GCTs have a fascinating biology which relates to the pluripotent nature of their cell of origin, the developing germ cell. A brief review of male gonadal development, spermatogenesis, and embryogenesis can be helpful to understanding the biology and range of histologies seen in GCTs. Arising from the embryonic ectoderm, the primordial germ cell (PGC) is first recognized during gastrulation based on its expression of alkaline phosphatase [34]. PGCs subsequently migrate to the genital ridge, where they further develop into gender-distinct gonads, a process dependent upon the presence (male) or absence (female) of stromal expression of the SRY gene, located on the Y chromosome. In males, PGCs in combination with Sertoli cells form seminiferous cords which, along with Leydig cells, subsequently organize into the embryonic gonads by about 2 months of gestational age. PGCs differentiate into gonocytes, which then cease proliferation until after birth. Postnatally, gonocytes begin proliferating again, and mature into undifferentiated Type A spermatogonia by about 3 months of age. Prior to and culminating with the initiation of puberty under stimulation from gonadotrophins, Type A spermatogonia mature into Type B spermatogonia. In AYA men, Type A spermatogonia are postulated to comprise the gonadal stem cells, existing as either Type Ad (dark type), a non-dividing germ cell reserve in case of destruction or loss, or Type Ap (pale type), an actively dividing, possibly self-renewing germ cell population. Following a single mitotic division, Type B spermatogonia become primary spermatocytes, which in turn undergo DNA replication and then two meioses, ultimately resulting in 4 haploid gametes. Spermiogenesis ensues, leading to the formation of mature spermatozoa.

GCTs arise when developing germ cells undergo malignant transformation. The earliest recognizable abnormal histology during this transformation is ITGCNU which is thought to represent the non-invasive precursor to all GCTs [35]. Upon becoming invasive, GCTs are separated into the two histologic categories, seminoma and nonseminoma.

Seminomas are more similar to ITGCNU than nonseminomas, morphologically resembling undifferentiated spermatogonial germ cells and expressing proteins common to germ cells in early development such as placental alkaline phosphatase (PLAP), KIT, and POU5F1 (OCT3/4) [36–38]. Seminomas typically display low mitotic and apoptotic rates and tend to be clinically diagnosed in Stage I (limited to the testis), often curable with orchiectomy alone [1]. Nonseminomas include four distinct histologies (embryonal carcinoma [EC], yolk sac tumor [YST], choriocarcinoma [CC], and teratoma [T]), each of which parallels a different stage of embryonic or extraembryonic development and differentiation. Normal germ cells destined to become gametes are subject to inhibitory signaling that prevents them from undergoing differentiation until fertilization with ova is achieved. Thus, nonseminoma formation can be explained by germ cells undergoing reprogramming during malignant transformation resulting in the acquisition of the capacity for embryonic and extraembryonic differentiation, although in a spatially and temporally aberrant manner. In vitro evidence supports this view as normal PGCs isolated from the mouse and human can be converted into pluripotent cells (embryonic germ cells) following exposure to KIT ligand (stem cell factor), leukemia inhibitory factor (LIF), and basic fibroblast factor (bFGF) [39]. Epigenetic modifications such as DNA methylation and chromatin acetylation may play a role in the reprogramming process [40]. In contrast, SGCTs lack the ability to initiate differentiation.

Differentiation of EC along embryonic (T) or extraembryonic (YST, CC) pathways leads to the decline of pluripotency of the transformed germ cell paralleling the process in embryonic development. For example, expression of POU5F1 in EC is downregulated in T, CC, and YST. [41] EC is considered the malignant counterpart of an early embryo and is pluripotent. ECs display the highest mitotic and apoptotic rates of any GCT histology and have been demonstrated to be genetically similar to embryonic stem cells [42]. The normal zygote at this stage is comprised of an inner cell mass surrounded by trophectoderm. The inner cell mass gives rise to the fetal tissues and the extraembryonic endoderm, whereas the trophectoderm gives rise to the placenta, consisting of an outer syncytiotrophoblast layer and an inner cytotrophoblast layer. CC represents malignant transformation of the placenta, and by definition must contain both the syncytiotrophoblast and cytotrophoblast layers. In contrast, malignant syncytiotrophoblast cells that appear in the absence of cytotrophoblast cells are not considered CC and in fact, can occur in combination with seminoma in the absence of any other nonseminoma component; indeed, they are still considered to be pure seminomas. CCs, like the placenta, produce HCG and are highly vascular in nature.

