Human Reproduction Update Advance Access originally published online on March 15, 2006
Human Reproduction Update 2006 12(3):303-323; doi:10.1093/humupd/dmk006
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Developmental model for the pathogenesis of testicular carcinoma in situ: genetic and environmental aspects
University Department of Growth and Reproduction, Copenhagen University Hospital (Rigshospitalet), Copenhagen, Denmark
To whom correspondence should be addressed at: University Department of Growth and Reproduction, Copenhagen University Hospital (Rigshospitalet), Section GR-5064, 9 Blegdamsvej, DK-2100 Copenhagen, Denmark. Email: erm{at}rh.hosp.dk
Submitted on February 3, 2005; resubmitted on December 21, 2005; accepted on January 10, 2006
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Abstract |
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Abstract
IntroductionCarcinoma in situ testis (CIS), also known as intratubular germ cell neoplasia (ITGCN), is a pre-invasive precursor of testicular germ cell tumours, the commonest cancer type of male adolescents and young adults. In this review, evidence supporting the hypothesis of developmental origin of testicular germ cell cancer is summarized, and the current concepts regarding aetiology and pathogenesis of this disease are critically discussed. Comparative studies of cell surface proteins (e.g. PLAP and KIT), some of the germ cell-specific markers (e.g. MAGEA4, VASA, TSPY and NY-ESO-1), supported by studies of regulatory elements of the cell cycle (e.g. p53, CHK2 and p19-INK4d) demonstrated a close similarity of CIS to primordial germ cells and gonocytes, consistent with the pre-meiotic origin of CIS. Recent gene expression profiling studies showed that CIS cells closely resemble embryonic stem cells (ESCs). The abundance of factors associated with pluripotency (NANOG and OCT-3/4) and undifferentiated state (AP-2
) may explain the remarkable pluripotency of germ cell neoplasms, which are capable of differentiating to various somatic tissue components of teratomas. Impaired gonadal development resulting in the arrest of gonocyte differentiation and retention of its embryonic features, associated with an increasing genomic instability, is the most probable model for the pathogenesis of CIS. Genomic amplification of certain chromosomal regions, e.g. 12p, may facilitate survival of CIS and further invasive progression. Genetic studies, have so far not identified gene polymorphisms predisposing to the most common non-familial testicular cancer, but this research has only recently begun. Association of CIS with other disorders, such as congenital genital malformations and some forms of impaired spermatogenesis, all rising in incidence in a synchronous manner, led to the hypothesis that CIS might be a manifestation of testicular dysgenesis syndrome (TDS). The aetiology of TDS including testicular cancer remains to be elucidated, but epidemiological trends suggest a primary role for environmental factors, probably combined with genetic susceptibility.
Key words: carcinoma in situ / germ cell differentiation / embryonic stem cells / testicular cancer / testicular dysgenesis syndrome
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Introduction |
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Testicular cancer is in most cases considered a disease of adults. Seeing a young man presenting with a testicular tumour or with symptoms of disseminated cancer disease, few clinicians would think that their patient’s disease had been initiated long time before, during fetal development. However, evidence gathered over the last three decades and the newest findings support this hypothesis, as will be critically discussed in this review.
The early origin is only one of the unique features of testicular germ cell cancer. This neoplasm is unlike any other solid tissue cancer for a number of reasons, including unusual epidemiological and biological features. Epidemiological hallmarks include the peak incidence in a very young adult age, a markedly increasing incidence worldwide but with striking geographic and ethnic differences, and association with other reproductive conditions. Among particular biological features are the unusual histology characterized by extreme heterogeneity with components mimicking any tissue type of the body, including caricatural reflection of early embryos in teratomas, and the extreme sensitivity to irradiation and cytotoxic treatment.
One of the possible explanations for the unique biology of testicular germ cell cancer is that it is derived from germ cells, which are different from any other cells in the body because of their special function of exchanging and transferring hereditary information as gametes. Germ cells are the only cells that use two different types of cell division (mitosis and meiosis), and for that they require different regulation of cell cycle and DNA repair. The regulation of gene expression appears to be different as well, including waves of epigenetic activation and silencing, and a final selective chromosomal condensation during the process of spermiogenesis. In contrast to other cell types, germ cells retain embryonic stem cell (ESC)-like features and pluripotency for a long time during development. For reasons not yet fully understood, perhaps because of this special hereditary role, germ cells and the reproductive system serving them appear to be exquisitely sensitive to changes in micro- and macro-environment. Research on these aspects has been energized in recent years after adverse epidemiological trends in male reproduction were observed worldwide, with a rise in testicular cancer the first trend to be noted. As will be discussed in detail in this review, studies on the origin and biology of the early stage of this neoplasia played a key role for the understanding of the association between male reproductive disorders and their possible link to changing environment and lifestyle.
