The three main categories of ovarian tumours are: surface epithelium-stromal tumours, sex-cord tumours and germ cell tumours (GCTs; benign and malignant). Malignant ovarian (OGCTs) have a median age at onset of 18 years and represent approximately 3% of all ovarian cancers in Western countries 1. The following histological sub-types exist: dysgerminoma, yolk sac tumour, embryonal carcinoma, polyembryoma, choriocarcinoma, immature teratoma, and mixed GCTs. Bilateral tumours occur in up to 10% of cases 12. The correct differential diagnosis is imperative since the prognosis and choice of therapy remain different among the various ovarian cancer types. A treatment consisting of a combination of surgical resection and platinum-based chemotherapy cures the majority of malignant OGCT patients 2. The fact that OGCTs often affect women in their reproductive years further imply the importance of optimal therapy in order to maximize the number of women in which ovarian function can be conserved.

Many similarities exist between GCTs of the ovary and testis, including a morphological resemblance and a similar pattern of chromosomal alterations 34. Furthermore, families with both ovarian and testicular GCTs have been reported, suggesting a possible association/common genetic aetiology 56. Ovaries and testes develop similarly until approximately 2 months of embryonic life, which is also consistent with a common origin of, at least some cases of, ovarian and testicular GCTs. In this study, recent knowledge of underlying mechanisms in development of testicular GCTs was used as a guide to investigate patterns of in situ protein expression in OGCTs. As a close resemblance between testicular GCTs and embryonic stem cells has been shown 7 particular focus was on stem cell-related factors including KIT (also known as c-Kit, tyrosine kinase receptor for stem cell factor (SCF)), OCT-3/4 (POU5F1, a POU-family transcription factor), NANOG, and AP-2γ (TFAP2C, transcription factor activator protein-2), and focus was also on germ cell-specific proteins (including MAGE-A4 and NY-ESO-1 belonging to the cancer/testis gene family) with a cell differentiation related biological function or a developmentally regulated expression pattern 8. KIT is involved in the migration of primordial germ cells (PGCs) 9 and there has been reported a frequent presence of KIT mutations in GCTs and in particular bilateral testicular GCTs 1011121314151617. Therefore KIT mutation status was also determined in the same samples and we established the expression pattern of proteins not previously studied in the ovary during foetal ovarian development. Finally, patients with intersex disorders and dysgenetic ovaries have an increased risk of harbouring a GCT, and the ovarian malignancies were therefore investigated for the presence of Y-chromosome material. Gonadoblastomas are rare neoplasms composed of germ cells and immature granulosa/Sertoli cells that develop nearly exclusively in males and phenotypic females harbouring Y-chromosome material. We chose TSPY as a marker, because this gene has been mapped to a smallest region of the Y chromosome consistently present in females with gonadoblastoma, and TSPY has been proposed to be responsible for the origin of this tumour 18. We attempted to analyse the various steps from the hypothesised development of some dysgerminomas from gonadoblastoma, considering why these neoplasias develop and progress.

In this study, we provide novel evidence for distinct, partly overlapping mechanisms acting in the pathogenesis of OGCTs. To our knowledge, this study is the first comprehensive analysis of an in situ protein expression profile of pluripotency genes and germ cell differentiation markers in these relatively rare malignant tumours. The expression of the transcription factors OCT-3/4 and NANOG were of particular interest, as they are key regulators of self-renewal and pluripotency of embryonic stem cells. In addition to the role in early embryonic development, these proteins are only present in PGCs, oogonia and gonocytes and in the testis only in malignant GCTs 202122. In the present study, OCT-3/4 expression was abundant in all gonadoblastomas and most dysgerminomas, in accordance with previously published data 23. NANOG has not previously been evaluated in the ovary. Approximately half of the dysgerminomas and gonadoblastomas expressed NANOG, whereas tumours with somatic differentiation were largely negative. We did not detect NANOG expression in foetal ovaries, but cannot exclude expression in first trimester. Murine foetal ovary expresses Nanog in early foetal development with earlier downregulation than in male gonads 24. Our results indicate that in analogy to the mouse model, NANOG could to be earlier down-regulated in human foetal ovary than testis, where gonocytes abundantly express NANOG until GW 20 22.

