Immature Endodermal Teratoma of the Ovary: Embryologic Correlations and lmmunohistochemistry

FRANCISCO F. NOGALES, MD, ISABEL RUIZ AVILA, MD, ANGEL CONCHA, MD, AND EMIL10 DEL MORAL, MD

Two grade 2 ovarian immature, predominantly endodermal tera- tomas are reported. The teratomas were in stage I and occurred in two girls, 9 and 10 years of age, who were treated with triple che- motherapy. These neoplasms differed from the usual immature ovarian teratoma as they contained no neuroectodermal components and had high a-fetoprotein and low human chorionic gonadotropin levels as their serum markers despite the absence of other concom- itant germ cell tumors. The epitbelia of the teratomas demonstrated

exclusively the embryologic development of endoderm, ranging from early endoderm to tissues similar to esophagus, liver, and intestinal structures. All epithelial derivatives were positive for a- fetoprotein and a,-antitrypsin. Liver and esophagus expressed fi- brinogen, while intestine and esophagus were positive not only for carcinoembryonic antigen and chromogranins but also for thyro- globulin, thus reflecting yet another type of endodermal differen- tiation into thyroid. Focal human chorionic gonadotropin positivity associated with primitive intestinal and esophageal epithelia may reflect the early embryologic relationships between endoderm and trophoblast. These cases demonstrate that simultaneous a-fetopro- tein and human chorionic gonadotropin secretion may occur in

immature teratoma. The mesenchymal component also showed a wide range of differentiation, from primitive mesoblastic cells to differentiated cells, such as hemopoietic foci, smooth muscle, bone, and cartilage. Both the primitive endoderm and the mesenchyme co-expressed vimentin and keratin, reflecting their intimate devel- opmental relationships and possibly supporting the hypothesis of mesenchyme originating from endoderm, as suggested by previous embryologic studies. Since endodermal and mesenchymal areas similar to those described here are found in association with yolk sac tumors and embryonal carcinoma, it is possible that the present cases may represent an endodermal differentiation accomplished by either of these developmentally related germ cell tumors. HUM PATHOL 24~364-370. Copyright 0 1993 by W.B. Saunders Company

Immature teratoma of the ovary contains tissues derived from all three germ layers, although neuroec- todermal derivatives in different stages of embryonic differentiation predominate over those of mesenchymal and endodermal origin.’ Endodermal derivatives in ter- atomas of the ovary are usually limited to isolated areas of bronchial, gastrointestinal, and thyroid tissues.

From the Departments of Pathology and Pediatrics, University of Granada School of Medicine and Virgen de las Nieves Hospital, Granada, Spain. Accepted for publication July 13, 1992.

Key words: ovary, endoderm, teratoma, trophoblast, intestine, esophagus, thyroid, embryology, a-fetoprotein, at-antitrypsin, human chorionic gonadotropin, thyroglobulin, keratin, vimentin, antigen co- expression, histogenesis, development.

Address correspondence and reprint requests to Francisco F. Nogales, MD, Departamento de Anatomia Patologica, Facultad de Medicina, 18012 Granada, Spain.

Copyright 0 1993 by W.B. Saunders Company 0046-8177/93/2404-0005$5.00/O

Two ovarian immature teratomas not associated with any other germ cell neoplasms, composed exclu- sively of early embryonic endodetrnal and mesenchymal structures, and producing high levels of n-fetoprotein (AFP) and low levels of human chorionic gonadotropin (hCG) are reported. The purpose of this paper is to establish a correlation between the morphology of neo- plastic tissues and the early embryogenesis of endoderm and to consider a possible relationship with other en- dodermal germ cell tumors.

