GCNIS is defined as the presence of malignant germ cells within the seminiferous tubules of the testis. Classically, these neoplastic germ cells are characterized by having abundant clear cytoplasm and a prominent enlarged nucleus, and they are usually located in the basal layers of the tubule (Fig. 7.2). It was originally described by Skakkebaek in 1972 [77]. Because of its undifferentiated nature, the term intratubular germ cell neoplasia unclassified type has been historically preferred in the USA over the term carcinoma in situ, more popular in European countries, as the neoplastic cells do not show epithelial differentiation nor necessarily give rise to invasive carcinomas. Recently, the term germ cell neoplasia in situ has been proposed for this lesion and was incorporated in the 2016 WHO classification [76]. This terminology will be used throughout this chapter.

The neoplastic nature of GCNIS has been demonstrated by epidemiological, morphological, and biological studies. Fifty percent of patients with GCNIS will develop an invasive form of germ cell tumor within 5 years [47]. Similarly, more than 90 % of patients with an invasive form of GCT show GCNIS in the adjacent testicular parenchyma [78–80]. GCNIS is found in patients with a high risk of developing invasive GCT, including patients with cryptorchidism, gonadal dysgenesis, contralateral GCT, infertility, and androgen insensitivity syndrome [46, 48, 49]. Morphologically, the cells of GCNIS are markedly similar to the individual cells of seminoma and share a similar immunophenotype, including the expression of c-KIT, OCT4, SALL4, and placental-like alkaline phosphatase (PLAP) [81–83]. Molecularly, they are aneuploid and share allelic losses with invasive GCT [57].

No specific findings are grossly detected in cases of GCNIS that are associated with an invasive tumor. Given that it is commonly associated with infertility, changes of atrophy are not uncommon. Microscopically, GCNIS is characterized by the presence of large cells with clear cytoplasm, conspicuous cell membranes, enlarged and hyperchromatic nuclei, and prominent nucleoli (Fig. 7.2) [58]. These cells stand out in low power and are usually found in the basal layer of seminiferous tubules with atrophic features. In fact, it is extremely rare to find it in tubules with ongoing spermatogenesis. The involved tubules tend to be small and have a thickened basement membrane. Aside from the GCNIS cells, they usually contain only Sertoli cells. The distribution of GCNIS is usually patchy, and affected tubules may be located immediately adjacent to completely unremarkable ones. Peritubular lymphoid and granulomatous inflammation may also be present. GCNIS may show pagetoid spread into the rete testis (Fig. 7.3) [84] and less frequently into the epididymis and vas deferens [85].

PAS stains demonstrate abundant intracytoplasmic glycogen and may be useful as a screening stain. GCNIS is almost always positive for PLAP with a membranous/cytoplasmic pattern (Fig. 7.4a) and is quite useful, as normal spermatogonia are rarely positive for this marker. OCT4 is also highly specific, displaying nuclear staining only in neoplastic germ cells (Fig. 7.4b). c-KIT is highly sensitive, but its specificity is rather low, as nonneoplastic germ cells may react with this marker. The same can be said for SALL4 [82].

While GCNIS shares most of its molecular profile with invasive GCT, particularly seminoma, 12p amplification, usually in the form of i(12p), is rarely present in GCNIS, contrary to the case in invasive GCT. It is thus believed that the presence of i(12p) is probably involved in the ability of GCNIS to invade the stroma (see below) [82].

GCNIS must be distinguished from other intratubular forms of GCT, such as intratubular seminoma (Fig. 7.5a). These result for the most part from intratubular spread of invasive GCT, as they are rarely seen in the absence of an invasive component. Intratubular seminomas share many of the features of GCNIS; however, they fill and distend the involved tubules. They may be seen without an invasive seminoma counterpart, but usually they are accompanied by some kind of invasive GCT [61, 62]. Whether they represent an exaggerated form of GCNIS or an intratubular spread of an invasive seminoma is unresolved. Intratubular EC shows the epithelial differentiation and high-grade cytologic features of its invasive counterpart, including necrosis (Fig. 7.5b) [60]. Intratubular teratoma [86] and YST [87] have also been reported. Intratubular ST shows the three characteristic types of cells of the invasive counterpart (see below) and is always associated with an invasive component [58]. Metastatic carcinomas to the testis may show involvement of seminiferous tubules resembling GCNIS [88].

Prepubertal testes tend to show enlarged, slightly atypical germ cells with abnormal chromatin. In this setting, particularly in young patients (less than 2 years), the atypical cells may share an immunophenotype with GCNIS. They are thought to represent delayed maturation of germ cells, frequently associated with DSD [89]. However, they are likely to be present diffusely throughout the parenchyma and are not limited to the basal layer within the tubules. Distinction between these two settings is clinically relevant, as these abnormal germ cells in prepubertal patients do not convey a high risk to progression to an invasive neoplasm. OCT4 is the preferred marker to use in the workup of this differential diagnosis [82].

