Two-dimensional mapping by the high-performance liquid chromatography of oligosaccharides released from glycosphingolipids by endoglycoceramidase.

A two-dimensional sugar mapping method has been developed by which sensitive, reproducible, and simple analysis can be carried out on the structures and compositions of oligosaccharides released from glycosphingolipids by endoglycoceramidase. The oligosaccharides were labeled quantitatively with an ultraviolet-absorbing compound, p-aminobenzoic acid ethyl ester (ABEE). The ABEE-oligosaccharides were separated first on an amide-silica column and then on a C4-silica column by high-performance liquid chromatography. The acidic ABEE-oligosaccharides were eluted as a group at the start of the chromatography while the neutral ABEE-oligosaccharides were separated according to size and structure on an amide-silica column using an eluent without salt. The acidic oligosaccharides were separated according to size and structure when rechromatographed on the same column using an eluent containing KH2PO4. NeuAc-containing ABEE-oligosaccharides were extensively separated from the corresponding NeuGc derivatives. The ABEE-oligosaccharides separated on an amide-silica column were then chromatographed on a column of C4-silica on which lactotriose and neolacto-series oligosaccharides were clearly shown to be separated from the others. On the basis of the retention times of the individual ABEE-oligosaccharides on two separate columns, 9 neutral and 15 acidic oligosaccharides derived from glycosphingolipid standards were two-dimensionally mapped without overlapping. The gangliosides of a human chondrosarcoma tissue and glycosphingolipids of tumor tissue of FBJ virus-transformed murine osteosarcoma cells were analyzed by this method in conjunction with exoglycosidase treatment. At least 11 species of glycosphingolipids were identified in both cases.

Immunohistochemical analysis of bone morphogenetic protein (BMP) in osteosarcoma.

The monoclonal antibody against bovine bone morphogenetic protein (bBMP-McAb) was first used for demonstration of bone morphogenetic protein (BMP) in osteosarcoma. The avidin-biotin complex method (ABC) demonstrated that of the 18 osteosarcomas, 15 stained positive, while all 6 fibrosarcomas were negative. The results showed that BMP mainly exists in the tumor cell plasma and some tumor-like bone tissues. Using this staining method, we can not only differentiate osteosarcoma from fibrosarcoma and other non-bone-derived tumors, but also classify osteosarcoma according to the content and distribution of BMP and the patient's clinical situation, thus providing a scientific basis for clinical treatment.

Identification of the monocyte chemotactic protein from human osteosarcoma cells and monocytes: detection of a novel N-terminally processed form.

The chemotactic activity for monocytes in culture supernatants from double-stranded RNA-stimulated human MG-63 osteosarcoma cells and from LPS-stimulated human monocytes was purified to homogeneity and characterized by amino acid sequence analysis. The chemotactic protein derived from the fibroblastoid osteosarcoma cells had a blocked N-terminus but sequencing of tryptic fragments showed that it was identical with a recently identified monocyte chemoattractant designated MCP-1 or MCAF isolated from glioma or myelomonocytic cells, respectively. Preparations of monocyte -derived chemotactic activity appeared to contain not only the blocked protein, but also a novel N-terminally processed form of this molecule, lacking 5 amino acid residues.

Metal-induced sarcoma. A case report and literature review.

The overall biocompatibility characteristics of metallic implants are important considerations in orthopedic surgery. A review of the literature shows very few reports of neoplasms in association with metallic implants. This case report demonstrates osteogenic sarcoma at the site of a Smith-Petersen nail that had been implanted for nine years in a 65-year-old woman for fixation of a femoral neck fracture. Gross examination revealed debris at the tumor site, with a concentration of 14 ppm of nickel within the tumor tissue. Experimental investigations support the possibility of neoplastic induction by heavy metals, particularly cobalt, cadmium, and nickel. Circumstantial evidence shows osteogenic sarcoma that developed at the site of this device, possibly in response to metal or factors at the site of metal.

[The membrane sialoglycolipids of tumor cells and the metastasis of bone tumors]. / Sialoglikolipidy membran opukholevykh kletok i metastazirovanie opukholei kostei.

