Increased fucosylation of high-molecular-weight glycoproteins accompanies retinoic-acid-induced differentiation of F-9 embryonal carcinoma cells.

Retinoic acid (RA) treatment of F-9 embryonal carcinoma cells resulted in cell flattening and increased production of laminin B1 chain, both indicating differentiation to endoderm-like cells. In addition, RA caused a time- and dose-dependent decrease in growth rate in monolayer culture and a dose-dependent decrease in the ability of the cells to form colonies in soft agarose. Differentiation was accompanied by an increase in the fucosylation of specific high-molecular-weight cellular and cell-surface glycoproteins. The fucosylation of glycoproteins of Mr 175,000 (gp175), 250,000 (gp250), and 400,000 (gp400) increased as early as 24 hr after the addition of 5 x 10(-6) M RA to the culture medium. These changes preceded both growth inhibition and the induction of laminin B1 expression, which were detected 48 to 72 hr after addition of RA. The increased fucosylation of these glycoproteins showed a distinct dose-response relationship. Both gp175 and gp250 showed the greatest increase in fucosylation at 10(-5) M, which was also the dose at which RA induced laminin maximally, while the fucosylation of gp400 was greatest at 10(-8) M RA and declined at higher concentrations. The overall synthesis of large fucosylated glycopeptides decreased in RA-treated cells, in spite of the increases in the fucosylation of specific cellular glycoproteins. RA-induced differentiation of F-9 cells was also accompanied by a time- and dose-dependent increase in fucosyltransferase activity. Although the functions of these glycoproteins are not currently known, the early increase in their fucosylation can be considered as a marker of differentiation in this system.

Two homologous mixed müllerian tumor lines of the ovary and their characteristics.

Two cell lines, NF and JoN, derived from human ovarian carcinosarcomas, were established in tissue culture and in nude mice. Both lines, growing in monolayers, showed morphologic features of adenocarcinoma cells (NF being aneuploid with a modal number of 53, and JoN being pseudodiploid with a modal number of 44). Intermediate filaments were demonstrated immunohistochemically; the JoN line expressed keratin, but not vimentin or desmin, whereas the NF line expressed vimentin and desmin, but not keratin. Plasminogen activator activity was found in both lines. It is concluded that both of these lines are potentially useful models for studying the diverse characteristics of malignant mixed Müllerian tumors.

Neoplastic embryoid bodies of embryonal carcinoma C44 as a source of blastocele-like fluid.

There is a cytotoxic activity in blastocele fluid that kills embryonal carcinoma cells with trophectodermal potential but spares those with embryonic potential. This activity is present when programmed cell death occurs in the inner cell mass (ICM), and the ICM loses its trophectodermal potential. Because of the paucity of blastocele fluid, cystic embryoid bodies of embryonal carcinoma C44 were examined ultrastructurally and in tissue culture to determine if they corresponded to late blastocysts and if their fluid corresponded to blastocele fluid. No trophectoderm was demonstrated in the embryoid bodies, but embryonal carcinoma and endoderm were present, leading to the conclusion that the embryonal carcinoma corresponded to late ICM that had expressed endodermal potential. As a result the cyst fluid might have contained the toxic activity of blastocele fluid. The cyst fluid of C44 embryoid bodies did contain a soluble, low-molecular-weight, cytotoxic activity that preferentially killed embryonal carcinoma cells with trophectodermal potential while sparing those with embryonic potential. Enough of this fluid was available to determine the chemical nature of this toxic activity.

The abnormal occurrence and the differentiation-dependent distribution of N-acetyl and N-glycolyl species of the ganglioside GM2 in human germ cell tumors. A study with specific monoclonal antibodies.

