Ultrastructural study of intracranial yolk sac tumor: With special reference to the oncologic phylogeny of germ cell tumors

Ultrastructural Study of Intracranial Yolk Sac Tumor:

With Special Reference to the Oncologic Phylogeny of Germ Cell Tumors

YOSHIO TAKEI, MD,' AND GARY S. PEARL, MD, P m t

Ultrastructural studies of yolk sac tumors (YST) have generally demonstrated the resemblance of this neoplasm to the primitive human yolk sac. However, these studies have failed to explain the common occurrence of mixed germ cell tumors with yolk sac elements. This report describes the ultrastructural features of a suprasellar YST. This tumor resembled those previously described. Also demonstrated, however, was the presence of primitive germ cells admixed within the neoplasm. A new schema is proposed based on these observations for the interrelationship of germ cell tumors, which may explain the common occurrence of mixed germ cell tumors.

Cancer 4832038-2046. 198 1.

OI.K SAC TUMOR (YST), or endodermal sinus tu- Y mor as defined by Teilum,' is primarily a gonadal neoplasm, but may also arise extragonadally in midline structures of the body, such as the sacrococcygeal, ret- roperitoneal or mediastinal regions.2 Rare primary intracranial YST arising in suprasellar or pineal locations have also been reported.j-' Regardless of the site of origin, Y STs are characterized histologically by the presence of structures called Schiller-Duval bodies and by alpha-fetoprotein droplets. I t has been pointed out, however, that YST often exhibits a wide range of histologic appearances and is commonly admixed with or found within other histologically distinct types of tumors, such as germinoma (seminoma in the testis or dysgerminoma in the ovary), choriocarcinoma, embryonal carcinoma, or teratomas with varying degrees of differentiation. 2,6

This concomitant occurrence of tumors of diverse histologic types has also been stressed in cases of primary intracranial Y STs.',' Teilum' emphasized the cy- togenetic kinship of these seemingly unrelated tumors, viewing them as a group of neoplasms which share a common origin from primitive germ cells endowed with totipotency for differentiation. Their selective or concomitant overgrowth in different directions of differ-

From Emory University School of Medicine. Atlanta. Georgia. * Chief, Division of Neuropathology, Department of Pathology and

1.aboratory Medicine. Emory University School of Medicine. + Chief Resident in Neuropathology, Department of Pathology and

Laboratory Medicine, Emory University School of Medicine. Address for reprints: Yoshio Takei, MD. Division of Neuropath-

ology. Department of Piithology and 1.aboratory Medicine. Iimory University School of Medicine. Atlanta, GA 30322.

Acccpted for publication September 25. 1980.

entiation gives rise to the histologic diversity of germ cell tumors. This holistic approach has also been ad- vocated by other

Recent ultrastructural studies of YST" l 6 have demonstrated its close resemblance to the primitive human yolk thereby supporting the concept that YSTs are derived from germ cells differentiating into yolk sac membrane. However, these studies have not clearly elucidated the relationship of YST to other tumors of germ cell origin, particularly with regard to possible maturation or differentiation into other tumor types. Since YST recapitulates the pimitive yolk sac, in which cellular elements naturally undergo rapid evolutionary changes, ultrastructural characterization of this tumor may pose some difficulties. When YSTs of gonadal origin are studied, the difficulties are compounded by the complicated embryologic development of the gonad, where primitive, multipotential cellular elements normally persist even in the adult. Some of these baffling problems, however, may be alleviated by a careful study of YST of extragonadal origin.

This communication describes the ultrastructural features of a suprasellar YST, with some new observations not previously reported. Based on a detailed study of this neoplasm, as well as on a review of the literature, we also propose a schema for the interrelationship and histogenesis of germ cell tumors in general, which may offer an explanation for the common occurrence of other germ cell-derived elements in association with YST.

