REVIEW ARTICLE
Intratubular Germ Cell Neoplasia of the Testis: A Brief Review Turki Al-Hussain, MD, Nasir Bakshi, MD, and Mohammed Akhtar, MD, MD, FCAP, FRCPA, FRCPath
Abstract: Germ cell tumors of the testis may be divided into 3 broad categories according to age at presentation. The tumors in the pediatric age group include teratoma and yolk sac tumor. These tumors are generally not associated with convincing intratubular neoplasia. The second group consists of tumors presenting in third and fourth decade of life and include seminoma, embryonal carcinoma, yolk sac tumor, choriocarcinoma, and teratoma as well as mixed germ cell tumors. The precursor cell for these tumors is an abnormal gonocyte that fails to differentiate completely into spermatogonia. These abnormal cells stay dormant in the gonad during intrauterine life as well as infancy and childhood, but undergo proliferation during puberty and can be identified as intratubular germ cell neoplasia unclassified (IGCNU). These tumor cells continue to manifest protein expression pattern that resembles primitive germ cells (PLAP, c-KIT, OCT3/4). After a variable interval following puberty, IGCNU cells may acquire ability to penetrate the seminiferous tubules and present as an overt germ cell tumor. Acquisition of isochrome 12 and other genetic abnormalities are usually associated with this transition. The level of DNA methylation generally determines the phenotype of the germ cell tumor. The third type of germ cell tumors is spermatocytic seminoma, which is a rare tumor encountered later in life usually in fifth and sixth decade. The cell of origin of this tumor is probably postpubertal mature spermatogonia which acquire abnormal proliferative capability probably due to gain of chromosome 9 resulting in activation and amplification of genes such as DMRT1. The tumor cells manifest many of the proteins normally expressed by mature sperms such as VASA, SSX2, and occasionally OCT2. Although spermatocytic seminoma may also have an intratubular growth phase, it completely lacks features of IGCNU. Key Words: germ cell tumor, intratubular, testis, seminoma, yolk sac, teratoma, embryonal carcinoma
incidence of testicular germ cell tumors increases shortly after the onset of puberty and peaks in the fourth decade of life with a median age of 34 years at diagnosis.1–6 Testicular germ cell tumors are generally grouped into 3 broad categories. Type I testicular germ cell tumors are encountered in young prepubertal male children and consist of benign teratomas and malignant yolk sac tumors (Table 1). These tumors are generally not associated with any intratubular neoplasia. Type II testicular germ cell tumors (including seminoma, embryonal carcinoma, teratoma, yolk sac tumor, choriocarcinoma, and mixed germ cell tumors) affect men aged between 20 and 40; although the inception of these tumors is believed to occur during intrauterine life at the time of development of fetal gonads. All type II testicular germ cell tumors develop from a preinvasive lesion termed intratubular germ cell neoplasia unclassified (IGCNU), which in the past has been called carcinoma in situ. Type III testicular germ cell tumors is called spermatocytic seminoma (SS), which is mostly a benign tumor usually affecting men above 50 years of age. SS may manifest an intratubular growth pattern, although the significance of this finding in relation to tumor histogenesis is not clear.6–10 The purpose of this review is to examine embryologic and neonatal development and differentiation of normal germ cells and highlight potential areas of this differentiation that are modified during the development of testicular germ cell tumors. We also discuss the genetic and epigenetic factors that contribute to inception of germ cell neoplasia within the testicular tubules as well as factors responsible for its progression to invasive germ cell tumors.
(Adv Anat Pathol 2015;22:202–212)
INTRATUBULAR GERM CELL NEOPLASIA OF THE TESTIS: A BRIEF REVIEW Testicular tumors are the most common malignancies among men between the ages of 20 and 40 years. These tumors comprise approximately 1% of all cancers in men, but only about 0.1% of cancer deaths in males because the majority of these tumors are curable. More than 90% of testicular neoplasms originate from germ cells. The From the Department of Pathology and Laboratory Medicine, King Faisal Specialist Hospital and Research Centre, Riyadh, Kingdom of Saudi Arabia. The authors have no funding or conflicts of interest to disclose. Reprints: Mohammed Akhtar, MD, FCAP, FRCPA, FRCPath, Department of Pathology and Laboratory Medicine (MBC 10), King Faisal Specialist Hospital and Research Centre, P.O. Box 3354, Riyadh 11211, Kingdom of Saudi Arabia (e-mails: [email protected]; [email protected]). All figures can be viewed online in color at http://www.anatomic pathology.com. Copyright r 2015 Wolters Kluwer Health, Inc. All rights reserved.
