Original Article

Subversion of antigenic expression in neoplasia: Lessons fromimmunohistology of teratomas

Amit Goel, R.K. Vasishta and Kusum Joshi

Department of Histopathology, Post Graduate Institute of Medical Education and Research, Chandigarh, India

astrocytic neoplasms.4 Vimentin is expressed by malignantepithelial tumors such as breast, ovarian and coloniccarcinoma.5

These aberrations in the expression of antigens in neo-plastic tissue closely simulate the varying expression ofantigens in immature tissues during development, whichsuggests that the phenomenon of antigenic subversion in neoplasia is related to the process of maturation anddifferentiation.6 Teratomas are unique tumors that are derivedfrom pluripotent stem cells and show a wide array of tissuesrepresenting the three germ layers: ectoderm, mesodermand endoderm. These tumors may be gonadal or extra-gonadal, sacrococcygeal being the commonest extragonadalteratoma.7,8 Teratomas may contain only mature tissue orhave juxtaposed mature and immature elements (usually of neural origin) or have an associated carcinoma. Themorphological hallmark of teratomas is a recapitulation ofembryonic histogenesis. Thus, these tumors can provide anexcellent model of maturation, differentiation and malignanttransformation for studying the subversions of antigenexpression in neoplasia.

MATERIALS AND METHODS

Ten specimens of teratomas were selected from the files of the Histopathology Department, PGIMER (Chandigarh,India) from 1987 to 1994. Each tumor was adequatelysampled (five to eight blocks). Hematoxylin and eosin-stained sections were reviewed microscopically and thetumors were classified into mature (two cases) and immatureteratomas according to the World Health Organizationclassification.9 One of the mature teratomas was sacro-coccygeal in origin, while all other tumors were testicular inorigin. Four of the cases were associated with carcinoma,three being embryonal carcinomas and one endodermalsinus tumor.

Finally, two blocks were selected from each specimen suchthat the sections showed maximum representation of the dif-

Pathology International 1999; 49: 141–146

Many examples of inappropriate expression of intermediatefilaments and other cell identity markers are known to occurin diagnostic tumor pathology. In the present study, thissubversion of antigenic differentiation in tumors has beeninvestigated using teratomas as a model, as these uniquetumors not only mimic developing tissues but also showcarcinomatous elements. Ten cases of teratoma (eightimmature, two mature) were studied immunohistochemi-cally using a panel of 13 commonly used cell identitymarkers, including intermediate filaments. Examples ofantigenic coexpression and transbarrier expression invarious morphologically mature and immature tissues werenoted to be similar to those seen in various tumors.Carcinomatous elements in teratomas were non-reactiverather than showing antigenic aberrations. Hence, thisstudy strengthens the proposition that antigenic subversionin neoplasia is related to the process of maturation anddifferentiation, rather than malignant transformation.

Key words: immunohistology, teratomas, tumor markers

INTRODUCTION

Subversion of antigenic differentiation in neoplasia is awidespread phenomenon, of which many examples are citedin the literature. Thus, anomalous cytokeratin expression canoccur in leiomyomas and leiomyosarcomas.1 Coexpressionof keratin and neurofilament proteins has been seen inbronchial carcinoids.2 Coexpression of cytokeratin (CK),vimentin and neurofilaments have been noted in a largenumber of neuroendocrine neoplasms, including pheochro-mocytoma, medullary carcinoma of thyroid and pancreaticislet cell tumors.3 Simultaneous expression of glial fibrillaryacidic protein (GFAP), keratin and vimentin occurs in

Correspondence: Professor K. Joshi, Department of Histopathol-ogy, PGIMER, Chandigarh 160 012, India. Email:<medinst@pgi.chd.nic.in>

Received 2 April 1998. Accepted for publication 26 August 1998.

ferent types of mature and immature tissues as identified byhistology alone, for immunohistochemical analysis.

Immunohistochemistry

Immunohistochemistry was done by the indirect immunoper-oxidase method. A battery of the following 13 primary antibodies was used in different dilutions: monoclonal anti-cytokeratin peptide 7 (1 : 100, CK7); monoclonal anti-cytokeratin peptide 13 (1 : 100, CK13); monoclonal anti-cytokeratin peptide 18 (1 : 100, CK18); monoclonal anti-vimentin (1 : 40); monoclonal anti-desmin (1 : 20); monoclonalanti-glial fibrillary acidic protein (1 : 100, GFAP); monoclonalanti-neurofilament protein (1 : 60); polyclonal anti-myoglobin(1 :100); monoclonal anti-skeletal muscle myosin (1 : 100);monoclonal anti-smooth muscle myosin (1 : 100); monoclonalanti-S-100 protein (1 : 50); polyclonal anti-Factor VIII-relatedantigen (1 : 500); and monoclonal anti-chromogranin (1 : 100).All antibodies were procured from Sigma Chemical Co. (StLouis, MO, USA) with the exception of anti-neurofilamentprotein and anti-chromogranin (Dako Corporation, Carpin-teria, CA, USA).