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A bit of history: histopathology of germ cell neoplasia |
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Germ cell tumours have fascinated several generations of pathologists because of their histological heterogeneity and seemingly unlimited ability to differentiate into all somatic tissues (totipotency). Moreover, germ cell-like tumours were noticed in remote extragonadal locations, including intracranial sites, usually near the midline of the body. Histological complexity of germ cell tumours constituted a diagnostic conundrum and contributed to the chaos with numerous classifications and nomenclatures. Because classification is not the topic of this review, the readers are referred to specialist reviews and monographs (Grigor, 1993
; Ulbright et al., 1999; Eble et al., 2004). An easy and logical division of testicular germ cell tumours follows three age groups: tumours of newborns and infants (teratomas and yolk sac tumours), tumours of adolescents and young adults (seminomas and non-seminomas, which may also occur simultaneously as combined tumours) and the spermatocytic seminoma of elderly men (Oosterhuis and Looijenga, 2005). In addition, individuals with intersexual phenotype and dysgenetic gonads can harbour gonadoblastoma, a clinically benign but potentially malignant lesion (Scully, 1970). The tumours of infants and elderly are very rare.
One of the most important advances in the understanding of the biology and natural history of germ cell neoplasms, which led to a substantial revision of previous classifications, was the first description of testicular carcinoma in situ (CIS) in patients who subsequently developed testicular cancer, by a paediatric endocrinologist with a keen interest in testicular development and function in various pathologies (Skakkebæk, 1972). The cells described by Skakkebæk as a precursor for overt germ cell tumours were seen previously, however, others did not recognize their biological significance and considered them as ‘degenerate forms’ secondary to a tumour or ‘intratubular spread of tumour cells’ (Azzopardi et al., 1961; Mark and Hedinger, 1965), even several years after the Skakkebæk’s description of CIS (Teilum, 1976; Pugh and Parkinson, 1981). Skakkebæk himself acknowledged those earlier descriptions (Skakkebæk, 1981), but it required an intervention by Gondos (1990) and a recent gracious commentary by Parkinson and Harland (2002) to put the earlier history of the discovery of CIS in the correct context. After a few years of denials and discussions, CIS has been commonly accepted as a precursor for all germ cell tumours of the adolescents and young adults, both seminomas and non-seminomas (Ulbright et al., 1999). Other synonyms for CIS have been proposed: intratubular germ cell neoplasia (ITGCN), also called unclassified (ITGCNU) (Ulbright et al., 1999), testicular intraepithelial neoplasia (Loy and Dieckmann, 1990) and gonocytoma in situ (Grigor, 1993). As will be evident from the discussion below, the last term may be the most accurate from the biological point of view.
Already some of the early studies of Skakkebæk and his group provided evidence that CIS was the pre-invasive lesion for the tumours of the adolescents and young adults but not for the infantile tumours or spermatocytic seminoma (Müller et al., 1987; Skakkebæk et al., 1987; Jørgensen et al., 1995a). Biological differences in the pathogenesis of these rare tumours have been confirmed subsequently by studies of genomic aberrations and gene expression patterns (Hawkins et al., 1997; Kraggerud et al., 1999; Perlman et al., 2000; Schneider et al., 2001; Stoop et al., 2001; Rajpert-De Meyts et al., 2003b; Looijenga et al., 2006).
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Phenotypic features of CIS in relation to germ cell differentiation |
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Morphological features of CIS cells (Figure 1) have been described in numerous previous articles and pathology textbooks (Skakkebæk, 1972
; Ulbright et al., 1999; Rørth et al., 2000; Eble et al., 2004). Close morphological similarity between CIS cells and human fetal gonocytes (as well as seminoma cells and the neoplastic germ cells of gonadoblastoma) was noticed soon after the first description of this lesion and later confirmed by ultrastructural studies (Holstein and Körner, 1974; Nielsen et al., 1974; Gondos, 1993). Subsequent studies provided supporting evidence for these similarities based on the comparison of immunohistochemical markers (Hustin et al., 1987; Jørgensen et al., 1993, 1995b, 1997; Honecker et al., 2004). Over the years, more and more proteins/antigens were identified in CIS cells (a partial list is presented in Table I). The status of knowledge on the emerging phenotype of the CIS cell up to year 2002 was summarized in my previous review (Rajpert-De Meyts et al., 2003a). Here, only the most important earlier findings are briefly highlighted, whereas the most recent advances are described in greater detail.
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CIS markers, including the KIT receptor, are also expressed in human gonocytes
Early studies focussed on finding clinically useful marker to facilitate the detection of CIS in testicular biopsies. A classic example is placental-like alkaline phosphatase (PLAP, Figure 1), the first identified marker of murine primordial germ cells (PGCs) with still unknown biological function, which remains to this day the most commonly used marker for CIS and seminoma in testicular biopsies and other pathological tissue samples (Jacobsen and Nørgaard-Pedersen, 1984; Hustin et al., 1987; Rajpert-De Meyts et al., 2003a; references therein).
Over the years, the list of markers for CIS steadily grew; the early markers were usually identified serendipitously, e.g. by testing of an antibody against a glycoprotein abundant in a tumour cell line. Two of these markers, TRA-1-60 (Giwercman et al., 1993; Badcock et al., 1999) and M2A (Giwercman et al., 1988; Marks et al., 1999), which are abundant in CIS but undetectable in the normal adult testis, were detected in normal fetal and infantile germ cells, thus giving the first evidence supporting the hypothesis of the prenatal origin of CIS (Jørgensen et al., 1993, 1995b).