Adult testis may also harbor occasional atypical germ cells, which usually show hyperchromatic nuclei, multinucleation, or enlarged size [90]. They lack the classic prominent nucleoli and typical distribution of GCNIS and do not share their immunophenotype (Table 7.3). While they may represent a manifestation of testicular dysgenesis, and thus may be present in the background of GCT, by themselves, they do not convey the high-risk implications of GCNIS.

During embryological development, germ cells, once located in the gonadal ridges where they are surrounded by mesenchymal cells, are termed gonocytes and are characterized by the expression of stem cell markers, including PLAP, NANOG, c-KIT, SOX2, and OCT4. Gonadal stromal cells express transcription factor SRY, which results in early development of Sertoli cells through the activation of SOX9 [58]. Sertoli cells create a microenvironment that allows differentiation of gonocytes into spermatogonia. This process marked by the gradual loss of expression of the stem markers mentioned above, the acquisition of expression of germ cell-specific proteins MAGE4A, VASA, TSPY, OCT2, and SSX2, and the migration toward the basement membrane of the seminiferous tubule. Disturbances of the microenvironment result in arrest of fetal germ cell differentiation. It is in this arrested stage that mutations in oncogenes or tumor-suppressing genes are believed to occur, resulting in transformation into a neoplastic cell [58]. In fact, GCNIS widely shares the immunophenotype of gonocytes, suggesting an arrest at this stage [91, 92].

With the acquisition of additional mutational events, likely potentiated by the changes in hormonal milieu at puberty, the neoplastic cells eventually acquire the capacity to invade through the basement membrane. The 12p abnormalities are consistently present in invasive tumors and are absent in GCNIS [74, 75]. It is thus likely that the abnormal region on 12p harbors genes that enable the tumor cells to survive, proliferate, and develop invasive growth independent of signals from the intratubular Sertoli cells and the adjacent Leydig cells. Candidate genes include KITLG, NANOG, BCAT1, and CCND2, but their exact definition remains elusive [58, 93]. The pathway to this may include intratubular transformation to either intratubular seminoma or intratubular EC (nonlinear progression) [94] or development first of an invasive seminoma and then transformation into EC (linear progression) [57]. YST, teratoma, and choriocarcinoma (CC) appear to evolve by differentiation from EC (Fig. 7.6).

Seminoma is the most common GCT comprising approximately 50 % of these tumors in the postpubertal setting. The average age at presentation is 40 years with most patients presenting between 35 and 45 years of age. Seminoma is unusual in childhood, and after the fifth decade [95], however, in a study by Berney et al. [96], seminoma accounted for 82 % of cases of GCT in the elderly. Bilateral involvement is seen in up to 5 % of cases. Seminoma has shown the highest incidence of bilaterality among GCT in several studies [97, 98]. Patients with seminoma often present with a painless testicular mass or dull aching sensation. Up to 2–3 % of patients present with symptoms related to metastatic disease in the retroperitoneum. Typically serum levels of AFP are normal; however mild elevations have been described in pure seminomas [99]. Significant elevations are regarded as evidence of non-seminomatous elements and should prompt a careful search for these components. Liver disease including metastasis of pure seminoma may explain the presence of mild to moderate elevations of AFP. Mild serum β-hCG elevation is observed in seminomas with syncytiotrophoblasts and can be associated with the development of gynecomastia [100, 101].

Grossly seminoma usually has a nodular configuration with well-circumscribed borders. The cut surface is lobulated, cream, tan, or white gray and shows variable consistency, reflecting the amount of fibrous tissue within the tumor (Fig. 7.7). Punctate foci of hemorrhage are often associated with the microscopic presence of syncytiotrophoblasts. Larger foci of hemorrhage and necrosis can be seen in tumors of large size. Invasion into the testis mediastinum and spermatic cord are uncommon with a reported frequency of 5–8 % [95].

Seminoma often has a diffuse sheetlike, nested, or trabecular growth. Characteristically the tumor is traversed by bands of connective tissue of variable thickness containing mature lymphocytes, predominantly of T cell type (Fig. 7.8a). These lymphocytes are also seen interspersed among tumor cells, but can be very prominent, occasionally obscuring the tumor cells. Cytologically the tumor cells show a uniform appearance with polygonal to round configuration and moderate amounts of clear, amphophilic, or eosinophilic cytoplasm with fairly distinct cell membranes (Fig. 7.8b). The latter attribute helps differentiate this tumor from EC, which exhibits a syncytial growth pattern. The nucleus is usually centrally placed and shows an evenly distributed chromatin with prominent nucleoli. The nuclear membrane is not perfectly round, with tendency to show angulated or flat contours, a term described by some as “squared-off” nuclei [102]. Some seminoma cells may have an eccentrically placed nucleus resulting in a plasmacytoid appearance. In contrast to plasma cells, a perinuclear hof is not observed. Mitotic activity is variable and may be quite brisk. Importantly, the degree of mitotic activity does not appear to correlate with tumor behavior. The term “anaplastic seminoma” was used in the past to designate seminomas with a mitotic rate equal or greater than three mitoses per high-power field and increased pleomorphism. This terminology has been discouraged due to insufficient data supporting a worsened prognosis in this subset of tumors [103, 104]. Similarly, necrosis, even when extensive, does not suggest a more aggressive behavior (Fig. 7.9). Prominent cytological atypia and pleomorphism, suggestive of early transition to EC, can be seen in seminoma [105]. In general most authors advice against designating these foci as EC, unless clear epithelial features such as glands or papillae are observed [106]. Up to 50 % of seminomas show granulomatous inflammation (Fig. 7.10a) [107]. This may range from scattered clusters of epithelioid histiocytes to an exuberant and diffuse reaction that may masquerade the tumor cells mimicking granulomatous orchitis. A subset of seminomas may exhibit ossification and calcification.