Method

The tissue used for ultrastructural study was obtained at the time of surgical biopsy of a suprasellar tumor in

No. 9 YOLK SAC TLMOR . Takei and Pearl 2039

FIG. 1 , Semithin section of YST shows elements of both YST and germinoma. Germi- nomatous region consists of cells with large round nuclei, prominent nucleoli, and abundant cytoplasm with interspersed smaller lymphocytes. The YST consists of glandlike spaces lined by cuboidal cells (X325).

a 19-year-old woman. The clinical and light microscopic features of this case have been described (Case 2).5 This specimen consisted largely of a classical YST, with minute foci of admixed germinoma and rare apparent trophoblastic cells. The tissue was minced, fixed in phos- phate-bufered 4% glutaraldehyde, post-fixed in phos- phate-buffered 2% osmium tetroxide, and embedded in epon 812. After light microscopic examination of to-

luidine blue-stained, 1 -pm-thick sections, ultrathin sections were cut on a Porter-Blum MT2 ultramicrotome, stained with uranyl acetate and lead citrate, and examined on an RCA MU-4 electron microscope.

Since the time of the biopsy, the patient has died with aspiration pneumonitis and Serratia mercescens septi- cemia. Postmortem examination of the brain revealed recurrent YST in the suprasellar region, intraventric-

Fie;. 2. Germinoniatous region consisting of primitive cclls with round nuclei with a lucent nucleoplasm and prominent nucleoli with a thread- like nucleonema. A. Few organelles are present within these germ cells lying adjacent to a vitelline vessel (X4845). B. Other germ cells have somewhat more organelles and form poorly developed intercellular junctions (arrowhmd) (X3200).

2040 CANCER November 1 198 1 VOl. 48

FIG. 3 . A . Vitelline vessels are lined by plump endothelial cells with prominent nuclei lying on a thick basal lamina containing mesenchymal cells ( M ) (X3200). H. The endothelial cells contain abundant rough cndoplasmic reticulum and are joined by junctional complexes. Mesenchymal csIIs M ) have cytoplasmic niicroliliinit'nts occasionally forming focal condensations (X4180).

ular and subarachnoid spread of the neoplasm and a nodule of metastatic YST within the mesencephalon. The pineal gland was free of neoplastic involvement and was unremarkable. At the time of postmortem examination. all of the tumor exhibited the classical features of a YST, including prominent Schiller-Duval bodies. PAS-positive hyaline droplets were present throughout the tumor, and the droplets and some of the neoplastic

cells demonstrated positivity for alpha-fetoprotein on immunofluorescent studies.

Results

Toluidine blue stained, semithin sections included foci of YST with admixed germinoma (Fig. 1 ). The germinomatous region consisted of round cells with

FIG. 4. A. Endodermal cells form glandlike spaces containing debris and flocculent material. The cells have prominent microvillous formations on their apical surface. Vesicles contain amorphous material representing alpha-fetoprotcin (AF) (X4970). B. Microvilli have core rootlets and cells are joined by tight junctions and desmosomes. AF: alpha-fetoprotein (X 10,730).

No. 9 YOLK SAC TUMOR . Takei and Pearl 204 I

FIG. 5 . A. Mesothelial cells have broad, branching microvilli on their surface and contain abundant intracytoplasmic rough endoplasmic reticulum, ribosomes, and glycogen. The cells are joined by well-developed junctional complexes ( X 4520). B. The microvilli do not have core rootlets. One vesicle ( V ) containing flocculent material is present in this mesothelial cell, which forms a junctional complex (arrowhead) on one surface (X8450).

prominent, round nuclei and large nucleoli. The cytoplasm was abundant. Lymphocytes and plasma cells were interspersed with these cells. The YST included Schiller-Duval bodies and gland-like spaces lined by cuboidal cells with round or irregularly shaped nuclei and clearly defined nucleoli.