Level and profile of gangliosides were studied in osteogenic and chondrosarcoma cells. Level of lipid-binding sialic acids in bone- and cartilage-producing tumors proved different. Most osteogenic sarcoma samples showed higher level of lipid-binding sialic acids as compared to chondrosarcoma. In the latter tumor, level of lipid-binding sialic acids was related to grade of tumor cell differentiation, peak levels being observed in undifferentiated neoplasms as compared to those showing grade I-II cell anaplasia. Chondro- and osteogenic sarcoma revealed different profiles of sialoglycolipids, particularly, due to markedly reduced set of gangliosides and nearly complete loss of polysialogangliosides in the latter tumor.

Monoclonal antibody to human osteosarcoma: a novel Mr 26,000 protein recognized by murine hybridoma TMMR-2.

Three murine hybridomas (TMMR-1-3) were developed by repeated immunizations of mice with four different human osteosarcoma cell lines in an alternating sequence of inoculations. The monoclonal antibodies were screened for reactivities to cultured cell lines and tissue sections of osteosarcomas using flow cytometry and immunohistochemical techniques. TMMR-2 is a highly specific antibody (IgG1) that reacted with all 14 osteosarcoma tumors and eight human osteosarcoma cell lines tested, including the established human osteosarcoma cell lines HOS and Saos-2. Benign neoplastic cells from two osteoblastomas, osteoblasts from regions of reparative osteoid formation and neonatal new bone, are also reactive with TMMR-2. TMMR-1 has mesenchymal specificity while TMMR-3, although reactive with osseous differentiated cells, also reacted with mitotic cells of all cell types. Characterization of antigen structure by Western immunoblotting revealed that TMMR-2 reacted with a 100 degrees C heat labile mercaptoethanol-sensitive Mr 26,000 protein, and TMMR-3 recognized a mercaptoethanol-resistant Mr 97,000 protein whereas TMMR-1 reacted with a series of bands from 65,000 to 85,000 molecular weight, all of which were mercaptoethanol sensitive. TMMR-1 and TMMR-2 monoclonal antibodies showed complement-independent inhibition of [3H]thymidine incorporation into DNA, but did not exhibit cytotoxic activity. The results suggest that TMMR-2 is a specific antibody that recognizes an osteoblast/osteocyte surface antigen present in normal, reactive, and neoplastic disorders of bone. The inhibitory effects on DNA synthesis in cultured osteosarcoma cells by TMMR-2 indicate an important cell growth/proliferation role of this surface antigen. These monoclonal antibodies, in combination with other known antibodies, can be used to characterize mesenchymal cell surface antigenic structure and differentiation.

[Breast carcinoma with features of osteosarcoma--a light and electron microscopic and immunohistochemical study].

Reported is the case history of a 58-year-old woman with a lump in the left breast. The tumor was composed of two nodules, sharing a common portion. Grossly, one was an irregular hard tumor that was grayish in color, and the other was a well circumscribed cystic tumor that showed necrosis and a hemorrhage that had filled it with a reddish soft mass. Light microscopically, combined features of a scirrhous carcinoma and of an osteosarcoma were observed in each nodule, but the common portion was consistent with a metaplastic carcinoma, with cancer cells and sarcoma-like cells closely mingled. An ultrastructural study showed that the sarcoma-like cells were composed of polymorphic cells that resembled osteoblasts, myofibroblasts, osteoclasts, histiocytes, and undifferentiated tumor cells. Immunohistochemically, vimentin and alpha 1-antitrypsin in the sarcoma-like cells were positive, suggesting these cells were of a mesenchymal rather than of an epithelial origin.

Primary osteosarcoma of the lung. A reappraisal following immunohistologic study.

Five neoplasms were initially believed to represent primary osteosarcomas of the lung. Of the five cases, two were reinterpreted as carcino-sarcomas based on the identification of epithelial elements on further histologic sectioning and immunohistologic study in one and on immunohistologic study alone in the second. The differential diagnosis of carcinosarcoma should be considered for any lesion believed to represent a primary sarcoma of the lung, and in some cases carcinomatous elements may only be demonstrable by immunohistologic means.

Histochemical detection of osteocalcin in normal and pathological human bone.