As normal embryological development continues, the morula undergoes repeated cell divisions and eventually the inner cell mass separates into two layers, an outer epiblast, which gives rise to the three fetal tissue layers (ectoderm, mesoderm, endoderm), and an inner hypoblast, consisting of extraembryonic endoderm, which forms the yolk sac. In the embryo, the yolk sac serves as the initial hematopoietic organ as well as a source of protein synthesis and nutrient transport. Differentiation of malignant germ cells along the yolk sac lineage leads to formation of YST, also known as endodermal sinus tumor. YSTs typically express AFP but not HCG in contrast to CCs, which express HCG but not AFP. EC, as a pluripotent neoplasm, is capable of differentiating into either of these tumor types, and can express both HCG and AFP.

Sperger and colleagues demonstrated the genetic similarity between EC and embryonic stem cells (ESCs) by comparing gene expression signatures of human ESC lines, EC cell lines and primary tumors, yolk sac tumor cell lines and primary tumors, seminomas, somatic cell lines, and normal testis. Upon hierarchical clustering, ESC lines clustered closest with EC tumors as compared to any of the other cell lines or tissues [42]. The original definition of ESCs was based on the expression of specific genes associated with pluripotency regulation including FGF2, POU5F1, THY1, SOX2, EBAF1, ZFP42, and TDGF1. Studies from our lab supported the similarity between ESCs and ECs; we demonstrated all of the aforementioned genes to be expressed by ECs whereas seminomas lacked expression of SOX2, FGF4, EBAF1, and TDGF1 [43, 44]. These data are consistent with the notion that SOX2, FGF4, EBAF1, and TDGF1 play a specific role in pluripotency. Furthermore, the transcription factors known to be important for maintenance of the undifferentiated state, such as POU5F1 (OCT3/4) and NANOG, were upregulated in both ECs and seminomas [43, 44].

Teratomas display somatic differentiation of the three tissue layers of the embryo. Typically, two or three of these layers are represented in a given teratoma. Differentiation can be complete, appearing identical to adult tissue types in the case of mature T, or incomplete, resembling fetal tissue in the case of immature T. Both mature and immature Ts tend to have low rates of mitosis and apoptosis, although this can be more variable in the case of immature Ts. On occasion, Ts can undergo malignant transformation, developing into a secondary somatic malignancy derived from a particular T tissue type. Common secondary somatic malignant histologies include rhabdomyosarcomas, adenocarcinomas, and primitive neuroepithelial tumors (PNET). Tumors that recur after prolonged remissions, known as late relapses, and mediastinal primary nonseminomas tend to have a higher propensity to undergo malignant transformation.

The most frequent nonseminomatous histology is a mixed form, comprised of more than one component (e.g., EC plus T) or a combination of a nonseminoma component with a seminoma [1, 9]. The most common pure nonseminoma histology is EC [1, 9]. Regardless of histologic subtype, a hallmark of all seminomas and nonseminomas is the presence of increased copies of 12p, usually as an isochromosome, (i[12p]) [45].

Although it is widely agreed that GCTs arise from malignant transformation of germ cells along their development, there is disagreement over the precise time point at which this occurs. Two models have been proposed. One, proposed by Skakkebaek and colleagues [46], postulates that PGCs or gonocytes, while still in utero, but after reaching the genital ridge, initiate abnormal cell proliferation under the direction of KIT pathway activation, leading to ITGCNU. This premalignant lesion remains dormant until puberty when, under stimulation from gonadotrophins, undergoes further transformation, acquires extra copies of 12p, and evolves to an invasive GCT. As such, this theory supposes that ITGCNU precedes acquisition of i[12p] and is supported by common expression patterns between PGCs/gonocytes and ITGCNU, of genes such as PRDM1/BLIMP1 and PRMT5, as well as the observation that not all ITGCNU may contain extra copies of 12p [47, 48]. In addition, epidemiologic data and the characteristics of germ cells in developmental abnormality syndromes that predispose patients to GCT such as testicular feminization and testicular dysgenesis, are cited in support this hypothesis. Another model, proposed by Chaganti and Houldsworth [49], suggests that the zygotene/pachytene spermatocyte with a 4n DNA content is the cell of origin. The error-prone homologous recombination at this stage of germ cell development allows acquisition of increased 12p copy number, which leads to aberrant gene expression, increased mitosis, re-establishment of pluripotentiality, and genomic instability that support malignant transformation to GCT. Evidence supporting this theory includes the shared chromosomal aneuploidy between GCTs and zygotene/pachytene spermatocytes and the abundant expression of wild-type p53, a hallmark of germ cells and GCTs. However, neither hypothesis has been experimentally validated.