Ultrastructurally, germinoma cells had round nuclei with a lucent nucleoplasm and prominent nucleoli with a thread-like nucleonema (Fig. 2). The cytoplasm was generally organelle sparse, containing mitochondria, rough endoplasmic reticulum, ribosomes and annulate lamellae. Some cells, however, had more cytoplasmic organelles, including glycogen. These cells were occasionally joined by poorly developed intercellular junctions. Plasma cells and lymphocytes were scattered among the germinoma cells.

The regions of YST recapitulated many features of the human yolk sac. The vessels resembled immature, vitelline vessels and were lined by prominent, protruding endothelial cells with large, irregularly shaped nuclei and occasionally prominent nucleoli (Fig. 3). Occa- sional mitotic figures were present. The cytoplasm contained mitochondria, rough endoplasmic reticulum and microfilaments, and occasionally formed microvillous projections from the lumenal surface. Many of the ves-

sels were fenestrated, but endothelial cells were focally joined by junctional complexes. The endothelial cells rested on a thickened and reduplicated basal lamina, which occasionally surrounded the processes of elongate mesenchymal cells. These cells had abundant cytoplasmic microfilaments and clusters of pinocytotic vesicles, and were surrounded by a thick basal lamina. This basal lamina was more attenuated when in apposition to germ cells than when the vessel apposed endodermal or mesothelial cells.

The YST included endodermal cells that showed a varying degree of differentiation. Occasional primitive cells with round nuclei, prominent nucleoli and rela- tively scant, organelle sparse cytoplasm were present, as were cells resembling those seen in the germinomatous region. Other cells formed glandlike spaces and had numerous microvilli with core rootlets a t their lumenal surface (Fig. 4). These cells generally had irregularly shaped nuclei with prominent nucleoli and their cytoplasm contained abundant rough endoplasmic reticulum, ribosomes, and mitochondria. Some of these endodermal cells showed degenerative changes, while others contained amorphous material of variable electron density within membrane bound vesicles, appar- ently representing alpha-fetoprotein (Fig. 4). The cells

2042 CANCER November I 1981 VOl. 48

FIG. 6 . A. Trophoblastic cells have large, irregularly shaped nuclei with prominent nucleoli and abundant intracytoplasmic rough endoplasmic reticulum, ribosomes, and mitochondria (X9685). B. Some trophoblastic cells form junctions (arrowhead) with adjoining gcrm cells (X4685).

surrounding the lumens were joined at their apical surface by tight junctions and at several points along their lateral borders by well-developed desmosomes. Degen- erating debris and amorphous material were present within some of the lumens. The hyaline droplets consisted of material with the same appearance as the intracellular droplets of alpha-fetoprotein.

Mesothelial cells formed a thin layer in some areas. These cells had broad, branching microvilli without core rootlets at their free surface (Fig. 5) . The cytoplasm contained abundant glycogen, rough endoplasmic reticulum, ribosomes, mitochondria, occasional Golgi complexes and scattered aggregates of microfilaments. Nu- clei were irregularly shaped and flattened and contained occasionally prominent nucleoli. The cells were joined by tight junctions at the border of their free surface and by desmosomes at their lateral borders. Occasional foci were observed where cells with the features of mesothelial cells were interspersed with and formed junctional complexes with endodermal cells.

Scattered, large trophoblastic cells with irregularly shaped, lobulated nuclei and prominent nucleoli were seen within the YST (Fig. 6 ) . Short microvilli projected from the surface, which was joined to adjacent cells by infrequent desmosomes. The cytoplasm contained abundant cisterns of rough endoplasmic reticulum, numerous polyribosomes, and scattered mitochondria.

Occasional cells were observed that had the ultra-

structural features of histiocytes. These cells were admixed with endodermal cells and had irregularly shaped nuclei, often with prominent nucleoli and marginated chromatin (Fig. 7). The cytoplasm formed filopodial processes and contained membrane-bound lipid, rough endoplasmic reticulum, mitochondria and well-developed Langerhans granules.