The tissue specific adult stem cell-related factors AP-2γ and KIT are highly expressed in immature gonocytes, testicular carcinoma in situ (the pre-invasive stage of testicular GCTs) and in seminomas, and AP-2γ, but not KIT, is expressed in embryonal carcinoma 252627. AP-2γ expression was previously reported in ovarian carcinomas 28 and in 5/5 dysgerminomas 29. In contrast to the male pattern, AP-2γ was only expressed in 33% of dysgerminomas and 38% of dysgerminomas containing gonadoblastoma. Higher oestrogen levels in the ovaries may in part explain the observed difference, as AP-2γ has been suggested involved in oestrogen signalling 27. The role of AP-2γ in the gonads has not yet been elucidated but disruption of the gene in mice leads to complete loss of germ cells, probably because of failure of KIT induction, which is hypothesised to be a main target for AP-2γ30.

The KIT/SCF system is of particular interest in regard to the origin of GCTs, due to a role in PGC proliferation and survival in the developing human gonad 9. KIT expression was previously described in oogonia and oocytes 253132 and in some OGCTs, predominantly dysgerminomas 333435. In accordance with these reports, we found KIT expressed in 80–100% of neoplastic cells in dysgerminomas and gonadoblastomas, and in 15% of yolk sac tumours. The high expression of KIT in dysgerminoma prompted us to investigate the possibility of a gene mutation. A gain-of-function mutation in the KIT gene was first reported in human GCTs, including one ovarian dysgerminoma/yolk sac tumour by Tian et al. 10 and later studies have shown KIT mutations in a varying proportion of phenotypically indistinguishable testicular seminomas 12141536, primary mediastinal seminomas 11 and intracranial germinomas, both in males and females 1317. Among 11 OGCTs previously analysed for KIT mutations two (one unilateral case – for the other such info was not given) had a GAC to CAC change in codon 816 101416. We detected five KIT mutations in the same codon, but found three different base changes in five OGCTs (four pure dysgerminomas and one dysgerminoma with gonadoblastoma). Strikingly, all mutations were found in unilateral cases and none among four analysed bilateral cases. This could suggest that KIT mutations occur after completion of PGC migration to the gonadal ridges in females, and may be in contrast to what has been suggested for males, where some reports have shown KIT mutations more frequently in bilateral seminomas 1215. We hypothesise that activating KIT mutation(s) in early oogonia may stimulate their proliferation and delay meiotic entry, which in normal ovaries occurs gradually in foetal life, thus increasing the pool of immature germ cells.

It has been proposed that dysgerminoma may evolve from gonadoblastoma 3738. We analysed the stages of tumour development, from pure gonadoblastoma, through dysgerminoma with gonadoblastoma, to pure dysgerminoma, using the TSPY gene, a proposed GBY gene, as a marker. Besides gonadoblastoma, TSPY is expressed in male pre-meiotic germ cells and testicular GCTs that retain germ cell phenotype 183940. We found abundant TSPY in 5/7 cases of gonadoblastoma or dysgerminoma with gonadoblastoma, but not in pure dysgerminoma (n = 11). Consistent results were detected with TSPY protein and FISH Y-centromere analyses, suggesting that TSPY is included in the sub-region of the Y-chromosome involved. In one case of combined dysgerminoma/gonadoblastoma we detected a KIT mutation. Taken together, these results indicate that development of dysgerminoma with gonadoblastoma versus pure dysgerminoma may follow different molecular routes, with a role for one or more Y-chromosome genes in the development of gonadoblastoma in phenotypic females. Sex-chromosome alteration leads to gonadal dysgenesis and partial masculinisation in at least some cells of the developing female ovary. This may lead to a delay of the normal differentiation of germ cells in foetal ovaries and a failure of the onset of meiotic prophase, thus increasing the chance of mutational events (e.g. in KIT) and neoplastic transformation in the mitotically dividing germ cells, perhaps stimulated by pleiotrophic action of TSPY.