Adult GCTs and testicular cancer have become synonymous since more than 95 % of testicular cancers are GCTs and more than 90 % of GCTs originate in the testis. Nevertheless, 5–10 % of GCTs arise from extragonadal locations, with the most common sites including the mediastinum, retroperitoneum, and the pineal gland. With the exception of tumors that arise in the pineal gland and show a predominance of seminomatous histology (often referred to as germinomas), the majority of extragonadal GCTs are nonseminomas [5]. The concept of retroperitoneal primary tumor remains controversial as many believe these cases to represent metastatic lesions from testicular tumors that were not able to be identified by ultrasound or at orchiectomy. Changes in the testicular parenchyma where a tumor has undergone spontaneous regression are referred to as having a “burnt out” appearance and could explain the failure to identify a gonadal primary tumor in some cases of solitary retroperitoneal GCT masses [50, 51].

Two mechanisms have been proposed to explain how GCTs of extragonadal primary sites other than the retroperitoneum arise. The conventional hypothesis suggests that PGCs or gonocytes get “left behind” while migrating through the embryo to the genital ridges and eventually transform. While this model is easy to conceptualize, misplaced germ cells at the PGC or gonocyte stage have never been identified in developing human embryos. Such cells have been observed in mouse embryos but are not viable due to a predilection to rapid apoptosis [52]. Finally, extragonadal GCTs have been identified to have chromosomal changes highly similar to gonadal GCTs with increased 12p copy number and aneuploidy [45]. These alterations are thought to be acquired later in GC development (meiosis of primary spermatocytes) than during the migratory stage of gonocytes, raising doubt to this theory and instead supporting a common cell of origin for gonadal and extragonadal GCT. An alternative explanation involves the potential of transformed germ cells to undergo reverse migration to the mediastinum or pineal gland, where stromal environments left over from embryological development could remain fertile for transformed germ cell proliferation. At present, this remains an open area of controversy.

GCTs are one of only a handful of malignancies (e.g., CML, GIST) that contain a pathognomonic genetic abnormality, present in nearly all cases. In GCT, this abnormality is the i(12p), which was first described in 1982 by Atkin and Baker during karyotyping of metaphase chromosomes from cases of GCTs [53]. Subsequently, several studies have documented approximately 85 % of GCTs to contain this chromosomal abnormality, and in cases where i(12p) was absent, extra copies of part or all of 12p occur as tandem duplications in situ or within other chromosomes [54]. As such, this assay has provided diagnostic utility for poorly differentiated midline tumors of unknown histogenesis, allowing the diagnosis of GCT to be made, and permitting administration of potentially curative chemotherapy [55]. Several studies have indicated that i(12p) is evident as early in GCT neoplasm development as ITGCNU, yet others have indicated that the appearance of this marker is associated with tumor invasion out of the tubules [48, 49, 56, 57].

Regardless of whether or not chromosome 12p gain is present only in fully malignant GCT or also ITGCNU, its omnipresence in invasive disease strongly suggests a role in the pathogenesis of GCT. Initially, it was thought that aberration of a single gene within 12p would be found responsible for GCT pathogenesis/progression. However, with more than 400 genes located on chromosome 12p and no overwhelming evidence to support one gene in particular, conventional wisdom now asserts that multiple genes on 12p, possibly in conjunction with other chromosomal anomalies, enable invasive GCT development. One gene of particular interest is cyclin D2 (CCND2), whose protein product is involved in regulation of DNA replication at the G1/S transition of the cell cycle. Overexpression of this protein leads to increased cell cycling and has been identified in ITGCNU, seminoma, and EC [58]. In contrast, normal spermatogonial cells in the adult testis rarely express cyclinD2, although expression of this protein has been observed in neonatal spermatogonial cells of the mouse.

