Significance of immunohistochemistry in neuro-oncology. V. Keratin as a marker for epithelial differentiation of primary and secondary intracranial and intraspinal tumors

Intermediate filament keratin is regarded as a good marker for epithelial and mesothelial tumors. In the intracranial and intraspinal spaces keratin has been demonstrated only in the endocrine cells of the adenohypophysis, squamous epithelial islands in the pars tuberalis of the hypophysis and in the choroid plexus epithelium. Since gliomas and meningiomas do not express keratin, this marker provides an additional help for differentiating between primary and secondary CNS tumors. Indirect immunofluorescence using an anti-keratin serum was used in a retrospective search for keratin in 80 tumors of the cranium and intraspinal space. Of the primary CNS tumors keratin positivity occurred in craniopharyngiomas, epidermoid tumors, pituitary adenomas, chordomas, a plexus papilloma as well as in the majority of germ cell tumors. Only 3 renal cell carcinoma metastases of 21 metastatic epithelial cell tumors (7 bronchial carcinomas, 6 breast cancers, 6 renal carcinomas, 1 rectum carcinoma, 1 cervix carcinoma) were keratin-negative. Similar findings were made in two melanoma metastases which we examined, whereas in a seminoma metastasis a few keratin expressing cells were found. Primary CNS tumors such as myxopapillary ependymomas, medulloepitheliomas, malignant meningiomas and paragangliomas which are often difficult to distinguish from these metastases proved to be keratin negative.     

Importance of immunohistochemistry for neuro-oncology. II. Localization of factor VIII-associated proteins and glial fibrillary acidic protein (GFAP) in angiomatous and sarcomatous tumors

Immunhistochemical methods utilizing specific antibodies against Factor VIII-related antigen and glial fibrillary acidic protein were employed in studies of 48 intracranial and intraspinal tumors. Factor VIII-related antigen occurred only in endothelial cells of the vascular wall and is therefore not of importance for the differential diagnosis of CNS tumors. Isolated Factor VIII positive cells in the stroma of hemangioblastomas turned out to be mast cells which may also normally contain this substance. The GFAP positive cells in hemangioblastomas are believed to all be of astrocytic lineage. Many of the multinuclear giant cells present in monstrocellular sarcomas contained GFAP but were Factor VIII negative. Genuine fibroxanthoma of the meninges can apparently exist next to pleomorphic xanthoastrocytomas. As demonstrated by one of our cases, the demonstration of GFAP alone can successfully distinguish between them.     

Placental alkaline phosphatase immunohistochemistry of intratubular malignant germ cells and associated testicular germ cell tumors

Two hundred three testicular germ cell tumors were studied immunohistochemically for the presence of placental alkaline phosphatase (PLAP). Special emphasis was placed on the pattern and incidence of positive staining of intratubular malignant germ cells (ITMGCs) adjacent to tumors. 99% of cases with adjacent ITMGCs showed a positive staining reaction in some or all IT-MGCs present. Other germ cell elements showed at least a focal positive staining reaction in the following proportions: seminomas, 96%; embryonal carcinomas, 96%; yolk sac tumors, 25%; mature teratomas, 5%; immature teratomas, 4%; choriocarcinomas, 45%; and syncytiotrophoblasts, 43%. The staining pattern for seminomas tended to be diffuse, whereas for embryonal carcinomas the staining pattern was more focal. Yolk sac tumors stained inconsistently for PLAP and a positive reaction was limited to a small percentage of cells. Syncytiotrophoblasts, singly or in choriocarcinomas, also showed variable positivity. These results corroborate the fact that PLAP is a sensitive marker for ITMGC, seminoma, and embryonal carcinoma.     

A model for karyotypic evolution in testicular germ cell tumors

Testicular germ cell tumor karyotypes are characterized by near-triploidy, with chromosome numbers ranging from 50 to 70, and by the frequent appearance of i(12p). The high chromosome number has been attributed to the formation of tetraploid carcinoma in situ cells followed by chromosomal losses that ultimately lead to tumor forms that are more advanced. In the present investigation, we show by analysis of the accumulated cytogenetic data on testicular germ cell tumors and computer simulations that two distinct processes are operating in the karyotypic evolution of these tumors. The results suggest that whole-chromosome changes originate from a multipolar cell division of a tetraploid cell, whereas imbalances caused by structural changes accumulate in a stepwise manner.     