Scattered syncytiotrophoblasts can be seen in up to 20 % of seminomas (Fig. 7.10b) [108]. These are characterized by multinucleation, large size, and usually denser cytoplasm than the adjacent seminoma cells. Some may show prominent vacuolization of the cytoplasm. As mentioned above, these may be associated with small foci of hemorrhage. A distinction should be made with CC, which will require the presence of both syncytiotrophoblasts and cytotrophoblasts. The presence of syncytiotrophoblasts should thus be documented in the pathology report as it may explain the presence of mild elevations of β-hCG [109].

Some seminomas show a tendency to form tubular, cribriform, or microcystic structures (Fig. 7.11), by virtue of developing spaces in between the solid growth of tumor cells [110–114]. These spaces are often associated with prominent intratumoral edema, but in other cases it may be artifactually induced by poor fixation or tumor degeneration. The result is a neoplasm that deviates from the classical appearance of seminoma and introduces the possibility of other diagnosis, such as YST or Sertoli cell tumor [112]. Usually typical areas of solid growth can be seen nearby. Difficult cases may require immunohistochemistry to resolve this differential diagnosis. Occasionally, seminomas may have prominent signet-ring cell change, a finding that may elicit the differential diagnosis with metastatic carcinoma (Fig. 7.11) [115]. Some seminomas may show exclusively or predominantly an intertubular growth pattern, without forming a discrete mass or nodule [116]. Intratubular seminoma was discussed in the section of GCNIS. It is not infrequently seen in association with invasive seminoma. Finally, some seminomas may have a very prominent lymphoid infiltrate that not only obscures the neoplastic cells but may even simulate a lymphoma involving the testis [117, 118]. Awareness of this phenomenon should prompt the careful search of typical seminoma cells and the use of appropriate immunohistochemical markers.

Seminoma is typically positive for OCT4, PLAP, c-KIT, podoplanin, SOX17, and SALL4 (Table 7.4). Keratin A1/AE3 expression is usually focal and weak, although, in our institutional experience, we have come across rare cases with diffuse reactivity. Seminoma lacks expression of CD30, SOX2, AFP, glypican-3, and ß-hCG [82].

The differential diagnosis of seminoma includes other forms of GCT, Sertoli cell tumors, and lymphoma. Solid variants of EC and YST are the most common types of GCT confused with seminomas. EC displays more pleomorphism than seminoma, and typically there is at least focal glandular differentiation. When both types of tumors are present, comparison of the nuclear characteristics may help in classifying an area of solid growth. Additionally, nuclei of EC frequently overlap, and cell borders are difficult to differentiate, imparting a “syncytium” appearance. Cytoplasm in EC tends to be more amphophilic and dense. Differential expression of cytokeratin, CD30, c-KIT, and SOX17 may help in difficult cases. YST may be confused with seminoma, specially its solid and microcystic growth patterns. Solid YST lacks the fibrous septae and lymphocytic infiltrate of seminoma and is rarely unaccompanied by more classical patterns, except in biopsy specimens. Cytologically, they show more variation in size and shape of the nuclei and are accompanied by hyaline globules and basement membrane material deposition. In tumors with microcystic architecture, attention should be paid to the cells lining the microcysts. In seminoma, they maintain their size and shape, while in YST they tend to be flattened, forming a lining [112]. A potentially critical misdiagnosis occurs when Sertoli cell tumors grow with a solid growth pattern mimicking seminomas, sometimes even with an associated lymphocytic infiltrate. Attention should be paid to the much lower nuclear grade of Sertoli cell tumors. The absence of associated GCNIS is an important diagnostic clue that should prompt appropriate immunohistochemical stains [119]. As stated above, some lymphoid stroma-rich seminomas may mimic lymphomas, while those associated with a prominent granulomatous reaction may lead to a misdiagnosis of granulomatous orchitis. Seminomas are the prototype of testicular neoplasms with a solid growth pattern. The differential diagnosis of this morphologic category is presented in Table 7.5.

