Pediatric Germ Cell Tumors

Specification of Primordial Germ Cells

Much of what is known about early molecular events in the mammalian germline comes from examination of germ-cell development in the mouse. Similar molecular mechanisms of germ cell development appear to operate in humans, based on studies of human embryos and human embryonic stem cells differentiating into germ cells in vitro. Development of the germline begins at the time of blastocyst implantation, when the extraembryonic ectoderm and the visceral endoderm send signals to cells in the proximal epiblast, also known as the embryonic ectoderm ( Fig. 63-1 ). The major inductive signals are the bone morphogenetic proteins (BMPs), which are members of the transforming growth factor β (TGF-β) superfamily. In response to BMPs, some of the epiblast cells begin to express the marker fragilis , signaling their competence to become germ cells. Of these fragilis -expressing cells, a few begin to express the transcriptional repressor Blimp1/Prdm1 . These cells, in which expression of somatic genes such as Hoxb1, T/Brachyury, and Snail is repressed, will become the primordial germ cells (PGCs). Unlike other epiblast-derived cells, PGCs regain or maintain expression of certain genes associated with pluripotency such as STELLA, OCT3/4, and NANOG . Certain pluripotency genes can be reactivated in GCTs and may contribute to malignant potential; for example, NANOG and OCT3/4 have been used as sensitive markers of malignant germ cells in studies of GCTs. In humans, PGCs can be identified in the wall of the yolk sac by their intrinsic alkaline phosphatase activity beginning at about day 24. The PGCs begin to proliferate as they migrate out of the yolk sac into the embryo.

Primordial Germ Cell Migration

In humans, as in many other organisms, the PGCs arise in an extraembryonic location, distant from the eventual site at which the gonad will form. This physical separation may serve to insulate the germ cells from various proliferation and differentiation signals in the developing embryo or to enforce a quality control by selecting for healthy PGCs capable of successfully navigating to the developing gonad. Proper PGC migration is critical to the survival of the germ cells and formation of the gonad, and failure of this migration can result in ectopic germ cells. Persistence and malignant transformation of these ectopic germ cells is one possible mechanism by which extragonadal GCTs are thought to arise.

Toward the end of gastrulation, morphogenetic movements in the developing embryo bring the PGCs in proximity to the hindgut. Invading the endoderm, the PGCs colonize the hindgut and begin vigorous, apparently random migratory movements, remaining confined to the gut. The receptor tyrosine kinase c-kit, expressed in PGCs, and its ligand, Steel, expressed in somatic cells, are required for the colonization of the hindgut and for the survival and migration of PGCs within the gut. Wylie and co-workers have shown that in mice, PGCs deficient in c-kit signaling undergo apoptosis through the action of the p53-target gene, Bax . At 5 to 6 weeks after fertilization, the PGCs upregulate expression of the adhesion factor E-cadherin and exit the hindgut on the dorsal side to begin colonization of the genital ridge primordia. The genital ridges are bilateral swellings of mesenchyme covered by coelomic epithelium and situated on either side of the dorsal aorta on the posterior wall of the embryo in the lower thoracic and lumbar regions. The genital ridges appear to attract the PGCs because of their expression of stromal cell–derived factor 1 (SDF-1, or CXCL12), which is a ligand for the chemokine receptor CXCR4, which is expressed in the PGCs. As the PGCs exit the hindgut, they divide into two lateral streams to colonize the genital ridges. PGCs that fail to colonize the ridges and remain in the midline are eliminated by apoptosis, as the midline cells downregulate expression of Steel. Once they have entered the genital ridges, PGCs become much less motile but continue several more rounds of division. This proliferation is dependent on continued Steel/c-kit signaling.

Erasure of Imprinting

Imprinting refers to the epigenetic modification of certain genes (typically by cytosine methylation) such that only the maternal or paternal allele of the gene is expressed. Lineage-specific patterns of imprinting are established in different tissues, including the germline, around the time of gastrulation. Upon entering the gonadal ridges, however, PGCs actively erase these genomic methylation patterns. This erasure is necessary in order to allow the maternal and paternal imprinting patterns to be established in the oocytes and sperm, respectively. Imprinting is reestablished in sex-specific patterns during gonadogenesis. GCTs exhibit partial or total erasure of imprinting, implying their origin from early germ cells.