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EMBRYOLOGIC ORIGIN OF GERM CELLS Germ cells are responsible for the passage of genetic material from one generation to the next and are therefore crucial to the survival and perpetuation of the species. Fertilization of oocytes by sperm promotes the formation of a zygote that undergoes cell division and cleavage to form a blastocyst. The outer layer of blastocyst gives rise to the trophectoderm, whereas the inner cell mass contains embryonic stem cells. By day 9 the inner cell mass differentiates into epiblast and hypoblast, setting the stage for gastrulation (day 15), during which the blastocyst develops the 3 germ layers (ectoderm, mesoderm, and endoderm) and cavitates further to develop yolk sac and the amniotic cavity (Fig. 1). The first glimpses of formation of germ cells called primordial germ cells are observed in the embryo after implantation, shortly before and during gastrulation. Some of the cells within the epiblast, as a result of specific signals from neighboring cells (inductive signaling), are induced to acquire the commitment and specification of primordial Adv Anat Pathol
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Volume 22, Number 3, May 2015
TABLE 1. Germ Cell Tumors of the Testis: 3 Broad Categories
Type 1
Type II
Prepubertal Teratoma
Postpubertal Classic seminoma
Yolk sac tumor
Embryonal carcinoma Yolk sac tumor Teratoma Choriocarcinoma Mixed germ cell tumors
Type III Usually >50 Spermatocytic seminoma
germ cells. Later, in response to molecular cues from the yolk sac, these cells segregate from the epiblast and migrate to localize in the wall of the yolk sac, at the base of the allantois.11–15 Two factors play a significant role in inducing the primordial germ cells to maintain the commitment to germ cell line and thus preventing these cells from acquiring somatic differentiation. First factor is the expression of BLIMP (B lymphocyte-induced maturation protein) within the primordial germ cells, which prevents the activation of somatic genes such as HOX, FDF8, and SNAIL. Secondly, the highly proliferative nature of the primordial germ cells results in lack of maintenance of adequate levels of DNA methyl transferase within these cells. This causes general demethylation of the DNA in primordial germ cells leading to complete loss of parental imprinting. The suppression of somatic cell phenotype along with demethylation results in expression of pluripotency factors such as NANOG and OCT 3/4. The exact roles of OCT 3/4 and NANOG in primordial germ cells are currently unknown. It is likely that they maintain pluripotency in germ cells and prevent somatic differentiation in these cells. OCT 3/4 is also believed to control the survival of the primordial germ cells, whereas NANOG expression facilitates further maturation of germ cells once they reach the developing gonads.14
MIGRATION OF PRIMORDIAL GERM CELLS During the fourth gestational week, when the embryonic disc undergoes a process of folding, primordial germ cells are passively incorporated into the embryo together with the yolk sac wall. They become segregated as single cells among the lining epithelial cells of the primitive hindgut and midgut. The germ cells travel within the
Intratubular Germ Cell Neoplasia of the Testis
mucosa until they reach midline near the aorta in the vicinity of the developing gonadal ridges, which are visible as distinct structures within the intermediate mesoderm at the beginning of the fifth week. At this time, primordial germ cells penetrate the mesenchyme surrounding the gut epithelium through breaks in the basal lamina, reach the dorsal mesentery, and continue to move laterally, beyond the primitive mesonephros and eventually enter the gonadal ridges.11–15
THE GONOCYTES Within the developing gonads the germ cells proliferate and are surrounded by Sertoli cells. The primordial germ cells are now designated as the gonocytes (Fig. 2). Primordial germ cells and gonocytes are both undifferentiated embryonic germ cells which can be identified by embryonic and stem cell markers including PLAP, NANOG, c-KIT, SOX2, and OCT3/4. Both cells have their original genomic imprinting pattern completely erased by DNA demethylation, which allows development of sexspecific germ cell lineages. In the presence of a Y chromosome, the gonadal stromal cells express transcription factor SRY and its target gene SOX9 resulting in the early development of Sertoli cells, which surround the gonocytes and form the seminiferous tubules (Fig. 2). The Sertoli cells create a microenvironment that allows differentiation of gonocytes into spermatogonia. During the remainder of the fetal life and into early postnatal period the germ cells gradually lose expression of PLAP, NANOG, SOX2, and OCT3/4, partially lose expression of KIT, and acquire expression of germ cell–specific proteins including MAGE4A, VASA, TSPY, OCT2, and SSX2 as they gradually transition from gonocytes to spermatogonia (Fig. 3). This transition occurs in an asynchronous manner such that cells at different stages of development may be present in an individual seminiferous tubule. Thus during this period some of the cells may coexpress gonocyte and spermatogonial markers. During the late phases of gestation, the gonocytes gradually migrate peripherally from the center of the seminiferous tubules toward the basement membrane to become either spermatogonia or spermatogonial stem cells. This process continues until about a year after birth and culminates in spermatogonia ultimately losing all the pluripotency and embryonal markers and instead expressing germ cell markers.11–15
THE RISK FACTORS FOR TYPE II TESTICULAR GERM CELL TUMORS
FIGURE 1. Diagram depicting sequence of events following fertilization leading to the segregation of primordial germ cells from the remaining cells which are destined for somatic differentiation. By day 15 the germ cells have moved to the wall of yolk sac. For further detail please see the text.
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Germ cell tumors of the testis may result from derangement of the normal process of germ cell maturation. A variety of risk factors for the development of testicular germ cell tumors have been identified. These include family predisposition, disorders of sexual development, cryptorchidism, high maternal estrogen during fetal development, HIV infection, environmental exposures, subfertility or infertility, and previous history of testicular germ cell tumors.1,14,15 Genetic factors, including deletions on the Y chromosome, androgen insensitivity, and KIT mutations, negatively impact normal germ cell development, which increases the risk of neoplastic transformation. Patients lacking the SRY gene on the Y chromosome required for development and differentiation of Sertoli cells have a high risk of type II testicular germ cell tumors, as the lack of
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FIGURE 2. A, Photomicrograph featuring developing kidneys and testes in a 10-week-old fetus. B, Higher magnification photomicrograph showing seminiferous tubules containing gonocytes surrounded by Sertoli cells. Please see this image in color online.
properly differentiated Sertoli cells inhibits germ cell development and maturation. Furthermore, genetic association studies have identified mutations in genes encoding KIT, KIT ligand, and its downstream signaling molecules such as KRAS, SPRY4, and BAK1, as likely predisposition genes for testicular germ cell tumors. A combination of both genetic and environmental factors may also prevent or delay the maturation of gonocytes into spermatogonia by retarding the development and maturation of the supporting Sertoli and Leydig cells, thus perturbing the microenvironment required for normal germ cell development.1,15,16
INTRATUBULAR GERM CELL NEOPLASIA UNCLASSIFIED The precursor lesion of almost all type II testicular germ cell tumors is IGCNU, defined as malignant germ cells
FIGURE 3. Sequence of events involved in transformation of primordial germ cells (primordial germ cells) to gonocytes and then spermatogonia. Maturation of gonocytes to spermatogonia starts during intrauterine period and continues into infancy. Primordial germ cells and gonocytes undergo demethylation of DNA and express markers of pluripotency such as PLAP, NANOG, OCT3/4, and AP-2g. These cells also express C-KIT which ensures their survival. Following maturation of gonocytes to spermatogonia, the pluripotency markers are switched off and there is expression of germ cell markers including MAGE4A, TSPY, and VASA among others. The spematogonia undergo male-specific remethylation as part of the process of maturation. Please see this image in color online.
