 134 missense mutation in 53 of 56 adult GCTs (94.6%) and two of the 16 thecomas (12.5%), but none in other tumours. Histologically, FOXL2 mutation‐negative adult GCT showed that GCT cells were admixed with fibrothecomatous cells, and FOXL2 mutation‐positive thecomas showed that luteinized theca cells were predominant. However, immunostaining of either inhibin α or FOXL2 did not differentiate the FOXL2 mutation status of adult GCTs and thecomas. There was no FOXL1 mutation and no common oncogenic mutation in the adult GCTs and thecomas. Our data indicate that the FOXL2 codon 134 mutation occurs exclusively in GCT and thecoma, and suggest the possibility that the development of most GCTs and a fraction of thecomas may be dependent on this mutation. Our data also suggest that the FOXL2 mutation status, as well as some histological features, may be important in the diagnosis of ovarian sex cord‐stromal tumours. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

The C134W (402 C>G) FOXL2 mutation is absent in ovarian gynandroblastoma: insights into the genesis of an unusual tumour

Oparka R, Cassidy A, Reilly S, Stenhouse A, McCluggage W G & Herrington C S
(2012) Histopathology  60, 838–842
The C134W (402 C>G) FOXL2 mutation is absent in ovarian gynandroblastoma: insights into the genesis of an unusual tumour Aims: Ovarian gynandroblastomas are rare tumours that, by definition, comprise a combination of components resembling both female, typically granulosa cell tumour (GCT), and male, typically Sertoli or Sertoli/Leydig cell tumour (ST/SLT), sex cord/stromal differentiation. The histogenesis of these tumours is unknown and, in view of the very strong association between the C134W (402 C>G) FOXL2 mutation and adult‐type GCT, we analysed a series of gynandroblastomas for this mutation. Methods and results: Both components of each lesion were isolated by laser capture microdissection and the C134W (402 C>G) FOXL2 mutation was analysed by polymerase chain reaction sequencing. No mutation was identified in either the GCT or ST/SLT component of six cases, three of which contained adult‐type GCT. Conclusions: This suggests that, despite their similar morphological appearances, the GCT‐like component of gynandroblastoma has a different molecular basis from conventional adult‐type GCT. This finding underscores a more general principle that morphological similarity does not necessarily indicate molecular identity.     

Practical roles for molecular diagnostic testing in ovarian adult granulosa cell tumour,Sertoli–Leydig cell tumour,microcystic stromal tumour and their mimics

Within the last decade, molecular advances have provided insights into the genetics of several ovarian sex cord–stromal tumours that have otherwise been enigmatic. Chief among these advances are the identification of FOXL2, DICER1 and CTNNB1 mutations in adult granulosa cell tumours, Sertoli–Leydig cell tumours (SLCTs), and microcystic stromal tumours (MCSTs), respectively. As access to molecular diagnostic laboratories continues to become more widely available, the potential roles for tumour mutation testing in the pathological diagnosis of these tumours merit discussion. Furthermore, links to inherited cancer susceptibility syndromes may exist for some women with SLCT (DICER1 syndrome) and MCST [familial adenomatous polyposis (FAP)]. This review will address practical issues in deciding when and how to apply mutation testing in the diagnosis of these three sex cord–stromal tumours. The pathologist's role in recommending referral for formal risk assessment for DICER1 syndrome and FAP will also be discussed.     

Recently characterized molecular events in uncommon gynaecological neoplasms and their clinical importance