Production of beta-human chorionic gonadotropin by germ cell tumors in vivo and in vitro

Production of the beta-subunit of human chorionic gonadotropin (beta-hCG) by human germ cell tumors was studied in surgical specimens, cultured cells and xenografted tumors. beta-hCG was identified most frequently in the syncytiotrophoblastic and intermediate trophoblastic components of choriocarcinoma, and it was also detected in syncytiotrophoblastic giant cells, occasionally in association with embryonal carcinoma, and less frequently with yolk sac tumor, seminoma and dysgerminoma. Our experimental data suggest that beta-hCG production by syncytiotrophoblastic giant cells in association with embryonal carcinoma is a result of trophoblastic differentiation of embryonal carcinoma. Moreover, it was proved that the production of beta-hCG by human embryonal carcinomas is regulated by more factors than is the case for human choriocarcinomas.     

Importance of immunohistochemistry for neuro-oncology. III. Demonstration of glial fibrillary acidic proteins (GFAP) in anaplastic astrocytomas and glioblastomas

Expression of gliofibrillary acidic protein in 23 anaplastic astrocytomas and 33 glioblastomas has been investigated and correlated with tumor behavior as reflected in both the length of the preoperative history and in the post-operative survival time. Three degrees of positive immunoreactivity to anti-GFAP can be distinguished: positive GFAP reaction in more than 2/3 of cells; in 1/3 to 2/3 of all cells; in less than 1/3 of all cells; negative reaction. All anaplastic astrocytomas and 27 of 33 glioblastomas showed GFAP positive reactions. The proportion of highly reactive tumors is higher by anaplastic astrocytomas than by glioblastomas (7 of 33). For both astrocytomas and glioblastomas there is a tendency for a decrease in the expression of GFAP to be associated with a shorter preoperative history and with a shorter survival time. This is more prominent for astrocytomas than for glioblastomas. This finding supports the opinion expressed in previous publications that the GFAP expression is reversely related to the level of tumor anaplasticity.     

Pathogenesis of adult testicular germ cell tumors. A cytogenetic model

In essence, two models exist of the pathogenetic relationship between seminomas and nonseminomatous germ cell tumors (NSGCTs). In the first model, the histogenesis of seminomas is assumed to diverge from that of the other testicular germ cell tumors (TGCTs) at an early stage. The neoplastic pathway of seminomas and NSGCTs is different, with limited or no crossover. The second model suggests that seminomas and NSGCTs have a common origin with a single neoplastic pathway on which seminomas are an intermediate stage in development of NSGCTs. Our data on the cytogenetics and ploidy of seminomas, combined tumors, and NSGCTs lend support to the model of pathogenesis of seminomas and NSGCTs in which all TGCTs (with the possible exception of spermatocytic seminoma and infantile yolk sac tumor) have a single origin and neoplastic pathway, with seminomas representing an intermediate stage in development of NSGCT components, as opposed to the model in which seminomas and NSGCTs develop separately. The progression of TGCTs probably proceeds from high to lower numbers of chromosomes and is therefore accompanied by a net loss of chromosomal material. This decrease will be the end result of loss of specific chromosomes, gain of some other chromosomes (or part of chromosomes), and development of structural abnormalities.     

Histochemically demonstrable changes in cell surface carbohydrates of human germ cell tumors

Cell surface carbohydrate chains of human germ cell tumors were investigated histochemically using peanut agglutinin (PNA), Dolichos biflorus agglutinin (DBA), Ulex europaeus agglutinin I (UEA-1), and anti-I-(Ma) antibody. Of 16 cases of embryonal carcinoma in adult testis, peanut agglutinin, a lectin specific for terminal beta-galactosyl residues, bound to the surface of tumor cells in 11 cases, D. biflorus agglutinin, a lectin specific for terminal alpha-N-acetyl galactosamine residues, in 10, and U. europaeus agglutinin, a lectin specific for terminal alpha-L-fucosyl residues in 7. I-(Ma) antigen, a branched carbohydrate chain composed of three N-acetyl lactosamine units, was found in 12 cases as well. The cell surface of tubular or organoid architecture tended to be positive for some of these four reagents, whereas that of a papillary pattern tended to be negative. All yolk sac tumors (three in adult testis, 10 in infantile testis, and four in ovary) were positive for peanut and U. europaeus agglutinins and anti-I-(Ma) antibody but were negative for D. biflorus agglutinin. Seventeen of 18 seminomas (in adult testis) and one dysgerminoma (in ovary) did not react with these four reagents. Four choriocarcinomas (three in testis and one in ovary) were not positive for any of them either. In 10 cases of immature teratoma, the appearance of the binding sites to these lectins and antibody depended on the direction of differentiation and the degree of maturity. These findings suggest that carbohydrate chains of human germ cell tumors change greatly in the process of differentiation from embryonal carcinoma cells to somatic cell (teratoid) component or yolk sac carcinoma component, and that histochemical detection of the binding sites described may assist in the classification of germ cell tumors, in spite of the lack of precise knowledge about the biologic role of cell surface carbohydrates.     