Clinical stage I seminoma has a cure rate of 99 %. Relapse rates at this stage are 15–19 % [120], with most posttreatment relapses occurring within 3 years post-orchiectomy [121]. Metastases typically occur first in the retroperitoneal lymph nodes and subsequently at supradiaphragmatic sites. Metastatic seminoma within the good prognosis group as determined by the International Germ Cell Cancer Collaborative Group has an overall 95 % survival rate. Intermediate prognosis seminoma is rare; therefore survival rates reported are limited by low patient numbers [120]. There is contradictory data with regard to prognostic value of rete testis invasion, tumor size, and invasion into the tunica albuginea in stage I seminoma [122–126].

In its pure form, EC represents 3 % of testicular neoplasms, but nearly 40 % of testicular GCT have a component of EC. The peak incidence of this tumor is in the second to third decade with an average of 32 years, about a decade earlier than seminomas. EC is rare in the prepubertal setting and appears to be associated with DSD [102]. The most common presentation is that of a palpable testicular mass (80 %) followed by hormonal-related symptoms, such as gynecomastia (10 %) and symptomatic metastatic disease (10 %) [127]. Metastases occur via hematogenous and lymphatic pathways and primarily affect the periaortic lymph nodes, lung, and liver [63]. Although older reports have noted serum AFP elevations in patients with pure EC, current literature suggests this may be the result of unrecognized YST elements [128]. ß-hCG elevation is commonly seen in EC and reflects the presence of syncytiotrophoblasts.

EC has a bulging soft, pale tan, pink, or dark-brown cut surface with areas of hemorrhage and necrosis (Fig. 7.12). The tumor borders are often ill defined. Tumor extension into the mediastinum testis and testicular adnexa is observed in up to 25 % of cases [106]. Compared to seminoma, EC are smaller at presentation, with average tumor size of 2.5 cm in diameter.

EC is composed of large pleomorphic cells with indistinct borders and nuclear crowding or overlap. Most tumor cells show vesicular chromatin and macronucleoli. The cytoplasm is predominantly basophilic although foci of amphophilic or clear cytoplasm are not unusual. Single-cell necrosis with frequent apoptotic bodies and mitosis are also readily encountered. Architecturally solid, papillary, and glandular patterns are the most common (Figs. 7.13, 7.14, and 7.15). The majority of EC show coexistence of two or more of the abovementioned patterns. Syncytiotrophoblasts are common in EC with a reported frequency of 46 % [129]. They are typically not associated with hemorrhage, in contrast with CC. Small amounts of undifferentiated spindled cellular stroma are accepted by some authors as part of EC, while others regard this as immature teratomatous mesenchyme [102, 127]. A granulomatous response of varied severity may be seen in EC [129]. Angiolymphatic invasion is commonly encountered at the periphery of the tumor. In fact, EC is the most common tumor identified in foci of lymphovascular invasion in mixed GCT. Intratubular EC is also similarly found at the edge of the tumor and is characterized by smudged hyperchromatic tumor cells admixed with abundant eosinophilic necrotic debris. Recognition of a thickened tubular basement membrane and residual Sertoli cells is helpful in avoiding misdiagnosis of intratubular EC as angiolymphatic invasion. Coarse calcifications are often seen in intratubular EC (Fig. 7.5b).

The morphologic spectrum of EC is relatively broad [129]. It includes patterns commonly associated with YST (see below) or that may mimic seminoma or other neoplasms. An important pattern to recognize is the so-called appliqué pattern. This results in smudged degenerated cells located at the periphery of tumor nests. These cells closely resemble syncytiotrophoblasts and may lead to confusion with CC. Secretory-type change is characterized by subnuclear vacuoles similar to secretory endometrium. The pseudo-endodermal sinus pattern includes the presence of structures similar to the Schiller-Duval bodies of YST [130]. Sievelike pattern is reminiscent of the microcystic pattern of YST. In these patterns, differentiation from YST is based on the degree of pleomorphism of the tumor cells. In cases difficult to categorize, immunohistochemical staining with CD30 and OCT4 would favor EC. Solid forms may show tumor cells with prominent cell membranes and even mild lymphocytic infiltrate, resembling seminoma. Again, these EC variants will present higher degrees of pleomorphism than seminoma, and in difficult cases keratin and CD30 staining should resolve the problem. Other less common patterns include nested pattern, micropapillary pattern, anastomosing glands, necklace pattern, and a blastocyst-like pattern, characterized by large vesicles with edema fluid. Given the importance of reporting the percentage of EC in a mixed GCT (see below), accurate recognition of these patterns is mandatory to not under or overestimate the proportion of EC.

EC is positive for cytokeratin cocktails, OCT4, CD30 (Fig. 7.13, inset), SALL4, SOX2, and PLAP. The latter is usually weaker than that observed in seminoma. AFP may be focally positive. CD30 may be negative in metastatic EC [99]. In this setting coexpression of OCT4, SALL4, and strong and diffuse expression of cytokeratins may help support the diagnosis [131]. hCG expression is limited to syncytiotrophoblasts [97].