Human primary germ cell tumors were analyzed for the presence of the ganglioside GM2 using three specific monoclonal antibodies which can distinguish the molecular species of the sialic acid moiety: the antibody MK1-16 is specific for N-acetyl GM2, MK2-34 is specific for N-glycolyl GM2, and MK1-17 detects both N-acetyl and N-glycolyl GM2. When the occurrence of the GM2 antigen was tested in 107 cases of human germ cell tumors by the immunohistochemical technique using these antibodies, seminoma was characterized as having the highest frequency of N-acetyl GM2 (89.4%, 42 of 47 cases) among germ cell tumors, followed by embryonal carcinoma (40.0%), and teratocarcinoma (26.6%). Compared with this, yolk sac tumors and choriocarcinoma had a much lower positive incidence of the N-acetyl GM2 antigen. On the other hand, the N-glycolyl GM2 antigen was not found at all in 47 cases of seminoma (0%), and the positive incidence was very low in embryonal carcinoma (6.6%), although considerably higher incidences were obtained with choriocarcinoma (25.0%), yolk sac tumor (22.2%), and teratocarcinoma (13.3%). The presence and molecular species of the GM2 antigens in these human germ cell tumors were also ascertained chemically by the thin-layer chromatography (TLC) immunostaining of the ganglioside fractions prepared from primary germ cell tumors. These results indicate that seminoma specifically contains N-acetyl GM2 and no N-glycolyl GM2, suggesting that N-acetyl GM2 could be a good marker for seminoma. On the other hand, non-seminomatous germ cell tumors were characterized by the presence of N-glycolyl GM2, one of the Hanganutziu-Deicher antigens (H-D antigens). Moreover, the positive occurrence of N-glycolyl GM2 correlated very well with the degree of differentiation of non-seminomatous germ cell tumors, i.e., the differentiated tumors such as yolk sac tumors, choriocarcinoma, and teratocarcinoma had a higher positive incidence of N-glycolyl GM2 type H-D antigen but a lower positive incidence of N-acetyl GM2 when compared with embryonal carcinoma, the most undifferentiated tumors among non-seminomatous germ cell tumors.

Flow cytometry measurement of nuclear RNA content in uveal melanoma.

We retrospectively studied the nuclear RNA content in uveal melanomas with known, long-term follow-up. Thirty patients had spindle cell melanomas, 25 had mixed cell tumors, and 9 had epithelioid cell neoplasms. Epithelioid cell types had a higher nuclear RNA content compared to mixed or spindle cell types (p less than 0.05). Using the Cox proportional hazards model, the nuclear RNA content appeared to be an independent prognostic indicator in uveal melanomas, and an increased nuclear RNA content was associated with a worse prognosis (p = 0.001).

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.

Molecular markers of primitive neuroectodermal tumors and other pediatric central nervous system tumors. Monoclonal antibodies to neuronal and glial antigens distinguish subsets of primitive neuroectodermal tumors.

Seventy-one tumors of the central nervous system in children were studied immunohistologically. Thirty-seven were classified histologically as PNETs, of which 35 were located in the cerebellum (medulloblastomas), one in the cerebrum, and one in the spinal cord. The 34 non-PNETs included five ependymomas, seven gangliogliomas, 15 astrocytomas, and seven tumors of other histology. We used monoclonal antibodies specific for neurofilament (NF) triplet proteins, for microtubule associated protein 2 and tau protein and for glial fibrillary acidic protein (GFAP) and myelin basic protein. In addition, a monoclonal antibody to epithelial membrane antigen was applied. The presence or absence of these antigens defined four major groups of PNETs: 1) PNETs not otherwise specified (10 cases), 2) PNETs with neuronal differentiation (eight cases), 3) PNETs with astrocytic differentiation (six cases), and 4) PNETs with both neuronal and astrocytic differentiation (12 cases). One case showed ependymal differentiation. The pattern of expression of NF isoforms in PNETs was reminiscent of that seen during normal mammalian development, such that phosphorylated NF-H was only present in combination with NF-M and NF-L. Among the other central nervous system tumors, all astrocytomas and gangliogliomas were positive for GFAP, and the gangliogliomas also expressed all NF isoforms. Three atypical teratoid tumors and two rhabdoid tumors showed strong positivity for epithelial membrane antigen and also for GFAP. We conclude that the differentiation antigens described here serve to distinguish PNETs from other pediatric central nervous system tumors and to identify subsets of PNETs. Accordingly, PNETs represent a heterogeneous group of pediatric brain tumors capable of neuronal and glial differentiation.

Isolation and characterization of a multipotent clone of human embryonal carcinoma cells.