Loss of Fhit expression in testicular germ cell tumors and intratubular germ cell neoplasia.

The FHIT gene, located at human chromosome 3p14.2, is frequently deleted in a number of human cancers, and interstitial deletions at this site were recently described in a significant proportion (41%) of testicular germ cell tumors. We studied the expression of Fhit protein in the progression and differentiation of testicular germ cell tumors to further elucidate its role in this type of malignancy. Forty-five patients with testicular germ cell tumors and intratubular germ cell neoplasia (identified in 42/45 cases) were included in the study. Immunohistochemical staining with polyclonal rabbit IgG antibody to Fhit (ZR44, Zymed Laboratories) on formalin-fixed, paraffin-embedded tissues was used. Fhit was constitutively expressed in germ cells, Sertoli cells, and Leydig cells. All 42 cases of intratubular germ cell neoplasia revealed no expression of this protein. No expression of Fhit was observed in any case of pure seminoma or in the seminomatous component of mixed germ cell tumors. Unexpectedly, Fhit expression was frequently (16/18) observed in the glandular tissue of mature teratomatous component of mixed germ cell tumors, despite the absence of Fhit in the intratubular germ cell neoplasia, the presumed precursor lesion. The loss of Fhit expression is a consistent characteristic of intratubular germ cell neoplasia, which suggests a potential role in a maturation/differentiation defect early in the development of testicular germ cell tumors. Likewise, the lack of expression in seminomas is supportive of this view. However, re-expression of Fhit in well-differentiated glandular epithelium of teratomatous component of mixed germ cell tumors suggests that there is no loss of FHIT gene in this subset of neoplasia but rather that Fhit protein expression is differently regulated through the phases of germ cell tumor progression.     

Immunohistochemical localization of murine stage-specific embryonic antigens in human testicular germ cell tumors.

Monoclonal antibodies raised against and/or recognizing stage-specific antigens on preimplantation mouse embryos and stem cells of murine teratocarcinoma were used to localize these antigens immunohistochemically on human testicular germ cell tumors. SSEA-1, the antigen found on mouse embryonal carcinoma (EC) cells and embryonic cells from the 8-cell stage embryo onward, including the fetal primordial germ cells, was detected on yolk sac carcinoma components of human tumors, but not on EC cells. SSEA-3, the antigen found on follicular ova, fertilized eggs, early cleavage stage embryonic cells, and visceral endodermal cells of the mouse embryo, but not on mouse EC cells, was detected on human EC cells. Both antigens were found on the cell surface of fetal testicular germ cells but not in the seminiferous tubules of adult human testes. These data point out differences between human and murine EC cells suggesting that human EC cells correspond developmentally to a less mature embryonic cell than the murine EC cells. The possible histogenesis of human germ cell tumors from primordial and/or fetal germ cells is briefly discussed.     

Significance of immunohistochemistry for neuro-oncology. VI. Occurrence, localization and distribution of glial fibrillary acid protein (GFAP) in 820 tumors

In a retrospective study 820 tumors were immunohistochemically examined with anti-GFAP. All 224 astrocytomas and 105 of 112 glioblastomas were, at least focally, positive. 72% of ependymomas and 64% of oligodendrogliomas contained tumor cells which expressed GFAP. In such entities the reaction is dependent on the histologic subtype. Only 26 of 114 medulloblastomas (22.8%) demonstrated scattered GFAP positive cells. GFAP was also demonstrated in the CNS in gangliogliomas, monstrocellular sarcomas, 3 of 6 PNET, one non-classifiable tumor in a child, 1 plexus papilloma, in scattered stromal cells in 15 of 26 hemangioblastomas as well as in the mature glial component of intracranial germ cell tumors. Outside of the CNS there was evidence of GFAP in 3 cases with nasal glial heterotopy and in the myxoidal part of a pleomorphic salivary gland adenoma. Neoplasms which proved negative to GFAP in our series included purely neural differentiated tumors meningioma, neurolemmomas, chordomas, paragangliomas, sarcomas, lymphomas, melanomas and carcinoma metastases. Separating GFAP-positive reactive astrocytes from the actual tumor cells has proved to be a problem in the routine use of GFAP in differential diagnosis. Absence of an immunohistochemical response does not exclude a tumor of glial origin. Tissue samples which are too small, particularly in the case of anaplastic astrocytomas and glioblastomas can give false negative results.     