Gonadogenesis

Beginning shortly before the arrival of the PGCs in the genital ridges, the coelomic epithelium begins to proliferate and invade the underlying mesenchyme, forming the primitive sex cords. Migrating PGCs entering the gonad are surrounded by the cords. At this early stage, the appearance of the developing gonad is identical in males and females, and the tissue is referred to as the indifferent gonad. Subsequently, changes occur in both the germ cells and the gonadal somatic cells according to the genetic sex of the embryo. In genetic males, sex determination occurs under the influence of the testis-determining SRY gene on the Y chromosome. After the initial rounds of cell division, male germ cells enter a mitotic arrest that persists until after birth. In males, the primitive sex cords proliferate and penetrate deeper into the mesenchyme, forming the testis or medullary cords, which connect proximally to form the rete testis. By the fourth month of development, the cords consist of germ cells and the supporting Sertoli cells, which are derived from the surface epithelium of the genital ridge. The cords remain solid until puberty, when they form lumens to become seminiferous tubules. By the eighth week of development the mesenchyme of the gonadal ridge gives rise in males to Leydig cells, which secrete testosterone. Secretion of antimüllerian hormone by the Sertoli cells and testosterone by the Leydig cells leads in males to degeneration of the paramesonephric ducts and the development of the mesonephric ducts into the vas deferens and epididymis. Testosterone is also necessary for male differentiation of the external genitalia.

Female gonadal development proceeds along different lines. In the absence of the Y chromosome, the sex cords initially degenerate and are replaced by a new set of cortical sex cords derived from the surface epithelium. By the fourth month the cortical sex cords have become isolated clusters with each cluster surrounding a single germ cell. In females the primitive germ cells, called oogonia, continue rapid proliferation, reaching maximal numbers by the seventh month. After that point, most of the oogonia degenerate; the remaining cells, now called primary oocytes, enter meiosis and arrest at the diplotene stage of the first meiotic prophase. Granulosa cells (derived from the surface epithelium) and thecal cells (derived from the mesenchyme) together form the follicle cells that surround each primary oocyte. Beginning at adolescence, groups of oocytes periodically resume meiosis. In females, the paramesonephric (müllerian) ducts develop into the oviducts, uterus, cervix, and upper part of the vagina. The mesonephric ducts degenerate.

In summary, development of the germline requires the proper specification of PGCs and their migration through the embryo to the gonadal ridges, where the gonads are formed through interactions of the germ cells with somatic cells. Concomitant with this process, the inherited pattern of genomic imprinting is erased, and a new, sex-specific pattern is formed. The complex process of gonadal organogenesis is subject to both genetic and environmental influences. Abnormal development of the gonads during the embryonic and fetal periods leads to defects such as cryptorchidism and gonadal dysgenesis, which are strongly associated with the risk of developing GCTs.

Overview of Testicular Germ Cell Tumors

Before puberty in infants and children, teratoma and/or YST account for the vast majority of GCTs. Seminoma (the term used for a testicular germinoma), embryonal carcinoma, and choriocarcinoma are very rare in this age group. GCTs of the prepubertal testis differ from their adult counterparts not only in the distribution of histologic subtypes, but also in their spectrum of cytogenetic abnormalities. After the early peak of testicular YST and teratoma in the 0-to-4–years age group, testicular tumors are relatively rare until the onset of puberty. Between ages 10 and 40 years, there is a second peak in testicular tumor incidence, comprised of seminoma and nonseminomas (including embryonal carcinoma, teratoma, YST, and choriocarcinoma). Postpubertal tumors demonstrate a wider array of histopathologic differentiation than do the GCTs of the prepubertal testis. Moreover, adolescent and adult GCTs share a common precursor cell of origin (the carcinoma in situ [CIS] or intratubular germ cell neoplasia, unclassified [IGCNU]), as well as characteristic cytogenetic abnormalities including the near-universal presence of amplifications of chromosome 12p. These histopathologic and molecular cytogenetic differences have lent support to the hypothesis that different pathogenic mechanisms underlie juvenile and adult testicular GCTs.

Overview of Ovarian Germ Cell Tumors

In neonates and infants, the most common ovarian lesions are ovarian cysts. Cysts can occur any time from late gestational stages onwards, and they occur at a greater rate in babies of mothers with diabetes, preeclampsia, and Rh immunization. Ovarian cysts in children often resolve spontaneously without intervention. GCTs account for 75% of ovarian tumors in the first 2 decades of life and up to 90% of ovarian tumors in premenarchal girls. The majority of these (95%) are benign, mature teratomas, mostly commonly “desmoid” tumors. Five percent of ovarian GCTs are malignant; these include dysgerminomas, immature teratomas, mature teratomas with somatic malignancies, YSTs, choriocarcinoma, embryonal carcinoma, polyembryoma, and mixed GCTs. Finally, gonadoblastoma is a mixed germ cell–sex cord stromal tumor occurring in cases of mixed gonadal dysgenesis with ambiguous genitalia, or 45,X Turner syndrome with Y chromosome material. The histopathology of ovarian GCT types is similar to that of their testicular counterparts. After the third decade of life, ovarian GCTs occur rarely, and carcinomas derived from the coelomic epithelial covering of the ovary are much more common.