Histopathological studies suggest that the stem cells of human teratomas may be classified into two major categories: nullipotent stem cells, and multipotent stem cells, capable both of self-renewal and differentiation into a wide range of somatic and extraembryonic cell types. We have isolated a multipotent stem cell clone from the human teratoma cell line GCT 27, and compared its properties to a nullipotent clone derived from the same strain. The multipotent clone GCT 27 X-1 gave rise to colonies of mixed cell morphology in vitro. Analysis of cell surface, cytostructural and extracellular matrix markers in GCT 27 X-1 cells showed that the stem cells of this line were very similar in phenotype to nullipotent cells. The two cell clones were predominantly hypotriploid, and contained several marker chromosomes in common. GCT 27 X-1 was feeder-cell-dependent for continuous growth in vitro; removal of the feeder layer resulted in differentiation of the stem cells into a variety of cell types, some with characteristics of extraembryonic endoderm, others showing neuronal properties. When transplanted into nude mice, GCT 27 X-1 cells gave rise to teratocarcinomas containing embryonal carcinoma stem cells, and many other cell types: yolk sac carcinoma cells; cells producing alphafetoprotein or human chorionic gonadotrophin; glandular, columnar, cuboidal, and squamous epithelium; primitive mesenchyme and cartilage; neuroectodermal cells. Nullipotent GCT 27 C-1 cells could form colonies in the absence of feeder layers, but multipotent GCT 27 X-1 cells could not. While a range of known growth factors and related substances failed to substitute for feeder layers in supporting the growth of GCT 27 X-1 stem cells, supernatants from yolk sac carcinoma cell line GCT 44 could partially replace the feeder cell requirement. Thus, the results revealed a basic difference in growth control between these multipotent and nullipotent human embryonal carcinoma cells, and suggested a possible paracrine regulatory pathway between multipotent stem cells and yolk sac carcinoma cells.

Mixed tumors of the skin. A histological and immunohistochemical study.

Sixty-four specimens of mixed tumors of the skin were studied by conventional microscopy. Sections from all 64 specimens were stained by hematoxylin and eosin, and sections from 18 of those specimens were stained by immunoperoxidase techniques for the presence of S-100 protein, carcino-embryonic antigen (CEA), keratin, actin, vimentin, epithelial membrane antigen (EMA), and gross cystic disease fluid protein-15 (GCDFP-15). Two distinctive histopathological patterns of mixed tumors of the skin became apparent, namely, apocrine and eccrine. Mixed tumors with apocrine features are by far the most common. Immunoperoxidase techniques, in our experience, do not enable differentiation between apocrine and eccrine types of mixed tumors.

Expression of c-myb in embryonal carcinoma cells and embryonal stem cells.

Mouse c-myb has been implicated in the regulation of differentiation and proliferation of haematopoietic cells. Analysis of the chromatin structure of the promoter region of c-myb in embryonal carcinoma (EC) cells and embryonal stem (ES) cells reveals a DNAse I-hypersensitive site coincident with a site found in c-myb-expressing haematopoietic cells, but absent in murine fibroblasts (which do not express c-myb). EC and ES cells were found to express c-myb mRNA, albeit at a level lower than found in haematopoietic cells. Differentiation of ES cells into embryoid bodies resulted in an elevated level of c-myb expression.

The expression of intermediate filaments in canine mammary glands and their tumors.

Monoclonal antibodies specific for different types of intermediate filaments (cytokeratin, vimentin, desmin and neurofilaments) were used to study the histogenesis of canine mammary glands and 57 canine mammary tumors by immunocytochemistry. The intra- and interlobular duct epithelium, acinar, and intralobular myoepithelial cells stained positively for cytokeratin. Peripheral ductal and acinar cells, as well as interstitial cells, stained positively for vimentin. A similar staining pattern was seen in adenomas, complex adenomas, benign mixed tumors, ductular carcinomas, and one myoepithelioma-like tumor. Additionally, cytokeratin positive cells were scattered interstitially in one single adenoma, most complex adenomas, some benign mixed tumors, complex carcinomas, and in the malignant mixed tumors. All stromal cells stained positively for vimentin. The fibrosarcomas were positive only for vimentin, while the following expressed both desmin and cytokeratin: epithelial-like cells in one adenoma, three complex adenomas, the myoepithelioma-like tumor, the single comedo carcinoma, two complex carcinomas, the single lobular carcinoma, one malignant mixed tumor, and three osteosarcomas. Epithelial-like cells in one adenoma, six complex adenomas, two benign mixed tumors, two complex carcinomas, the lobular carcinoma, and the malignant schwannoma stained for neurofilaments. Three tumors, one adenoma, one complex adenoma, and the lobular carcinoma expressed both desmin and neurofilaments in addition to cytokeratin and vimentin. The results show the expression of different types of intermediate filaments and indicate that there might be a stem cell origin in most of the canine mammary tumors.

[Malignant mesodermal mixed tumor of the bladder: report of a case].