Malignant germ cell tumors of the ovary.

This article reviews 281 malignant germ cell tumors of the ovary from the Armed Forces Institute of Pathology and highlights their distinctive clinical and pathologic features. Emphasis is placed on the importance of a combined therapeutic approach utilizing surgery, chemotherapy, and radiation. The rationale for unilateral salpingo-oophorectomy in conjunction with chemotherapy for certain types of neoplasm confined to one ovary (stage 1a) is emphasized, and the role of human chorionic gonadotropin and alpha-fetoprotein as tumor markers in the management of patients with these tumors is discussed.     

MAGE-1 and MAGE-3 tumor rejection antigens in human germ cell tumors.

The MAGE-1 and MAGE-3 genes are members of the melanoma antigen-encoding gene family. These genes encode for HLA-restricted tumor-associated rejection antigens recognized by cytotoxic T lymphocytes. MAGE-1 and MAGE-3 gene expression has been identified in a number of human malignancies, and MAGE-1 and MAGE-3 antigenic peptides are potential targets for tumor-specific immunotherapy. The only normal tissues known to express these genes are testicular germ cells and placental tissue. The objective of this study was to examine MAGE-1 and MAGE-3 antigens immunohistochemically in testicular germ cell tumors, including seminoma, a germ cell tumor frequently associated with a lymphoid infiltrate. Forty-three germ cell tumors (24 seminomas, six embryonal carcinomas and 13 mixed germ cell tumors), and 10 Leydig cell tumors were selected for study, and standard immunohistochemical techniques were used on formalin-fixed paraffin-embedded tissues using mouse monoclonal antibodies to MAGE-1 (clone M454) and MAGE-3 (clone 57B) antigens. MAGE-1 antigen was identified in 16.6% of seminomas. No embryonal carcinomas, yolk sac tumors, or teratomas contained MAGE-1 protein. MAGE-3 antigen was identified in 41.8% of seminomas, and this protein was not identified in embryonal carcinomas, yolk sac tumors, or teratoma. Spermatogonia and primary spermatocytes contained MAGE-1 and MAGE-3 antigen, and more mature forms, including spermatids, were weakly positive to negative. Leydig cell tumors were negative for MAGE-1 and MAGE-3. In seminoma, the presence of MAGE-1 and MAGE-3 antigens did not correlate with tumor size, tumor stage, the presence of a lymphoid infiltrate, or patient outcome. The low frequency of MAGE-specific HLA alleles in the population, the loss of the HLA class I antigens in neoplastic germ cells, and the finding that the majority of seminomas and all non-seminomatous germ cell tumors lacked MAGE-1 and MAGE-3 antigenic peptides indicate that immunotherapy directed towards MAGE-1 and MAGE-3 antigen is not a likely treatment option for seminoma and nonseminomatous germ cell tumors. The significance of MAGE-1 and MAGE-3 proteins in normal spermatogonia and primary spermatocytes will require additional study.     

Keratin 7 is a marker for a subset of trophoblastic cells in human germ cell tumors.

Human testicular germ cell tumors were studied immunohistochemically with the monoclonal antibody to the 54-kd keratin polypeptide (keratin 7) to determine whether this antibody could be used selectively to identify trophoblastic cells. The antibody reacted with the intermediate filaments in the cytoplasm of some cells in choriocarcinoma cell lines, and in trophoblastic cells in mixed germ cell tumors and a seminoma. It did not react with classic seminoma cells, embryonal carcinoma, yolk sac carcinoma, or somatic tissues of mixed germ cell tumors. On the basis of these data we conclude that monoclonal antibody to keratin 7 is a marker for a subset of trophoblastic cells in human germ cell tumors.     

Alpha-fetoprotein in germ cell tumors. An immunohistochemical study

Total 40 cases of testicular and ovarian germ cell tumors and one case of extragonadal germ cell tumor were studied for the presence of alphafetoprotein (AFP) by indirect immunoperoxidase technique. All seminomas (7 cases) and teratomas (13 cases) were negative for AFP; while 85% of the pure embryonal carcinomas, (E.C.) all pure yolk sac tumors (Y.S.T.) (7 cases) and all embryonal carcinoma and yolk sac components in mixed tumors were AFP positive. Immunostaining of tumor marker appeared to help only in differentiating seminomatous and nonseminomatous tumors and hence does not provide any additional information for classification of these tumors.     

