Although it is the GCT with the highest rate of bilaterality, this only occurs in 10 % of cases [28]. Tumors are usually solid and well encapsulated; on cut section they are homogeneous or lobulated (Fig. 6.1) but rarely cystic. They are usually white or tan in color, except in congestive, torsioned tumors. Heterogeneous and gritty areas should be sampled in order to demonstrate other histologic components. Punctate red, hemorrhagic foci are often associated with syncytiotrophoblastic differentiation. Marked necrosis should prompt an extensive sampling of the specimen.

An extensive microscopic description of its testicular counterpart, seminoma, is found in Chap. 7. Here we will review histopathologic features present in dysgerminomas in females and dysgenetic gonads that are relevant to differential diagnosis with other ovarian tumors.

At low power, it is necessary to search for coarse or morular microcalcifications that may remain from a preexisting gonadoblastoma, overgrown by the malignant germ cells of dysgerminoma. Dysgerminoma cells are arranged in a variety of patterns: diffuse or solid, lobular, trabecular, etc. They have uniform nuclei with prominent nucleoli and well-defined membranes. A variable degree of atypia is present but lacks any prognostic significance.

Cells of dysgerminoma have a labile structure due to a poorly developed cytoskeleton, and consequently dysgerminoma histology is often subject to rapid autolytic changes being very sensitive to frozen section artifacts. Retraction of cytoplasmic gels during fixation is often reflected in the formation of clear cells. Invariably, the intervening septa show lymphocytes and, less often, histiocytic, epithelioid non-caseating granulomas, the consequence of an enhanced T-lymphocytic response induced by neoplastic germ cells [56]. This inflammatory response is an invaluable diagnostic aid in poorly fixed cases where identification of the large, clear cells is difficult. Only in rare instances may extensive chronic inflammatory infiltrates complicate diagnosis by effacing the dysgerminoma architecture.

Distortion due to autolysis may produce large empty tubular or pseudofollicular colloid-filled spaces that may mimic other ovarian tumors in the young, such as small cell carcinoma, hypercalcemic type (SCCH), and even struma ovarii (Figs. 6.2a, b). Differential diagnoses also include ovarian lymphoma and clear cell carcinoma (CCC). Dysgerminoma shares many similarities with SCCH since it involves the same age group and may also present with serum hypercalcemia. Especially in poorly fixed cases that imitate pseudofollicular spaces, the diagnosis is made by the absence of lymphocytic infiltrate in SCCH and the characteristic immunophenotype of dysgerminoma. CCCs involve an older age group but can show solid areas with cytoplasmic clearing and even plasma cell stromal infiltrates [57]. Usually, a thorough sampling will reveal the tubulocystic areas of CCCs. Lymphoma in young patients is a more remote differential and when an adequate immunohistochemical panel is used, diagnosis should not present any problems, although in rare cases, ovarian lymphomas can be negative for B and T-lymphocyte (CD3) markers [58] and thus may mimic an undifferentiated tumor.

Syncytiotrophoblastic focal differentiation occurs in about 3–5 % of cases. Syncytiotrophoblasts are found focally either alone or accompanied by isolated mononuclear cells (Fig. 6.3a). Often they are surrounded by erythrocytes from burst capillaries.

In the same way as germinomas of other locations (see Chap. 12), rarely dysgerminoma may be complicated by somatic malignancies (see Chap. 12). A case of coexisting fibrosarcoma [59] has been reported in the ovary (Fig. 6.3b).