An 81-year-old woman was admitted to our clinic due to gross hematuria. A large bulky pedunculated mass was found in the bladder by cystoscopic examination. Subtotal cystectomy and bilateral cutaneostomy was performed on January 12, 1987. Histologically the tumor was composed of carcinomatous and sarcomatous elements. The carcinomatous element was composed fundamentally of grade 2, transitional cell carcinoma with numerous foci of squamous metaplasia. The sarcomatous element was composed of myxosarcomatous, chondro-sarcomatous pattern and non-differentiated malignant spindle cell component. Immunohistochemical examination demonstrated the presence of cytokeratin and epithelial membrane antigen in the spindle cell and more obvious carcinomatous regions, using the avidin-biotin conjugated immunoperoxidase technique. The patient died 3 months after operation. Autopsy findings showed multiple organ metastasis which were composed of carcinomatous and sarcomatous elements.

Immunocytochemical and ultrastructural studies of the histogenesis of Ewing's sarcoma and putatively related tumors.

The histogenesis of Ewing's sarcoma (EW) and extraskeletal Ewing's sarcoma (EEW) is still disputable. Their relationship to the so-called Askin's tumor, neuroectodermal tumor of bone, and peripheral neuroblastoma remains to be established. In an attempt to clarify these points, immunocytochemical and ultrastructural studies were done on tissues from 14 cases of EW, 4 cases of EEW, and 9 cases of primitive neuroectodermal tumor (PNET) and compared with neuroblastoma and olfactory neuroblastoma. Six tumors categorized initially as EW and EEW on biopsy, turned out to be PNET by extensive histologic and/or ultrastructural observations. Abundant glycogen was recognized not only in 16 of 18 cases of EW and EEW, but also in seven of nine cases of PNET. Fine fibrillar cell processes were seen between tumor cells, at least in limited areas even in cases of EW and EEW. Immunocytochemically, neuron-specific enolase (NSE), neuroblastoma cell surface antigen (NBCA), neuron cell surface antigen (NCSA), and neurofilament (NF) were demonstrated not only in neuroblastoma, but also frequently in cases of EW, EEW, and PNET. The results seem to suggest that EW and EEW represent the most immature forms of neuroectodermal tumor. Electron microscopic study showed predominantly primitive cells with occasional areas of cell processes, neurosecretory granules, and microtubules, suggesting a neuroectodermal origin.

[Clinicopathologic and immunohistochemical study on 8 cases of malignant mixed müllerian tumor].

The clinico-pathological features of eight cases of malignant mixed müllerian tumor, a rare neoplasm composed of an admixture of epithelial and stromal elements, are reported. Four of the tumors located in the endometrium, three in the cervix and one in the ovary. Microscopically, the epithelial elements ranged from poorly to well differentiated adenocarcinoma. Homologous stromal sarcoma cells were present in six tumors and heterologous elements were also seen in the other two tumors. Immunohistochemical studies showed diffuse cytoplasmic staining of keratin in the epithelial elements of all the eight cases. Sarcomatous cells were positive focally for keratin in four spindle cell sarcoma cases. Desmin immunoreaction was moderately positive in the sarcomatous element of four neoplasms. Follow-up result was available in 7 patients of whom 3 survived and 4 died from recurrence or metastasis. Immunohistochemical findings support the stem cell origin of this tumor.

Solid teratomas and mixed müllerian tumors of the ovary: a clinical, histological, and immunocytochemical comparative study.

The differential diagnosis between solid teratomas and malignant mixed Müllerian tumors (MMTs) can be difficult, because both tumours have an epithelial as well as a mesenchymal component. Sometimes an MMT is incorrectly diagnosed as a solid teratoma. This has prognostic and therapeutic consequences, because MMTs have a worse prognosis. In this study 20 solid ovarian teratomas and 15 MMTs of the ovary have been compared according to their clinical, histomorphological, and immunocytochemical characteristics. Teratomas occur in the younger age group and are characterized by the presence of argyrophil cells which are immunoreactive to a wide range of neurohormonal peptides and are in addition characterized by the presence of GFAP immunoreactive tissues (glial fibrillar acidic protein). MMTs occur in the older age group and are characterized by the presence of sarcomatous elements and by the absence of an organoid arrangement, neuroectoderm, skin, argyrophilia, and immunoreactivity to GFAP and neurofilament.

[Gonadotropin, as a tumor marker, in body fluid and tumor tissues of germ cell tumors].