Further evidence for our hypothesis was provided by investigations of the expression of c-KIT in germ cell neoplasms. This gene encodes a cell membrane tyrosine kinase receptor for stem cell factor, a signalling system essential for early germ cell survival, as was first observed in mutant mice with either W or Sl phenotype (Chabot et al., 1988; Huang et al., 1990; Yarden et al., 1987). Differential expression of KIT was first described in germ cell tumours by Strohmeyer et al. (1991a) and detected in CIS cells (Figure 2) by Rajpert-De Meyts and Skakkebæk (1994), followed by several other studies (Izquierdo et al., 1995; Strohmeyer et al., 1995; Bokemeyer et al., 1996). As expected, KIT was also strongly expressed in fetal and infantile gonocytes (Jørgensen et al., 1995b; Robinson et al., 2001; Gaskell et al., 2004; Honecker et al., 2004) but very low or undetectable in adult spermatogonia in the adult human testis, although this has been somewhat dependent on the specificity of the antibodies and tissue fixation used (Rajpert-De Meyts et al., 2003b). The ontogeny of expression of KIT in the human testis demonstrated that it is present at a very high level in the majority of gonocytes during the first trimester of gestation, thereafter the KIT expression was gradually down-regulated (Jørgensen et al., 1995b; Gaskell et al., 2004; Honecker et al., 2004). The retention of a very high expression of KIT beyond a normal window was noted in dysgenetic fetal gonads of some intersex cases (Rajpert-De Meyts et al., 1996a). As KIT is a potent pro-survival factor, its prolonged expression could give a growth advantage to the surviving undifferentiated cells. This observation, along with a known association of CIS with poor gonadal development (Table II), led to a new hypothesis that a delay in differentiation could be of one of the mechanisms of neoplastic transformation of germ cells (Rajpert-De Meyts et al., 1998a). This is in the line with reports on ‘gain-of-function’ mutations in the c-KIT gene in virtually all sporadic bilateral tumours, both seminomas and non-seminomas (Looijenga et al., 2003a), and in a subset of familial and sporadic unilateral testicular tumours but, interestingly, less frequently in non-seminomas (Tian et al., 1999; Madani et al., 2003; Kemmer et al., 2004; Rapley et al., 2004). The high frequency of mutations of KIT in bilateral tumours suggests that the mutations most probably had occurred in PGCs, before their migration to the gonadal regions has taken place (Looijenga et al., 2003a).
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Stem cell-like features: is CIS a fossil from the embryonic past?
The high expression of KIT (the receptor for the stem cell factor), which is present in different types of tissue-specific stem cells, turned our attention into stem cell-like characteristics of CIS cells. Previous studies of embryonal carcinoma-derived cell lines have demonstrated that they closely resemble human ESCs, including such hallmark features, as pluripotency and ability to differentiate when stimulated with retinoic acid (Andrews, 1984, 1998). Among the above-mentioned early markers for CIS cells was TRA-1-60, one of the best known markers for embryonal carcinoma and human ESC (Andrews et al., 1984; Giwercman et al., 1993; Badcock et al., 1999; Henderson et al., 2002; Park et al., 2004). More recently, OCT-4 (or OCT-3/4) encoded by POU5F1, the first transcription factor associated with pluripotency and specific for ESC (Schöler et al., 1989) was detected in CIS cells, gonadoblastoma and overt germ cell tumours, with the exception of differentiated teratomas (Palumbo et al., 2002; Gidekel et al., 2003; Looijenga et al., 2003b; Jones et al., 2004; Rajpert-De Meyts et al., 2004). Interestingly, OCT-4 was highly expressed by virtually all CIS cells in all these studies, whereas other markers, TRA-1-60 and to lesser extent KIT, were present in a subset of CIS cells only, preferentially in those in the vicinity of non-seminomas or seminomas, respectively, thus demonstrating a remarkable heterogeneity of CIS cells (Rajpert-De Meyts et al., 1996b). Heterogeneity of the expression of certain embryonic and germ cell-specific markers in CIS cells indicates plasticity of the phenotype of CIS cells, which may begin invasive transformation while still in situ.
Recent development of high throughput methods sped up markedly the characterization of gene expression in germ cell tumours and CIS at the RNA level. Most of the published studies analysed gene expression profiles in overt tumours or tumour-derived cell lines, focusing first on genes on certain chromosomal regions, e.g. 17q and 12p (Skotheim et al., 2002; Rodriguez et al., 2003), and later on a genome-wide analysis (Okada et al., 2003; Sperger et al., 2003; Skotheim et al., 2005). The Norwegian group investigated also gene expression at the protein level in a large array of tissues, including CIS, and confirmed the expression of JUP (plakoglobulin) in all CIS samples studied (Skotheim et al., 2003).
The first study that focussed on the expression profile of CIS (Hoei-Hansen et al., 2004a) used differential display and identified several genes that function in fetal life and thus supported the hypothesis of fetal origin of CIS. A substantial advance was the study by Almstrup et al. (2004), which using a genome-wide cDNA microarray, identified a large number of genes not previously reported in CIS. Importantly, the gene expression profile of CIS revealed a remarkable similarity to ESC (Almstrup et al., 2004). Among the genes over-expressed in CIS were NANOG, POU5F1 (OCT-3/4), KIT, SFRP1, TFAP2C and several members of the DPPA family, which all have been identified in human ESC (Sato et al., 2003; Sperger et al., 2003; Clark et al., 2004), and more recently, also in embryonal carcinoma (Skotheim et al., 2005). A more detailed analysis of NANOG in CIS and germ cell tumours demonstrated a pattern of expression essentially identical to that of OCT-3/4 (Hart et al., 2005; Hoei-Hansen et al., 2005b). A common feature of these genes is their link to pluripotency; they prevent further differentiation of the cell and ensure a ‘stock’ of undifferentiated cells to renew the tissue. Outside the early embryonic development, NANOG and OCT-3/4 are only found in immature germ cells. A high expression of these genes is a probable explanation of the ability of CIS cells to undergo reprogramming to pluripotent embryonal carcinoma and further differentiation to teratomas, which may contain all types of somatic tissues.
