Christina E. Hoei-Hansen

Embryonic Stem Cell-Like Features of Testicular Carcinoma in Situ Revealed by Genome-Wide Gene Expression Profiling

Carcinoma in situ (CIS) is the common precursor of histologically heterogeneous testicular germ cell tumors (TGCTs), which in recent decades have markedly increased and now are the most common malignancy of young men. Using genome-wide gene expression profiling, we identified >200 genes highly expressed in testicular CIS, including many never reported in testicular neoplasms. Expression was further verified by semiquantitative reverse transcription-PCR and in situ hybridization. Among the highest expressed genes were NANOG and POU5F1, and reverse transcription-PCR revealed possible changes in their stoichiometry on progression into embryonic carcinoma. We compared the CIS expression profile with patterns reported in embryonic stem cells (ESCs), which revealed a substantial overlap that may be as high as 50%. We also demonstrated an over-representation of expressed genes in regions of 17q and 12, reported as unstable in cultured ESCs. The close similarity between CIS and ESCs explains the pluripotency of CIS. Moreover, the findings are consistent with an early prenatal origin of TGCTs and thus suggest that etiologic factors operating in utero are of primary importance for the incidence trends of TGCTs. Finally, some of the highly expressed genes identified in this study are promising candidates for new diagnostic markers for CIS and/or TGCTs.

Stem cell pluripotency factor NANOG is expressed in human fetal gonocytes, testicular carcinoma in situ and germ cell tumours.

Aims : NANOG is a key regulator of embryonic stem cell (ESC) self‐renewal and pluripotency. Our recent genome‐wide gene expression profiling study of the precursor of testicular germ cell tumours, carcinoma in situ testis (CIS), showed close similarity between ESC and CIS, including high NANOG expression. In the present study we analysed the protein expression of NANOG during normal development of human testis and in a large series of neoplastic/dysgenetic specimens.

The emerging phenotype of the testicular carcinoma in situ germ cell

This review summarises the existing knowledge on the phenotype of the carcinoma in situ (CIS) cell. CIS is a common pre‐invasive precursor of testicular germ cell tumours of adolescents and young adults. These tumours display a variety of histological forms. Classical seminoma proliferates along the germ cell lineage, whereas embryonal carcinoma retains embryonic features and readily differentiates into teratomas that resemble various somatic cell lineages. A thorough review of the gene expression in CIS cells in comparison to normal testicular germ cells and overt tumours supports the view that CIS is a common precursor for both tumour types. Impaired cell differentiation resulting in a partial retention of the embryonic features, associated with an increasing genomic instability may be responsible for a remarkable phenotypic heterogeneity of CIS cells. Depending on the degree of differentiation and pluripotency, CIS cells found in adult patients seem to be predestined for further malignant progression into one or the other of the two main types of overt tumours. A new concept of phenotypic continuity of differentiation of germ cells along germinal lineage with a gradual loss of embryonic features based on the analysis of gene expression in all types of germ cells during their ontogeny is presented in this review. The data point out that despite the phenotypic continuum of gene expression, there are two periods of rapid changes of gene expression: first at the transition from primordial germ cells to pre‐spermatogonia, and later during the pubertal switch from the mitotic to meiotic cell division. The persistent expression of embryonic traits in CIS cells, and the high expression of the cell cycle regulators that are typical of mitotic germ cells support our long‐standing hypothesis that CIS cells originate from primordial germ cells or gonocytes and not from germ cells in the adult testis.

Histological evidence of testicular dysgenesis in contralateral biopsies from 218 patients with testicular germ cell cancer

This study was prompted by a hypothesis that testicular germ cell cancer may be aetiologically linked to other male reproductive abnormalities as a part of the so‐called ‘testicular dysgenesis syndrome’ (TDS). To corroborate the hypothesis of a common association of germ cell cancer with testicular dysgenesis, microscopic dysgenetic features were quantified in contralateral testicular biopsies in patients with a testicular germ cell tumour. Two hundred and eighty consecutive contralateral testicular biopsies from Danish patients with testicular cancer diagnosed in 1998–2001 were evaluated retrospectively. Two hundred and eighteen specimens were subsequently included in this study, after 63 patients who did not meet inclusion criteria had to be excluded. The presence of carcinoma in situ (which is believed to originate from transformed gonocytes) was detected in 8.7% of biopsies. The incidence of other dysgenetic features was immature tubules with undifferentiated Sertoli cells, 4.6%; microcalcifications (microliths), 6.0%; and the presence of a Sertoli‐cell‐only pattern in at least a few tubules, 13.8%. The cumulative incidence of one or more signs of testicular dysgenesis was 25.2%. In a few patients, areas with immature and morphologically distorted tubules were also noted. Spermatogenesis was qualitatively normal in 51.4%, whereas 11.5% had very poor or absent spermatogenesis. It is concluded that microscopic testicular dysgenesis is a frequent feature in contralateral biopsies from patients presenting with testicular germ cell neoplasms of the adolescent and young type. The findings therefore support the hypothesis that this cancer is part of a testicular dysgenesis syndrome. The presence of contralateral carcinoma in situ was higher in the present study than previously reported. Copyright