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confined to the seminiferous tubules, which usually lack normal spermatogenesis. Testicular IGCNU is characterized histologically by proliferation of basilar malignant germ cells with enlarged hyperchromatic nuclei, often 1 or 2 prominent nucleoli, and clear cytoplasm due to abundant glycogen (Fig. 4). Sertoli cells are often displaced luminally. Affected seminiferous tubules usually have thickened peritubular basement membranes. Pagetoid spread of IGCNU into the rete testis is common. The interstitium may show hyperplasia of the Leydig cells. Although IGCNU can be diagnosed in the testes of prepubertal individuals, the histologic diagnosis can be challenging because IGCNU cells resemble normal infantile gonocytes.17 IGCNU cells were first identified in 1972 in biopsies of infertile men, and a causal link was subsequently established during follow-up, when the men with this lesion developed tumors, whereas control subjects exhibited no cases of TGC.18 Intratubular germ cell neoplasia is seen adjacent to the invasive tumors in 72% to 98% of the cases or in contralateral testis in 4.9% to 6.6% of cases.2 In addition, IGCNU can be a sporadic finding in approximately 0.4% to 0.8% of the testes of otherwise healthy males.19–27 The widely accepted theory of testicular germ cell tumors tumorigenesis is that development of IGCNU starts in utero.14,28 Transformation of fetal gonocytes to spermatogonia is a crucial step in proper maturation of the germ cells for future progression to spermatogenesis. The disturbance of germ cell development at this stage may result in arrest of fetal germ cell differentiation, making them susceptible to neoplastic transformation. IGCNU cells appear to be committed to germ cell differentiation at the level of the gonocytes but are blocked from differentiating further into spermatogonia and entering spermatogenesis (Fig. 5). Fetal gonocytes such as primordial germ cells have a completely erased genomic imprinting pattern by DNA demethylation, and are therefore susceptible to mutational events accumulating during cell replication. These mutations may activate oncogenes as well as silence some of the tumor-suppressing genes. The combined effect of these changes may be transformation of some of the gonocytes to neoplastic cells. These cells, however, stay dormant until puberty and early adulthood when proliferation is believed to occur in the setting of hormonal
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Intratubular Germ Cell Neoplasia of the Testis
FIGURE 4. A and B, Two tubules with intratubular germ cell neoplasia unclassified (IGCNU). The tumor cells are larger than normal spermatogonia and have large nuclei and mostly clear cytoplasm due to abundant glycogen. The tubule in (A) has markedly thickened lamina propria. Please see this image in color online.
stimulation thus resulting in IGCNU (Figs. 6, 7). The IGCNU cells proliferate and gradually replace normal germ cells within the affected tubules but continue to rely on Sertoli cell–derived growth/survival signals and production of testosterone by Leydig cells.28,29 Morphologically, IGCNU cells resemble primordial germ cells/gonocytes and express a number of similar markers, in that they lack parental imprinting and express OCT 3/4, PLAP, and c-KIT (Fig. 8). In fact, the transcriptome of IGCNU cells is very similar to that of isolated normal human gonocytes, demonstrating a close relationship between these cell types.12 IGCNU subsequently progresses to invasive testicular germ cell tumors with differentiation into the various histologic subtypes of testicular germ cell tumors either before or after invasion.
PROGRESSION OF INTRATUBULAR GERM CELL NEOPLASIA UNCLASSIFIED With the acquisition of additional mutational events, the neoplastic cells break their dependence on the signals from Sertoli cells for proliferation and enter a phase of independent proliferation. The tumor cells now fill the
FIGURE 5. Development of intratubular germ cell neoplasia unclassified (IGCNU) starts during intrauterine life when a gonocyte fails to undergo proper maturation into spermatogonium and continues to express pluripotency markers. These cells stay dormant until puberty but during and after puberty they undergo proliferation and replace the normal germ cells within the seminiferous tubules. Please see this image in color online.
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tubular lumina, acquire the phenotype of one of the TGT (usually seminoma and/or embryonal carcinoma), and subsequently break through the tubular wall and invade the gonadal stroma thus transforming into an overt testicular germ cell tumors (Figs. 9–13). It has been estimated that approximately 70% of patients with ITGCNU will develop an invasive germ cell tumor within 7 years, which may be extrapolated to 100% at 10 years.23 Intratubular germ cell tumors with morphologic features of seminoma and embryonal carcinoma are frequently observed in testes with invasive germ cell tumors.30,31 Rarely intratubular seminoma or embryonal carcinoma may be seen in the absence of any invasive component. Intratubular syncytiotrophoblastic and cytotrophoblastic cells have been seen occasionally but as reported by Berney et al,31 may actually be more common than previously thought. These cells are usually seen in association with seminoma and less frequently with embryonal carcinoma. These cells are encountered as isolated cells or small clusters within the tubules. Their recognition is greatly facilitated by immunohistochemical staining. Syncytiotrophoblastic cells are strongly immunoreactive for human chorionic gonadotropin, whereas cytotrophoblasts may be negative or stain only lightly. The invasive component in cases with
FIGURE 6. A, Diagram depicting normal development and maturation of germ cells during intrauterine period, infancy, and childhood. The gonocytes undergo a gradual maturation so that all the gonocytes are ultimately transformed to spermatogonia. B, In individuals who are destined to develop type 2 germ cell tumors, some of the gonocytes are abnormal and fail to make the transformation to spermatogonia. The abnormal cells, however, stay dormant within the tubules until the onset of puberty.