In the past, diagnostic immunohistochemistry for dysgerminomas has relied on the classic membrane staining of placental-like alkaline phosphatase (PLAP). In our experience, however, PLAP is relatively unreliable, especially in poorly fixed material; furthermore, it may stain positively in a high percentage of cases of both embryonal carcinoma and YST [18]. CD117 (c-kit) [60], however, is a conspicuous membrane marker, and c-kit mutations have been shown to occur in a third of dysgerminoma cases [39]. Clone D2–40 of podoplanin is a stable cytoplasmic and membrane marker [61], working well in poorly fixed and necrotic material [62]. OCT4 is regularly expressed in dysgerminoma and in gonadoblastoma, often a precursor lesion, but also in embryonal carcinoma [63]. However, considering the exceptionality of embryonal carcinoma in the ovary, OCT4 can be considered as a selective marker of ovarian dysgerminoma [18], especially in cases of poorly fixed tissue with tubular structures, helping to differentiate them from SCCH and even struma ovarii. OCT4 also identifies isolated dysgerminoma cells engulfed by fibrosis or effaced by inflammation [18] (Fig. 6.4). SALL4 represents a sensitive marker for all malignant ovarian GCT being also expressed in dysgerminoma. Cytokeratin expression does not exclude the diagnosis of dysgerminoma, since positives ranging from strongly diffuse to focal and dot-like can be seen in up to a third of cases [18]. Frequently used cocktails such as CAM 5.2 and AE1/AE3 are positive in 19.2 % and 7.7 %, respectively [64]. Cytokeratin 7 is occasionally focally positive; consequently, it is not advisable to differentiate dysgerminoma from embryonal carcinoma by cytokeratin expression alone, as initially proposed [65]. Expression of cytokeratins has unknown significance, but it may be related to an eventual differentiation of primitive germ cells into somatic-type cells. This assumption is partly supported by the focal positivity of blood group-related antigens in the cytoplasm of dysgerminomas [66], which may reflect a differentiation into somatic-type tissues or yolk sac tumor. Additionally, trophoblastic cell differentiation in dysgerminoma is always cytokeratin positive. Finally, rare cases may be positive for neuron-specific enolase (NSE) [67].

Tumors are unilateral and large, with an average size of 15 cm; their weight may reach 5 kg [25]. They are usually well encapsulated, although large herniations and rupture may be present. On cut section, the tumor tissue is gray or yellow with numerous irregular zones of hemorrhage and liquefaction. Cysts are usually situated at the periphery and contain mucinous or gelatinous material. Rarely, the tumors are unicystic [86] or uniformly multicystic; the latter appearance correlates with a histological pattern of polyvesicular type [87, 88] (Fig. 6.5a). Solid tumors with a fibrous or elastic consistency correspond to YST with an abundant mesenchymal component (Fig. 6.5b). One-seventh of cases are associated with an otherwise typical mature cystic teratoma in the ipsi- or contralateral ovary [89].

Tumors usually present an admixture of endodermal extraembryonal and somatic patterns in variable proportions.

The heterogeneous immunophenotype of YST is a consequence of the various differentiations and developmental stages that coexist in these tumors. For this reason, the use of a broad immunohistochemical diagnostic panel covering various endodermal phenotypes is advised [96].

Comparative expression of various proteins in the human yolk sac (HYS) and YST has been attempted. As a referential point, the HYS immunophenotype has been recently studied by analysis of the expression of pluripotentiality and endodermal proteins [107]. The HYS (Fig. 6.7a) has a consistent expression of immunohistochemical markers associated with hepatic (HepPar-1 and GPC3) and intestinal (villin and CDX2) functions. In addition, it expresses SALL4 during all its functional period. In contrast, LIN28, another pluripotentiality protein, is only expressed in early yolk sacs up to the 5th week of development. Additionally, AFP is secreted by endodermal cells via a transient canalicular network that represents the substrate of a transport system functioning during the period of maximum activity of the HYS [107].

Alpha-fetoprotein (AFP) has been considered a gold standard for the diagnosis of YST, although it is known that this embryonal protein can be expressed, among others, in ovarian clear cell carcinoma [108] and in ovarian metastases of gastric carcinoma. In the normal yolk sac, AFP is present as early as the 5th week showing a granular cytoplasmic positivity, which is concentrated in the intra- and intercellular lumina and tubular surfaces that constitute a complex transfer system. In classic YST patterns, AFP expression is constant but heterogeneous in the epithelium, (Fig. 6.7b) where it also delineates occasional cellular lumina. In somatic glandular or solid [95] patterns, however, its distribution tends to be focal or absent [109]. Thus, it can be said that AFP negativity does not necessarily preclude a diagnosis of YST.