Some of the genes associated with ‘stemness’ are present not only in ESC but also in various tissue-specific stem cells, e.g. KIT and TFAP2C. TFAP2C (mapped to chromosome 20q13.2), which encodes the transcription factor activator protein-2 (AP-2), was previously known as a possible oncogenic factor in other neoplasms, e.g. breast cancer (Turner et al., 1998) but never detected in testis. We established AP-2 as a novel marker for fetal gonocytes and neoplastic germ cells, including testicular CIS (Figure 2), with a role in pathways regulating cell differentiation and a possible involvement in testicular oncogenesis (Hoei-Hansen et al., 2004b). This was confirmed by another study (Pauls et al., 2005). Thanks to its abundance in nuclei of CIS cells; AP-2 is currently under investigation as a possible tool for the identification of CIS cells in semen samples in a clinical setting (Hoei-Hansen et al., 2005a).
Studies of the pattern of expression during development (Figure 3) demonstrated that OCT-4, AP-2, NANOG, as well as KIT, and probably a number of other CIS markers are abundant in early fetal gonocytes and the expression gradually decreases while gonocytes differentiate to infantile spermatogonia (Jørgensen et al., 1995b; Gaskell et al., 2004; Hoei-Hansen et al., 2004b, 2005b; Honecker et al., 2004; Rajpert-De Meyts et al., 2004). During human fetal testicular development, a rapid transition from PGCs (which in the testis are germ cells not yet enclosed in seminiferous cords) to gonocytes first takes place, later followed by much slower differentiation of gonocytes into pre-spermatogonia (also called infantile spermatogonia). At that time, germ cells gradually loose their embryonic characteristics while acquiring features of germ cells manifested by the expression of male-specific genes. It is important to underline here the continuum of the expression profile of germ cells, which are the only cell type in the body that retains for such a long time the high expression of genes necessary to maintain ESC-like pluripotency.
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In addition to ESCs and early fetal germ cells, CIS cells have also a lot in common with normal germ cells of the adult testis. Numerous of proteins/antigens present in normal spermatogonia were also found in CIS cells. The list of such proteins is growing practically by the day. Among the first published were globotriazol ceramide, Gb3 (Kang et al., 1995), and neuron-specific enolase, NSE (Kang et al., 1996), followed by many others, including some found also in spermatocytes and even in haploid spermatids, as listed in Table I (and reviewed in Rajpert-De Meyts et al., 2003a). One recent example is VASA, a gene-encoding DEAD-box RNA helicase, which is present in human germ cells throughout their development and maturation (Castrillon et al., 2000; Honecker et al., 2004) and is also expressed in CIS and overt tumours that retain germ cell-like morphology, such as testicular seminomas and ovarian dysgerminomas (Zeeman et al., 2002).
Recent advances in studies on germ cells uncovered a large number of genes that are germ cell-specific, but their biological function has not yet been elucidated, except that many of these genes appear to be involved in RNA processing and regulation, which is essential for spermatogenesis. As expected, quite a few of male germ cell-specific genes are located on the Y chromosome (Lahn and Page, 1997). Very little is known about the expression and function of these genes during early development of germ cells and even less about possible changes in testicular dysgenesis. An early study reported the expression of RBMY gene family both in the fetal and in the adult testis (Elliot et al., 1997), however, in more recent studies, RBMY was not detected by immunohistochemistry neither in CIS cells nor in overt tumours (Lifschitz-Mercer et al., 2000; Schreiber et al., 2003). Whether or not down-regulation of this gene family has something to do with neoplastic transformation of early germ cells into CIS remains to be elucidated. Another germ cell-specific gene family includes DAZ (on the Yq, usually consist of four copies) and closely related autosomal genes DAZL and BOULE. DAZ and DAZL have been described in mitotic germ cells, including PGCs and gonocytes (Reijo et al., 2000; Xu et al., 2001). Consequently, DAZL protein was detected in CIS, in seminomas but not in non-seminomas, consistent with its germ cell-specific function (Lifschitz-Mercer et al., 2002). Another multicopy gene, TSPY, was suggested as a candidate gene for gonadoblastoma (Salo et al., 1995; Tsuchiya et al., 1995). TSPY in the adult testis is expressed in spermatogonia, and its protein product was also described in immature germ cells in undifferentiated tubules of dysgenetic testes, CIS, seminoma (Schnieders et al., 1996) and gonadoblastoma (Lau et al., 2000; Kersemaekers et al., 2005). The function and biological role of TSPY remains to be elucidated. Likewise, it remains to be proven that TSPY is the only gene responsible for gonadoblastoma, as this tumour is frequently seen in mixed gonadal dysgenesis where there is a mosaic aneuploidy of sex chromosomes (46,XY/45,X). The presence of gonadoblastoma is thus most probably a result of male germ cells developing in an insufficiently masculinized gonad because of the lack of function of the Y-chromosome genes in somatic cells in the vicinity. As it will be discussed further, a similar pathogenesis is most probably responsible for CIS, except that CIS occurs in testes with development impaired to much lesser degree than is the case in mixed gonadal dysgenesis.