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FIGURE 7. A, In normal testis with the onset of puberty, the spermatogonia undergo proliferation and proceed to normal spermatogenesis. B, The abnormal gonocytes undergo proliferation during puberty and afterward and gradually replace the normal germ cells resulting in IGCNU. After a variable interval (usually several years), cells with morphologic features of seminoma and/or embryonal carcinoma may evolve from IGCNU. These cells later break through the tubular walls and produce overt germ cell tumors. IGCNU indicates intratubular germ cell neoplasia unclassified.
intratubular trophoblastic cells also contains these cells. However, no cases of intratubular choriocarcinoma have been reported. Similarly, elements of intratubular teratoma
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have not been recorded. These findings indicate that IGCNU may give rise to intratubular seminoma and embryonal carcinoma (with or without trophoblastic cells), which may subsequently invade the gonadal stroma. Although IGCNU is generally accepted as a form of a noninvasive precursor lesion for seminoma and embryonal carcinoma, the developmental relationship between seminoma and embryonal carcinoma remains poorly understood. Some studies suggest a single consecutive progression from IGCNU to seminoma and then to embryonal carcinoma (linear progression), whereas others suggest that seminoma and embryonal carcinoma independently derive from IGCNU (nonlinear progression). In contrast, yolk sac tumor, teratoma, and choriocarcinoma seem to evolve from a background of invasive embryonal carcinoma29,32 (Figs. 14, 15). Chromosome 12p alterations are the hallmark of transition of IGCNU into overt testicular germ cell tumors.6,7,14,16,28,33 It is identified in approximately 80% of all invasive testicular germ cell tumors, as well as in intratubular embryonal carcinoma and intratubular seminoma. In the remaining 20% amplification of shorter stretches of 12p may be present. Interestingly, IGCNU without adjacent invasive testicular germ cell tumors does not contain isochromosome 12p in most studies, which suggests that isochromosome 12p is not required for the development of IGCNU and may be indicative of progression beyond the stage of IGCNU.
FIGURE 8. The cells in IGCNU continue to manifest protein expression pattern of primordial germ cells and gonocytes. A: PLAP, B: CKIT, C: OCT3/4. Please see this image in color online.
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Intratubular Germ Cell Neoplasia of the Testis
FIGURE 9. A and B, Intratubular seminoma filling the seminiferous tubules. Please see this image in color online.
FIGURE 10. A and B, Intratubular embryonal carcinoma. Please see this image in color online.
FIGURE 11. Intratubular seminoma (lower part) and intratubular embryonal carcinoma (upper part). Please see this image in color online.
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FIGURE 12. Intratubular seminoma with stromal invasion. Please see this image in color online.
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FIGURE 13. Intratubular embryonal carcinoma with early stromal invasion. Please see this image in color online.
It has been hypothesized that amplified region on 12p may harbor genes which enable the tumor cells to survive, proliferate, and develop invasive growth independent of signals from the Sertoli cells. Several candidate genes have been suggested, including KITLG, NANOG (and its pseudogenes), KRAS2, BCAT1, and CCND2. However, the exact genes still have not been identified. Further studies are required before we fully understand the precise role of chromosome 12p in testicular germ cell tumors carcinogenesis. Less frequent cytogenetic aberrations found in testicular germ cell tumors include overpresentation of chromosomes 7, 8, 12, 17, and X, and underpresentation of chromosomes 4, 11, 13, 18, and Y. Mutations in single genes including KIT, TP53, KRAS/ NRAS, and BRAF have been implicated; however, the frequency of these mutations is relatively low.6–8,16,32 KIT is a protooncogene located on chromosome 4q11q12. Its gene product CD117 is a tyrosine kinase receptor that in the presence of KIT ligand dimerizes and becomes phosphorylated. KIT plays a crucial role in the survival, proliferation, and migration of normal primordial germ
FIGURE 14. Progression of IGCNU to embryonal carcinoma and/ seminoma involves development of isochromosome 12. The cells in seminoma continue to manifest demethylation; however, the development of embryonal carcinoma involves remethylation of DNA. Other germ cell tumors seem to arise from embryonal carcinoma and have even higher levels of DNA methylation. IGCNU indicates intratubular germ cell neoplasia unclassified. Please see this image in color online.
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cells and gonocyte, both of which are immunoreactive for KIT.34,35 KIT is also expressed in almost all IGCNU, most seminomas, and some NS germ cell tumors.6,16,28 Some of these tumors may additionally express KIT ligand, thus setting the stage for an autocrine or paracrine system to stimulate tumor cell proliferation. Gain-of-function mutations in KIT may be encountered in seminomas (19%) and uncommonly in nonseminomatous germ cell tumors (2%), resulting in constitutive activation of KIT which drives tumorigenesis. KIT-mutated testicular germ cell tumors may be potentially amenable to therapy by KIT inhibitors such as Imatinib mesylate.16,28,36 Activating mutations have been associated with several bilateral testicular germ cell tumors.6,16,28 In a retrospective study of 224 unilateral and 61 bilateral testicular germ cell tumors, Looijenga et al37 demonstrated significantly higher incidence of an activating KIT mutation in bilateral tumors compared with unilateral tumors (93% vs. 1.3%). However, recently, Coffey et al38 analyzed 175 unilateral and 40 bilateral testicular germ cell tumors and found no evidence of an increased frequency of KIT mutations in bilateral testicular germ cell tumors. Similar lack of KIT mutation in bilateral testicular germ cell tumors was reported by Sakuma et al39 from Japan. The reason for this discrepancy is not clear.
SEMINOMATOUS VERSUS NONSEMINOMATOUS GERM CELL TUMORS Although both seminoma and NS germ cell tumors arise from the IGCNU cells, they seem to exhibit distinct gene expression signatures with almost 90% of genes discordantly regulated between the 2 groups of tumors. Seminomas in general have upregulated genes such as oncogenes, genes encoding intracellular transducers, and genes related to DNA synthesis, proliferation, and repair. In NS germ cell tumors these genes are mostly downregulated. Nonseminomatous germ cell tumors are typically hypotriploid and seminomas are usually hypertriploid. This would indicate that despite a common origin, these tumors seem to follow very different pathways of differentiation. Epigenetic changes play a crucial role in normal development, maturation, and differentiation of germ cells.27,28,39–41 Primordial germ cells and gonocytes DNA are generally hypomethylated, whereas more mature germ cells such as spermatogonia and spermatocytes are fully methylated. IGCNU and seminomas exhibit low levels of DNA methylation and permissive chromatin structure associated with high transcriptional and proliferative activity. More differentiated testicular germ cell tumors (yolk sac tumors, choriocarcinomas, and teratomas) show a higher degree of methylation with embryonal carcinomas exhibiting an intermediate pattern. Thus it seems that different levels of global methylation in testicular germ cell tumor subtypes may determine the final phenotype of the tumor.