Peroxidase-conjugated swine anti-rabbit and anti-mouseimmunoglobulins (Dako) were used in a dilution of 1 : 100 as secondary antibodies. Diaminobenzidine hydrochloride(Sigma) was used as the chromogen. A positive and anegative control section from normal human adult tissueswas used with each batch of slides stained.

Poly-L-lysine was used as an adhesive to bind the sectionsto the slides at a concentration of 0.01%. This prevented thefloating of sections.

RESULTS

All ten cases of teratoma studied were of male patients andtheir age varied from neonate to 29 years.

The tissues seen in the ten cases included stratified squamous, cuboidal and columnar epithelia, cartilage, bloodvessels, muscles, pancreatic acini, pancreatic islets andnerves. Mature glial tissue was seen in two cases. The imma-ture elements, as determined by histology, consisted of undif-ferentiated epithelial areas comprising of round to polygonalshaped cells with large vesicular nuclei, inconspicuous nucle-oli and scant to moderate cytoplasm forming glands or solidclusters. The immature mesenchyme was seen as undif-ferentiated round to oval cells in a loose stroma. Immatureneural elements forming tubules and rosettes were also seenin places. Embryonal carcinoma was seen along with otherelements in three cases and endodermal sinus tumor in onecase.

Stratified squamous epithelium was seen in five cases andwas positive for CK7, 13 and 18 with some areas showing allthree cytokeratins. Two cases, however, showed expressionof S-100 protein and GFAP. In one case, a focus was seenshowing epithelial tissue merging into neuroglial tissue,which showed coexpression of CK18, GFAP and S-100protein in both epithelial and glial tissue. The epithelialelement was seen to cover fibrous connective tissue over anodular protuberance. The epithelium was three to four cellsthick and did not show intercellular bridges or surfacekeratinization. In one focus, an area of glial tissue was indirect continuity of the epithelium, where the latter wasindistinguishable from ependymal cells. It lacked basementmembrane and the cells showed foot processes and cilia.

142 A. Goel et al.

Figure 1 Photomicrograph showing the neuroepithelium (arrow)continuing into the neuroglial tissue (O). The epithelium and deeperneural tissue are strongly reactive for glial fibrillary acidic protein.Neuroglia (O) is weakly reactive (immunoperoxidase).

Figure 2 Morphological details of the epithelium seen in Fig. 1. (a)Neuroepithelium showing stratification without intercellular bridges isreactive with cytokeratin 18 (immunoperoxidase). (b) Epitheliumshowing multilayering with cilia (S-100 protein). (c) Ependymaldifferentiation with cilia and absent basement membrane is stronglyreactive with glial fibrillary acidic protein (immunoperoxidase).

A continuity between stratified squamous epithelium toependymal lining, with a gradual change from one to theother was noticed. Mature glial tissue was strongly positivefor GFAP, S-100 protein and weakly positive for cytokeratin.Epithelium was strongly reactive for cytokeratin, GFAP andS-100 protein. This area was not well recognized on thehematoxylin-eosin sections but was well highlighted byimmunostaining, and is interpreted as neuroectodermaltissue continuous into neuroglial tissue (Figs 1,2).

Cystic spaces and glands lined by cuboidal or columnarepithelial cells were seen in all ten cases. Intestinal lining wasseen in some places. Cytokeratin 18 was found to be positivein all cases, whereas CK13 was found in only one case andCK7 in none. Vimentin expression was seen in the basalregion of cuboidal epithelium in two cases; desmin in theapical region in two cases; myoglobin, S-100 protein, FactorVIII-related antigen in two cases; and skeletal muscle myosinin one case. Morphologically, well-differentiated columnarepithelium showed interesting coexpression of vimentin,GFAP and S-100 protein in one case.

Cartilage was seen in five cases, with areas showingimmature chondrocytes. Vimentin expression was seen in allcases as in mature adult tissue. S-100 protein, CK18 andmyoglobin expression was seen in three cases, CK18 beingpresent in chondrocyte nuclei.