MATERIALS AND METHODS

Two endodermal immature teratomas of the ovary were selected from 84 immature ovarian teratomas. The number of available paraffin blocks ranged from 65 in case no. 1 to 12 in case no. 2. Immunohistochemical studies were carried out in selected blocks by the avidin-biotin complex method using the following primary antisera and antibodies: mono- clonal Bl keratin (Immustain, Los Angeles, CA), monoclonal keratin 8.18.19 (Biodesign, Kennebunkport, ME), AFP, &hCG, a,-antitrypsin (AtAT), carcinoembryonic antigen (Dako, Co- penhagen, Denmark), fibrinogen and chromogranin AB (Bio- genex, San Ramon, CA), monoclonal desmin and thyroglobulin (Eurodiagnostics BV, Holland), and monoclonal anti-smooth muscle-specific actin (Enzo, New York, NY). Periodic acid- Schiff, mucicarmine, and Grimelius stains also were carried out. In both cases the mitotic and cell counts were performed in 100 fields using a X 10 eyepiece and a X40 objective.

CASE REPORTS

Case No. 1

A g-year-old girl, 46Xx, with a history of familial sphe- rocytosis successfully treated by splenectomy at the age of 7 years, developed lower abdominal pain associated with a pal- pable hypogastric mass in February 1991. No other symptoms were present. a-Fetoprotein levels at this time were 500 ng/ mL (normal, 0 to 15 ng/mL), hCG levels were 30 mU/mL (normal, 0 to 10 mU/mL), and carcinoembryonic antigen levels were normal. Surgery revealed a 1 Z-cm stage I left solid ovarian mass and left salpingo-oophorectomy was performed. Rupture occurred during the surgical procedure. One month after sur- gery the AFP and hCG levels fell to basal values. Triple che- motherapy (iphosphamide, vincristine, and actinomycin D) was given for 9 months and was well tolerated. Monthly analysis of serum markers was consistently negative. The patient is free of disease 2 years postoperatively.

Case No. 2

A 12-year-old premenarchal female presented with a painful, hypogastric mass. Preoperatively, a level of 730 ng/

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FIQURE 1. Section of tumor from case no. 2. Note the microcystic oppear- ante.

mL of AFP was detected and a pregnancy test was positive. A RESULTS g-cm stage I right ovarian mass was found during laparotomy and right salpingo-oophorectomy was performed. Three cycles Macroscopy of vinblastine/actinomycin-D/cyclophosphamide triple che- motherapy were administrated, but were not well tolerated Both tumors had an analogous appearance, with a

and were discontinued. Levels of AFP and hCG have since maximum diameter of 10 and 12 cm, respectively, and

been consistently negative. The patient had a normal menarche an intact capsule. On section, the tumors were composed

with subsequent regular menstrual cycles and is alive and well of a gelatinous, spongy tissue, honeycombed by numer- 10 years after the initial operation. Repeat ultrasound scans ous small cysts with clear mucous content (Fig 1). Small have been normal. areas of subcapsular, coagulative-type necrosis were

FIGURE 2. (Left) Small cyst lined by pseudostratifted co- lumnar cells with marked apical and subnuclear vac- uoles. (Hematoxylin-eosin stain; magniitcation X279.) (Right) a-Fetoprotein is fo- cally but intensely positive in some glands. In other glands (inset) positivity is diffuse and intense. (Immunoperoxidase stain; magnification X344.)

HUMAN PATHOLOGY Volume 24, No. 4 (April 1993)

FIQURE 4. Esophageal-type squamous epithelium. Basal vac- uolation is still present at the periphery. (Hematoxylin-eosin stain; magniitcation X340.)

FIGURE 3. (Left) An early intestinal structure with focal syncytial trophoblastic dif- ferentiation (arrow). (He- matoxylin-eosin stain; mag- nification x227.) (Right) Focal hCG positivity in endodermal epithelium. (Immunoperoxidase stain; magnification x340.)

present in case no. 1, No large hemorrhagic areas were present in either case.

Microscopy

The histologic appearance was strikingly homoge- nous in all of the numerous slides taken from both tu- mors. Two well-defined components were seen, one ep- ithelial and the other mesenchymal.