The solid pattern of EC may be confused with seminoma, lymphoma, ST, or solid YST (Table 7.5). YST is also a differential diagnosis in papillary and glandular patterns of EC (Table 7.6). The cytological features of EC may be sufficient to discriminate against other germ cell tumors in most cases; however, difficult cases can be easily resolved with the aid of immunohistochemical studies. Diffuse reactivity for OCT4, CD30, and cytokeratin is usually diagnostic (Table 7.4). Metastatic carcinomas to the testis may pose difficulty in distinguishing them from EC, but the absence of GCNIS, the usually older age of the patients, and commonly an intertubular growth pattern can raise the suspicion of a metastatic process [88].

EC is considered an aggressive form of GCT. In fact, the percentage of EC in mixed germ cell tumors has been regarded as a risk factor for metastatic spread [132, 133] (see “Mixed GCT” below).

YST is a malignant germ cell tumor that recapitulates the primitive endodermal structures, including embryonic yolk sac, allantois, and extraembryonic mesenchyme. A detailed review of the nomenclature, histogenesis, and histologic types is included in Chap. 6. In the testis, pure YST has a bimodal distribution with most cases occurring in the prepubertal period, while it is uncommon in the postpubertal period. Most reported cases of adult YST have occurred in the third and fourth decade. Elements of YST are present in up to half of mixed germ cell tumors. Clinically, YST presents as a painless palpable testicular mass. Elevation of serum AFP is noted in 95–100 % of cases [134].

YST appears poorly circumscribed and nonencapsulated and has a homogenous gray to white to tan gelatinous cut surface. Hemorrhage, necrosis, and cystic degeneration may be seen.

YST shows the most diverse histology among GCT with a multiplicity of architectural patterns, including microcystic (reticular), macrocystic, endodermal sinus, papillary, solid, glandular/alveolar (including so-called endometrioid and enteric), polyvesicular vitelline, myxomatous, sarcomatoid, hepatoid, and parietal. A more detailed description of the histologic types is presented in Chap. 6.

The microcystic or reticular pattern is the most common and consists of anastomosing cords of vacuolated to spindled cells resulting in a meshwork or spider web like appearance (Fig. 7.16). The cystic spaces often show basophilic secretions. Coalescence of the small cysts gives rise to the macrocystic pattern. The endodermal sinus pattern is characterized by the presence of Schiller-Duval bodies, which consist of papillary structures lined by a cuboidal to columnar malignant-appearing epithelium with a distinct central vessel (Fig. 7.17). The papillae are recessed in a cystic space lined by flattened epithelium. Papillary formations with or without fibrovascular cores that project into cysts are the hallmark of the papillary pattern. The lining cells often exhibit a hobnail appearance with high nuclear to cytoplasmic ratios. Exfoliated cells forming cell clusters can be seen in proximity of these papillae. The solid pattern consists of sheets of polygonal cells with variable nuclear yet with well-defined borders and moderate amounts of clear to amphophilic cytoplasm (Fig. 7.18). A variant form of solid YST with scant cytoplasm may have a blastema-like appearance. Glands lined by columnar epithelium with intestinal and secretory-phase endometrium-like features comprise the glandular pattern. These glands may be simple or branching and often present in a background of other YST patterns including microcystic, macrocystic, solid, or polyvesicular vitelline. The lining epithelium usually lacks cytological atypia and therefore it could be easily mistaken as teratomatous epithelium. Glandular branching and absence of an encircling smooth muscle layer are features that help differentiate YST from teratoma. The polyvesicular vitelline pattern is exceptional in the testis. It consists of vesicles/cysts lined by a bland epithelium present in an edematous to fibrous stroma. The vesicles may show an area of constriction which gives rise to an eight-shaped structure. The epithelium lining the vesicles often transitions from flat to cuboidal to columnar at the site of constriction. The myxomatous pattern consists of scattered innocuous spindle or stellate cells in a loose, myxoid stroma, which often features a rich capillary network (also called angioblastic pattern). Focal differentiation into skeletal muscle and cartilaginous elements is allowed in YST and should not prompt a diagnosis of teratoma [106]. The term sarcomatoid YST has been used to describe tumors with spindled cellular stroma that retains cytokeratin reactivity. A recent study [135] has proposed that some somatic sarcomatoid malignancies may actually represent sarcomatoid YST. Hepatoid YST consists of cells with abundant eosinophilic cytoplasm, large central nucleus, and prominent nucleolus arranged in nests, cords, or trabeculae. Bile secretion and canaliculi have been observed in these tumors. Parietal YST is characterized by bland neoplastic cells embedded in dense eosinophilic basement membrane material that recapitulates the parietal layer of the murine yolk sac (Reichert’s membrane). As noted above, cytological features of YST may vary across histological patterns. Significant nuclear atypia may be seen in solid, glandular, and sarcomatoid patterns of YST. Mitotic activity and single-cell necrosis can also be noted but are typically less prominent than in EC. A characteristic yet not pathognomonic feature of YST cells is the presence of intracytoplasmic eosinophilic globules, which are PAS-positive (Fig. 7.19).