As the OGCTs may develop in dysgenetic ovaries with some testicular differentiation, we examined the expression of AMH, a glycoprotein involved in involution of Müllerian ducts in the foetus leading to male differentiation 19, but did not detect expression in any OGCTs. We furthermore examined MAGE-A4 and NY-ESO-1 41, two representatives of the germ cell-specific "cancer/testis" gene family on the X-chromosome, which appear in gonocytes from around gestational week 17–18 at differentiation into pre-spermatogonia, and are expressed in adult spermatogonia and primary spermatocytes, as well as in some testicular tumours and a broad range of somatic cancers 4243. In accordance with previous studies 4344 we detected expression of MAGE-A4 and NY-ESO-1 in foetal oogonia/oocytes of 13–40 weeks of gestational age. MAGE-A4 and NY-ESO-1 have been reported in ovarian epithelial and serous carcinomas 45, but this is the first study regarding OGCTs. We found a heterogeneous expression of MAGE-A4 but not NY-ESO-1 in dysgerminomas, analogous to the pattern in testicular seminomas.

In this study, we included also some small cell carcinomas of the hypercalcaemic type that occasionally develop in the ovaries of young patients. None of the applied markers were positive, and therefore it is unlikely that small cell carcinomas have a germ cell progenitor, as has been suggested 4647. With regard to the immature teratomas, they were also negative for all analysed markers, which may support the observation that this tumour, which is usually diploid and does not have the isochromosome (12p) imbalance, probably develops in a different manner than other malignant OGCTs 34. No KIT mutations or presence of Y-chromosome material was detected in any of the small cell carcinomas or immature teratomas.

We detected many similarities, but also some notable differences in the protein expression profiles of ovarian and testicular GCTs, but nevertheless propose a comparable developmental model, as depicted in Fig. 3. Genetic alterations and biological mechanisms may be identical, with abnormal persistence of undifferentiated PGCs in both sexes, with the large difference in incidence reflecting the much lower number of susceptible cells in females at the time of puberty 48. The proposed stepwise progression from gonadoblastoma to dysgerminoma in dysgenetic ovaries 38, and the similarities of gonadoblastoma and testicular carcinoma in situ 49, could indicate that gonadoblastoma – analogous to carcinoma in situ in the testes – is a precursor stage, which probably only occurs in phenotypic females with Y-chromosome material. As the stem cell-related markers were abundantly expressed, our hypothesis is that in 46, XX females, the OGCTs probably are derived from PGCs or oogonia. We speculate that despite the lack of the Y-chromosome material, some degree of gonadal dysgenesis leading to a delay in the differentiation of PGC/oogonia and initiation of gonadoblastoma-like lesions, may be due to changes in hormonal environment, perhaps caused by endocrine disrupters or by lifestyle changes, e.g. obesity. However, the low incidence of OGCTs complicates establishment of epidemiological associations.

We provide in this study new evidence for two partially overlapping mechanisms that may contribute to the pathogenesis of ovarian germ cell neoplasms. One mechanism is related to the presence of Y-chromosome material and ensuing gonadal dysgenesis. The other mechanism is primarily genetic and involves spontaneous mutation(s) in the KIT gene, leading to increased survival and proliferation of undifferentiated oogonia. We expect this mechanism not to be limited to KIT, but demonstrate that mutations in KIT are responsible for a sizeable number (5/17) of cases containing dysgerminoma. The neoplastic transformation must occur before oocytes enter meiosis, most likely after the migration to the gonadal ridges, as the mutations were detected in unilateral tumours.

The author(s) declare that they have no competing interests.

C.E.H.H. performed and co-analysed most immunohistochemical stainings, supervised FISH analysis, performed part of the data analysis and drafted the manuscript. S.M.K. coordinated and designed the tissue microarray, performed part of the mutation analysis and data analysis, and contributed to writing of the manuscript. V.M.A. performed pathology evaluation of the tumours and guided tissue selection for microarray preparation, co-analysed immunohistochemical stainings, and contributed to writing of the manuscript. J.K. was responsible for inclusion of the clinical data and performed the clinical follow-up of many of the patients. E.R.M. supervised immunohistochemical stainings contributed to the planning, data analysis, and writing of the manuscript. R.A.L. initiated the project, supervised KIT mutation analysis, contributed to the planning, data analysis, and writing of the manuscript. All authors read and approved the final manuscript.