Association between testicular dysgenesis syndrome (TDS) and testicular neoplasia: Evidence from 20 adult patients with signs of maldevelopment of the testis

Based on a well established association between testicular cancer and undescended testis and more recent publications on epidemiological links between these disorders and male infertility, we proposed the existence of a testicular dysgenesis syndrome (TDS). In most cases TDS presents with impaired spermatogenesis, only in rare cases the full range of its signs, including genital malformations and testicular cancer can be seen in one patient. In order to further corroborate our hypothesis about the presence of testicular dysgenesis in patients with testicular abnormalities, we decided to re‐analyse recent testicular biopsies derived from patients with infertility, hypospadias and undescended testis. We searched for histological signs of testicular dysgenesis: microliths, Sertoli‐cell‐only tubules, immature seminiferous tubules with undifferentiated Sertoli cells, and tubules containing carcinoma in situ (CIS) cells. We identified 20 patients who fulfilled the histological criteria for testicular dysgenesis, 9 of whom were diagnosed with uni‐ or bilateral testicular germ cell neoplasia, and the remaining ones with subfertility. The presence of CIS was detected in 5 patients (3 of them with overt contralateral germ cell tumours). In all but one of the CIS cases, at least one additional sign of testicular dysgenesis was detected. Clinical records of all patients were subsequently analysed. The majority of cases had oligozoospermia or azoospermia. Their reproductive hormone profiles correlated with the results of semen sampling and testicular histology. In conclusion, our study of 20 patients with various reproductive abnormalities provided evidence that TDS is a real clinical entity. We speculate that most of these abnormalities are caused by adverse environmental effects rather than specific gene mutations.

Transcription Factor AP-2γ Is a Developmentally Regulated Marker of Testicular Carcinoma In situ and Germ Cell Tumors

Purpose: Transcription factor activator protein-2γ (TFAP2C, AP-2γ) was reported previously in extraembryonic ectoderm and breast carcinomas but not in the testis. In our recent gene expression study we detected AP-2γ in carcinoma in situ testis (CIS, or intratubular germ cell neoplasia), precursor of testicular germ cell tumors. In this study we aimed to investigate the expression pattern of AP-2γ and to shed light on this factor in germ cell differentiation and the pathogenesis of germ cell neoplasia. Experimental Design: We analyzed expression pattern of AP-2γ at the RNA and protein level in normal human tissues and a panel of tumors and tumor-derived cell lines. In the gonads, we established the ontogeny of expression of AP-2γ in normal and dysgenetic samples. We also investigated the regulation of AP-2γ by steroids and retinoic acid. Results: We detected abundant AP-2γ in testicular CIS and in testicular germ cell tumors of young adults and confirmed differential expression of AP-2γ in somatic tumors. We found that AP-2γ expression was regulated by retinoic acid in an embryonal carcinoma cell line (NT2). The investigation of ontogeny of AP-2γ protein expression in fetal gonads revealed that it was confined to oogonia/gonocytes and was down-regulated with germ cell differentiation. In some prepubertal intersex cases, AP-2γ was detected outside of the normal window of expression, probably marking neoplastic transformation of germ cells. Conclusions: AP-2γ is developmentally regulated and associated with the undifferentiated phenotype in germ cells. This transcription factor may be involved in self-renewal and survival of immature germ cells and tissue-specific stem cells. AP-2γ is a novel marker of testicular CIS and CIS-derived tumors.

Ovarian dysgerminomas are characterised by frequent KIT mutations and abundant expression of pluripotency markers

BackgroundOvarian germ cell tumours (OGCTs) typically arise in young females and their pathogenesis remains poorly understood. We investigated the origin of malignant OGCTs and underlying molecular events in the development of the various histological subtypes of this neoplasia.ResultsWe examined in situ expression of stem cell-related (NANOG, OCT-3/4, KIT, AP-2γ) and germ cell-specific proteins (MAGE-A4, NY-ESO-1, TSPY) using a tissue microarray consisting of 60 OGCT tissue samples and eight ovarian small cell carcinoma samples. Developmental pattern of expression of NANOG, TSPY, NY-ESO-1 and MAGE-A4 was determined in foetal ovaries (gestational weeks 13–40). The molecular genetic part of our study included search for the presence of Y-chromosome material by fluorescence in situ hybridisation (FISH), and mutational analysis of the KIT oncogene (exon 17, codon 816), which is often mutated in testicular GCTs, in a subset of tumour DNA samples. We detected a high expression of transcription factors related to the embryonic stem cell-like pluripotency and undifferentiated state in OGCTs, but not in small cell carcinomas, supporting the view that the latter do not arise from a germ cell progenitor. Bilateral OGCTs expressed more stem cell markers than unilateral cases. However, KIT was mutated in 5/13 unilateral dysgerminomas, whereas all bilateral dysgerminomas (n = 4) and all other histological types (n = 22) showed a wild type sequence. Furthermore, tissue from five phenotypic female patients harbouring combined dysgerminoma/gonadoblastoma expressed TSPY and contained Y-chromosome material as confirmed by FISH.ConclusionThis study provides new data supporting two distinct but overlapping pathways in OGCT development; one involving spontaneous KIT mutation(s) leading to increased survival and proliferation of undifferentiated oogonia, the other related to presence of Y chromosome material and ensuing gonadal dysgenesis in phenotypic females.