According to traditional knowledge, genes on the Y chromosome were considered to play the principal role in male reproduction, whereas the X chromosome was more linked to the female fertility. Female ovarian failure is frequently caused by the monosomy (Turner syndrome) or deletions of the X chromosome (reviewed in Zinn and Ross, 2001; Laml et al., 2002; Schlessinger et al., 2002). Recent years provided new evidence that the X chromosome contains a large number of genes expressed in male germ cells and is apparently essential not only for the female but also for the male germ cell function (Wang et al., 2001; Wang, 2004). Only a few of these genes have been studied so far in germ cell neoplasms. Of particular interest is large family of the so-called ‘cancer/testis’ genes, most of them mapped to the X chromosome, which were given this name because—apart from germ cells—they were only detected in various somatic cancers, e.g. melanoma and breast cancer (reviewed in Scanlan et al., 2002). Two members of this family, MAGE-A4 and NY-ESO-1, are highly expressed at the protein level in normal fetal gonocytes at the transition period to infantile pre-spermatogonia, in adult spermatogonia as well as in a subset of CIS cells and germ cell tumours, including in spermatocytic seminoma but not in non-seminomas (Jungbluth et al., 2000; Aubry et al., 2001; Yuasa et al., 2001; Satie et al., 2002; Rajpert-De Meyts et al., 2003b). Such a pattern of expression is consistent with a physiological function of these genes in germ cells, in analogy to the above-mentioned germ cell-specific genes of the Y chromosome. The lack of expression of MAGE-A4 and NY-ESO-1 in non-seminomatous tumours is poorly understood but may be explained by differences in the genome methylation, which is much more pronounced in non-seminomas (Koul et al., 2002; Smith-Sorensen et al., 2002; Smiraglia et al., 2002; Honorio et al., 2003). The re-expression of cancer/testis genes in somatic tumours is probably also linked to changes in DNA methylation of promoter regions (Maio et al., 2003) but may be a result of other regulatory mechanisms. The X chromosome is the most tightly controlled in this aspect because of the need to compensate for the double dosage effect in females. The process is controlled by the X-inactivation centre, which produces the XIST transcript, which in turn triggers chromatin changes by Polycomb group proteins and DNA methylation (Csankovszki et al., 2001; Heard, 2004). In male germ cells, XIST is transcribed, but the X chromosome remains largely active. Interestingly, the XIST transcript is also over-expressed in testicular germ cell tumours and in CIS cells, perhaps partly because of a frequent increase in the copy number of X chromosomes in aneuploid neoplastic germ cells (Looijenga et al., 1997; Kawakami et al., 2003; Hoei-Hansen et al., 2004a).
Studies of the cell cycle and DNA repair are consistent with the pre-meiotic origin of CIS
Profound differences in the biology of germ cell neoplasms in comparison with the somatic tumours are undoubtedly related to a very special feature of germ cells—their ability to switch from mitotic cell division to the meiotic division, which is required for gamete formation. Regulatory mechanisms involved in the two types of cell division differ, and a number of studies provided evidence supporting the pre-meiotic origin of germ cell tumours, including CIS. Cell division is a final step in the cell cycle, which has to be exquisitely regulated to maintain the balance between proliferation and differentiation, a disturbance of this balance may lead to cancer or cell death. Closely related to the cell cycle regulation are the mechanisms of DNA repair, which are essential to prevent cell death or neoplastic transformation, especially in cells subjected to adverse environmental effects. Germ cells appear to have inherently high sensitivity to cytotoxic drugs and irradiation. This feature is further magnified in germ cell-derived tumours (reviewed in Masters and Koberle, 2003; Spierings et al., 2003). This is, of course, with great benefit for the patients with germ cell neoplasms, who can be efficiently treated by cisplatin-based regimens (Einhorn, 1997) or, in certain cases of isolated CIS, even by irradiation alone (Von der Maase et al., 1986). The processes of DNA repair are regulated differently in mitotically dividing immature germ cells during testicular development, and different mechanisms are specifically triggered when the meiotic division starts at puberty, because the meiotic crossover requires double-strand DNA breaks. As far as CIS is concerned, the evidence accumulated so far unequivocally demonstrates that a high expression of the key tumour suppressors involved in the DNA repair, such as p53 (Bartkova et al., 1991) and CHK2 (Bartkova et al., 2001), is a persistent developmental feature. Both proteins are abundant in normal fetal gonocytes (see p53 in Figure 2); p53 is then down-regulated in spermatogonia, whereas CHK2 remains highly expressed in spermatogonia but disappears at the onset of meiosis (Quenby et al., 1999; Bartkova et al., 2001; Rajpert-De Meyts et al., 2003b). A recent study demonstrated that after the onset of meiosis, a rapid activation of the ATM kinase takes place in spermatocytes to process multiple DNA double-strand breaks (Bartkova et al., 2005).