The introduction of new sequencing technologies has resulted in the discovery of commonly mutated genes in uncommon cancers, including non‐epithelial ovarian neoplasms and other rare gynaecological tumours, such as cervical embryonal rhabdomyosarcoma. In some of these neoplasms, mutations in certain genes are both frequent and specific enough for the genomic mutations and sometimes their associated protein loss or overexpression to be used as an aid to diagnosis. In this review, we contrast previous gene identification methods with newer ones, and discuss how the new sequencing technologies (collectively referred to as ‘next‐generation sequencing’) have permitted the identification of specific molecular events that characterize several rare gynaecological neoplasms. We highlight the value of using sequencing to complement traditional pathological methods when diagnosing certain tumours, and provide practical advice to pathologists dealing with these neoplasms. We focus on adult granulosa cell tumours (somatic monoallelic mutations in FOXL2), Sertoli–Leydig cell tumours, gynaecological embryonal rhabdomyosarcomas (germline and somatic mutations in DICER1), and small‐cell carcinoma of the ovary, hypercalcaemic type (biallelic mutations in SMARCA4). The new genetic findings provided by next‐generation sequencing in these uncommon neoplasms have brought these disorders back into focus, and point the way towards new diagnostic, preventive and therapeutic avenues.     

RNF43 is a tumour suppressor gene mutated in mucinous tumours of the ovary

Mucinous carcinomas represent a distinct morphological subtype which can arise from several organ sites, including the ovary, and their genetic characteristics are largely under‐described. Exome sequencing of 12 primary mucinous ovarian tumours identified RNF43 as the most frequently somatically mutated novel gene, secondary to KRAS and mutated at a frequency equal to that of TP53 and BRAF. Further screening of RNF43 in a larger cohort of ovarian tumours identified additional mutations, with a total frequency of 2/22 (9%) in mucinous ovarian borderline tumours and 6/29 (21%) in mucinous ovarian carcinomas. Seven mutations were predicted to truncate the protein and one missense mutation was predicted to be deleterious by in silico analysis. Six tumours had allelic imbalance at the RNF43 locus, with loss of the wild‐type allele. The mutation spectrum strongly suggests that RNF43 is an important tumour suppressor gene in mucinous ovarian tumours, similar to its reported role in mucinous pancreatic precancerous cysts. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Aberrant FGF-2, FGF-3, FGF-4 and C-ERB-B2 Gene Copy Number in Human Ovarian,Breast and Endometrial Tumours

Abstract

The important role of oncogene amplification and tumour suppressor gene deletion in human tumours is becoming increasingly apparent. However, extensive screening of human tumours is required before the prognostic significance of such genetic abnormalities can be fully appreciated. The present investigation describes a rapid non-radioactive and largely automated procedure for the analysis of aberrant gene copy number in large numbers of tissue samples of different human rumours. This procedure is based on the sequential use of the polymerase chain reaction (PCR) and high performance ion exchange liquid chromatography (HPIEX). Using this rapid PCR/HPIEX technique, we have identified amplification and deletion of the FGF-2 gene and the FGF-3, FGF-4 and c-erb-B2 oncogenes in human tumours of the breast, ovary and endometrium. Comparison of the data with tumour pathology has revealed possible associations between aberrant gene copy number and tumour type, invasiveness and metastases.     

Disparate genomic characteristics of concurrent endometrial adenocarcinoma and ovarian granulosa cell tumor,revealed by targeted next-generation sequencing

Concurrence of both endometrial adenocarcinoma and ovarian adult granulosa cell tumor (aGCT) is believed to be related to high estrogen milieu, but genomic alterations of the concurrent endometrial adenocarcinoma and aGCT are not known. For this, we analyzed an uterine endometrial adenocarcinoma and an ovarian aGCT in a same patient by a targeted next generation sequencing (NGS). We found a germline mutation in STK11 (p.L113fs). The endometrial adenocarcinoma harbored FGFR2 and TP53 mutations and the aGCT harbored a FOXL2 (p.C134?W) mutation. These germline and somatic mutations have been reported in non-concurrent tumors. These two tumors harbored 20 CNAs but only one CNA was exactly overlapped in the tumors. Our findings indicate that the concurrent endometrial adenocarcinoma and aGCT in this patient might not be genetically related to each other at germline or somatic level and suggest that such concurrence might be originated from non-genetic backgrounds including stimulated estrogen milieu.     