The blood vessels showed vimentin and Factor VIII-relatedantigen expression in all of the seven cases in which theywere seen.

Muscle was seen in four cases and showed expression ofmyoglobin and skeletal muscle myosin in all cases. Desminreactivity was seen in two cases, and CK7 in morphologicallymature-looking smooth muscle was seen in one case.

Pancreatic tissue was seen in only two cases. The aciniwere positive for CK7 and 18 and islets showed chromo-

Immunohistology of teratomas 143

Figure 3 Localization of desmin by immunostaining in apicalregions of pancreatic acinar cells (immunoperoxidase).

granin expression. Desmin (Fig. 3), vimentin myoglobin,skeletal muscle myosin and focal GFAP reactivity were seenin the apical region of acinar tissue. Chromogranin expres-sion was also seen in acinar tissue (Fig. 4). The pancreaticislets also showed GFAP and CK7 reactivity in one case.

Nerve tissue was seen in only one case and showedneurofilament protein positivity, as expected. In one caseneuroglial tissue showed differentiation into histologicallymature looking brain parenchyma with coexpression ofvimentin, GFAP and S-100 protein in the same areas.

Undifferentiated epithelial areas showed positivity forCK18, thus confirming their epithelial nature. Focal reactivityfor vimentin was also seen in these areas (Fig. 5).

Immature mesenchyme in eight cases showed vimentinGFAP and S-100 protein reactivity. Focal areas of neuraldifferentiation with neural rosettes and tubule formation wereseen to be reactive for S-100 protein and GFAP in one case,and CK18 in two cases (Fig. 6). The endodermal sinus tumorwas positive for CK18, vimentin and S-100 protein, whereasembryonal carcinoma was negative for all markers. Resultsare summarized in Tables 1 and 2.

DISCUSSION

The present study is an investigation into the widely preva-lent subversion of antigenic expression seen in neoplastictissues.1–5

We have previously shown that the aberrant expression ofantigens in neoplastic tissues simulates the varying expres-sion of antigens in immature tissues of human fetuses andembryos.6 Hence, it was hypothesized that the phenomenonof antigenic subversion in neoplasia is related to the processof maturation and differentiation.

Figure 4 Reactivity for chromogranin in the islets of Langer-hans’ cells as well as in the surrounding pancreatic acini(immunoperoxidase).

Teratomas are unique tumors with phenotypic expressionof the three classical germ layers and are considered to bethe neoplastic counterpart of embryonal tissues. These may,in addition, show carcinomatous elements. These tumorsencompass maturation, differentiation and malignant trans-formation, and thus can be used as a model to study thesephenomena.

In the present study, one case of teratoma showed a con-tinuity of neuroepithelium into glial tissue both morpho-logically and immunohistochemically. Both the epitheliumand glial tissue showed reactivity for cytokeratin, vimentin,GFAP and S-100 protein. This finding confirmed theneuroectodermal derivation of glial tissue. Another two cases

showed GFAP and S-100 protein reactivity in stratifiedsquamous epithelium, and S-100 protein was localized incolumnar epithelium. Cytokeratin 18 positivity was noted inglial tissue in two cases. These findings can be explained onthe basis of neuroectodermal derivation of glial tissue. Vanceet al.10 found GFAP reactivity in teratomas in the margins ofglial masses and immature ependymal rosettes, but not inprimitive neuroepithelial tubules or mature ependymoblas-tomatous foci. The present study also found that immatureneural tissue and primitive neuroepithelial tubules werereactive for vimentin rather than GFAP.

Coexpression of cytokeratin and vimentin was seen incolumnar epithelium in two instances in the present series.

144 A. Goel et al.

Table 1 Normal and aberrant expression of cell identity markers in different tissues in teratomas

Normal No. cases Aberrant No. casesNo. cases cell marker with normal cell marker with aberrant

Tissues in which seen expression expression expression expression

Mature elementsStratified squamous epithelium 5 CK7 4/5 S-100 2/50

CK13 4/5 GFAP 2/50CK18 3/5

Cuboidal/columnar epithelium 10 CK7 0/10 Vim. (basal) 2/10CK13 1/10 Des. (apical) 2/10CK18 10/10 F VIIIrAG 2/10

Myo. 2/10SKM 1/10S-100 2/10

Cartilage 5 Vim. 5/5 CK18 3/50S-100 3/5 Myo. 3/50

Muscle 4 Myo. 4/4 CK7 (smooth 1/40SKM 4/4 muscle)Des. 2/4

Pancreatic acini 2 CK18 2/2 Des. (apical) 1/20Vim. (apical) 1/20SKM (apical) 1/20GFAP (focal) 1/20Chromo. 2/20