SPERMATOCYTIC SEMINOMA SS is a relatively rare tumor representing
Intratubular Germ Cell Neoplasia of the Testis: A Brief Review Turki Al-Hussain, MD, Nasir Bakshi, MD, and Mohammed Akhtar, MD, MD, FCAP, FRCPA, FRCPath
Abstract: Germ cell tumors of the testis may be divided into 3 broad categories according to age at presentation. The tumors in the pediatric age group include teratoma and yolk sac tumor. These tumors are generally not associated with convincing intratubular neoplasia. The second group consists of tumors presenting in third and fourth decade of life and include seminoma, embryonal carcinoma, yolk sac tumor, choriocarcinoma, and teratoma as well as mixed germ cell tumors. The precursor cell for these tumors is an abnormal gonocyte that fails to differentiate completely into spermatogonia. These abnormal cells stay dormant in the gonad during intrauterine life as well as infancy and childhood, but undergo proliferation during puberty and can be identified as intratubular germ cell neoplasia unclassified (IGCNU). These tumor cells continue to manifest protein expression pattern that resembles primitive germ cells (PLAP, c-KIT, OCT3/4). After a variable interval following puberty, IGCNU cells may acquire ability to penetrate the seminiferous tubules and present as an overt germ cell tumor. Acquisition of isochrome 12 and other genetic abnormalities are usually associated with this transition. The level of DNA methylation generally determines the phenotype of the germ cell tumor. The third type of germ cell tumors is spermatocytic seminoma, which is a rare tumor encountered later in life usually in fifth and sixth decade. The cell of origin of this tumor is probably postpubertal mature spermatogonia which acquire abnormal proliferative capability probably due to gain of chromosome 9 resulting in activation and amplification of genes such as DMRT1. The tumor cells manifest many of the proteins normally expressed by mature sperms such as VASA, SSX2, and occasionally OCT2. Although spermatocytic seminoma may also have an intratubular growth phase, it completely lacks features of IGCNU. Key Words: germ cell tumor, intratubular, testis, seminoma, yolk sac, teratoma, embryonal carcinoma
incidence of testicular germ cell tumors increases shortly after the onset of puberty and peaks in the fourth decade of life with a median age of 34 years at diagnosis.1–6 Testicular germ cell tumors are generally grouped into 3 broad categories. Type I testicular germ cell tumors are encountered in young prepubertal male children and consist of benign teratomas and malignant yolk sac tumors (Table 1). These tumors are generally not associated with any intratubular neoplasia. Type II testicular germ cell tumors (including seminoma, embryonal carcinoma, teratoma, yolk sac tumor, choriocarcinoma, and mixed germ cell tumors) affect men aged between 20 and 40; although the inception of these tumors is believed to occur during intrauterine life at the time of development of fetal gonads. All type II testicular germ cell tumors develop from a preinvasive lesion termed intratubular germ cell neoplasia unclassified (IGCNU), which in the past has been called carcinoma in situ. Type III testicular germ cell tumors is called spermatocytic seminoma (SS), which is mostly a benign tumor usually affecting men above 50 years of age. SS may manifest an intratubular growth pattern, although the significance of this finding in relation to tumor histogenesis is not clear.6–10 The purpose of this review is to examine embryologic and neonatal development and differentiation of normal germ cells and highlight potential areas of this differentiation that are modified during the development of testicular germ cell tumors. We also discuss the genetic and epigenetic factors that contribute to inception of germ cell neoplasia within the testicular tubules as well as factors responsible for its progression to invasive germ cell tumors.
(Adv Anat Pathol 2015;22:202–212)
INTRATUBULAR GERM CELL NEOPLASIA OF THE TESTIS: A BRIEF REVIEW Testicular tumors are the most common malignancies among men between the ages of 20 and 40 years. These tumors comprise approximately 1% of all cancers in men, but only about 0.1% of cancer deaths in males because the majority of these tumors are curable. More than 90% of testicular neoplasms originate from germ cells. The From the Department of Pathology and Laboratory Medicine, King Faisal Specialist Hospital and Research Centre, Riyadh, Kingdom of Saudi Arabia. The authors have no funding or conflicts of interest to disclose. Reprints: Mohammed Akhtar, MD, FCAP, FRCPA, FRCPath, Department of Pathology and Laboratory Medicine (MBC 10), King Faisal Specialist Hospital and Research Centre, P.O. Box 3354, Riyadh 11211, Kingdom of Saudi Arabia (e-mails: [email protected]; [email protected]). All figures can be viewed online in color at http://www.anatomic pathology.com. Copyright r 2015 Wolters Kluwer Health, Inc. All rights reserved.