Allelic losses on chromosome arm 10q and mutation of the PTEN (MMAC1) tumour suppressor gene in primary and metastatic malignant melanomas

Malignant melanomas frequently show loss of alleles on the long arm of chromosome 10. The PTEN (MMAC1) gene has been identified as a tumour suppressor gene at 10q23.3 that is mutated in various types of advanced human cancers. We have investigated a series of 40 sporadic melanomas from 37 patients (15 primary cutaneous melanomas and 25 melanoma metastases) for allelic losses on chromosome 10, as well as for deletion and mutation of the PTEN gene. Microsatellite analysis revealed loss of heterozygosity at loci located on 10q in tumours from 15 of 34 patients investigated (44%). Somatic PTEN mutations were identified in melanomas from 4 of 37 patients (11%), all of whom had metastatic disease. In two of these patients, the tumours had additionally lost one PTEN allele, indicating complete loss of wild-type PTEN in the tumour cells. Our findings corroborate that loss of heterozygosity on chromosome 10 is a frequent aberration in malignant melanomas and implicate PTEN as a tumour suppressor gene inactivated by somatic mutation in a fraction of these tumours. Received: 1 September 1999 / Accepted: 22 December 1999     

Arid1a inactivation in an Apc‐ and Pten‐defective mouse ovarian cancer model enhances epithelial differentiation and prolongs survival

Inactivation of the ARID1A tumour suppressor gene is frequent in ovarian endometrioid (OEC) and clear cell (OCCC) carcinomas, often in conjunction with mutations activating the PI3K–AKT and/or canonical Wnt signalling pathways. Prior work has shown that conditional bi‐allelic inactivation of the Apc and Pten tumour suppressor genes in the mouse ovarian surface epithelium (OSE) promotes outgrowth of tumours that reflect the biological behaviour and gene expression profiles of human OECs harbouring comparable Wnt and PI3K–AKT pathway defects, although the mouse tumours are more poorly differentiated than their human tumour counterparts. We found that conditional inactivation of one or both Arid1a alleles in OSE concurrently with Apc and Pten inactivation unexpectedly prolonged the survival of tumour‐bearing mice and promoted striking epithelial differentiation of the cancer cells, resulting in morphological features akin to those in human OECs. Enhanced epithelial differentiation was linked to reduced expression of the mesenchymal markers N‐cadherin and vimentin, and increased expression of the epithelial markers Crb3 and E‐cadherin. Global gene expression profiling showed enrichment for genes associated with mesenchymal–epithelial transition in the Arid1a‐deficient tumours. We also found that an activating (E545K) Pik3ca mutation, unlike Pten inactivation or Pik3ca H1047R mutation, cannot cooperate with Arid1a loss to promote ovarian cancer development in the mouse. Our results indicate that the Arid1a tumour suppressor gene has a key role in regulating OEC differentiation, and paradoxically the mouse cancers with more initiating tumour suppressor gene defects had a less aggressive phenotype than cancers arising from fewer gene alterations. Microarray data have been deposited in NCBI's Gene Expression Omnibus (GSE67695). Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Foxl-2 in gonad development and pathology

The Foxl-2 gene is involved in eyelid and ovary development. Mutations can lead to a shortened protein and malformations such as BPES associated or not to POF. Forkhead point mutation C134W is a marker of adult type granulosa cell tumors only. Foxl-2 dysregulation is also present in DSD and DSD associated tumors such as Gonadoblastoma and gonadoblastoma like intratubular undetermined germ cell neoplasia. A similar spectrum of pathology involvement is also found for WT1 and RET and gives a new insight into the relationship between development, malformations and oncogenesis.     

Mutations in the coding region of the FOXL2 gene are not a major cause of idiopathic premature ovarian failure

Premature ovarian failure (POF) is a heterogeneous disorder whose aetiology is still unknown. Recently, the autosomal FOXL2 gene, highly expressed in the adult ovary, has been correlated with the disorder. FOXL2 mutations, causing a truncation of the FOXL2 protein in the forkhead domain or in the poly-Ala tract lead to blepharophimosis-ptosis-epicanthus-inversus syndrome associated with POF (BPES I). Interestingly, in two out of 70 idiopathic POF patients, a 30 bp deletion (898-927del) and a missense mutation (1009T-->A) were identified. To further evaluate the correlation between POF and FOXL2 mutations, 120 phenotypically normal women affected by POF were analysed by direct sequencing of the FOXL2 coding region. The analysis did not reveal any mutation in the 240 analysed chromosomes, indicating that mutations in the FOXL2 coding region are rarely associated with non-syndromic POF.     