Pancreatic islets 2 Chromo. 2/2 GFAP 1/20CK7 1/20

Mature glial tissue 2 GFAP 1/2 Vim. 1/20S-100 1/20

Nerve 1 NFP 1/1 – –Immature elements

Immature epithelium 2 CK 2/2 Vim. 2/20Immature mesenchyme 9 Vim. 8/9 – –

GFAP 8/9 – –S-100 8/9 – –SKM 1/9 – –

Immature neural tissue 4 Vim. 2/4 CK18 2/40GFAP 1/4S-100 1/4

Carcinomatous elementsEmbryonal carcinoma elements 3 CK18 0/3 Vim. 0/30

S-100 0/30Endodermal sinus tumor 1 CK18 1/1 Vim. 1/10

S-100 1/10

CK, cytokeratin; Myo., myoglobin; Vim., vimentin; GFAP, glial fibrillary acidic protein; Des., desmin; FVIIIrAG, factor VIII-related antigen; Chromo.,chromogranin; SKM, skeletal muscle myosin; NFP, neurofilament protein.

Vimentin was localized to the basal regions of the cells,whereas cytokeratin was present diffusely. This suggests atrue coexpression rather than a sharing of common epitomesby the two antibodies. Azumi and Battifora5 have previouslyreported differing cytoplasmic distributions of cytokeratin andvimentin, again suggesting a true coexpression.

The finding of vimentin and S-100 protein reactivity incartilage in cases of teratoma is similar to human embryosand fetuses,6 and consistent with the study by Notohara et al.11 However, Notohara and colleagues also found GFAPpositivity in cartilage close to neural tissue, which was notfound in the present study. We also found CK18 in carti-laginous areas in three cases of teratoma, which has pre-

viously not been mentioned in the literature. This may,however, be explained on the basis of the theory proposed byStefansson et al. of mesodermal chondrocytes being devel-opmentally close to the neuroectoderm.12

Pancreatic acini showed positive immunostaining withCK18 as in fetal tissue. An interesting finding, however, wasthe presence of GFAP, desmin, vimentin and skeletal musclemyosin focally in pancreatic acini of teratomas. Lee et al.have also found GFAP positivity in the salivary gland acini of15–18-week-old fetuses, which was explained on the basis ofneuronal cytodifferentiation of the ductal reserve cells.13 Theislets also showed interesting expressions of GFAP and CK7in addition to chromogranin.

Desmin was seen in columnar epithelium in another case.Thus, it appears that myogenic cytodifferentiation of theepithelial cells can also occur.

Smooth muscle in teratomas shows occasional CK7expression, as was found in the present study on humanfetuses.6 Immature mesenchyme was reactive with vimentin,S-100 protein, GFAP and, occasionally, with skeletal myosin,signifying its pluripotent nature.

Thus, the varied expression of cell markers in themorphologically mature components of teratomas mimics the aberrant expression of antigens in the various tumorsmentioned earlier. The results are similar to those found inhuman fetuses.6

However, the carcinomatous elements in three teratomacases were non-reactive, with most antibodies implying thatmalignant transformation leads to loss of antigens rather than the aberrant expression of antigens. The present studyfurther strengthens the hypothesis that antigenic subversionin neoplasia is related to the processes of maturation anddifferentiation.

Immunohistology of teratomas 145

Table 2 Aberrant expression of cell identity marker in humanteratomas

Markers showing Tissues showing No. casesaberrant expression aberrant expression (n 5 10)

Cytokeratin 7 Smooth muscle 1Cytokeratin 18 Cartilage 3

Immature neural tissue 2Glial fibrillary Pancreatic islets 1

acidic protein Pancreatic acini 1Stratified squamous 1

epithelium 1 keratinMyoglobin Columnar epithelium 2

Cartilage 3Myoglobin 1 skeletal Pancreatic acini 1

myosin 1 desmin (apical)S-100 protein Stratified squamous 1

epithelium Mature glial tissue 1

(1) Coexpression with.Figure 5 Reactivity for vimentin in undifferentiated epithelial cellsand in the surrounding immature mesenchyme (immunoperoxidase).

Figure 6 Reactivity for cytokeratin 18 in the neurotubules(immunoperoxidase).

ACKNOWLEDGMENTS

The authors acknowledge the financial support from theIndian Council of Medical Research and the guidance of Professor B.N. Datta, ex-Chairman, Department of Pathology, PGIMER (Chandigarh, India).

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