Histogenesis

Gonadal Germ Cell Tumors

There is now a consensus that seminomas and ter-atomas (or NSGCTs) arise from germ cells, based on their morphologic, histochemical, functional, and ultrastructural features. However, the precise stage of development of the germinal tissue that gives rise to germ cell tumors remains uncertain. Electron microscopy shows that seminomas are composed of a broad spectrum of cells with features of spermato-cytes, spermatogonia, and undifferentiated cells, and it has thus been proposed that seminoma is a tumor of seminiferous epithelium, arising from stem cells that are committed to spermatocytic differentiation.8

In opposition to the generally accepted concept of a germ cell origin of classic seminoma, Masson ascribed the features of the precursors of spermatozoa to spermatocytic seminoma and suggested that this variant was derived directly from these cells.9 Today, the consensus is that classic seminoma is derived from primordial germ cells and that the origin of spermatocytic seminoma is different (but as yet undefined). It is likely that spermatocytic semi-noma also originates from the germ cell but perhaps at a different stage of embryologic development.

The histogenesis of malignant teratomas has been a more controversial subject although it is now agreed that these varied tumors also arise from germ cells (Figure 1-3); this has led to their designation as NSGCTs. Their unusual histologic appearance, with a range of tissues of varied differentiation and with the presence of elements of up to three germinal layers, initially led to many different theories of histogenesis. For example, in 1926, Budde10 suggested that the formation of teratomas was the result of a malfunction of the primary embryonic organizers, with cells being released from normal developmental control at the primitive stalk stage. It was also suggested that ter-atomas represented suppressed twins or were caused by dysfunction of the cellular organizers during embryogenesis. Willis11 showed that teratomas lack somatic distribution of tissues and that there is no organization in relation to the spinal column, thus discounting these theories, but he did believe that blas-tomeres, displaced in early embryonic development and having escaped the influence of cellular organizers, give rise to teratomas. However, for many years, the British school of pathologists viewed seminomas and teratomas as having distinct histogenetic origins.12

embryonal cell carcinoma (MTU)

(with/without hCG containing tumor cells)

teratomas with somatic differentiation (MTI, TD)

Figure 1-3. Schema of histogenesis of germ cell tumors. Note interrelationships between pathways of development of seminoma and nonseminomatous germ cell tumor (NSGCT). 1, Origin of seminoma from an atypical germ cell. 2, Overlap of seminoma and solid variant of yolk sac carcinoma (morphologic?/functional?). 3, Concurrent evolution of seminoma plus STGC. 4, Developmental linkages (potentially reversible) between variants of NSGCT. 5, Atypical germ cell giving rise directly to NSGCT. 6, Potential for seminoma to be the originator of all tumor types, as intermediary after rise of atypical germ cells. (Reproduced with permission from Raghavan D, Neville AM.1)

embryonal cell carcinoma (MTU)

(with/without hCG containing tumor cells)

teratomas with somatic differentiation (MTI, TD)

Figure 1-3. Schema of histogenesis of germ cell tumors. Note interrelationships between pathways of development of seminoma and nonseminomatous germ cell tumor (NSGCT). 1, Origin of seminoma from an atypical germ cell. 2, Overlap of seminoma and solid variant of yolk sac carcinoma (morphologic?/functional?). 3, Concurrent evolution of seminoma plus STGC. 4, Developmental linkages (potentially reversible) between variants of NSGCT. 5, Atypical germ cell giving rise directly to NSGCT. 6, Potential for seminoma to be the originator of all tumor types, as intermediary after rise of atypical germ cells. (Reproduced with permission from Raghavan D, Neville AM.1)

Askanazy, in 1907, was the first to propose that totipotential undifferentiated cells could undergo a metamorphosis to produce teratomas.13 Several clin-icopathologic studies in the following half century supported this fundamental concept,11416 as did a series of preclinical studies (see below). In particular, support for the germ cell origin of testicular cancer has been provided by the demonstration of a preinvasive intratubular carcinoma in situ, or "atypical germ cell."1718 These atypical cells, which differ from normal germ cells in their increased size, irregular chromatin patterns, increased deoxyribonucleic acid (DNA) content, and mitotic index,19 have been found in infertile males,19,20 in the contralateral testes of patients with germ cell tumors,21 in cryp-torchid and ectopic testicles,22 and in "normal" tissue adjacent to germ cell tumors.23