Familial rhabdoid tumour 'avant la lettre'—from pathology review to exome sequencing and back again

Here we provide compelling evidence that next‐generation sequencing will revolutionize diagnostics. We reappraised a case from 1991, published in 1993, describing the unique occurrence of an ovarian immature teratoma arising in a young woman and a clonally distinct intracerebral immature teratoma developing in her daughter. We conducted whole‐exome sequencing on constitutional DNA from the mother and her daughter and identified a previously unreported nonsense mutation (c.3533G>A; p.Trp1178^*) in the chromatin remodelling gene, SMARCA4, that was present in both individuals and was subject to nonsense‐mediated decay. Tumour analysis by Sanger sequencing revealed a somatic SMARCA4 mutation in both the mother (c.2438+1G>T) and her daughter (c.3229C>T; p.Arg1077^*), which are predicted to be truncating. As immature teratomas are classified as germ cell tumours, we performed a comprehensive mutation survey of 106 apparently sporadic germ cell tumours, but did not find any other clearly deleterious SMARCA4 mutations. Recently, inactivating mutations in SMARCA4 have been found in two cases of rhabdoid tumour predisposition syndrome type 2. In the light of these findings, renewed efforts to locate previously unobtainable tumour samples were successfully undertaken. Histopathological and immunohistochemical re‐analysis of the daughter's tumour revealed that it was indeed a rhabdoid tumour (atypical teratoid/rhabdoid tumour). In this context, the original pathology report of the mother's ovarian tumour was re‐interpreted as describing a malignant rhabdoid tumour of the ovary. This report raises the question as to whether molecular genetic analysis should be included in tumour classification, alongside more traditional microscopy‐based methods. The use of new sequencing technologies, particularly when applied to archived samples, will lead to many more 'molecular rediagnoses'. This is the earliest known case of rhabdoid tumour predisposition syndrome type 2 and the first described case with an autosomal dominant pattern of inheritance, only discovered through an exome sequencing project. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Impact of oviductal versus ovarian epithelial cell of origin on ovarian endometrioid carcinoma phenotype in the mouse

Endometrioid carcinoma (EC) is a relatively indolent ovarian carcinoma subtype that is nonetheless deadly if detected late. Existing genetically engineered mouse models (GEMMs) of the disease, based on transformation of the ovarian surface epithelium (OSE), take advantage of known ovarian EC driver gene lesions, but do not fully recapitulate the disease features seen in patients. An EC model in which the Apc and Pten tumour suppressor genes are conditionally deleted in murine OSE yields tumours that are biologically more aggressive and significantly less differentiated than human ECs. Importantly, OSE is not currently thought to be the tissue of origin of most ovarian cancers, including ECs, suggesting that tumour initiation in Müllerian epithelium may produce tumours that more closely resemble their human tumour counterparts. We have developed Ovgp1‐iCreER^T2 mice in which the Ovgp1 promoter controls expression of tamoxifen (TAM)‐regulated Cre recombinase in oviductal epithelium – the murine equivalent of human Fallopian tube epithelium. Ovgp1‐iCreER^T2;Apc^fl/fl;Pten^fl/fl mice treated with TAM or injected with adenovirus expressing Cre into the ovarian bursa uniformly develop oviductal or ovarian ECs, respectively. On the basis of their morphology and global gene expression profiles, the oviduct‐derived tumours more closely resemble human ovarian ECs than do OSE‐derived tumours. Furthermore, mice with oviductal tumours survive much longer than their counterparts with ovarian tumours. The slow progression and late metastasis of oviductal tumours resembles the relatively indolent behaviour characteristic of so‐called Type I ovarian carcinomas in humans, for which EC is a prototype. Our studies demonstrate the utility of Ovgp1‐iCreER^T2 mice for manipulating genes of interest specifically in the oviductal epithelium, and establish that the cell of origin is an important consideration in mouse ovarian cancer GEMMs. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Sequence variants of DLC1 in colorectal and ovarian tumours

