Interaction between CD44 and hyaluronate is directly implicated in the regulation of tumor development.

CD44 is implicated in the regulation of tumor growth and metastasis but the mechanism by which expression of different CD44 isoforms determines the rate of primary and secondary tumor growth remains unclear. In the present study we use a human melanoma transfected with wild-type and mutant forms of CD44 to determine which functional property of the CD44 molecule is critical in influencing tumor behavior. We show that expression of a wild-type CD44 isoform that binds hyaluronic acid augments the rapidity of tumor formation by melanoma cells in vivo, whereas expression of a CD44 mutant, which does not mediate cell attachment to hyaluronate, fails to do so. The importance of CD44-hyaluronate interaction in tumor development is underscored by the differential inhibitory effect of soluble wild-type and mutant CD44-Ig fusion proteins on melanoma growth in vivo. Whereas local administration of a mutant, nonhyaluronate binding, CD44-Ig fusion protein has no effect on subcutaneous melanoma growth in mice, infusion of wild-type CD44-Ig is shown to block tumor development. Taken together, these observations suggest that the tumor growth promoting property of CD44 is largely dependent on its ability to mediate cell attachment to hyaluronate.

Glycosylation of CD44 is implicated in CD44-mediated cell adhesion to hyaluronan.

CD44-mediated cell adhesion to hyaluronate is controlled by mechanisms which are poorly understood. In the present work we examine the role of N-linked glycosylation and Ser-Gly motifs in regulating CD44-hyaluronate interaction. Our results show that treatment of a panel of human cell lines which constitutively express CD44 with the inhibitor of N-linked glycosylation tunicamycin results in the loss of attachment of these cells to hyaluronate-coated substrate. In contrast, treatment of the same cells with deoxymannojirimycin, which inhibits the conversion of high mannose oligosaccharides to complex N-linked carbohydrates, results in either no change or an increase in CD44-mediated adhesion to hyaluronate, suggesting that complex N-linked oligosaccharides may not be required for and may even inhibit CD44-HA interaction. Using human melanoma cells stably transfected with CD44 N-linked glycosylation site-specific mutants, we show that integrity of five potential N-linked glycosylation sites within the hyaluronate recognition domain of CD44 is critical for hyaluronate binding. Mutation of any one of these potential N-linked glycosylation sites abrogates CD44-mediated melanoma cell attachment to hyaluronate-coated surfaces, suggesting that all five sites are necessary to maintain the HA-recognition domain in the appropriate conformation. We also demonstrate that mutation of serine residues which constitute the four Ser-Gly motifs in the membrane proximal domain, and provide potential sites for glycosaminoglycan side chain attachment, impairs hyaluronate binding. Taken together, these observations indicate that changes in glycosylation of CD44 can have profound effects on its interaction with hyaluronic acid and suggest that glycosylation may provide an important regulatory mechanism of CD44 function.

Regulation of CD44 binding to hyaluronan by glycosylation of variably spliced exons.

The hyaluronan (HA)-binding function (lectin function) of the leukocyte homing receptor, CD44, is tightly regulated. Herein we address possible mechanisms that regulate CD44 isoform-specific HA binding. Binding studies with melanoma transfectants expressing CD44H, CD44E, or with soluble immunoglobulin fusions of CD44H and CD44E (CD44H-Rg, CD44E-Rg) showed that although both CD44 isoforms can bind HA, CD44H binds HA more efficiently than CD44E. Using CD44-Rg fusion proteins we show that the variably spliced exons in CD44E, V8-V10, specifically reduce the lectin function of CD44, while replacement of V8-V10 by an ICAM-1 immunoglobulin domain restores binding to a level comparable to that of CD44H. Conversely, CD44 bound HA very weakly when exons V8-V10 were replaced with a CD34 mucin domain, which is heavily modified by O-linked glycans. Production of CD44E-Rg or incubation of CD44E-expressing transfectants in the presence of an O-linked glycosylation inhibitor restored HA binding to CD44H-Rg and to cell surface CD44H levels, respectively. We conclude that differential splicing provides a regulatory mechanism for CD44 lectin function and that this effect is due in part to O-linked carbohydrate moieties which are added to the Ser/Thr rich regions encoded by the variably spliced CD44 exons. Alternative splicing resulting in changes in protein glycosylation provide a novel mechanism for the regulation of lectin activity.

Expression of CAR-3 and TAG-72 macromolecules in normal and transformed endometrium: potential diagnostic application in postmenopausal patients.

Mass cytoscreening for the early diagnosis of endometrial carcinoma has thus far been hampered by the low diagnostic accuracy of current cytopathology. In this study we have analyzed the reactivity of the two monoclonal antibodies, AR-3 and B72.3, recognizing two distinct glycosylated high molecular weight carcinoma associated antigens on histological specimens from normal, hyperplastic, and transformed endometrium with the aim of establishing their diagnostic potential. Because women with a high risk of endometrial cancer are frequently postmenopausal, where normal endometrium is characterized by atrophy and cystic glandular hyperplasia, the following findings were of interest. Both antibodies reacted with variable and apical staining patterns with a minority of specimens of normal cycling endometrium from premenopausal women. However, they were constantly negative when tested on normal atrophic postmenopausal endometrium, and only monoclonal antibody AR-3 occasionally stained glandular cystic hyperplasia. By contrast, lesions with atypical hyperplasia, which represent a preneoplastic condition, were stained by both antibodies in 89 or 67% of the cases depending on the monoclonal antibody used (100% if used in combination). Furthermore, 98% of the endometrial carcinomas tested were found to react with the combination of two monoclonal antibodies. If these findings are confirmed in a multicentric study, the use of the two reagents could be a valuable adjunct in the cytodiagnosis of endometrial cancer, especially in providing a guideline to selecting patients for endometrial curettage and additional diagnostic procedures.