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EMBRYOLOGIC ORIGIN OF GERM CELLS Germ cells are responsible for the passage of genetic material from one generation to the next and are therefore crucial to the survival and perpetuation of the species. Fertilization of oocytes by sperm promotes the formation of a zygote that undergoes cell division and cleavage to form a blastocyst. The outer layer of blastocyst gives rise to the trophectoderm, whereas the inner cell mass contains embryonic stem cells. By day 9 the inner cell mass differentiates into epiblast and hypoblast, setting the stage for gastrulation (day 15), during which the blastocyst develops the 3 germ layers (ectoderm, mesoderm, and endoderm) and cavitates further to develop yolk sac and the amniotic cavity (Fig. 1). The first glimpses of formation of germ cells called primordial germ cells are observed in the embryo after implantation, shortly before and during gastrulation. Some of the cells within the epiblast, as a result of specific signals from neighboring cells (inductive signaling), are induced to acquire the commitment and specification of primordial Adv Anat Pathol
Volume 22, Number 3, May 2015
Adv Anat Pathol
Volume 22, Number 3, May 2015
TABLE 1. Germ Cell Tumors of the Testis: 3 Broad Categories
Type 1
Type II
Prepubertal Teratoma
Postpubertal Classic seminoma
Yolk sac tumor
Embryonal carcinoma Yolk sac tumor Teratoma Choriocarcinoma Mixed germ cell tumors
Type III Usually >50 Spermatocytic seminoma
germ cells. Later, in response to molecular cues from the yolk sac, these cells segregate from the epiblast and migrate to localize in the wall of the yolk sac, at the base of the allantois.11–15 Two factors play a significant role in inducing the primordial germ cells to maintain the commitment to germ cell line and thus preventing these cells from acquiring somatic differentiation. First factor is the expression of BLIMP (B lymphocyte-induced maturation protein) within the primordial germ cells, which prevents the activation of somatic genes such as HOX, FDF8, and SNAIL. Secondly, the highly proliferative nature of the primordial germ cells results in lack of maintenance of adequate levels of DNA methyl transferase within these cells. This causes general demethylation of the DNA in primordial germ cells leading to complete loss of parental imprinting. The suppression of somatic cell phenotype along with demethylation results in expression of pluripotency factors such as NANOG and OCT 3/4. The exact roles of OCT 3/4 and NANOG in primordial germ cells are currently unknown. It is likely that they maintain pluripotency in germ cells and prevent somatic differentiation in these cells. OCT 3/4 is also believed to control the survival of the primordial germ cells, whereas NANOG expression facilitates further maturation of germ cells once they reach the developing gonads.14
MIGRATION OF PRIMORDIAL GERM CELLS During the fourth gestational week, when the embryonic disc undergoes a process of folding, primordial germ cells are passively incorporated into the embryo together with the yolk sac wall. They become segregated as single cells among the lining epithelial cells of the primitive hindgut and midgut. The germ cells travel within the
Intratubular Germ Cell Neoplasia of the Testis
mucosa until they reach midline near the aorta in the vicinity of the developing gonadal ridges, which are visible as distinct structures within the intermediate mesoderm at the beginning of the fifth week. At this time, primordial germ cells penetrate the mesenchyme surrounding the gut epithelium through breaks in the basal lamina, reach the dorsal mesentery, and continue to move laterally, beyond the primitive mesonephros and eventually enter the gonadal ridges.11–15
THE GONOCYTES Within the developing gonads the germ cells proliferate and are surrounded by Sertoli cells. The primordial germ cells are now designated as the gonocytes (Fig. 2). Primordial germ cells and gonocytes are both undifferentiated embryonic germ cells which can be identified by embryonic and stem cell markers including PLAP, NANOG, c-KIT, SOX2, and OCT3/4. Both cells have their original genomic imprinting pattern completely erased by DNA demethylation, which allows development of sexspecific germ cell lineages. In the presence of a Y chromosome, the gonadal stromal cells express transcription factor SRY and its target gene SOX9 resulting in the early development of Sertoli cells, which surround the gonocytes and form the seminiferous tubules (Fig. 2). The Sertoli cells create a microenvironment that allows differentiation of gonocytes into spermatogonia. During the remainder of the fetal life and into early postnatal period the germ cells gradually lose expression of PLAP, NANOG, SOX2, and OCT3/4, partially lose expression of KIT, and acquire expression of germ cell–specific proteins including MAGE4A, VASA, TSPY, OCT2, and SSX2 as they gradually transition from gonocytes to spermatogonia (Fig. 3). This transition occurs in an asynchronous manner such that cells at different stages of development may be present in an individual seminiferous tubule. Thus during this period some of the cells may coexpress gonocyte and spermatogonial markers. During the late phases of gestation, the gonocytes gradually migrate peripherally from the center of the seminiferous tubules toward the basement membrane to become either spermatogonia or spermatogonial stem cells. This process continues until about a year after birth and culminates in spermatogonia ultimately losing all the pluripotency and embryonal markers and instead expressing germ cell markers.11–15
THE RISK FACTORS FOR TYPE II TESTICULAR GERM CELL TUMORS
FIGURE 1. Diagram depicting sequence of events following fertilization leading to the segregation of primordial germ cells from the remaining cells which are destined for somatic differentiation. By day 15 the germ cells have moved to the wall of yolk sac. For further detail please see the text.
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Germ cell tumors of the testis may result from derangement of the normal process of germ cell maturation. A variety of risk factors for the development of testicular germ cell tumors have been identified. These include family predisposition, disorders of sexual development, cryptorchidism, high maternal estrogen during fetal development, HIV infection, environmental exposures, subfertility or infertility, and previous history of testicular germ cell tumors.1,14,15 Genetic factors, including deletions on the Y chromosome, androgen insensitivity, and KIT mutations, negatively impact normal germ cell development, which increases the risk of neoplastic transformation. Patients lacking the SRY gene on the Y chromosome required for development and differentiation of Sertoli cells have a high risk of type II testicular germ cell tumors, as the lack of
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FIGURE 2. A, Photomicrograph featuring developing kidneys and testes in a 10-week-old fetus. B, Higher magnification photomicrograph showing seminiferous tubules containing gonocytes surrounded by Sertoli cells. Please see this image in color online.
properly differentiated Sertoli cells inhibits germ cell development and maturation. Furthermore, genetic association studies have identified mutations in genes encoding KIT, KIT ligand, and its downstream signaling molecules such as KRAS, SPRY4, and BAK1, as likely predisposition genes for testicular germ cell tumors. A combination of both genetic and environmental factors may also prevent or delay the maturation of gonocytes into spermatogonia by retarding the development and maturation of the supporting Sertoli and Leydig cells, thus perturbing the microenvironment required for normal germ cell development.1,15,16
INTRATUBULAR GERM CELL NEOPLASIA UNCLASSIFIED The precursor lesion of almost all type II testicular germ cell tumors is IGCNU, defined as malignant germ cells
FIGURE 3. Sequence of events involved in transformation of primordial germ cells (primordial germ cells) to gonocytes and then spermatogonia. Maturation of gonocytes to spermatogonia starts during intrauterine period and continues into infancy. Primordial germ cells and gonocytes undergo demethylation of DNA and express markers of pluripotency such as PLAP, NANOG, OCT3/4, and AP-2g. These cells also express C-KIT which ensures their survival. Following maturation of gonocytes to spermatogonia, the pluripotency markers are switched off and there is expression of germ cell markers including MAGE4A, TSPY, and VASA among others. The spematogonia undergo male-specific remethylation as part of the process of maturation. Please see this image in color online.