Additional genes of interest have been identified through gene expression profiling, including a group of stem cell associated genes all mapping to a 200 Kb region at 12p13.31. These genes include STELLAR, NANOG, and GDF3, all of which demonstrate elevated expression in seminomas and embryonal carcinomas [44]. The overexpression of these genes through gain of 12p may be responsible for the undifferentiated phenotype observed in these two GCT histologies. Furthermore, exposure of EC cell lines to differentiating agents such as all-trans retinoic acid (ATRA) or bone morphogenic protein 2 (BMP2) lead to downregulation of these genes and resultant loss of pluripotency [44, 59, 60].

Evaluations of chromosomal changes within GCTs, primarily seminomas, by comparative genomic hybridization (CGH) revealed the frequent presence of a high-level amplification of the 12p11-12.2 region in addition to gain of the entire short arm of chromosome 12 [61, 62]. However, attempts to identify the specific target gene within this amplicon using molecular cytogenetic studies and global genomic screening have not been conclusive [63–65].

In addition to i[12p], conventional karyotype analyses have demonstrated that GCTs are aneuploid in DNA content, typically hypertriploid or tetraploid. Specific chromosomal abnormalities have been identified as recurrent across GCTs, some correlating with particular histologic subtypes [66, 67]. For example, breakpoints at 1p32-36 and 7q11.2 have been associated with teratoma whereas breakpoints at 1p22 correlated with yolk sac tumor histology [67]. Deletion or rearrangement of 12q and deletion of 6q13-25 constitute other frequently observed chromosomal changes in GCTs [66].

Interrogation of ITGCNU demonstrated frequent gain of portions of chromosomes 1, 5, 7, 8, 12p, and X and loss of DNA content from chromosome 18 [48, 56]. Adjacent invasive tumors also exhibited many of these changes but in addition, frequently had gains of portions of chromosomes 2, 3, 4, 6,13q, 14q, 17q, 18q, 20, and 21 and losses of portions of chromosomes 1p, 4, 6q, 9, 11, 13q, and 19 [48]. In the case of 17q gain, GRB7 and JUP were identified as potential target genes through microarray analysis [46].

Potential tumor suppressor genes involved in GCT pathogenesis have also been identified, primarily through loss of heterozygosity (LOH) studies [68, 69]. These studies demonstrated GCTs frequently contain loss of regions including the known tumor suppressor genes, RB1, DCC, and NME. In addition, loss of heterozygosity was demonstrated in regions where other proposed tumor suppressor genes are located (1p, 3p, 5q, 10q, 11p, 11q, and 17p) as well as new sites not previously identified as containing tumor suppressor genes (1q, 2q, 3q, 5p, 9q, 12q, 18p, and 20p). Epigenetic modifications such as promoter methylation might also contribute to loss of heterozygosity for tumor suppressor genes involved in GCT histopathogenesis. For example, seminomas have been demonstrated to contain lower levels of promoter methylation than nonseminomas. In addition, methylation of MGMT correlated with loss of its expression [70, 71]. However, methylation changes and expression of other genes have not correlated well in other studies [72].

In contrast to most malignancies, GCTs are believed to contain relatively few driver mutations. However, mutations in KRAS [73], KIT [74, 75], and SMAD4 [76] have been identified in some GCTs and have been proposed as important in germ cell transformation. KIT is perhaps the most well studied of these genes. In one series, activating KIT mutations were found in a large proportion of bilateral GCTs, particularly bilateral seminomas [74]. However, other studies did not support this claim [77, 78]. As discussed earlier, aberration of KIT signaling was also recently identified as increasing susceptibility to GCT development [25, 26]. More recent efforts by our group and others have identified additional mutations within a subset of GCTs, particularly those that demonstrate cisplatin resistance [111].