Expression of AFP is variable and in many cases entirely absent. Intense staining for AFP is often seen in hepatoid pattern and in intracytoplasmic eosinophilic globules. Villin, glypican-3, SALL4, and low-molecular-weight keratins are often positive (Fig. 7.20) [82, 136, 137]. Rarely, c-KIT, SOX2, PLAP, and podoplanin positivity may be seen in YST [138]. This could represent a pitfall in the differentiation of the solid variant from seminoma. OCT4 and CD30 are negative. Hep-Par-1 reactivity has been documented in areas with or without hepatoid differentiation [136].

YST is the prototypic testicular neoplasm with glandular differentiation, a differential that is summarized in Table 7.6. Glandular YST may be confused with immature teratoma, as previously discussed. Solid YST may resemble seminoma. The lack of fibrous septa with lymphocytic infiltration may help in their differentiation. Immunohistochemical reactivity for OCT4 excludes YST and therefore is useful in their distinction. OCT4 may prove useful when glands or papillary structures show cytological atypia that overlaps with EC.

Prognostic data for adult pure YST is limited due to the rarity of this entity. A series of 12 patients with stage I and II adult pure YST revealed a similar clinical behavior with respect to other non-seminomatous testicular GCT [139].

Teratomas are GCT that display differentiation into somatic elements, including components of variable proportions from the ectoderm, mesoderm, and endoderm layers of the developing embryo. In the testis, they occur in two settings, based on the developmental stage of the surrounding testis: prepubertal or postpubertal. Contrary to their ovarian counterpart, the vast majority of postpubertal testicular teratomas are malignant, independent of the degree of maturity of its constituent elements. This is explained by the different histogenesis in both neoplasms. While in the ovary, teratomatous transformation of the germ cell occurs via parthenogenesis, in the testis it is most often an event that takes place after malignant transformation of a germ cell. As most other GCT in the male gonad, teratoma arises in association with GCNIS and, as explained before, likely corresponds to a terminally differentiated invasive component derived from more primitive forms, such as EC. Thus, postpubertal teratoma in the testis represents a form of differentiation of an already malignant neoplasm with a type II histogenesis. This explains its metastatic potential, independent of its degree of immaturity, and the fact that it is most frequently seen in association with other forms of GCT. Pediatric teratomas are reviewed in Chap. 10. However, occasionally, some teratomas in the postpubertal testis have a histogenesis and morphology comparable to those of the prepubertal gonad (type I) and thus behave in a benign fashion [140]. They are currently classified as prepubertal-type teratomas by the WHO [76] and will be considered separately (see below).

Gross features of teratomas reflect their more prevalent somatic components (Fig. 7.21). Cysts filled with mucinous material or keratinaceous laminated debris are common. However, testicular teratomas tend to be more solid than the ovarian counterpart. More fleshy solid areas usually represent less differentiated components and may be associated with hemorrhage or necrosis. Bone, fat, cartilage, or even teeth material may be seen. Hair is rarely seen, and its presence should suggest a prepubertal-type teratoma.

In contrast to mature ovarian teratomas and prepubertal teratomas, postpubertal teratomas lack a well-organized organoid distribution of the different elements and display significant cytologic atypia and mitotic activity. Because of the lack of organoid arrangement, the exact somatic structure being replicated is sometimes difficult to determine. Representation from all three embryological layers is usually, but not always, present (Fig. 7.22). Glandular elements often include enteric-type epithelium, with variable amount of goblet cells, or mucinous glands (Fig. 7.23). Ciliated respiratory-type epithelium is also common. Frequently, glands with cylindrical cells with no particular differentiation are seen. Specialized glandular elements such as the liver, thyroid, or pancreas are rare [141]. Squamous nests are frequently present, displaying varying degrees of keratinization (Fig. 7.24). Commonly encountered mesenchymal elements include the cartilage, skeletal and smooth muscle, and rarely fat. Some of these elements may not be fully mature and may resemble mesenchymal tissue of the fetus and embryo.

More primitive, embryonic-type tissues, such as neuroepithelium and nephroblastic-type tissue, are commonly seen (Fig. 7.25). The presence of these elements does not impact prognosis or diagnostic terminology, unless frank overgrowth is present, in which case one must consider the possibility of a secondary malignancy [135] (see Chap. 12). Neuroectodermal tissue usually includes formation of neural-type tubules, rosettes, or sheets of undifferentiated primitive neuroectodermal small cells. Transition to better-differentiated glial type tissue may be seen. Other neural related tissues include meninges and retina-type pigmented epithelium. Nephroblastoma-type elements include primitive tubules, primitive spindle cells, and blastema elements. As in other types of postpubertal germ cell neoplasia in the testis, GCNIS is seen in adjacent tubules.

Another feature in testicular teratomas that differs from the ovarian counterpart is that all elements, including those with complete maturity, show some degree of cytologic dysplasia. This is easily seen in the epithelial elements, which frequently show nuclear hyperchromasia, irregular chromatin, and mitotic features, and can also be seen in the chondrocytes of the cartilage islands and other mesenchymal elements (Figs. 7.22 and 7.23).