A wealth of evidence indicates that the G1/S-phase transition of the cell cycle is primarily controlled by the retinoblastoma protein (pRB) pathway, which is commonly involved in the pathogenesis of various malignancies (Mihara et al., 1989; Bartek and Lukas, 2001; Sherr, 2004; references therein). The pRB pathway regulation appears to be different in germ cells and deregulated in germ cell tumours but without structural aberrations (mutations) typical for somatic cancers (reviewed in Bartkova et al., 2003b). The observed changes are most likely due to a direct transcriptional regulation, an increased promoter methylation, or a more recently discovered regulatory mechanism by micro-RNAs (reviewed in Ambros, 2001; Zamore and Haley, 2005). As far as the CIS cells are concerned, the first interesting observation was the lack of pRB in CIS, seminoma and embryonal carcinoma, with a normal expression in teratomas (Strohmeyer et al., 1991b). This surprising finding is consistent with developmental regulation of pRB, which is apparently physiologically down-regulated in fetal gonocytes but active in mature spermatogonia (Bartkova et al., 2003a). As pRB is a tumour suppressor, the lack of pRB in fetal germ cells and CIS may render these cells more vulnerable to oncogenic stimuli but simultaneously also more prone to apoptosis (Bartkova et al., 2003b).
The second interesting feature of CIS and overt germ cell tumours is the over-expression of a protooncogenic cyclin D2 (encoded by CCND2 mapped to chromosome 12p), significance of which will be discussed below (Sicinski et al., 1996; Houldsworth et al., 1997; Bartkova et al., 1999; Schmidt et al., 2001). The third feature, important for our discussion on the origin of germ cell neoplasms in relation to the meiotic switch, is the lack of the cyclin-dependent kinase (CDK) inhibitor p19-INK4d in CIS and overt germ cell tumours. P19-INK4d is abundant in normal spermatocytes and detectable in spermatids but completely absent from fetal gonocytes (Bartkova et al., 2000). Similarly, cyclin A1—which was described in spermatocytes—has not been detected in CIS or seminomas (Liao et al., 2004). Taken together, the studies of the regulatory machinery of the cell cycle strongly support the origin of CIS from early fetal and pre-meiotic germ cells.
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Genomic aberrations in CIS: 12p or not 12p? |
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The question addressed soon after the discovery of a remarkable resemblance of CIS cells and fetal germ cells was whether CIS cell is a truly neoplastic cell or simply an immature gonocyte persisting in an adult testis. While substantial knowledge concerning genomic aberrations of the overt germ cell tumours was accumulated, the studies of CIS lagged behind, mainly because of technical difficulties due to a low number of CIS cells, their relatively low rate of proliferation (Höfken and Lauke, 1996
) and poor growth in culture (Rajpert-De Meyts et al., 1998b). Only after the advent of a new technology of the comparative genomic hybridization (Kallioniemi et al., 1992), the genome of CIS cells has been better characterized. A detailed overview of genomic aberrations in the germ cell neoplasms, including CIS, is beyond the scope of this article, therefore, the reader is referred to recent excellent review articles on this topic (Skotheim and Lothe, 2003; von Eyben, 2004). I shall discuss here only the aberrations that are probably the most informative with regard to the possible mechanism of neoplastic transformation, namely polyploidization and regional amplification of chromosome 12p.
Like nearly all neoplasms, CIS cells found in the adults are aneuploid with a mean DNA content in the hyper-triploid to hypo-tetraploid range (Skakkebæk, 1972; Müller and Skakkebæk, 1981; de Graaff et al., 1992). The longest lasting controversy concerned the presence in CIS of an isochromosome of the short arm of chromosome 12, i(12p), an aberration first described by Atkin and Baker (1982) and considered a hallmark of overt germ cell tumours (Castedo et al., 1988; Rodriguez et al., 1992; Van Echten et al., 1995a). Even in germ cell tumours without apparent presence of i(12)p, some amplification of the 12p material have been reported (Castedo et al., 1988; Rodriguez et al., 1993; Suijkerbuijk et al., 1993). The i(12)p has usually identical arms and is probably caused by an erroneous centromeric division during mitotic anaphase (Sinke et al., 1993). However, some loci on 12q in i(12)p-positive tumours retain heterozygosity, and thus polyploidization has to precede the formation of i(12p) (Geurts van Kessel et al., 1989).
The i(12p) in CIS was sporadically demonstrated by karyotyping (Vos et al., 1990; Van Echten et al., 1995b), but this has been disputed as the majority of the subsequent molecular studies did not detect genomic amplification of that region in CIS (Rosenberg et al., 2000; Summersgill et al., 2001). It was, therefore, proposed that the formation of i(12p) was not involved in the early pathogenetic process, but the relative gain of 12p sequences was associated with survival of CIS independently of Sertoli cells leading to their transformation to invasive tumours (Looijenga et al., 2003c). Our own study performed on the microdissected CIS cells by the comparative genomic hybridization added a missing link in this puzzle: we demonstrated that there indeed was no gain of 12p in two cases of CIS found as an isolated pre-invasive lesion, however, a clear genomic amplification in this region was detected in nearly all cases of CIS present in the vicinity of invasive tumours (Figure 4), suggesting clonal heterogeneity and possibly genomic instability of CIS cells (Ottesen et al., 2003). A subsequent analysis performed on CIS cells flow-sorted according to the DNA ploidy (Ottesen et al., 2004a) supported a hypothesis first suggested by Oosterhuis et al. (1989, 1990) that the polyploidization (tetraploidization) probably precedes the gain of 12p and other chromosomal aberrations. Some allelic losses detected in CIS resemble quite closely those in seminoma and, to a lesser extent, those in non-seminomas (Faulkner et al., 2000). However, the pattern of chromosomal aberrations/imbalances in overt germ cell tumours reported in numerous studies is quite similar despite morphological differences among germ cell tumour types (reviewed in Van Echten et al., 1995a; Skotheim and Lothe, 2003; von Eyben, 2004). A recent analysis of a large number of germ cell tumour karyotypes proposed that a multipolar cell division with non-disjunction of a tetraploid precursor cell, combined with some secondary imbalances/structural changes, is the most likely model of the karyotypic evolution of germ cell tumours (Frigyesi et al., 2004). Overall, genetic evidence gathered so far supports the progression of these tumours from a polyploid precursor cell, such as CIS (Oosterhuis et al., 1989, 1990), but the mechanisms of polyploidization remain to be elucidated.