SNRPN methylation patterns in germ cell tumors as a reflection of primordial germ cell development.

Studies examining altered imprinted gene expression in cancer compare the observed expression pattern to the normal expression pattern for a given tissue of origin, usually the somatic expression pattern for the imprinted gene. Germ cell tumors (GCTs), however, require a developmental stage-dependent comparison. To explore using methylation as an indicator of germ cell development, we determined the pattern of methylation at the 5' untranslated region of SNRPN in 89 GCTs from both children and adults. Fifty-one of 84 tumors (60.7%) (12/30 (40%) of cultured pediatric GCTs, 23/36 (63.9%) of frozen adult GCTs, and 16/23 (69.5%) of frozen pediatric GCTs, with five samples having results from both cultured and uncultured material) demonstrated a nonsomatic methylation pattern after dual digestion with XbaI, NotI, and Southern blot analysis. In contrast, only 2 of 18 (11%) control samples (16 non-GCTs and 2 normal ovaries) exhibited a nonsomatic pattern. In both cases, the result was shown to be due to copy number differences between maternal and paternal homologs, unlike the GCTs in which there was no evidence of an uneven homolog number. A comparison of the data for only the gonadal GCTs and the control data showed a highly significant difference in the proportion of tumors with methylation alterations at this locus (P = 0.0000539). Since there is no published evidence of the involvement of SNRPN methylation changes in the development of malignancy, the data suggest that the methylation pattern of SNRPN in GCTs reflects that of the primordial germ cell giving rise to the tumor.     

Characteristics of "embryoid body" in human gonadal germ cell tumors

The characteristics of so-called "embryoid body" appearing in gonadal germ cell tumors were studied histologically and immunohistochemically on serial sections of three cases (one ovary and two testes). The embryoid bodies were usually observed to be contiguous with immature or mature intestinal ducts, hepatic nests, or epidermal cell nests on serial sections, though they appeared to be isolated in one section. The "amniotic cavity"-like structure of embryoid body was continuous with intestinal duct, and rarely with squamous cell nests, while the "yolk sac" was continuous with hepatic tissue. In these immature or mature structures, differentiation was always found independently of "disc," and portions of "ectoderm" and "endoderm" remained less differentiated in comparison with others. These findings were in contrast with a normal embryo in which immature and/or mature structures are derived from the embryonic disc. The amniotic cavity connected frequently with yolk sac. From the present results, the embryoid body is not considered to be a real or teratomatous embryo, but only a product during a divergent differentiation into intestine and liver from the plastic epithelium, which seems to be derived from an embryonic gut.     

Histologically confirmed intracranial germ cell tumors; an analysis of 62 patients in a single institute

This study was undertaken to document the clinicopathologic characteristics of histologically verified, primary intracranial germ cell tumors (GCTs), determine treatment outcomes, and to identify prognostic factors. The records of 62 patients (45 males and 17 females) with a primary intracranial GCT were retrospectively analyzed. Mean patient age was 18 years, and median follow-up was 41 months. The most common histological subtypes were germinoma (48.4%), followed by mixed GCT (27.4%), and teratoma (19.4%). In 23 patients (37.1%), disease onset occurred between 16 and 20 years. Germinomas and malignant non-germinomatous germ cell tumors were most prevalent in the pineal gland, suprasellar region, and basal ganglia, whereas teratomas dominated at other sites. Synchronous bifocal GCTs were found in six patients. Five-year overall survival (OS) rates according to a therapeutic classification proposed by Sawamura were 82.93%, 83.08%, and 64.71% in the good, intermediate, and poor prognosis groups, respectively (P = 0.2839). Five-year OSs in patients with normal tumor marker (αFP or βHCG) and patients with elevated marker were 85.26% and 66.96%, respectively (P = 0.0568). Five of six patients with alpha-fetoprotein (α-FP) of >1,000 ng/ml succumbed to disease, whereas all five patients with a beta-human chorionic gonadotropin of >1,000 mIU/ml survived. Mixed GCTs are more common in Korea than in the West. Sawamura’s classification of intracranial GCT may be a fine tool for stratifying patients’ survival. Patients with elevated tumor marker levels may appear to have poorer OS independent of histology. In particular, high titers of α-FP seem to impact prognosis.