Intratubular Germ Cell Neoplasia, Unclassified

IGCNU, also called testicular CIS, is considered the precursor for all invasive testicular GCTs (TGCTs) of postpubertal males except spermatocytic seminoma. According to one source, if left untreated IGCNU has a up to a 50% probability of progressing to invasive TGCT, either seminoma or nonseminoma, within 5 years. Nonseminomas can be composed of embryonal carcinoma (the stem cell component), teratoma (somatic differentiation), and YST and choriocarcinoma (extraembryonic lineages). In IGCNU, germ cells with abundant vacuolated cytoplasm; large, irregular nuclei; and prominent nucleoli are found within the seminiferous tubules, showing similarities to PGCs and early gonocytes. IGCNU is found in 1% of cases of male infertility and in 2% to 4% of cryptorchid testes in adults. In men with a TGCT, the prevalence of IGCNU is about 80% in the ipsilateral testis (range, 63% to 99%], predominantly in men with nonseminomas, and 5% in the contralateral testis. Various markers such as placental alkaline phosphatase (PLAP), c-kit, transcription factor AP-2γ, OCT3/4 (POU5F1), and testis-specific protein Y-encoded (TSPY) have been extensively used for immunohistochemical detection of IGCNU. All these markers are also found in PGCs and/or gonocytes, which supports the model that IGCNU derives from PGCs or gonocytes.

Whether an identifiable precursor lesion exists for GCTs of children is much less clear. In prepubertal children, the incidence of IGCNU appears to be very low, although several cases have been reported. These data must be interpreted with caution, however, because of the difficulty of distinguishing IGCNU in the developing gonad, in which primitive germ cells normally express markers such as OCT3/4 and c-KIT . The precursor lesion for the teratomas and YSTs of neonates and infants is yet to be identified. These pediatric tumors show partially erased imprinting, suggesting that the cell of origin is most likely a germ cell at an earlier development stage than that of IGCNU.

IGCNU has been reported in a high percentage of gonadal biopsy specimens from children with disorders of sexual development (DSDs) who are at risk for the development of germ cell malignancies. These disorders include gonadal dysgenesis, partial androgen insensitivity syndrome (AIS), and less often, complete AIS. Development of these tumors in patients with DSDs is linked to the presence of a specific fragment of the Y chromosome, known as the GBY region. For these patients with an increased risk of germ cell malignancy, prophylactic gonadectomy is often indicated. However, overdiagnosis of IGCNU is possible in this setting because of the common maturation delay of germ cells in patients with DSDs, resulting in retained presence of c-kit -positive germ cells. Therefore careful examination of the distribution pattern of OCT3/4 -positive cells and their position within the seminiferous tubule are essential to making the diagnosis of IGCNU.

Histopathology of Teratoma

Teratomas are tumors composed of multiple tissues that are normally foreign to, and able to proliferate in excess of, the site in which they occur. In the usual definition of teratoma, tissue elements of all three germ layers (endoderm, mesoderm, and ectoderm) are present; however, teratomas also occur in which only one or two of the germ layers are present (referred to as monodermal or bidermal teratomas, respectively). The clinical and biologic behavior of teratomas varies significantly with differing anatomic location, degree of maturity of the tumor tissue, and age of onset. Grossly, teratomas are nodular and heterogeneous with solid and cystic areas depending on the types of differentiated tissue present. Cartilage, bone, hair, and pigmented areas may be recognizable ( Fig. 63-2 ). At the histopathologic level, ectodermal derivatives such as neuroepithelium, skin, and hair are the most common tissues in teratomas of infants and children. However, practically any tissue type can be seen, including muscle, bone, cartilage, and well-differentiated glands ( Fig. 63-3 ).

Mature teratomas are typically cystic and contain differentiated adult-type tissue from one or more of the germ layers. The term dermoid cyst refers to the common finding in mature teratomas of tissue resembling the adult epidermis and its appendages. Immature teratomas are those in which embryonal-appearing tissue is present, typically in the form of neuroepithelial rosettes and tubules and often mixed with mature tissue ( Fig. 63-4 ). A loose, myxoid stroma of immature mesenchyme with focal differentiation into osteoid, fat, cartilage, and rhabdomyoblasts may be present. Hypercellularity, increased mitotic index and nuclear atypia can be present. The prognostic significance of these findings is different for ovarian and testicular teratomas, and these are discussed separately in the next sections.