Epithelium. The lining of the numerous microcysts was variable and reproduced the derivatives of endo- derm at different stages of embryogenesis. Most cysts were lined by columnar cells with centrally located, slightly irregular nuclei with abundant mitoses. Their cytoplasm showed consistent apical and subnuclear vac- uolation similar to early endodermal derivatives (allan- tois, lung of the glandular period, early intestine, and esophagus) (Fig 2). Cytoplasm stained strongly for AFP and AiAT (Fig 2), and co-expressed keratin and vimen- tin. Isolated foci of large, atypical cells (l/80 high-power fields) with a strong reactivity to hCG in their cytoplasm (Fig 3) arose abruptly from this lining, a phenomenon that occurred exclusively in the epithelium. Another type of lining consisted of large clear cells with regular nuclei and sharp cellular borders, sometimes arranged in sheets with occasional small lumina (Fig 4) closely resemblin

8 the developing esophagus of the seventh-week embryo. This squamous type of epithelium was positive for AFP and AiAT as well as for low molecular weight cytoker- atins; it also was positive focally for vimentin, fibrinogen, hCG, and thyroglobulin.

Other cysts were lined by stratified columnar cells that exhibited differentiation into periodic acid-Schiff, apical-membranous carcinoembryonic antigen-positive goblet cells (Fig 5), and interspersed basal cells showing intense chromogranin-positive cytoplasmic granules

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IMMATURE ENDODERMAL TERATOMA OF THE OVARY (Nogales et al)

FIQURE 5. Higher differen- tiation of intestinal areas. (Left) Goblet cells are pres- ent (arrow). (Right) Numer- ous basal chromogranin- positive cells (arrows) are seen interspersed between columnar ceils. (Hematoxy- lin-eosin and immunoperox- idase stains; magnification x286.)

(Fig 5), and resembled the early differentiated intestinal epithelium. In case no. 1 the cells of some of these cysts also showed strong cytoplasmic positivity to thyroglob- ulin (Fig 6). Present in the stroma and in the vicinity of the cysts were anastomosing cords of polyhedral cells with undefined cell borders, mitotically active nuclei, and cytoplasm that was strongly positive for fibrinogen and stained weakly for AFP, having an appearance sim- ilar to the early differentiated liver tissue of a fifth-week embryo (Fig 7). No marked cellular atypia was noted, except for foci of isolated trophoblasts.

Mesenchym. The predominant tissue was of prim- itive mesoblastic type, composed of actively mitotic, stellate cells within an abundant basophilic matrix and with a cytoplasm that was strongly positive for both vi- mentin and keratin (Fig 8). Actin and AiAT also were positive in some cells. Differentiation into smooth mus- cle bundles had occurred around esophageal and intes- tinal organoid tissues (Fig 9); the muscle cells had fre- quent mitoses and were positive for desmin, actin, vimentin, and keratin. Cartilage was seen in only two slides and bone in only one. Cartilage was positive for both keratin and vimentin. Isolated foci of hemopoiesis with nucleated red blood cells were found in the stroma of case no. 2. There were no other mesenchymally de- rived tissues, such as striated muscle or fat. We were unable to find neuroectodermal derivatives, such as skin, adnexa, or neural tissue, or any of the recognized pat- terns of human yolk sac tumor (YST), hyalin globules, or embryonal carcinoma (EC).

DISCUSSION

Both tumors reported here are unusual since their epithelial component only reproduced endodermal tis-

sues in the various stages of differentiation found in the first trimester of embryonal life, ranging from primitive gut to differentiated intestine. The epithelial component also produced high levels of AFP despite the absence of any of the known patterns of YST or EC.

To date, no cases similar to these have been re- ported, although an example of a mature cystic teratoma of the ovary composed entirely of respry-type ep- ithelium occurring in a 5-year-old girl was interpreted as an endodermal monophyletic benign teratoma. In the same category of differentiated endodermal tumors, pure struma ovarii and perhaps some carcinoids could be included. Perrone et al4 reported clinical AFP pro- duction in three cases of pure immature teratomas of the ovary; immunohistochemical AFP positivity was confined to endodermal tissues. However, no infor- mation was provided about the presence of other com- ponents, such as neuroectoderm.