Friedman believed that the germinoma (semi-noma) was the precursor of undifferentiated malignant teratoma (embryonal carcinoma), which, in turn, gave rise to malignant teratomas with varying degrees of somatic differentiation and to tumors with extraembryonic differentiation.24 The frequent coexistence of elements of seminoma and NSGCT within extragonadal germ cell tumors is often interpreted as support for commonality of origin, but Dixon and Moore14 noted the absence of transition stages in these sites and suggested that this favored parallel origins for the two elements. However, transitional morphologic patterns were subsequently identified; for example, Friedman and Pearlman described the "seminoma with trophocarcinoma," a classic seminoma with widely dispersed small foci of embryonal carcinoma, which manifested radioresistance and had an intermediate prognosis between those of seminoma and embryonal carcinoma.25 This seems to reflect a phenomenon similar to that found in studies (that my colleagues and I conducted) of a solid variant of yolk sac tumor with AFP production that morphologically resembles classic seminoma, as noted above.2

Similarly, the place of anaplastic seminoma in the histogenetic scheme of testicular cancer is not clear. This pattern is less differentiated than classic seminoma, but there is considerable controversy as to whether it actually has a different natural history, therapeutic responsiveness, and prognosis26-29 (see also Chapters 18 and 19). Given the variability of criteria used to define this entity, it is likely that anaplastic seminoma actually represents a series of different tumors, from classic seminoma with reduced differentiation through the spectrum to embryonal carcinoma. This entity further supports the germ cell theory of the origin of these tumors.

Ultimately, it appears that the primordial germ cell gives rise to an atypical germ cell (or carcinoma in situ) that is associated with most types of testicu-lar tumors. Primordial germ cells are first seen in the gastrula,30 and they subsequently migrate to the genital ridges via the endoderm (Figure 1-4). In humans, they develop within the gonad early in the first trimester, giving rise to spermatogonia during the second and third trimesters. The spermatogonia then undergo a series of mitoses that produce type A, intermediate, and type B spermatogonial cells. The type B cells go on to form primary spermatocytes after further replication and meiosis and eventually produce four haploid cells that form spermatids and spermatozoa. Many of these cells die, and the process of apoptosis is prominently identified, both in the mouse and in humans. The factors that control evolution to malignancy or, alternatively, that lead to apoptosis and limitation of the process of germ cell division have not yet been defined. Several genes have been identified as being involved somehow in

Figure 1-4. The migration of germ cells from the endoderm to sites of genital ridges and the aberrant pathway of migration that leads to the development of extragonadal germ cell tumors. (Reproduced with permission from Raghavan D, Neville AM.1)

this process, including aberrations of chromosome 12, mutations of c-kit, and (possibly) expression of the D2 cyclins (see below).

Extragonadal Germ Cell Tumors

Although extragonadal germ cell tumors (EGCTs) are histologically identical to germ cell tumors derived from primary gonadal sites, their biology is substantially different, for reasons that are not clear. Initially, it was suggested that EGCTs arise from a local dislocation of tissues during embryogenesis, with neoplasia developing in primitive rests of totipotential cells left during the blastular or morular phase.31 Analogous to the theory of the origins of testicular germ cell tumors, this theory was subsequently discounted and replaced by a theory of germinal origin.24 The basic concept of the evolution of EGCTs is that they are derived from germ cells with an aberrant path of migration from the yolk sac to the genital ridges (see Figure 1-4). During the first trimester of pregnancy, germ cells normally migrate from the yolk to the genital ridges, leading ultimately to the formation of the gonads.

In the instance of EGCTs, it is postulated that these cells have an aberrant path of migration in the midline,32 as a consequence of which they lodge in the pineal gland, mediastinum, retroperitoneum, or sacrococcygeum.33 It has been proposed that this migration may be triggered by c-Kit and stem cell factor receptor-ligand interactions.34 A contrasting view (because of the chromosomal similarities) is that EGCTs actually represent tumors derived by retrograde migration of carcinoma in situ (ClS)-type lesions from the genital ridges or evolving gonads.35

EGCTs have been shown to have identical histo-logic patterns and production of tumor markers,33 as well as the replication of chromosome 12 or the elaboration of the isochromosome 12p marker (at least in the adult patient).36 However, nonseminoma-tous EGCTs appear (stage for stage) to be much more resistant to therapy although this does not seem to apply to the seminomatous or germinoma variants.33 In children, EGCTs appear to have different genetic characteristics, with alterations of chromosomes 1 and 6 and usually an absence of changes of chromosome 12p.37 It may well be that infantile and adult EGCTs develop from germ cells at different stages of their development.

Schneider and colleagues examined germ cell tumors (GCTs) of gonadal and extragonadal origin to assess whether loss of imprinting reflects origin from different stages of germ cell development.38 They studied DNA methylation of CpG dinucleotides as this represents the most significant biochemical marker of imprinting and allows the identification of maternal and paternal alleles. These workers hypothesized that primordial germ cells erase their inherited imprint and establish a new gender-specific imprinting pattern. If GCTs preserve the original imprinting status of their cell of origin, it would be reasonable to assume that GCTs arising from primordial germ cells before entry into the gonadal ridges and GCTs arising from premeiotic germ cells show erased imprinting; by contrast, if GCTs arise from primordial germ cells that have already entered meiosis (in the genital ridges), they should display a gender-specific gametic imprinting pattern.38 These studies showed that (1) both gonadal and extrago-nadal GCTs share a common cell of origin, the primordial germ cell, that has already erased its imprinting; (2) the pattern of erasure is different between adult and infant patients; and (3) ovarian and extragonadal teratomas may arise from germ cells that are more advanced in their development.