The value of gonadotropin in the body fluids of germ cell tumor patients is its usefulness as a tumor marker. It is also used for differential diagnosis and/or judgement of therapeutic effects. In order to clarify the most effective value of gonadotropin as a tumor marker in the body fluids, we compared the value in serum, liquor and urine with one another. The liquor contained highest (1650 IU/l) value of gonadotropin in the primary intracranial germ cell tumors, mostly in choriocarcinoma. But the gonadotropin value was highest (3050 IU/l) in the serum of secondary intracranial choriocarcinoma. Chiasmal germ cell tumor, except choriocarcinoma, which does frequently secrete gonadotropin (alpha, beta) showed moderate or very high values in the liquor. However, pineal germ cell tumors rarely secrete gonadotropin and sometimes mild high value are obtained in the serum without gonadotropin secretion immunohistochemically. In such cases, the gonadotropin may be increased by indirect mechanism of gonadotropin-secretion following pineal disorder. In most of such cases, the gonadotropin was not human chorionic gonadotropin (HCG) but lutein hormone (LH). Because alpha-subunit of such gonadotropin has the same structure, their antibodies show immunologic cross reaction. So, a count of beta-subunit gonadotropin in the serum or liquor is the best way for differential diagnosis or judgement of therapeutic effects. From our results, it is considered that the tumor secretes HCG if the serum beta-HCG value was higher than 30 IU/l, and that it doesn't secrete HCG if beta-HCG value was lower than 10 IU/l or non calculable. The mild increased HCG may be caused by hypothalamo-diencephalic disorder such as pineal tumor.(ABSTRACT TRUNCATED AT 250 WORDS)

Lectin histochemistry of human testicular germ cell tumors.

The lectin binding pattern (WGA, UEA-I, PNA, PSA, Con A, RCA and LCA) of 28 human testicular germ cell tumors (pure or combination tumors) was investigated. Lectin binding sites could be demonstrated in all germ cell tumor types. In classic and spermatocytic seminomas as well as seminomas with high mitotic index an equal distribution of lectin binding sites was observed. In embryonal carcinomas the lectin binding of UEA-I, PNA, WGA, LCA and RCA correlated to histological differentiation. A polarized staining of WGA, PNA, RCA and UEA-I, typical for embryonal carcinomas, yolk sac tumors and teratomas, was never seen in seminomatous tumors and may be of importance in differential diagnosis. By means of Con A decoration it was possible to distinguish cytotrophoblastic and syncytiotrophoblastic differentiation. Aspects of lectin histochemistry in tumor biology in general and in differential diagnosis of germ cell tumors in particular are discussed.

Immunohistochemical demonstration of pancreatic secretory trypsin inhibitor in gynecologic tumors.

Pancreatic secretory trypsin inhibitor (PSTI) is a specific trypsin inhibitor secreted by the acinar cells of the pancreas. Serum levels of immunoreactive PSTI have been reported elevated in patients with various malignancies including gynecologic tumors. The immunohistochemical localization of PSTI is studied in the comparison with argyrophilia and amylase immunoreactivity on 100 ovarian tumors, 35 endometrial carcinomas, and 34 cervical carcinomas. PSTI was noted in 19 of 85 common epithelial tumors of the ovary, being most frequently in mucinous tumors and less in endometrioid, but only occurred in immature pancreatic tissue of 1 of 15 germ cell tumors tested. In the uterus, 5 of 19 adenocarcinomas of the endometrium and 3 of 10 adenocarcinomas of the cervix were positive for PSTI immunoreactivity. PSTI was found more frequently in the tumors with argyrophil cells, especially of type I, but was related to the intestinal metaplasia and exocrine secretion rather than argyrophilia itself, judging from the different localization of PSTI and argyrophilic granules. No relationship was observed between amylase and PSTI immunoreactivity.

Detection of AFP and HCG in metastatic testicular cancer after treatment with chemotherapy or radiation therapy.

We used an indirect immunoperoxidase technique to detect alpha-fetoprotein (AFP) and human chorionic gonadotropin (HCG) in tissue sections of nine metastatic germ cell tumors excised after treatment with chemotherapy or radiation therapy, and correlated the results with the serum levels of AFP and HCG. In all but 1 case yolk sac tumor (YST) was the only histologic type that reacted for AFP (AFP+) and syncytiotrophoblasts (STB) were the only histologic type that reacted for HCG (HCG+). Among 5 cases with normalization of the serum AFP before surgery, 3 were associated with YST-/AFP-, 1 with YST+/AFP+, and 1 with YST+/AFP- metastases; and among 4 cases with normalization of the serum HCG all were associated with STB-/HCG- metastases. Among 3 cases with persistent elevation of the serum AFP, 1 was associated with YST+/AFP+, 1 with YST+/AFP-, and 1 with YST-/AFP- metastases; and of 2 cases with persistent elevation of the serum HCG, 1 was associated with STB-/HCG- and 1 with STB+/HCG+ metastases. These data suggest that marker normalization in the face of persistent tumor results primarily from eradication of YST and STB, but also from treatment-induced inhibition of AFP and HCG synthesis or secretion.