We investigated the immunohistochemical localization of osteocalcin in demineralized, paraffin-embedded normal and pathological human bone. Acid decalcification protocols appeared to be more suitable for osteocalcin detection than mild chelating agents. In normal lamellar bone, osteocalcin was detected in osteocytes and along the lamellar bone matrix in fine granular deposits. Under pathological conditions (osteomyelitis, neoplasia), appositional bone showed immunoreactivity in osteoblasts and osteocytes but not in the provisory woven bone matrix. Intense immunoreactivity could be seen at the cell borders of osteoclasts and the bone margins of Howship lacunae. In primary bone-forming tumors, osteocalcin immunoreactivity was detected in osteoblasts and their malignant counterparts. On the basis of these results, we conclude that optimal preservation of osteocalcin is obtained through mild acid decalcifiers. Osteocalcin is deposited in bone matrix, especially that of metabolically inactive bone. In neoplasms, osteocalcin could be a marker of osteoblastic differentiation.

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.

Estrogen receptors and human bone cells: immunocytochemical studies.

In this immunocytochemical study we have probed a number of human bone cell types and bone preparations for the presence of the estrogen receptor (ER) with two distinct monoclonal antibodies. Using a well-validated antibody (H222) that recognizes human ER and standard peroxidase-antiperoxidase methodology, we were unable to demonstrate nuclear staining for ER in cultured primary or transformed human bone-derived cells or in fetal bone sections. Attempts to visualize ER in osteosarcoma cell lines (TE85C and HTB96) using a silver enhancement procedure were also unsuccessful. Additionally, we failed to detect immunocytochemical staining for the progesterone receptor (using monoclonal antibody mPR1) in control or estrogen-treated human bone cell cultures. Estrogen and progesterone receptor staining was readily detectable in MCF7 human breast cancer cells. In contrast, with a monoclonal antibody that recognizes a 29 kDa cytoplasmic component (p29) closely related to human ER, we observed specific staining in all the osteoblastlike cells studied. Cytoplasmic staining for this p29 antigen was most intense in primary cultures of human bone-derived cells. It is possible that the relatively abundant but as yet undefined p29 antigen may act as a sensitive marker for the presence of ER in cells at levels below the detection limit of the anti-ER monoclonal antibody. If so, our results are consistent with the presence of ER in osteoblastlike cells at very low concentrations.

Prognostication including DNA analysis in osteosarcoma.

In a retrospective study of 83 osteosarcoma patients treated by surgery and adjuvant interferon from 1971 to 1986, the clinical course was related to different clinicopathologic features and tumor DNA content. DNA analysis was feasible in 60 cases. Four tumors were diploid and 56 hyperploid. The 7-year survival rate, estimated by life-table analysis, was 0.44 for the whole series. Multivariate analysis disclosed that male sex, proximal tumor location, and histologic Grade IV were independent risk factors--all approximately of equal strength. DNA analysis did not provide prognostic information, except for tumors with extreme abnormality of the DNA content, which was associated with a very poor prognosis. A prognostication model was created, based on the number of risk factors present. The 7-year survival rate for patients with none, one, two, or three risk factors was 0.80, 0.59, 0.42, and 0.13, respectively. The estimated 7-year rate of local recurrence was 0.29: 0.07 after ablative surgery and 0.54 after local surgery. Among patients who were free of metastasis 1 year after diagnosis, local recurrence reduced the 7-year survival rate from 0.86 to 0.48. In high-grade osteosarcoma, conventional clinicopathologic features and local tumor control remain the most important prognostic factors.

Dedifferentiated parosteal osteosarcoma of the femur with aneuploidy and lung metastases.

In this report, the pathologic findings and the results of cellular DNA measurements of a tumor that on first presentation seemed to be a classical parosteal osteosarcoma are described. After resection 8 months later, part of the tumor appeared to display highly malignant features. DNA flow cytometry of this part of the tumor showed an aneuploid cell population. The aggressive nature of the tumor was confirmed by the development of lung metastases approximately 1 year after resection of the primary tumor.

Immunohistochemical localization of type II collagen in cartilage-forming tumors.