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Why the gain of 12p is so interesting? A look at the list of genes located there explains that. A number of genes associated with pluripotency of ESC and human teratocarcinoma cell lines, e.g.. NANOG, STELLAR, DPPA-5 and GDF3 (Caricasole et al., 1998; Sato et al., 2003; Sperger et al., 2003; Clark et al., 2004; Skotheim et al., 2005), and with germ cell proliferation or increased survival, e.g. CCND2 and K-RAS (Sicinski et al., 1996; Houldsworth et al., 1997; Roelofs et al., 2000), are localized to the 12p region. This region constitutes also one of the hot spots of highly expressed genes in the profiling study of CIS (Almstrup et al., 2004). Interestingly, non-random gains of chromosomal material in the same region have been reported in human ESC maintained for a prolonged period in culture (Draper et al., 2004). That study, and a more recent investigation by Maitra et al. (2005), reported also non-random aberrations in cultured ESC in 17q, a region frequently rearranged in germ cell tumours (Kraggerud et al., 2002; Skotheim et al., 2002) where a cluster of genes highly expressed in CIS was detected as well (Almstrup et al., 2004). The observation of chromosomal aberrations in cultured ESC indicates that the microenvironment of growing ESC may be important for genomic stability. The molecular mechanisms are though poorly understood, and it is not known whether 12p and 17q are especially sensitive to chromosomal rearrangements. An alternative hypothesis is that the genome of CIS cells undergoes many random aberrations, and only the aberrations that render the cells better adapted to a changed microenvironment survive. This hypothesis postulates that the regions 12p, 17q and probably parts of X harbour genes with oncogenic potential, perhaps particularly oncogenic for germ cells. Some of the genes in these regions are indeed highly expressed in CIS cells, and we listed these candidate genes in a recent review article (Almstrup et al., 2005). I speculate that a similarity between ESC and CIS could indicate that CIS cells perhaps may originate from PGCs or gonocytes through a similar mechanism of ‘natural selection’ of cells that adapted themselves to their disturbed microenvironment in the developing gonad. How the development of the early gonad may be disturbed is the matter discussed in the remaining part of this review.
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Who is at risk for germ cell cancer? The importance of prenatal events and the concept of testicular dysgenesis syndrome |
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Conditions associated with germ cell cancer and factors which increase the risk of this cancer are numerous and surprisingly variable. A systematic and critical analysis of clinical epidemiology of testicular cancer was recently published by Dieckmann and Pichlmeier (2004)
. Here, only a partial list of the best documented risk factors is listed in Table II, and a short summary of this topic is presented, mainly to illustrate the concept of the testicular dysgenesis syndrome (TDS).
Severe but relatively rare genetic abnormalities which cause testicular dysgenesis and the intersex syndrome (e.g. 45X/46XY and androgen insensitivity) are associated with a high risk of testicular cancer, often in combination with undescended testis and hypospadias (Aarskog, 1970; Scully, 1981; Savage and Lowe, 1990). Skakkebæk was the first to notice CIS in the dysgenetic testes of children with the intersex syndrome (Skakkebæk, 1979; Müller and Skakkebæk, 1984; Müller et al., 1985). Subsequently, several reports described the presence of CIS or gonadoblastoma in dysgenetic gonads of subjects with various forms of the intersex syndrome with or without structural aberrations of chromosomes (Cassio et al., 1990; MacMahon and Cussen, 1991; Rutgers and Scully, 1991; Jacobsen and Henriques, 1992; Ramani et al., 1993; Slowikowska-Hilczer et al., 2001; Slowikowska-Hilczer et al., 2003). In addition to linking gonadal dysgenesis with germ cell neoplasia, these observations support the notion that CIS and CIS-derived germ cell tumours may occur in the pre-pubertal testes and speak against an alternative hypothesis that the post-pubertal zygotene–pachytene spermatocyte is the cell of origin for CIS (Chaganti and Houldsworth, 2000).