The immunophenotype of teratoma components recapitulates that of the somatic elements that are being reproduced. Pluripotentiality markers are expressed less consistently than in other types of GCT. SALL4 marks up to 80 % of elements within the teratoma. Glypican-3 is seen usually in more immature elements. PLAP and AFP are usually seen in only a subset of epithelial elements, while OCT4 is usually negative [82]. IMP3, an oncofetal protein that plays an important role in embryogenesis and carcinogenesis, is selectively expressed in postpubertal male teratomas and is negative in the female counterpart [142].

Teratoma needs to be differentiated from other GCT, particularly in the setting of a mixed GCT. While a large component of teratoma is hardly underrecognized, small amounts of somatic glands within extensive areas of EC or YST may be overlooked. Attention should be paid to the organization of the cells within the gland, which contrasts with the disorganized and primitive look of YST and EC elements. Some patterns of YST may be misinterpreted as teratoma, particularly the glandular, alveolar patterns, or the hepatoid patterns. Teratoma admixed with YST usually have abrupt transitions between both elements, while these YST patterns usually merge imperceptibly with more classical patterns. Also, teratomas will usually display other more easily recognizable components, such as cartilage or squamous epithelium. Regardless, misinterpretation of small amounts of teratoma as other GCT or vice versa, in the setting of a mixed GCT, would rarely have a significant clinical impact. Distinction from prepubertal-type teratomas, including dermoid cysts, which carry a markedly different prognosis, is explained below.

Postpubertal teratomas are rarely pure, and thus its behavior is usually compounded by that of the other elements associated with it. Cases of pure teratomas have been shown to present with metastases, and metastases from mixed GCT frequently contain teratoma components. Whether this represents and inherent metastatic potential of the teratoma elements or the teratoma represents a maturation process occurring in a metastasis from another GCT (particularly EC), is difficult to determine. Nevertheless, the association of teratoma with metastases and potential death due to disease in patients with GCT is well established. Up to 37 % of pure teratomas present with metastases [143]. Pure teratoma metastases are frequently found in patients that have undergone chemotherapy for metastatic mixed GCT, presumably because other GCT components have a better response to chemotherapy, or, alternatively, because chemotherapy induces other components to differentiate into teratoma. Regardless, outcome is generally favorable. Exceptions include the occurrence of secondary malignancy [135] (see Chap. 12) or cases where complete surgical removal is not possible. In this setting, progressive growth of the teratomatous metastases may result in lethal compression of vital structures with ultimate demise of the patient [144–146].

Choriocarcinoma (CC) is a GCT that shows trophoblastic differentiation and is composed of a variable mixture of mononucleated cytotrophoblastic and multinucleated syncytiotrophoblastic cells. A pure form of the former may be seen, more commonly in the metastatic setting, and occasionally in primary tumors [147]. In the testis, CC is usually a component of a mixed GCT, with pure forms corresponding to less than 1 % of all GCT [148]. Other variants of less common trophoblastic tumors, such as epithelioid trophoblastic tumor and placental site trophoblastic tumor, will be discussed separately.

As part of a mixed GCT, they usually present as a painless testicular mass. However, occasionally in this context or more frequently when extensive, predominant, or pure, CC presents as metastatic disease, with symptoms related to hemorrhagic metastasis (hemoptysis, melena, intracranial hemorrhage, etc.) [149–152]. In these cases, a clinically evident primary may not be readily apparent. Serum β-hCG levels are typically high (usually >100,000 mIU/mL). Gynecomastia and hyperthyroidism have been described, and this is secondary to structural and functional similarities between the alpha chain of hCG and TSH and FSH [153–157].

CC are frequently diagnosed while they are still small primary lesions in the testis, given their propensity to present clinically with symptoms of metastatic disease before a testicular mass is discovered. Even at this small size, tumors tend to be extensively necrotic and hemorrhagic, with usually solid residual tumor in the periphery. Larger tumors tend to be extensively cystic.

Histologically, classic CC shows a mixture of cyto- and syncytiotrophoblasts (Figs. 7.26 and 7.27). The latter is characterized by multinucleated giant cells with abundant eosinophilic cytoplasm. They may show vacuolated cytoplasm and other degenerative changes. Nuclei within them tend to have dense chromatin and occasional nucleoli. Cytotrophoblasts are polygonal or round cells with well-demarcated cell borders. They are usually small to medium sized, with irregular nuclei, vesicular chromatin, and visible nucleoli. Mitotic activity is easily seen. They usually cluster, forming tight aggregates. Some tumors contain cells of intermediate size, with more abundant and eosinophilic cytoplasm. Because these cells appear to morphologically and immunophenotypically imitate intermediate trophoblasts [158], some authors prefer to refer to all non-syncytiotrophoblast cells as “mononucleated trophoblast cells” to encompass both lines of differentiation (i.e., cytotrophoblast and intermediate trophoblast) [86]. Both mononuclear and multinucleated components are intimately associated, with syncytiotrophoblast cells frequently wrapping or capping aggregates of cytotrophoblast cells. Some cases have a relative paucity of syncytiotrophoblast cells and may appear monophasic, particularly when composed predominantly of the larger mononucleated cells. Extensive areas of necrosis and hemorrhage are frequently seen. Tumor cells tend to project into hemorrhagic areas with columns recapitulating the extravillous growth of trophoblast in the placenta. Vascular invasion is almost invariably seen in tumors with large components of CC. As with most of the other germ cell tumor types, GCNIS is seen in the adjacent seminiferous tubules.