The presence and distribution of type II collagen was studied in 36 cartilage and cartilage-related tumors, including five osteosarcomas and one chordoma. A monoclonal antibody prepared from chicken type II collagen was used with paraffin sections, employing the ABC (avidin biotinylated horseradish peroxidase complex) peroxidase technique. Fetal cartilage and fracture callus were used as control materials. Type II collagen was present in the matrix of all the cartilage tumors. The reaction was strongest in areas of well-differentiated cartilage and weakest in the poorly differentiated tissue of high-grade chondrosarcomas. Areas of mineralization or ossification, and areas of eosinophilic, fibrous, or degenerated cartilage gave a negative reaction.

[Steroid hormone receptors in osteogenic sarcomas]. / Issledovanie retseptorov steroidnykh gormonov v osteogennykh sarkomakh.

The method of hormone-receptor complex precipitation with protamine sulfate was used in 57 males suffering osteogenic sarcoma to identify and evaluate cytoplasmic androgen (AR) and estrogen receptor (ER) levels versus age and prior treatment as well as to assess their prognostic significance. AR were most often observed in younger patients and those pretreated with chemo- and radiotherapy whereas ER mostly occurred in older ones. Presence of AR (in untreated tumor or following preoperative therapy) adversely influenced prognosis. It took AR-positive tumors less time to disseminate to the lung after the start of treatment. The use of antiandrogen drugs for osteogenic sarcoma treatment is discussed.

[The relationship between the prostaglandin E level in the tumor and the times of the metastasis of primary osteogenic sarcoma]. / Sviaz' mezhdu urovnem prostaglandinov E v opukholi i srokami metastazirovaniia pervichnoi osteogennoi sarkomy.

Radioimmunoassay was used in 46 cases of osteogenic sarcoma to assess prostaglandin E (PgE) levels in tumor tissue. Those levels were found to vary with age. A correlation was established between the effect of preoperative chemoradiation treatment, on the one hand, and degree of treatment-induced pathomorphosis and PgE concentration in tumor, on the other. High PgE level in osteogenic sarcoma tissue proved prognostically unfavorable whatever age and prior chemoradiation treatment and were associated with shorter metastasis-free survival.

[A study of an immunosuppressive acidic protein (IAP) in patients with a bone tumor].

We evaluated an immunosuppressive acidic protein (IAP) as a tumor marker in cases of a tumor of the bone (benign, 21; primary malignant, 26; metastatic carcinoma, 12). The IAP positive rate of a primary malignant tumor of the bone was 60%, and the mean was 591 + 59.4 micrograms/ml. This rate of a benign tumor of the bone was 10%, and the mean was 382 +/- 31.3 micrograms/ml. IAP may represent a tumor marker in malignant tumors of the bone.

DNA analysis in the differential diagnosis of osteosarcoma.

The DNA content of osteosarcomas, and of giant cell tumors, osteoblastomas, aneurysmal bone cysts, and fibrous dysplasias was determined by cytophotometry. Of 158 tumors, 141 were histologically noncontroversial, whereas 17 posed diagnostic difficulties. In the noncontroversial group, all 41 benign tumors had a diploid (normal) DNA content. Ninety-two of 96 high-grade osteosarcomas were hyperploid (increased DNA content). The four analyzed low-grade parosteal osteosarcomas were diploid. Among 17 diagnostically controversial cases, nine were primarily diagnosed and treated as benign. Three of these patients, nevertheless, exhibited a malignant clinical course and two had local recurrence. All five proved to have hyperploid tumors. The four nonrecurrent lesions were diploid. Of eight patients primarily evaluated as malignant, one died and two developed local recurrence. These three patients had hyperploid tumors. Among the five nonrecurrent lesions, two were hyperploid and three diploid. In the diagnostically controversial group, recurrence or death was consistently related to hyperploidy. The present study shows that the vast majority of high-grade osteosarcomas are hyperploid. Benign bone tumors, which may be mixed up histologically with osteosarcoma, are diploid. Routine DNA analysis of primary bone tumors, as an adjunct to histopathologic assessment, can be employed to obtain diagnostic confirmation. In cases presenting histopathologic difficulties, ploidy determination may provide decisive diagnostic information.