Discussion Our ultrastructural study of this suprasellar YST

revealed the tumor to be composed of multiple cellular elements (Fig. 8). The endodermal and mesothelial cells are best identified by the villous formations character- istic of each cell type. Both of these lining cells are also characterized by well-developed junctional complexes and intracytoplasmic, membrane-bound material, pre- sumably alpha-fetoprotein, the latter being more commonly seen in endodermal cells than in mesothelial cells. The tumor vessels are of a vitelline type and many are formed by immature endothelial cells with proliferative features (Fig. 3) . These vitelline vessels are surrounded by a basement membrane of varying thickness, which, at times, forms amorphous masses in the extracellular space containing scattered mesenchymal cells (Fig. 3). Since all of these cellular elements are known to be a part of the human fetal yolk s a ~ , ’ ’ ~ ’ ~ ~ ” ~ ’ ~ the ultrastructure of this suprasellar neoplasm does appear to reca- pitulate that of the normal yolk sac membrane.

No. 9 YOLK SAC TUMOR . Takei and Pearl 2043

FIG. 7. Scattered histiocytes within YST contain well- developed Langcrhans granules, which can be seen forming at the cell surface (arrowheads) (X 13,960).

In humans, one of the most important functions of the fetal yolk sac is the production of primordial germ cell^.'^,^" Curiously, however, the presence of germ cell elements has not been described in the previous ultrastructural studies of YST, even though the structural recapitulation of the fetal yolk sac by this neoplasm has often been empha~ ized . ’~ . ’~ Instead, some of these au- thors describe “undifferentiated” or “poorly differen- tiated” cells within the YST.”*’2

Positive identification of neoplastic germ cells may be difficult within tissues such as YST, which are composed of multiple cellular elements in various stages of differentiation. Nevertheless, the ultrastructural characterization of neoplastic germ cells may best be made with studies on germinomas (seminomas, dysgerminomas), because their principal cellular component is the neoplastic germ cell. A review of the literature reveals that the ultrastructural features of germinoma cells are not necessarily uniform in all cases, and a variety of cytoplasmic electron densities and organelle composi- tions have been reported. The cells range from those with few organelles in an extremely lucent cytoplasm with little or no g l y ~ o g e n ~ ’ - ~ ~ to those characterized by abundant organelles, including masses of glycogen granules and numerous microfilaments, as well as fre- quent d e ~ m o s o r n e s . ~ ’ ~ ~ ~ ~ ~ ~ 29

In the present study, the tissue examined for electron microscopy contained a focus of germinoma showing the classical two-cell pattern (Fig. 2). In this area, the small cells were primarily lymphocytes, with some admixed plasma cells, whereas the large neoplastic germ

FIG. 8. Essential cellular elements of yolk sac tumors. The vitelline vcssels (Vv) are formed by immature and proliferating endothelial cells, surrounded by basement membrane-like material (b). Mesen- chymal cells (Ms) and occasional mast cells ( M ) lie within this basement membrane. Endodermal surface is composed of endodermal cells (En) with intracellular alpha-fetoprotein (af). glycogen (g), and ly- sosomes (ly). These cells are joined by desmosomes at their lateral borders and have slender, filament-containing microvilli at their free surface. Germ cells ( G ) with large, round nuclei and thread-like nucleoli may be admixed with endodermal cells or may aggregate in association with lymphocytes (L). Occasional germ cells are undergo- ing mitosis (mi). Mesothelial cells (MT) with broad. branching microvilli may be seen forming a single layer or may be admixed with endodermal cells. Occasional histiocytes ( H ) containing Langerhans granules and trophoblastic cells (Tr) may also be present.