Genome-wide gene expression profiling of testicular carcinoma in situ progression into overt tumours

The carcinoma in situ (CIS) cell is the common precursor of nearly all testicular germ cell tumours (TGCT). In a previous study, we examined the gene expression profile of CIS cells and found many features common to embryonic stem cells indicating that initiation of neoplastic transformation into CIS occurs early during foetal life. Progression into an overt tumour, however, typically first happens after puberty, where CIS cells transform into either a seminoma (SEM) or a nonseminoma (N-SEM). Here, we have compared the genome-wide gene expression of CIS cells to that of testicular SEM and a sample containing a mixture of N-SEM components, and analyse the data together with the previously published data on CIS. Genes showing expression in the SEM or N-SEM were selected, in order to identify gene expression markers associated with the progression of CIS cells. The identified markers were verified by reverse transcriptase–polymerase chain reaction and in situ hybridisation in a range of different TGCT samples. Verification showed some interpatient variation, but combined analysis of a range of the identified markers may discriminate TGCT samples as SEMs or N-SEMs. Of particular interest, we found that both DNMT3B (DNA (cytosine-5-)-methyltransferase 3 beta) and DNMT3L (DNA (cytosine-5-)-methyltransferase 3 like) were overexpressed in the N-SEMs, indicating the epigenetic differences between N-SEMs and classical SEM.

Identity of M2A (D2-40) antigen and gp36 (Aggrus, T1A-2, podoplanin) in human developing testis, testicular carcinoma in situ and germ-cell tumours

Testicular germ-cell tumours of young adults are derived from a pre-invasive intratubular lesion, carcinoma in situ (CIS). In a recent genome-wide gene expression screening using cDNA microarrays, we found PDPN over-expressed in CIS compared to normal adult testis. PDPN encodes podoplanin (Aggrus, human gp36, T1A-2), a transmembrane glycoprotein expressed in lymphatic endothelium and various solid tumours. To examine a potential role for PDPN in testicular neoplasms and during testicular development, we investigated its expression pattern during the development of human testis and in a series of testicular CIS, gonadoblastoma and overt germ-cell tumours. We established by RT-PCR and by immunohistochemistry with a gp36 antibody that PDPN mRNA and the protein product were expressed in testes with germ-cell neoplasms but not in the normal adult testis. We also found gp36 expression in early foetal gonocytes and immature Sertoli cells, similar to the expression pattern of M2A antigen, a previously identified marker for CIS and seminoma. This reinforced our previous proposal that M2A (D2-40) antigen was identical to gp36 (podoplanin, Aggrus, T1A-2). Our findings also suggest that podoplanin has a function in developing testis, most likely at the level of cell–cell interactions among pre-meiotic germ cells and immature Sertoli cells.

From Gonocytes to Testicular Cancer

Abstract: Testicular germ‐cell tumors occur primarily in young individuals, and the tumors in this age group (seminomas or nonseminomas) are derived from a preinvasive precursor cell called carcinoma in situ (CIS) or intratubular germ‐cell neoplasia. These tumors have been a growing problem, especially in highly developed industrialized countries. A hypothesis was put forward that CIS originates from arrested fetal germ cells, thus testicular cancer is a developmental disease of germ‐cell differentiation. This notion was supported by comparative studies of the gene expression at the protein and RNA level, which demonstrated a close similarity of CIS to primordial germ cells and gonocytes with many features of embryonic stem cells. The arrest of germ‐cell differentiation is thus the key first event, which may be followed by malignant transformation and overt germ‐cell cancer in young adult age, usually after puberty. In most cases the arrest/delay of germ‐cell differentiation is caused by testicular dysgenesis, a multifactorial and complex syndrome that has a broad spectrum of phenotypes ranging from moderate impairment of spermatogenesis to severe disorders of sexual development and differentiation. The most severe cases are a result of inherited genetic aberrations, but the etiology of the common sporadic testicular cancer must involve environmental factors, including maternal lifestyle and possibly an early exposure to endocrine disruptors. The effects of environmental factors are likely modulated by genomic variation (polymorphisms), thus explaining the individual susceptibility and population‐level differences in the incidence of testicular cancer.