Glypican-3 (GPC3). In the normal yolk sac, GPC3 displays a cytoplasmic or membranous expression (Fig. 6.7c) that also highlights inter- and intracellular tubules. GPC3 is also extensively or patchily expressed in YST [110]. GPC3 is diffusely expressed in classical YST patterns but has a heterogeneous distribution, and it is even absent in somatic glandular variants. Similar in distribution to AFP, it is a more sensitive antibody, although not as specific as AFP [111], being also expressed in tumors of the female genital tract such as the primitive neural tubules of immature teratoma, hepatocellular carcinoma, squamous cell carcinomas, carcinosarcomas, and placental site trophoblastic tumors, among many others. Some clear cell carcinomas may also express GPC3, and consequently its demonstration may not be that useful in the differential diagnosis [112] of YST originating from somatic tumors.

Hepatocyte paraffin-I (HepPar-1) is expressed throughout the lifespan of the human yolk sac, reflecting its vicarious hepatic role. HepPar-1 positivity has been reported in both the hepatoid areas of YST and in hepatoid carcinomas [113]. Our study suggests that the frequent, but focal, HepPar-1 positivity in YST does not necessarily mean that it identifies a hepatic tissue, but that it can also reflect a HYS differentiation. Well-differentiated glandular YST of intestinal type show an intense and diffuse HepPar-1 expression (Fig. 6.7d) similar to that found in the small intestine [114].

CDX2 expression is consistently present in the human yolk sac [107]. In classical patterns, CDX2 expression is focal [115, 116] (Fig. 6.7e), but in somatic glandular patterns, it displays a stronger, diffusely positive, staining [96]. Therefore, it would seem that CDX2 positivity will highlight both areas of HYS and intestinal differentiation, the latter being more evident in somatic glandular patterns, especially those with vacuolated epithelia resembling the embryonal gut.

Villin is consistently expressed during early embryogenesis in both HYS and early endoderm [107] and is a highly sensitive endodermal epithelial component marker in all cases of YST, both in classical and somatic glandular patterns (Fig. 6.7f), being absent in pure embryonal carcinoma and seminoma/dysgerminoma. Its diffuse cytoplasmic expression parallels that of the HYS [107] and intestinal adenocarcinomas, where both diffuse and apical staining patterns occur [117]. We believe that villin is an excellent marker of both primitive and differentiated YST variants. To our knowledge, villin has not been previously used as a marker for the epithelial components of YST.

SALL4, as a marker of cells retaining pluripotency, is a highly sensitive marker for YST (Fig. 6.7g), but with a low specificity, since it is also consistently expressed by all malignant primitive germ cell tumors of both gonadal and extragonadal locations [118–120]. It is also present throughout the life cycle of the HYS, reflecting that this temporary organ retains a degree of pluripotency in its endodermal cell component. This could explain the eventual differentiation of ectopic mature endodermal tissues such as liver cell and enteric tissue in the placenta, which may originate from displaced yolk sac remnants [121, 122].

LIN28 is also a highly sensitive marker for malignant GCT [108, 123–125] (Fig. 6.7h). However, its expression is not specific for YST, although it has been proposed that it might be useful for immunohistochemical detection of YST metastases and for differential diagnoses with clear cell carcinoma of the ovary [108, 124, 125], where both AFP and GPC3 can also be positive. Other stemness markers, such as OCT4 and SOX2, are not expressed in YST [126].

GATA3 [127] exhibits nuclear expression only in primitive patterns of YST being, however, often negative in glandular and hepatic variants [128]. Conversely, NUT (nuclear protein in the testis) expression is absent in primitive patterns but is positive in differentiated variants, as it seems to play a role in intestinal or hepatoid differentiations [129].