Testicular Teratoma

Ovarian Teratoma

Teratoma is the most common GCT of the ovary, comprising more than 95% of all ovarian GCTs. Ovarian teratomas differ in important ways from testicular teratomas. Teratomas in the prepubertal testis are uniformly benign. In adolescents and adults, testicular teratomas are nearly always malignant and occur as a component of mixed malignant GCTs (MGCTs), the exception being dermoid cysts of the testis. In the ovary, in contrast, the phenotype of teratomas does not vary as strikingly by age, and ovarian teratomas are usually pure tumors. The great majority of ovarian tumors are mature, benign tumors (often referred to as dermoid cyst or mature cystic teratoma ). Less than 5% of ovarian teratomas are malignant; these are discussed separately in the following sections.

Immature Teratoma

The term immature teratoma refers to teratomas containing immature-appearing tissues, principally neuroepithelium. Additionally, foci of mitotically active glia may be present. A grading system has been developed for immature teratomas based on the amount of neuroepithelial tissue present. Mature teratomas containing only fully differentiated tissue are considered grade 0, and immature teratomas range from grade 1 (comprised of less than 10% immature neuroepithelium) to grade 3 (more than 50% immature neuroepithelium present). A two-component grading system (low grade and high grade) has also been proposed and shown to have improved reproducibility. In adult ovarian teratomas, the finding of grade 2 or 3 immature teratoma predicts the likelihood of metastasis and confers a worse prognosis. In children, however, it is not clear that the same relationship of grade to outcome of immature teratomas holds true. A POG study concluded that surgery alone was curative in children and adolescents with stage I immature teratomas of any grade and that chemotherapy should be reserved for cases of relapse.

Immature teratomas may be associated with implants of glial tissue on the peritoneum, known as gliomatosis peritonei . If the implants are composed solely of mature tissues, the presence of gliomatosis peritonei does not “upstage” the patient. Interestingly, data suggest that the implants arise from metaplastic transformation of pluripotent müllerian stem cells in the peritoneum or underlying mesenchyme, rather than representing metastasis of the teratoma tissue.

Monodermal Teratomas

Monodermal teratomas consist largely or exclusively of a single type of tissue, such as thyroid, carcinoid, and neuroectodermal tissues. Monodermal teratomas with neuroectodermal differentiation include indolent types such as ependymoma and poorly differentiated forms such primitive neuroectodermal tumor (PNET), as well as anaplastic tumors resembling glioblastoma. These tumors are rare in children.

Teratoma with Malignant Transformation

Teratoma with malignant transformation makes up 0.2% to 1.4% of mature cystic tumors of the ovary. In these tumors, more commonly seen in older women, somatic-type malignancies are seen among the individual tissues in the teratoma. Squamous cell carcinomas are the most common tumor type, but others including melanoma, adenocarcinoma, small cell carcinoma, sarcomas (including PNET), and malignant glioma may also occur.

Histopathology of Yolk Sac Tumor

YST is the most common malignant GCT in infancy and childhood. The differentiation of the tumor cells is predominantly along endodermal lines and can take the form of both intraembryonic endodermal derivatives, such as primitive gut and liver, and extraembryonic structures such as allantois and yolk sac. There are many synonyms for YST, including orchioblastoma, Teilum's tumor, and clear cell adenocarcinoma. Although endodermal sinus tumor is commonly in use as a synonym for YST, some consider the term problematic, because the endodermal sinus is not a component of normal human embryogenesis. In macroscopic appearance, YSTs are yellowish, with multiple cysts and areas of hemorrhage, necrosis, and liquefaction. Microscopically, YSTs display a loose, myxoid stroma containing a reticulated pattern of microcystic spaces ( Fig. 63-5 ). The cysts are lined by a flattened, periodic acid-Schiff (PAS)–positive, diastase-resistant epithelium. About 20% of YSTs exhibit characteristic Schiller-Duval bodies (a clustering of cells around a small, central blood vessel) (see Fig. 63-5 ). In addition to the microcystic/reticular pattern, several variant histologies of YST are described, including the solid, polyvesicular, and parietal types (corresponding to primitive endoderm and extraembryonic structures such as the allantois and yolk sac) and the glandular and hepatic types (corresponding to the intraembryonic endodermal derivatives, primitive lung and liver).