Among more common urogenital abnormalities, cryptorchidism (undescended testis) is the best documented risk factor for testicular neoplasia, including CIS (Campbell, 1942; Morrison, 1976; Krabbe et al., 1979; Batata et al., 1982; Giwercman et al., 1989; Prener et al., 1996; Coupland et al., 1999; Weir et al., 2000). A recent meta-analysis evaluated the relative risk (RR) of testicular cancer in subjects with a history of cryptorchidism as 4.8 (95% CI = 4.0–5.7) (Dieckmann and Pichlmeier, 2004). There is also evidence for an association between testicular cancer and inguinal hernia or hypospadias (Morrison, 1976; Klein et al., 1996; Prener et al., 1996). Testes in cases with congenital urogenital malformations often are associated with some degree of maldevelopment, including clusters of poorly differentiated Sertoli-cell-only tubules and hyaline bodies (Sohval, 1954; Huff et al., 1993). More conspicuous but surprisingly common are histological signs of poor testicular development and function in adult patients with sporadic testicular tumours (Sohval, 1956), even in the seemingly ‘normal’ contralateral testes in patients with unilateral testicular cancer (Berthelsen and Skakkebæk, 1983; Hoei-Hansen et al., 2003). The degree of differentiation of Sertoli cells in adults with testicular cancer is variable depending on the grade of dysgenesis, but even morphologically immature Sertoli cells in most cases with complete spermatogenesis present elsewhere in the testis do not retain expression of the anti-Müllerian hormone, which is highly expressed before puberty (Rey et al., 1996; Rajpert-De Meyts et al., 1999). Hyaline bodies are frequently (but not always) seen on the ultrasound as testicular microlithiasis (reviewed in Holm et al., 2001). An association of microlithiasis with CIS and even testicular masses in the contralateral testis is so common that this ultrasonic abnormality should alert the attending physician about a possibility of testicular neoplasia, especially in patients with atrophic testes (Bach et al., 2003; Holm et al., 2003; de Gouveia Brazao et al., 2004).
Several studies documented that men with testis cancer had significantly reduced fertility before the development of their tumour, with a lower proportion of male children (decreased offspring sex ratio), and abnormal semen characteristics (Berthelsen and Skakkebæk, 1983; Møller and Skakkebæk, 1999; Jacobsen et al., 2000a,b; Richiardi et al., 2004c). On the contrary, men with subfertility have often a history of genital malformations and may harbour histological signs of testicular maldevelopment, including CIS, thus confirming an association between these conditions (Skakkebæk et al., 2003). Furthermore, an analysis of risk factors, such as low birthweight or intrauterine growth retardation (Depue et al., 1986; Morley and Lucas, 1987; Francois et al., 1997; Cicognani et al., 2002; English et al., 2003), suggested that the pathogenesis might be, at least partially, shared by germ cell tumours, cryptorchidism and male subfertility. Recently, a Norwegian study of risk factors for hypospadias found also, among others, a low birthweight and inguinal hernia (Aschim et al., 2004a). The epidemiological associations outlined above constituted the basis for a hypothesis of an aetiological link between the male reproductive disorders that are associated with impaired testicular development, within the so–called TDS presented schematically in Figure 5 (Skakkebæk et al., 2001; Asklund et al., 2004). The assumption that prenatal or perinatal factors are responsible for growing incidence of germ cell cancer and TDS is additionally corroborated by the birth cohort effects, meaning that the epidemiological trends are associated with the year of birth, and each subsequent cohort is more affected that the previous one. A birth cohort effect was, e.g., demonstrated for a decline in sperm concentrations of Scottish men (Irvine et al., 1996), one of the studies that followed the report on the possible decline of semen quality worldwide (Carlsen et al., 1992). One exception to the rule of the consecutive decline, which at the same time is a striking example of a birth cohort effect, was an unexplained decrease of the prevalence of testicular cancer among Scandinavian men born during wartime (Møller, 1993; Bergström et al., 1996).
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A strong corroborating evidence for the TDS concept—which simultaneously incriminates environmental factors—is the geographical association between various components of TDS. A very illustrative example is given by the comparison of the rates in Denmark and in Finland, and another nearby located Nordic country. The incidence of testicular cancer, which is high in Denmark, is markedly lower in Finland (Adami et al., 1994; Richiardi et al., 2004a). Studies of the incidence rates of testicular cancer in populations migrating from these two countries to Sweden, which is located in between, clearly demonstrated that the first generation immigrants retained the incidence as in their country of origin, whereas the second generation (born in Sweden) had the risk of testicular cancer similar to native Swedes (Hemminki and Li, 2002). Studies of semen quality found also all parameters better in Finland than in Denmark (Jensen et al., 2000; Jørgensen et al., 2001, 2002). The differences in rates of congenital genital malformations seemed also to be different, but less certain because of problems with definition and registry data (reviewed in Toppari et al., 2001). Data from other countries were confusing with some reporting an increase while other argued for a possible decline in cryptorchidism rates (Chilvers et al., 1984; Paulozzi, 1999; Toledano et al., 2003). Therefore, coordinated prospective studies of genital malformations have been launched in cohorts of infants, providing most telling evidence for the difference in the rates of cryptorchidism and hypospadias at birth in Denmark versus Finland (Boisen et al., 2004, 2005). At the same time, the Boisen et al. (2004) study demonstrated an increase of the incidence of cryptorchidism in Denmark over time (Buemann et al., 1961).
Geographical and ethnic differences have been noted much earlier for testicular cancer in other countries of the world, with unexplained high prevalence among Caucasians living in well-developed countries and notably lower prevalence among men of African descent and Asians, even inhabiting the same countries (English et al., 2003; Huyghe et al., 2003). The obvious question that arises is whether the reasons for the geographic and temporal differences in the prevalence of TDS are because of environmental differences or genetic variation/predisposition?
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