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confined to the seminiferous tubules, which usually lack normal spermatogenesis. Testicular IGCNU is characterized histologically by proliferation of basilar malignant germ cells with enlarged hyperchromatic nuclei, often 1 or 2 prominent nucleoli, and clear cytoplasm due to abundant glycogen (Fig. 4). Sertoli cells are often displaced luminally. Affected seminiferous tubules usually have thickened peritubular basement membranes. Pagetoid spread of IGCNU into the rete testis is common. The interstitium may show hyperplasia of the Leydig cells. Although IGCNU can be diagnosed in the testes of prepubertal individuals, the histologic diagnosis can be challenging because IGCNU cells resemble normal infantile gonocytes.17 IGCNU cells were first identified in 1972 in biopsies of infertile men, and a causal link was subsequently established during follow-up, when the men with this lesion developed tumors, whereas control subjects exhibited no cases of TGC.18 Intratubular germ cell neoplasia is seen adjacent to the invasive tumors in 72% to 98% of the cases or in contralateral testis in 4.9% to 6.6% of cases.2 In addition, IGCNU can be a sporadic finding in approximately 0.4% to 0.8% of the testes of otherwise healthy males.19–27 The widely accepted theory of testicular germ cell tumors tumorigenesis is that development of IGCNU starts in utero.14,28 Transformation of fetal gonocytes to spermatogonia is a crucial step in proper maturation of the germ cells for future progression to spermatogenesis. The disturbance of germ cell development at this stage may result in arrest of fetal germ cell differentiation, making them susceptible to neoplastic transformation. IGCNU cells appear to be committed to germ cell differentiation at the level of the gonocytes but are blocked from differentiating further into spermatogonia and entering spermatogenesis (Fig. 5). Fetal gonocytes such as primordial germ cells have a completely erased genomic imprinting pattern by DNA demethylation, and are therefore susceptible to mutational events accumulating during cell replication. These mutations may activate oncogenes as well as silence some of the tumor-suppressing genes. The combined effect of these changes may be transformation of some of the gonocytes to neoplastic cells. These cells, however, stay dormant until puberty and early adulthood when proliferation is believed to occur in the setting of hormonal
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Intratubular Germ Cell Neoplasia of the Testis
FIGURE 4. A and B, Two tubules with intratubular germ cell neoplasia unclassified (IGCNU). The tumor cells are larger than normal spermatogonia and have large nuclei and mostly clear cytoplasm due to abundant glycogen. The tubule in (A) has markedly thickened lamina propria. Please see this image in color online.
stimulation thus resulting in IGCNU (Figs. 6, 7). The IGCNU cells proliferate and gradually replace normal germ cells within the affected tubules but continue to rely on Sertoli cell–derived growth/survival signals and production of testosterone by Leydig cells.28,29 Morphologically, IGCNU cells resemble primordial germ cells/gonocytes and express a number of similar markers, in that they lack parental imprinting and express OCT 3/4, PLAP, and c-KIT (Fig. 8). In fact, the transcriptome of IGCNU cells is very similar to that of isolated normal human gonocytes, demonstrating a close relationship between these cell types.12 IGCNU subsequently progresses to invasive testicular germ cell tumors with differentiation into the various histologic subtypes of testicular germ cell tumors either before or after invasion.
PROGRESSION OF INTRATUBULAR GERM CELL NEOPLASIA UNCLASSIFIED With the acquisition of additional mutational events, the neoplastic cells break their dependence on the signals from Sertoli cells for proliferation and enter a phase of independent proliferation. The tumor cells now fill the
FIGURE 5. Development of intratubular germ cell neoplasia unclassified (IGCNU) starts during intrauterine life when a gonocyte fails to undergo proper maturation into spermatogonium and continues to express pluripotency markers. These cells stay dormant until puberty but during and after puberty they undergo proliferation and replace the normal germ cells within the seminiferous tubules. Please see this image in color online.
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tubular lumina, acquire the phenotype of one of the TGT (usually seminoma and/or embryonal carcinoma), and subsequently break through the tubular wall and invade the gonadal stroma thus transforming into an overt testicular germ cell tumors (Figs. 9–13). It has been estimated that approximately 70% of patients with ITGCNU will develop an invasive germ cell tumor within 7 years, which may be extrapolated to 100% at 10 years.23 Intratubular germ cell tumors with morphologic features of seminoma and embryonal carcinoma are frequently observed in testes with invasive germ cell tumors.30,31 Rarely intratubular seminoma or embryonal carcinoma may be seen in the absence of any invasive component. Intratubular syncytiotrophoblastic and cytotrophoblastic cells have been seen occasionally but as reported by Berney et al,31 may actually be more common than previously thought. These cells are usually seen in association with seminoma and less frequently with embryonal carcinoma. These cells are encountered as isolated cells or small clusters within the tubules. Their recognition is greatly facilitated by immunohistochemical staining. Syncytiotrophoblastic cells are strongly immunoreactive for human chorionic gonadotropin, whereas cytotrophoblasts may be negative or stain only lightly. The invasive component in cases with
FIGURE 6. A, Diagram depicting normal development and maturation of germ cells during intrauterine period, infancy, and childhood. The gonocytes undergo a gradual maturation so that all the gonocytes are ultimately transformed to spermatogonia. B, In individuals who are destined to develop type 2 germ cell tumors, some of the gonocytes are abnormal and fail to make the transformation to spermatogonia. The abnormal cells, however, stay dormant within the tubules until the onset of puberty.