YSTs are typically cytokeratin-positive, which can help differentiate the solid YST variant from germinomas. Alpha-fetoprotein (AFP) expression is characteristic of these tumors, and AFP is a valuable tumor marker. Neonates and infants have high physiologic levels of AFP in serum, which can confound the use of serum AFP levels for diagnosis of YST or for monitoring therapy. Tables of normal serum AFP levels in neonates and infants have been published ( Fig. 63-6 ). In addition, at least 70% of YSTs have detectable AFP expression by immunohistochemical assays.

Testicular Yolk Sac Tumor

Ovarian Yolk Sac Tumor

YST of the ovary is a clinically aggressive tumor that often presents at advanced stages with metastasis to lymph nodes or peritoneal structures. The tumors are rarely bilateral. The median age at diagnosis is 18 to 19 years. In a series of 26 pediatric patients, reticular type was the most common histology identified.

Histopathology of Seminoma and Dysgerminoma

Seminomas are a class of GCTs in which the tumor cells resemble primitive, undifferentiated germ cells, both in morphology and in expression of pluripotency genes such as OCT3/4, NANOG, and STELLAR/DPPA3 . These tumor are referred to as germinomas when arising in extragonadal locations (such as the pineal gland, the mediastinum, or the retroperitoneum) , dysgerminomas when arising in the ovary, and seminomas when arising in the testis. The histology of seminoma is identical regardless of the site in which the tumor occurs. Grossly, these are rounded, nodular tumors, separated into lobules by fibrous bands. Microscopically, seminomas are characterized by a monotonous proliferation of large, uniform cells with large central nuclei and prominent nucleoli ( Fig. 63-7 ). The cytoplasm is clear or granular and often PAS-positive because of high glycogen content. A lymphocytic infiltrate is a typical feature of seminomas.

Seminoma

Seminoma is extremely rare in children. In the postpubertal testis, seminoma can occur in pure form or as a component of mixed GCT. The peak age of onset of seminoma is 34 to 45 years, slightly later than that of nonseminomatous tumors. Several distinct variants of seminoma have been recognized. Up to 7% of classic seminomas contain syncytiotrophoblastic giant cells, and almost 25% of seminomas contain foci that stain positive for β-human chorionic gonadotropin (HCG). These tumors may be accompanied by an elevation in serum β-HCG; however, the presence of syncytiotrophoblasts or elevated β-HCG does not worsen the prognosis. Marked elevations of β-HCG or persistently elevated β-HCG after orchiectomy may indicate the presence of choriocarcinoma. Anaplastic or atypical seminoma is the designation used for seminomas displaying increased mitotic activity, nuclear pleomorphism, and sparse lymphocytic infiltrate on histologic examination. Whether these aggressive features portend a worse prognosis or a higher likelihood of metastasis remains controversial. Whereas some studies have suggested that anaplastic seminomas are clinically more aggressive tumors, others have found no evidence of a worse prognosis when these features are present.

Dysgerminoma

Dysgerminoma is the most common MOGCT of children and adolescents, and makes up one third of MOGCTs. Pathologically, dysgerminoma is the ovarian counterpart of the seminoma of the testis and the germinoma of extragonadal sites. Unlike seminomas of the testis, which are rare in the prepubertal period, dysgerminomas can occur at any age, though the peak incidence is age 15 to 19 years. Also unlike seminomas, which develop from IGCNU, dysgerminomas do not appear to arise from a precursor cell. Dysgerminomas stain positively for OCT4 on immunohistochemistry, which can be useful for distinguishing these tumors from nondysgerminomas. As with seminomas, the majority of dysgerminomas also stain positive for c-KIT.

Histopathology of Embryonal Carcinoma

Embryonal carcinoma is very rare in infants but can occur in prepubertal females and adults of both sexes. Embryonal carcinomas commonly have areas of hemorrhage and necrosis and can be locally invasive into the epididymis and spermatic cord in males. At the microscopic level, the tumors consist of large, epithelial-appearing cells that resemble the early embryonic cells of the inner cell mass ( Fig. 63-8 ). The tumor cells may grow in solid, papillary, and reticular patterns, forming many clefts and glandlike structures. The tumors are typically cytokeratin and CD30-positive, but negative for epithelial membrane antigen (EMA), carcinoembryonic antigen, and vimentin. Syncytiotrophoblast cells, when present in the tumor, can produce β-HCG and cause precocious pseudopuberty in premenarchal girls or vaginal bleeding in older women.