2044 CANCER November I 198 1 VOl. 48

Endodermal Predominance I

Germ Ceil Production

I I NORMAL FETAL YOLK SAC 1 I . I ( ; , 0. l’ohsihle phylogcnic relationhliip of germ cell tuniors. Neo-

plastic germ cells may proliferate as germinoma cella, which have lost their differentiating potential. or may differentiate along embryonal (teratonia) or extra-embryonal (choriocarcinoma. YST) lines. Neo- plastic germ cells may also form within YST and may proliferate or differentiate to form mixed germ cell elements within a YST.

cells exhibited a variety of ultrastructural appearances similar to those described in the cases of germinoma cited above. In addition, these neoplastic germ cells, as identified by their ultrastructural features, were also found in the areas of YST in close relationship with either endodermal cells or vitelline vessels (Fig. 2). In the normal yolk sac, by comparison, primordial germ cells are initially organelle-poor and electron lucent, but they develop an increasingly complex array of organelles and cytoplasmic electron density during their migration and subsequent residence within the gonadal folds.” Therefore, the heterogeneous ultrastructure of the neoplastic germ cells observed in some cases of germinoma, as well as i n the present case of YST, probably represent different stages of cellular development re- capitulating those of the primordial germ cells in the normal embryo. Furthermore, the neoplastic germ cells with sparse organelles in an electron lucent cytoplasm may be those in the most primitive stage of development. Hence our finding of such primitive forms of neoplastic germ cells within the YST tissue may be evi- dence that this neoplasm can reproduce germ cells as a functional recapitulation of the normal yolk sac.

Two additional, distinct cellular elements were also

identified in the present case of YST: trophoblastic cells (Fig. 6) and histiocytes with Langerhans granules (Fig. 7). The trophoblastic cells have ultrastructural features similar to those found in choriocarcinomas,3’32 indi- cating that YST is capable of differentiating along this extra-embryonal line. The presence of histiocytes with Langerhans granules is somewhat puzzling, however. They have rarely been described outside of histiocytosis X,33.34 and have never been reported in germ cell tumors. Although the phagocytic activity of macrophages has been emphasized with regard to lymphocyte-related immune mechanisms taking place in viwo and in witro in g e r m i n o m a ~ , ~ ~ . ~ ’ the significance of these histiocytes within YST remains unknown at the present time.

Teilum’s holistic concept regarding the origin of germ cell tumors’ has acquired support from other investi- gators, but the histogenesis and interrelationship of each type of tumor still appears to be a subject of consid- erable debate.6,’0.35 In the hope of clarifying this situ- ation, we propose a schema of the possible oncologic phylogeny of the tumors of germ cell origin (Fig. 9). This new concept has been derived from our present study and a review of the literature, in addition to our past experience with 34 cases of intracranial germ cell tumors ( 1 6 germinomas, 5 YSTs, 4 teratomas, 3 choriocarcinomas, and 6 mixed tumors).

In this schema, the primordial germ cells are pro- duced in the normal fetal yolk sac and then migrate toward the gonadal Germ cells that are to- pographically misplaced during migration normally do not survive and are probably eliminated by an immune mechanism. If they survive and acquire neoplastic prop- erties as a result of unknown oncogenic factors, they may be designated as “neoplastic germ cells with to- tipotentiality.” An analogous oncogenesis may occur within the gonadal folds, which is the normal destina- tion of the migrating, primordial germ cells. Such neoplastic germ cells have been found and referred to as “carcinoma in situ of the testis”36 or “intratubular malignant germ cells.’’6

Germinomas (seminomas, dysgerminomas) are formed by neoplastic germ cells without further differentiation. In fact, the ultrastructural characteristics of intracranial germinoma cells reported in the litera-

are uniformly similar to those of the primordial germ cells observed during migration within the normal human ernb~yo.~’ This observation may be interpreted to mean that the potentiality for differentiation of the neoplastic germ cells has been lost or suppressed at the developmental stage equivalent to the migrating, primordial germ cell. The interspersed lymphocytes in germinomas are an immune response to the neoplastic germ cells and may play a role in preventing their further differentiation.