As a summary, a diagnostic antibody panel, including both markers of pluripotentiality (SALL4 and LIN28) and endodermal identity (AFP, GPC3, and villin), is useful in recognizing the multiple differentiations present in YST. The overlapping immunophenotypes of primitive and differentiated YST areas lend support to the newly proposed term of primitive endodermal tumors [71]. This diagnostic panel will also prove useful in the differential diagnosis of unusual histological variants of YST in uncommon locations [130], as well as in the absence of classical diagnostic patterns and AFP expression. Furthermore, it helps both to identify YST arising from somatic neoplasms [131] and differentiate them from clear cell carcinomas, especially in elderly patients. Additionally, the negativity of CK7 and EMA is characteristic of primitive YST and discriminates them from endometrioid and clear cell carcinomas [132, 131]. In contrast, glandular somatic patterns often show an incomplete YST immunophenotype, being potentially negative for AFP or GPC3 and unexpectedly positive for CK7, EMA [133], HepPar-1, and CDX2, especially when they occur in association with somatic neoplasms (type VI GCT). Consequently, AFP or GPC3 negativity in the presence of other markers such as villin, SALL4, or LIN28 should not preclude the diagnosis of YST.

IT are usually bulky, solid tumors, rarely bilateral, that can be associated with MCT in a fourth of cases [158]. Three quarters show a smooth capsule, and only a third may present with rupture or herniations. On cut section, most tumors are solid, white, encephaloid (Fig. 6.11) and may present small cysts. Small foci of bone or cartilage may be present. A multilocular appearance occurs in a third of cases. Only rarely are tumors parvilocular or pedunculated within a large, solitary cyst, and up to a fourth of cases may show embedded or peripheral dermoid-like cysts containing sebum and hairs [158]. Hemorrhage and necrosis are often found and are especially evident in cases of torsion. Sampling must include a tissue block for every centimeter in diameter; it is mandatory that the mass should be adequately sampled as this is crucial for grading the tumor.

A mixture of mature and immature tissues with predominance of neural ones characterizes this tumor. Grading, assessed in the relative amounts and degree of immaturity of the neural tissues, is prognostically related as demonstrated in prechemotherapy series of IT [155, 157]. Grade 0, or mature solid teratomas, do not show the usual dermoid features and are composed of mature tissues of predominant neural origin. Grade 1 tumors contain rare foci of immature neural tissue (<1 low-power field (LPF) in any one slide), while grade 2 and grade 3 tumors contain 2–3 LPFs or 4 or more LPFs of immature neural tissue, respectively [28]. This system can be simplified into a two-tier classification with low grades referring to grade 1 teratomas and high grades encompassing grades 2 and 3 [163].

Structures relevant to histologic grading. The characteristic immature neural structures evaluated in grading are neural blastematous nodules containing rosettes and neuroepithelial tubules (Fig. 6.12a) with a crowded cell lining with abundant mitoses that may be occasionally pigmented. Their sharp-edged apical borders are well delineated by an inner limiting membrane (Fig. 6.12b) which differentiates them from other immature tubules of nonneural origin. It is important to take into account some other immature tubular structures that can mimic neuroepithelial tubules. Endodermal tubules lined by tall columnar vacuolated epithelium (Fig. 6.12c), similar to glandular YST, are, in our experience, intimately admixed with immature neural tubules in grade 3 tumors, especially in pediatric IT (type I GCT), where they are found in a third of cases [156]. While some authors may regard these tubules in high-grade IT as microscopic foci of YST, we believe that the presence of these does not necessarily signify a mixed GCT but yet another immature embryologic structure differentiated in a high-grade tumor and thus may influence prognosis [97]. Microscopic areas of primitive, microcystic YST can be found, especially in high-grade IT in children (Fig. 6.12d). Endodermal tubules are often identified by marked periglandular stromal basophilic rarefaction (Fig. 6.12e). Hence, the finding of endodermal tubules should also be taken into account when grading a teratoma. Solid, undifferentiated areas, rarely forming embryoid bodies, may be present in high-grade tumors and resemble EC, even sharing a similar immunophenotype (see below).