The epithelial derivatives reproduced here reflect the whole range of differentiation of embryonic endo- derm with varying degrees of maturity. The most prim- itive were spaces lined with cylindrical epithelium with apical and subnuclear vacuolation and strong AFP pos- itivity, characteristic of endoderm and similar to the early gut. Structures such as these have been described recently5 as an ectopic somatic component in the wall of mature, developmentally altered secondary human yolk sacs. A similar type of epithelium is reproduced in a papillary or glandular pattern in the “endometrioid- like” variant of ovarian YST.’ Other endodermal im- mature structures, albeit in a higher stage of differen- tiation, were reproduced in both our tumors; these were AFP- and AiAT-positive squamoid esophageal epithe- lium,’ fibrinogen-positive liver cell cords, and, most mature of all, an intestinal type of epithelium with mu-

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FIGURE 6. Glandular area with strong paranuclear (PN) and diffuse (arrow) positivity for thyroglobulin. (Immunoperoxidase stain; magnification X340.)

tin-producing cells with abundant interspersed chromo- granin and Grimelius-positive cells. These intestinal-type and squamoid cells also are capable of producing thy- roglobulin, thus reflecting a further specialization into yet another type of endodermal differentiation, namely, the thyroid, an organ that originates from the fore gut. Thyroglobulin secretion is present in the embryo at 10 to 12 weeks gestation’ and is dependent on thyroid- stimulating hormone secreted by the embryonic pitu- itary.

Focal differentiation of the primitive endodermal epithelium into trophoblast-type cells capable of se- creting hCG was seen in both of our cases; in one, weak serum expression of hCG was found and, in the other, urine gave a positive pregnancy test. This phenomenon may be explained by considering the early relationships between endoderm and trophoblast: in preimplantative stages the endodermal layer is found in close apposition under the trophoblast and in the earliest postimplan- tation embryos the endoderm is represented by a thin layer within the trophoblast constituting a bilaminar omphalopleure. Moreover, in the rhesus monkey there are intermediate cells found between endoderm and trophoblast8 This embryologic correlation supports the hypothesis of a close interrelationship between these

HUMAN PATHOLOGY Volume 24, No. 4 (April 1993)

two tissues that could be expressed both morphologically and functionally in tumors. Furthermore, it should be noted that epithelial neoplasms originating from organs derived from the fore, mid, and hind gut, such as lung,g stomach,” and colon,” are those that most frequently exhibit trophoblastic differentiation, although this dif- ferentiation also may be found in epithelial tumors of mesodermally derived organs.” Indeed, in lung’s and stomach9 tumors expression of both chorionic (hCG) and endodermal (AFP) markers may be simultaneously present.

The mesenchymal component in both cases also showed a wide range of differentiation, from the stellate mesoblasts to a full organoid smooth muscle coat sur- rounding immature intestine, as well as some cartilage and bone. Immunohistochemically, the endodermal primitive cells and the mesenchyme had a similarly strong expression of both cytokeratin and vimentin, a fact that has been reported recently in YSTs.14 This co- expression, which also may be present in many other tumors, has been interpreted as proof that the mesen- thyme of YST may be derived from the endodermal areas of the tumor and that such cells may serve as pro- genitors for further differentiated heterologous com- ponents, such as striated muscle.15 Recent embryologic

FIOURE 7. Liver cell nodule in the wall of an endodermal mi- crocyst. Nuclei are irregular and hyperchromatic. (Hematoxylin- eosin stain; magnification stain X340.)