MODELS OF GERM CELL DIFFERENTIATION: ONTOGENESIS AND ONCOGENESIS

Murine Germ Cell Evolution

Murine spermatogenesis potentially offers a model of cellular regulation in germ cell neoplasia. During mouse embryogenesis, primordial germ cells migrate from the yolk sac through the hindgut and dorsal mesentery to the genital ridges, where they are seen by the eleventh day of gestation. During migration, they undergo mitosis, and their number increases from around 100 to about 25,000.39 While incomplete, some data are available regarding molecular factors regulating this process. It appears that BMP4 is required for the generation of primordial germ cells40 and that OCT4 may be required in the establishment of their multipotentiality.41

The level of mitotic activity decreases after arrival at the genital ridges, and differentiation of the gonad begins. Subsequently, spermatogonia proliferate, and some undergo meiosis, producing haploid spermatids. These give rise to spermatozoa. Several different types of spermatogonia have been identified although the regulation of their proliferation and differentiation has not been clearly defined. In this context, it should also be noted that there are important differences between the development of germ cells in the female and in the male. Female germ cells proliferate by mitosis and enter meiosis in the embryo, arresting in prophase of the first meiosis and remaining dormant until puberty. At that time, the first meiotic division is completed, but growth is again arrested at the second metaphase, until fertilization.42 However, in males, the germ cells undergo a few mitotic divisions in the embryo and then remain dormant until puberty, when the spermatogonial stem cells divide by mitosis and then commence meiosis, culminating in the formation of haploid spermatids.

In the mouse, the presence of two mutant alleles initially identified as causing coat color mutations, at either the dominant-white spotting (W) locus (producing the c-Kit receptor) or the Steel (SI) locus (which controls the stem cell factor or c-Kit ligand), can cause sterility, marrow hypoplasia, depletion of mast cells, and reduction of melanocytes.43 As noted elsewhere in this chapter, c-Kit is a member of the type III receptor tyrosine kinase family; interaction of the c-Kit receptor with the Steel factor leads to activation of tyrosine kinase and phosphorylation of the receptor, which allows binding to sulfhydryl moieties.44 The mutation in the dominant-white spotting (W) locus leads to a defect in precursor cells that leads to the depletion of differentiated germ cells whereas the mutation of the Steel gene causes a defect in the tissue microenvironment, perhaps through a defect in the MGF gene, which encodes a growth factor similar to epidermal growth factor (EGF).45 Mutation of the Sl gene also leads to depletion of germ cells. Sertoli's cells produce stem cell factor in the testis whereas c-Kit is expressed on several evolving stages of spermatogonia (but not on the undifferentiated type A spermatogonia).46 This process can be studied by a technique in which donor mouse germ cells are injected into seminiferous tubules; when spermatogenesis results, the recipient mouse transmits the donor haplotype to the offspring.43 Such studies have confirmed the expression of c-Kit in evolving stages of spermatogenesis (with the exception of undifferentiated type A sper-matogonia) and suggest that stem cell factor may be a prerequisite for the maintenance of c-Kit-positive differentiated germ cells.43 Orr-Urtreger and colleagues showed that c-Kit is expressed in primordial germ cells before and after migration into the genital ridges during embryogenesis.47 The model developed by Ohta and colleagues also suggests that the undifferentiated type A spermatogonia in Sl-mutant testes may be the putative stem cells that can proliferate and differentiate.43 Potten and Loeffler48 proposed that these stem cells occupy a special environmental site, or niche, generated by adjacent cells, which facilitates self-renewal, and it appears that this niche may reside physically in the surrounds of the basement membrane of the testis. It may be no accident that atypical germ cells (carcinoma in situ) are characterized by a migration away from the basement membrane.

However, the process is complex, especially in a context in which the series of observations from Stevens and colleagues suggests that it is unlikely that simple inactivation of the MGF/c-kit pathway leads to increased susceptibility to germ cell tumors per se.54 It has been suggested that an additional relevant gene may reside in the region of the Sl deletions. This region of murine chromosome 10 is homologous to the critical 12q22 chromosomal locus in humans.