Elements which are not relevant to grading include metanephric tubules. Nevertheless, these may mimic neuroepithelial tubules as they are lined by crowded cells with scanty cytoplasm. However, they lack the inner limiting membrane characteristic of neurotubules and are associated with abortive glomerular structures (Fig. 6.12f). Other areas showing developmental immature features which are not relevant to grading are mesenchymal derivatives, such as immature skeletal muscle, which should be differentiated from true overgrowth of sarcoma [28]. Immature mesenchymal areas often resemble myxoid liposarcoma with a characteristic vascular arrangement. Other immature areas such as tooth buds, lack any importance other than providing a beautiful microphotograph. Marked vascular hyperplasia with both endothelial and adventitial proliferations, similar to that occurring in central nervous system tumors, is frequently found in any grade. However, it may be quite prominent in high-grade IT, where it may be associated with a malignant overgrowth of primitive neuroectodermal tumors (PNET), including malignant retinal anlage-type tumors [164].

Only when IT elements are a component of a type II mixed GCT, they may it show 12p chromosome abnormalities [145]. Some teratomas are almost exclusively composed of both mature and immature endodermal structures in various stages of differentiation. For these, the name immature endodermal teratoma has been used, and their relationship with YST is open to speculation [68].

The usually unilateral cysts show irregular, thickened, or ulcerated plaques and/or fungating intracavitary growths. Since they involve postmenopausal women, any teratoma in this age group warrants extensive sampling, especially when necrosis, hemorrhage, or thickened or pigmented areas (Fig. 6.20a, b) are present.

Squamous cell carcinoma is the most common histologic type of secondary malignant tumor found in MCT. Most originate from the epidermis, as demonstrated by the presence of in situ lesions (Fig. 6.20c) [153], but are not related to human papilloma virus. An origin from bronchogenic structures is also contemplated. A squamous papillomatous pattern (Fig. 6.20d), reminiscent of a schneiderian papilloma of the upper respiratory tract, should be differentiated from a proliferative Brenner tumor.

Differential diagnosis from rare primary squamous cell ovarian carcinomas [303], either of pure histological type or associated with endometrioid lesions or Brenner tumor, should be made if teratoid structures are not found after extensive sampling. Squamous cell carcinoma often invades the capsule and lymphatic vessels and has a poor prognosis (Fig. 6.20e).

Benign, malignant, and borderline intestinal-type mucinous tumors [300, 304] originating from mature teratoma exhibit well-differentiated intestinal epithelium and are often associated with pseudomyxoma ovarii (Fig. 6.20f). A recent study [106] has demonstrated the homozygosity of these tumors, suggesting that ovarian intestinal-type mucinous tumors may have a teratoid origin. Those showing appendiceal-type histology [305] may associate with a form of pseudomyxoma peritonei less aggressive than that originated in appendiceal tumors. Immunohistochemically, these tumors show a variable expression of CK20 and 7. In mucinous cystadenomas, a CK7+/CK20− phenotype occurs as commonly as a CK7−/CK20+ phenotype [300], being diffusely CK20 positive in over a third of cases and, more often, only focal or partially positive [304]. Borderline or malignant mucinous tumors tend to have a lack of expression of cytokeratin 7 [300]. Mucin (Muc) family antibodies, although often nonspecific, may be helpful in establishing an origin from teratoid tissues [306].

Melanoma (Fig. 6.20b). Although most ovarian melanomas are metastatic in nature, origin from teratoid tissues may occur, representing one of the rarest origins of metastatic melanoma. The only series dealing with ovarian melanoma originating in teratoma [307] revealed a wide age range from 18–72 years. All were unilateral and histologically showed large epithelioid cells with eosinophilic cytoplasm, small cells, spindle-shaped cells, or a combination of these. Melanin pigment was not always present, but their melanocytic nature was shown by electron microscopic demonstration of melanosomes and positivity for one or more melanocytic markers.

Potentially, practically any tumor pattern found in adults and children can occur in the tissues of a mature teratoma. An updated list of reported sarcomas, cutaneous, neural, and miscellaneous tumors, and their rare combinations is presented in Table 6.5.

SO is a term applied to teratomas which contain thyroid tissue as the unique or predominant component, usually 50 % or more. SO occurs in an older age group than simple MCT and presents as an abdominal mass that is unusually associated with hyperthyroidism or pseudo Meigs’ syndrome [399, 400]. The great majority of cases are benign. Malignant change is uncommon but may represent a challenge in the understanding of its clinical behavior and treatment. Ultrasonographic findings are nonspecific, usually revealing a heterogeneous, predominantly solid mass [401].