tUre24.25.27.29,37.38

No. 9 YOLK SAC TUMOR . Takei and Pearl 204 5

The totipotential neoplastic germ cells may mature along either embryonal or extra-embryonal lines. Em- bryonal differentiation results in teratomatous elements. The classical studies by Stevens4’ and by P i e r ~ e ~ ’ . ~ ’ have demonstrated that teratomas in the testis originate directly from germ cells. Teratomas have been subclassified according to the degree of maturity of their elements and/or to the presence of admixed malignant areas. This nosologic discrepancy has caused some unnecessary reduplication of terminology, result- ing in a confusing taxonomy. However, within the con- text of oncologic phylogeny, the degree of histologic maturity is of primary concern, and any concomitant, distinctly identifiable tissue elements which may affect the biologic behavior of the tumor should be so recorded that comparative studies may be possible.

Along extra-embryonal lines, trophoblastic differentiation results in the formation of choriocarcinoma. Mostofi‘ described syncytiotrophoblasts arising directly from intratubular germ cells in the testis. We have observed choriocarcinomatous foci in peritoneal shunt-related metastases from intracranial, “pure” YSTs. In these cases, repeated cytologic examination of the shunt fluid revealed only germ cell elements.

The other line of extra-embryonal differentiation is toward Y ST and embryonal carcinoma. The distinction between YST and embryonal carcinoma, however, has been somewhat confusing. In fact, these two names have often been used interchangably in the literature. On the basis of clinicopathologic studies, attempts have been made to distinguish the two both in ovarian4’ and tes- t i c ~ l a r ~ ~ tumors. These studies emphasize the location of the tumor and the age and sex of the patient as important factors. Nevertheless, the histologic distinction between YST and embryonal carcinoma does not appear to be as clear in every case as the descriptive definitions suggest, since they frequently exhibit some overlap in histologic patterns. This may account for some of the confusion in terminology. This is particularly true when the areas defined as either of these tumors are admixed with other types of germ cell tumors. Therefore, it may be logical to interpret that YST and embryonal carcinoma represent a “vertical” spectrum of differentiation in the same direction and that the distinction between the two becomes increasingly apparent only as they are compared at either end of the spectrum. A question then arises as to the “horizontal” convertibility between the two. Ultrastructurally, YST is composed of multiple cellular elements, as described in the present study, whereas the essential cellular component of embryonal carcinoma appears to be endodermal cells with varying degrees of anaplasia.** In other words, the major structural difference between the two is that embryonal carcinoma is composed pre-

dominantly of anaplastic endodermal cells, which are only one of multiple elements in YST. This endodermal predominance may fluctuate depending on factors such as primary or metastatic location of the tumor or the age and sex of the patient harboring the neoplasm. In the previously published concepts regarding the oncologic phylogeny of germ cell turn or^,'^,^^ embryonal carcinoma has been regarded as totipotential, and may progress along the extra-embryonal route to form Y ST. Recently, Mostofi‘ stressed the possibility that testicular YSTs can arise directly from neoplastic germ cells as an alternate route to differentiation from embryonal carcinoma. However, considering the ultrastructural composition of these neoplasms, it is almost inconceiv- able that those anaplastic endodermal cells that once elaborated such specialized structures as villi and junctional complexes are capable of “dedifferentiating” into multiple cellular elements, including the most primitive form of germ cells, to develop YST. It is more likely that embryonal carcinomas are formed as a result of the overgrowth and rapid anaplasia of endodermal elements within YST. In this regard, our ultrastructural confirmation of the presence of primitive, neoplastic germ cells within YST tissue becomes of cardinal im- portance, because the reproduction of such totipotential cells certainly increases the possibility for YST to be admixed with other types of tumors which can originate from them. This offers a plausible explanation for the common observation of mixed germ cell-derived elements associated with YST.

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Documents

Ultrastructural study of intracranial yolk sac tumor: With special reference to the oncologic phylogeny of germ cell tumors