Loss of heterozygosity occurs frequently on the short arm of chromosome 8 in many neoplasms, including colorectal and ovarian cancer. Monochromosome transfer experiments into colorectal tumour cell lines have provided functional evidence for a tumour suppressor gene located at 8p22-23. One of the genes from this region that is expressed by our suppressed hybrids is a candidate tumour suppressor gene, DLC1 (deleted in liver cancer), which has homology to rat RhoGAP. We have delineated the structure of the DLC1 gene and used single-stranded conformation polymorphism analysis (SSCP) to look for sequence variants in 126 colorectal and 33 ovarian primary tumours and cell lines. One exonic missense mutation and three intronic insertions/deletions were identified in primary colorectal tumours, as well as many polymorphisms present in germline DNAs. The rarity of exonic missense mutations, and the absence of protein-truncating mutations, indicates that DLC1 is not the target of 8p LOH in colorectal or ovarian tumours. The delineation of the gene structure allows mutation analysis of DLC1 in other tumour types for which it remains a candidate tumour suppressor gene based on its location and homology to rhoGAP.     

The site of inhibin production in ovarian neoplasms

Inhibin, a physiological product of ovarian follicle cells, normally absent in serum of postmenopausal women, is elevated in adult granulosa cell tumours of the ovary. Recently, high serum levels of inhibin were reported in carcinomas and, surprisingly, also in Krukenberg tumours of the ovary. This study attempted to determine the site of inhibin production in primary (111 cases), metastatic (13) and secondary (10) ovarian tumours by using immunohistochemistry. Positive staining in tumour cells was encountered in all cases of sex-cord- stromal cell tumours, adult (13) and juvenile (3) granulosa cell tumours, thecofibromas (10), in a lipid cell tumour (1) and a Sertoli-Leydig cell tumour (1). Primary and secondary tumours not derived from sex-cord stroma revealed no positivity in tumour cells, but in theca-like cells in the surrounding non-neoplastic ovarian stroma. A positive reaction was not observed in non-tumour-bearing ovaries of a control group. The ovarian inhibin of postmenopausal women is derived from activated sex-cord stroma or sex-cord-stromal neoplasms. Therefore, elevated serum inhibin concentrations in women with primary or secondary ovarian neoplasms with other histogenesis seem to be due to an activation of the non-neoplastic ovarian stroma. Inhibin will fail to be a tumour marker in these cases. By contrast, it will be useful in proving sex-cord differentiation by immunohistochemistry and might be used in surveillance of malignant sex-cord derived neoplasms by serum assays.     

Identification of ZDHHC14 as a novel human tumour suppressor gene

Genomic changes affecting tumour suppressor genes are fundamental to cancer. We applied SNP array analysis to a panel of testicular germ cell tumours to search for novel tumour suppressor genes and identified a frequent small deletion on 6q25.3 affecting just one gene, ZDHHC14. The expression of ZDHHC14, a putative protein palmitoyltransferase with unknown cellular function, was decreased at both RNA and protein levels in testicular germ cell tumours. ZDHHC14 expression was also significantly decreased in a panel of prostate cancer samples and cell lines. In addition to our findings of genetic and protein expression changes in clinical samples, inducible overexpression of ZDHHC14 led to reduced cell viability and increased apoptosis through the classic caspase‐dependent apoptotic pathway and heterozygous knockout of ZDHHC14 decreased cell colony formation ability. Finally, we confirmed our in vitro findings of the tumour suppressor role of ZDHHC14 in a mouse xenograft model, showing that overexpression of ZDHHC14 inhibits tumourigenesis. Thus, we have identified a novel tumour suppressor gene that is commonly down‐regulated in testicular germ cell tumours and prostate cancer, as well as given insight into the cellular functional role of ZDHHC14, a potential protein palmitoyltransferase that may play a key protective role in cancer. © 2014 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.