Most cases are unilateral and associated with teratoma and can reach up to 20 cm in size, although a small SO is not an unusual incidental finding (Fig. 6.21a). Larger tumors often have a pure thyroidal histology and may be malignant. Tumors are well encapsulated and multicystic and their contents are usually brown or green colloid. Rarely, they may present as a unicystic tumor that is only diagnosed in the microscopic study (Fig. 6.21b, c).

Thyroid tissue is readily identified with colloid-filled cystic follicles grouped in nodules. The full range of microscopic changes seen in the eutopic thyroid including microfollicular, solid, tubular, trabecular, Hürthle cell, pseudopapillary and mucinous [305] changes (Fig. 6.21d–f), hyperplasia, atrophy, lymphocytic infiltration, etc. can be present in the teratomatous thyroid. Cases exhibiting densely packed cellular follicles or pseudopapillary change have been termed proliferative struma [399], and although benign, they represent the main differential diagnosis with histologically malignant struma. Luteinized stromal cells are not uncommon in the periphery.

The enigmatic association with Brenner tumor (Fig. 6.22a, b) [402, 403] and mucinous cystadenoma suggests that, in these cases, the urothelial and mucinous components so intimately admixed with thyroid tissue would represent a combination of teratoid endodermal tissue differentiations rather than a coincidental association of a common epithelial neoplasm with teratoma.

The pathology of malignant SO parallels that of thyroid carcinomas [399, 404, 405]. Papillary carcinomas of both classic, tall cell, and follicular variants (Fig. 6.23a–e) are the most frequent, and they should be differentiated from proliferative struma using the same criteria applied in the eutopic thyroid to differentiate between papillary carcinomas of follicular variant and hyperplastic nodules (ground glass nuclei, vascular invasion (Fig. 6.23f), etc.) [399, 405]. True follicular carcinomas are unusual and poorly differentiated carcinomas (Fig. 6.23d, e) are even more infrequent. It must be borne in mind that true medullary carcinomas do not occur in the ovary, their closest histological relative possibly being represented by strumal carcinoid.

Malignant SO are usually large masses measuring over 10 cm in size, often with adhesions and ascites. Their behavior is indolent, with a good survival rate at both 10 (89 %) and 25 years (84 %) [406]; extraovarian spread or recurrences rarely develop. The hormonal milieu of pregnancy apparently may provoke more aggressive behavior [405, 407]. Slow growing peritoneal implants, previously called benign strumosis, are only examples of well-differentiated metastases. Histologically, both a papillary pattern and a poor degree of differentiation are associated with a more aggressive course [406].

The expression of specific markers such as thyroglobulin (Fig. 6.23e) and thyroid transcription factor-1 (TTF-1) is useful in identifying the thyroidal nature of the neoplastic tissue in cases when it is difficult to recognize, such as the thin cuboidal lining of the cavity of cystic SO (Fig. 6.21b, c) and in solid, poorly differentiated thyroid carcinomas with minimal follicular formation. These markers also help in establishing a thyroidal phenotype in the differential diagnosis with other ovarian tumors with tubular or trabecular arrangement, such as Sertoli-Leydig cell and carcinoid tumors.

Classic papillary carcinoma is a relatively straightforward diagnosis, but the more complex diagnosis of the follicular variant of papillary carcinomas may be facilitated by the expression of HMBE-1 and CK19 positivity [408]. BRAF point mutations of the type commonly observed in papillary carcinomas of the eutopic thyroid have also been reported in malignant struma [407, 409].

Histologic diagnosis of malignancy occurs in most cases without any evidence of extraovarian spread. In these, a conservative approach with salpingo-oophorectomy and analysis of thyroglobulin serum levels to exclude occult disease is advised [399, 404, 405]. Postoophorectomy treatment should follow similar protocols to those used in eutopic thyroid carcinoma, potentially including thyroidectomy or radioiodine ablation, and hormonal suppressive therapy. Long-term follow-up for both metastases and abdominal recurrences can be accomplished by serial serum thyroglobulin measurements and radioiodine scans [410].