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IMMATURE ENDODERMAL TERATOMA OF THE OVARY (Nogales et al)

FIGURE 8. (Left) Low mo- lecular weight, keratin-posi- tive mesenchymal cells. Pos- itively stained epithelium (top) serves as positive con- trol. (Right) Mesenchymal cells positive for vimentin. Small epithelial nodule (EP) serves as negative control. (Immunoperoxidase stain; magnification X387.)

FIGURE 9. Strong smooth muscle-specific actin positivity in fas- cicles surrounding intestinal structures. (Immunoperoxidase stain; magnification X340.)

data point toward the endodermal origin of mesenchyme in the very early stages of embryogenesis. The original assumption of Luckett” that the reticulum, an accu- mulation of stellate cells found between the endoderm of the primary yolk sac and trophoblast, originated from endoderm recent1 structural studies’ 7

has been substantiated by ultra- J’ in the rhesus monkey, in which

the reticulum develops from the endoderm and differ- entiates into mesenchymal cells. Furthermore, early mesenchymal cells have ultrastructural features similar to endoderm and even share junctional complexes with it. This may explain the biphasic expression of inter- mediate filaments of both epithelium and mesenchyme in the present cases and in mesenchymal YST. Similar co-expression also was seen in our cases in the small cartilaginous areas and smooth muscle. Hemopoietic foci were found in the mesenchyme of case no. 2; he- mopoiesis is an early developmental feature found in the mesenchyme adjacent to endoderm. It has been proposed that endoderm determines hemopoiesis’g and, indeed, in polyvesicular vitelline tumor hemopoiesis is related to endodermal vesicles.” Ex that endodermally derived tumors R

erimental evidence are able to differ-

entiate both mesenchyme and trophoblastz2 further emphasizes the differentiating capacity of the endoderm.

The tumors reported here share many clinical markers as well as immunohistochemical and histologic features with the ever-changing spectrum of YST, al- though neither the classically described visceral and pa- rietal patterns nor the recently described organoid pat- terns, such as intestina12’ or hepatic,24 were present. The highly primitive endodermal epithelia, hyalin glob- ules, or amorphous basement membrane material found in YST was absent in our tumors, while immature but developmentally correct epithelia of somatic type were

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found instead, making our tumors different from pre- viously reported YST. It may be possible that the full endodermal organoid differentiation present in these two cases originated from a YST, as it has been shown that human secondary into somatic structures. %

olk sac also may differentiate Another hypothesis is that the

tumor may have originated from an EC with which it shares the double clinical markers of AFP and hCG,25 in which case the undifferentiated EC component may have provided the stem cell that would have eventually differentiated into somatic endoderm. Both possibilities are speculative; however, data from experimental germ cell tumors demonstrate the relationship between EC, YST, and teratomas that appear to belong to the same family of neoplasms, only with different commitments to differentiation of somatic or extraembryonic cell types. 26 Similar endodermal glands and mesenchyme usually are found in both YST and EC, and are inter- preted as focal differentiations from a primitive cell line. The cases presented here could represent a fully accom- plished endodermal differentiation from any of those interrelated malignant tumors.

If the usual histologic grading of ovarian teratomas2? is used here, these tumors should be con- sidered as grade 2 since they have abundant embryonal tissue and mitotic activity, but little or no atypia. This may be true for the usual immature ovarian teratomas with marked predominance of neuroectodermal deriv- atives, which are the ones that more frequently metas- tasize and usually provide the immature tissues for grading procedure. While the biologic behavior of im- mature neural tissue is well known, no data of its en- dodermal counterpart is available, although it is known that in testicular teratoma endodermal tissues can dif- ferentiate in the metastases.28 The behavior of the pres- ent cases was benign, with a follow-up of 2 and 10 years. It is possible that this may have been due to the che- motherapy, although in case no. 2 the treatment was incomplete but the outcome was still favorable. Post- operative marker levels proved consistently negative. These cases also further indicate that AFP is not nec- essarily a marker exclusive to YST or EC and that ele- vated levels of AFP in immature teratoma4s2g.30 may lead to clinical misinterpretation as a more malignant tumor.

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