By immunohistochemistry, CC is negative for OCT4 [82]. Syncytiotrophoblastic giant cells are usually positive for β-hCG and glypican-3, while both components tend to show expression of EMA, MUC1, CEA, SALL4, GATA3, and α-inhibin [159]. Contrary to other epithelial GCT, CK7 is expressed in a subset of trophoblasts [160]. p63 is preferentially expressed in cytotrophoblastic cells [161]. hPL is expressed strongly in syncytiotrophoblastic giant cells, but may stain some of the larger mononucleated trophoblasts [158].

The main differential diagnosis is another GCT type associated with syncytiotrophoblastic giant cells. As explained before, it is not uncommon for other types of GCT to be associated with occasional syncytiotrophoblastic giant cells. Since these are not associated with cytotrophoblast, they do not represent a component of CC. This is particularly significant in seminoma, where a misinterpretation of isolated syncytiotrophoblastic cells as CC may exclude a patient from the treatment arm of seminoma. In other GCT types, an overdiagnosis of CC may incorrectly assign a patient an adverse prognosis. Attention should be paid to the morphologic features of the other components and the absence of cytotrophoblasts. The pleomorphism of this component is usually higher than what is seen in YST but less severe than what is seen in EC. Seminoma would present with typical uniformity throughout the tumor mass, with intervening fibrous septa and lymphocytic aggregates. Neither would show the classic wrapping or capping of syncytiotrophoblasts over the cytotrophoblast component. In difficult cases, immunohistochemical stains may be used to correctly identify each component.

A CC component within a mixed GCT is usually associated with more aggressive behavior. However, when predominant or exclusive, CC is associated with a particularly ominous prognosis. In a recent study, 15 cases of pure or predominant CC component presented with metastases to distant sites, including the lungs, liver, and brain. About 79 % of these patients died of disease-related complications, with a median survival of 13 months, despite current regimes of chemotherapy [148]. Pulmonary metastases have a better prognosis than other visceral metastases. In the above-referenced study, two patients with exclusive pulmonary metastases were disease-free after 60 and 72 months of follow-up, respectively [148].

Contrary to what occurs in the female genital tract, only a handful of cases of testicular placental site trophoblastic tumor have been described, three of them in a metastatic setting [147, 162–166]. The tumors are characterized by the presence of intermediate trophoblast cells exhibiting moderate amounts of dense eosinophilic cytoplasm with occasional vacuolization. The nuclei are smudged, and the majority of cells are mononuclear, with only occasional multinucleation of up to four nuclei per cell. Vessel wall invasion and small areas of hemorrhage are seen. The stroma is myxoid, and Alcian Blue positive. The tumor cells express HPL, and only focal β-hCG. Of the three primary tumors described, one was pure (in a 16-month old boy) [147], while the others were associated with a teratomatous component [163, 166]. Of the three tumors in the metastatic setting, one was chemotherapy naive and occurred in a pulmonary metastasis of a primary mixed GCT [162]. The other presented as a post-chemotherapy retroperitoneal recurrence 4 years after resection of a mixed GCT that included chorciocarcinomatous elements [164]. The third was also a retroperitoneal, post-chemotherapy recurrence of a mixed GCT in a 39-year-old man [165]. In this setting, the main differential diagnosis is a partially regressed CC, as treated CC may show numerous mononuclear intermediate trophoblastic cells. A hemorrhagic background with necrosis would favor the latter (Table 7.7).

Originally described in the uterus, epithelioid trophoblastic tumor (ETT) is a rare neoplasm of mononuclear cells that shows differentiation toward the chorionic-type intermediate trophoblast found in the chorion laeve. In the testis it was initially described as a component of a metastatic GCT [167], but five recently reported cases included this component in primary tumors [165]. They are characterized by nests of cells with squamoid appearance, with well-defined cytoplasmic membranes and intracytoplasmic vacuoles containing fibrinoid debris. Nuclei are mostly single, with occasional multinucleation. The tumor cells are positive for inhibin, GATA3, p63, PLAP, and variably for β-hCG while negative for SALL4, glypican-3, and OCT4. Serum β-hCG levels were normal or mildly elevated. Most tumors have been part of a mixed GCT with other components, although two cases corresponded to 95 % and 100 % of a recurrence, respectively. The ETT component did not appear to confer a different prognosis to the GCT [165].