Also of interest, the interactions between the spermatogonial precursors and the Sertoli's cells are very similar to the interactions between the hem-atopoietic stem cells and the surrounding stroma.43 Hematopoiesis may be mediated by the interaction of c-Kit with a transmembrane 4 superfamily protein complex and integrin family proteins.49 Steel factor is a potent co-stimulating cytokine that participates in growth stimulation of the hematopoietic progenitors. The similarity of these interactions between stem cells and the microenvironment may explain the occasional overlap of leukemia and germ cell malignancy in the same patient.

Another set of potential regulators of mammalian spermatogenesis are the A-type cyclins.50 Cyclins and cyclin-dependent kinases (CDKs) have important roles in DNA synthesis and cell division, functioning variously at Gi-S and mitosis.51 There are two vertebrate cyclin genes, cyclin A1 and cyclin A2, which have different patterns of expression; cyclin A1 functions in germline and early embryonic cells, and cyclin A2 functions in somatic cells.52,53 Immunohistochemical studies have identified cyclin A1 only in male germ cells prior to or during the first meiotic division but not associated with the second division. Cyclin A2 ribonucleic acid (RNA) has been identified in murine testicular germ cells, including spermatogonia and preleptotene sperma-tocytes (which are destined for meiosis) and in both ovarian germline and somatic cells.50 Cyclin A1 binds both cyclin-dependent kinases 1 (CDK1) and 2 (CDK2), but cyclin A2 binds only CDK2.50 Although it should not be forgotten that the mere identification of messenger RNA (mRNA) does not necessarily have functional implications, these data suggest different functions for cyclin A1 and cyclin A2 in the initiation of meiosis for germ cells and in the regulation of germ cell evolution. The marked differences in cyclin A1 and A2 expression during spermatogenesis may thus provide a model for the exploration of the regulation of the different types of germ cell tumors (Figure 1-5).

Teratomas in the 129 Strain Mouse

The 129/Sv inbred strain mouse model is characterized by a susceptibility to spontaneous germ cell tumors, with an incidence of up to about 10% in mice that are 3 to 4 weeks old. This model, developed by Leroy Stevens, was initially used to characterize the biology of teratomas and embryonic stem cells.54 55 Analogous to human NSGCTs, 129 strain murine teratomas are composed of disorganized aggregations of malignant cells arranged in sheets, rosettes, and organoid formations although the pattern varies with the age of the mice.55 For example, in fetal and neonatal mice, the tumors are composed predominantly of undifferentiated sheets of embryonal carcinoma whereas adult mice have tumors composed predominantly of differentiated tissues.

Kleinsmith and Pierce cloned these cells and demonstrated their multipotentiality by showing variable differentiation after their injection into different somatic sites.15 Pierce and colleagues initially demonstrated in this system the presence of embry-oid bodies (aggregates of embryonal carcinoma surrounded by mesenchymal and endodermal cells) that resembled mouse embryos.56 These aggregates, when transplanted subcutaneously, gave rise to ter-atomas and teratocarcinomas, also supporting the potential reversibility of the malignant process in this model. Mintz and Illmensee provided added evidence regarding the potential reversibility of malignancy by producing phenotypically normal but genetically mosaic mice by injection of 129 strain teratocarcinoma cells into murine blastocysts.57

Stevens55 demonstrated a time dependence of the ability to form GCTs by grafting genital ridges into ectopic sites, leading to the concept that the maximal time of tumorigenesis in this system is around the twelfth day of gestation. In addition, Stevens demonstrated the importance of mutation of the Steel (Sl) gene in this system: mice that are homozy-gous for mutation of this gene lack primordial germ cells and consequently fail to develop these murine teratomas. Sexual differentiation is also critical, as germ cell tumors arise in males in this model, perhaps because of the differences in the evolution of normal germ cells (see above) or perhaps because of hormonal factors. Also of relevance, primordial germ cells are still undergoing mitosis at the eleventh and twelfth days of gestation, but this decreases substantially after that. This time of maximum mitotic activity parallels the time at which GCTs arise maximally. Of relevance, the 129/Sv Ter strain, in which there is a very high incidence of GCTs, has an abnormally prolonged period of mitotic activity.58

Several genes and resulting proteins with involvement in tumorigenesis in other systems (such as tumor necrosis factor, transforming growth factor-P, and basic fibroblast growth factor ) are involved in germ cell proliferation and are candidates for being involved in the genesis of murine GCTs (see also below). Of particular interest, mutant genes that appear to be involved with susceptibility to murine GCTs include Ter, Steel, and TCGT1, and specific involvements have been mapped to chromosomes 13 and 19.58-60 The Ter mutation increases the risk of 129 strain mice developing GCTs, with more than 90% of Ter/Ter male mice being affected. Ter/Ter mutant mice also show a deficiency of primordial germ cells, but they also continue to exhibit prolonged mitosis, which suggests that normal Ter protein may modulate mitotic arrest.

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