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FIGURE 7. A, In normal testis with the onset of puberty, the spermatogonia undergo proliferation and proceed to normal spermatogenesis. B, The abnormal gonocytes undergo proliferation during puberty and afterward and gradually replace the normal germ cells resulting in IGCNU. After a variable interval (usually several years), cells with morphologic features of seminoma and/or embryonal carcinoma may evolve from IGCNU. These cells later break through the tubular walls and produce overt germ cell tumors. IGCNU indicates intratubular germ cell neoplasia unclassified.
intratubular trophoblastic cells also contains these cells. However, no cases of intratubular choriocarcinoma have been reported. Similarly, elements of intratubular teratoma
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have not been recorded. These findings indicate that IGCNU may give rise to intratubular seminoma and embryonal carcinoma (with or without trophoblastic cells), which may subsequently invade the gonadal stroma. Although IGCNU is generally accepted as a form of a noninvasive precursor lesion for seminoma and embryonal carcinoma, the developmental relationship between seminoma and embryonal carcinoma remains poorly understood. Some studies suggest a single consecutive progression from IGCNU to seminoma and then to embryonal carcinoma (linear progression), whereas others suggest that seminoma and embryonal carcinoma independently derive from IGCNU (nonlinear progression). In contrast, yolk sac tumor, teratoma, and choriocarcinoma seem to evolve from a background of invasive embryonal carcinoma29,32 (Figs. 14, 15). Chromosome 12p alterations are the hallmark of transition of IGCNU into overt testicular germ cell tumors.6,7,14,16,28,33 It is identified in approximately 80% of all invasive testicular germ cell tumors, as well as in intratubular embryonal carcinoma and intratubular seminoma. In the remaining 20% amplification of shorter stretches of 12p may be present. Interestingly, IGCNU without adjacent invasive testicular germ cell tumors does not contain isochromosome 12p in most studies, which suggests that isochromosome 12p is not required for the development of IGCNU and may be indicative of progression beyond the stage of IGCNU.
FIGURE 8. The cells in IGCNU continue to manifest protein expression pattern of primordial germ cells and gonocytes. A: PLAP, B: CKIT, C: OCT3/4. Please see this image in color online.
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Intratubular Germ Cell Neoplasia of the Testis
FIGURE 9. A and B, Intratubular seminoma filling the seminiferous tubules. Please see this image in color online.
FIGURE 10. A and B, Intratubular embryonal carcinoma. Please see this image in color online.
FIGURE 11. Intratubular seminoma (lower part) and intratubular embryonal carcinoma (upper part). Please see this image in color online.
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FIGURE 12. Intratubular seminoma with stromal invasion. Please see this image in color online.
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FIGURE 13. Intratubular embryonal carcinoma with early stromal invasion. Please see this image in color online.
It has been hypothesized that amplified region on 12p may harbor genes which enable the tumor cells to survive, proliferate, and develop invasive growth independent of signals from the Sertoli cells. Several candidate genes have been suggested, including KITLG, NANOG (and its pseudogenes), KRAS2, BCAT1, and CCND2. However, the exact genes still have not been identified. Further studies are required before we fully understand the precise role of chromosome 12p in testicular germ cell tumors carcinogenesis. Less frequent cytogenetic aberrations found in testicular germ cell tumors include overpresentation of chromosomes 7, 8, 12, 17, and X, and underpresentation of chromosomes 4, 11, 13, 18, and Y. Mutations in single genes including KIT, TP53, KRAS/ NRAS, and BRAF have been implicated; however, the frequency of these mutations is relatively low.6–8,16,32 KIT is a protooncogene located on chromosome 4q11q12. Its gene product CD117 is a tyrosine kinase receptor that in the presence of KIT ligand dimerizes and becomes phosphorylated. KIT plays a crucial role in the survival, proliferation, and migration of normal primordial germ
FIGURE 14. Progression of IGCNU to embryonal carcinoma and/ seminoma involves development of isochromosome 12. The cells in seminoma continue to manifest demethylation; however, the development of embryonal carcinoma involves remethylation of DNA. Other germ cell tumors seem to arise from embryonal carcinoma and have even higher levels of DNA methylation. IGCNU indicates intratubular germ cell neoplasia unclassified. Please see this image in color online.
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cells and gonocyte, both of which are immunoreactive for KIT.34,35 KIT is also expressed in almost all IGCNU, most seminomas, and some NS germ cell tumors.6,16,28 Some of these tumors may additionally express KIT ligand, thus setting the stage for an autocrine or paracrine system to stimulate tumor cell proliferation. Gain-of-function mutations in KIT may be encountered in seminomas (19%) and uncommonly in nonseminomatous germ cell tumors (2%), resulting in constitutive activation of KIT which drives tumorigenesis. KIT-mutated testicular germ cell tumors may be potentially amenable to therapy by KIT inhibitors such as Imatinib mesylate.16,28,36 Activating mutations have been associated with several bilateral testicular germ cell tumors.6,16,28 In a retrospective study of 224 unilateral and 61 bilateral testicular germ cell tumors, Looijenga et al37 demonstrated significantly higher incidence of an activating KIT mutation in bilateral tumors compared with unilateral tumors (93% vs. 1.3%). However, recently, Coffey et al38 analyzed 175 unilateral and 40 bilateral testicular germ cell tumors and found no evidence of an increased frequency of KIT mutations in bilateral testicular germ cell tumors. Similar lack of KIT mutation in bilateral testicular germ cell tumors was reported by Sakuma et al39 from Japan. The reason for this discrepancy is not clear.
SEMINOMATOUS VERSUS NONSEMINOMATOUS GERM CELL TUMORS Although both seminoma and NS germ cell tumors arise from the IGCNU cells, they seem to exhibit distinct gene expression signatures with almost 90% of genes discordantly regulated between the 2 groups of tumors. Seminomas in general have upregulated genes such as oncogenes, genes encoding intracellular transducers, and genes related to DNA synthesis, proliferation, and repair. In NS germ cell tumors these genes are mostly downregulated. Nonseminomatous germ cell tumors are typically hypotriploid and seminomas are usually hypertriploid. This would indicate that despite a common origin, these tumors seem to follow very different pathways of differentiation. Epigenetic changes play a crucial role in normal development, maturation, and differentiation of germ cells.27,28,39–41 Primordial germ cells and gonocytes DNA are generally hypomethylated, whereas more mature germ cells such as spermatogonia and spermatocytes are fully methylated. IGCNU and seminomas exhibit low levels of DNA methylation and permissive chromatin structure associated with high transcriptional and proliferative activity. More differentiated testicular germ cell tumors (yolk sac tumors, choriocarcinomas, and teratomas) show a higher degree of methylation with embryonal carcinomas exhibiting an intermediate pattern. Thus it seems that different levels of global methylation in testicular germ cell tumor subtypes may determine the final phenotype of the tumor.
SPERMATOCYTIC SEMINOMA SS is a relatively rare tumor representing