Increased expression of insulin-like growth factor I receptor in malignant cells expressing aberrant p53: functional impact.

We investigated the functional impact of p53 on insulin-like growth factor I receptor (IGF-IR) expression in malignant cells. Using the BL-41tsp53-2 cell line, a transfectant carrying temperature-sensitive (ts) p53 and endogenous mutant p53 (codon 248), we demonstrated a drastic down-regulation of plasma membrane-bound IGF-IRs on induction of wild-type p53. However, a similar response was obtained by treatment of BL-41tsp53-2 cells expressing mutant ts p53 with a p53 antisense oligonucleotide. Thus, even if the negative effect of wild-type p53 predominates under a competitive condition, these data indicate that mutant p53 may be important for up-regulation of IGF-IR. To further elucidate this issue, three melanoma cell lines (BE, SK-MEL-5, and SK-MEL-28) that overexpressed p53 were investigated. The BE cell line has a "hot spot" mutation (codon 248) and expresses only codon 248-mutant p53. SK-MEL-28 has a point mutation at codon 145. SK-MEL-5 cells did not exhibit any p53 mutations, but the absence of p21Waf1 expression suggested functionally aberrant p53. Our data suggest that interaction with Mdm-2 may underlie p53 inactivation in these cells. Using p53 antisense oligonucleotides, we demonstrated a substantial down-regulation of cell surface expression of IGF-IR proteins in all melanoma cell lines after 24 h. This was paralleled by decreased tyrosine phosphorylation of IGF-IR and growth arrest, and, subsequently, massive cell death was observed (this was also seen in BL-41tsp53-2 cells with mutant conformation of ts p53). Taken together, our results suggest that up-regulation of IGF-IR as a result of expression of aberrant p53 may be important for the growth and survival of malignant cells.

Galectin-3 and CD44v6 isoforms in the preoperative evaluation of thyroid nodules.

PURPOSE: Thyroid cancer is the most frequently occurring endocrine malignancy; however, preoperative diagnosis of some lesions, in particular those with follicular histology, is difficult, and a consistent number of not otherwise-specified "follicular nodules" are surgically resected more for diagnosis than therapeutic purposes. In this study, we investigated whether the lectin-related molecules CD44v6 and galectin-3, the expression of which is altered during deregulated cell growth and malignant transformation, could be potential markers for improving the diagnostic accuracy of conventional cytology. MATERIALS AND METHODS: A comparative immuno-chemical and molecular analysis was performed on 157 thyroid specimens representative of normal, benign, and malignant tissues, and on 36 cytologic samples obtained preoperatively by fine-needle aspiration biopsy from nonselected patients with palpable thyroid nodules. RESULTS: Normal thyrocytes did not express galectin-3 nor CD44v6. Although the expression of CD44v6 isnegligible in thyroiditis, these molecules are variably detected in benign and malignant proliferative lesions. Interestingly, galectin-3 is never expressed in benign lesions, but it is invariably detected in cancers. A comparative evaluation of CD44v6 and galectin-3 expression in thyroid malignancies demonstrated that these molecules are coexpressed at the messenger RNA and protein level in almost all lesions. CONCLUSION: Our findings suggest that CD44v6 and galectin-3 could be potential markers to preoperatively identify malignant transformed thyrocytes. Immunodetection of these molecules on cytologic specimens obtained by fine-needle aspiration biopsy is an accurate and improved method for selecting, on a molecular basis, those nodular lesions of the thyroid gland that need to be surgically resected.

Cloning and characterization of spliced fusion transcript variants of synovial sarcoma: SYT/SSX4, SYT/SSX4v, and SYT/SSX2v. Possible regulatory role of the fusion gene product in wild type SYT expression.

The synovial sarcoma translocation t(X;18)(p11.2; q11.2) results in the fusion of the SYT gene on chromosome 18 to exon 5 of either SSX1 or SSX2 genes on chromosome X. We recently reported that the SSX4 gene is also involved in such a translocation. In the present investigation we cloned and sequenced the full-length cDNA of SYT/SSX1, SYT/SSX2 and SYT/SSX4 from synovial sarcoma tissues. We isolated a novel fusion transcript type variant involving the fusion of SYT with exon 6 of the SSX4 gene (SYT/SSX4v). The SYT/SSX4 and SYT/SSX2 open reading frame also differed from previously reported SYT/SSX sequences by an in-frame addition of 93bp exon located in the junction between exon 7 and 8 of the SYT. This exon is identical to that reported for the murine SYT but has not been previously found in the human transcript. Two SYT transcripts, with and without the 93 bp exon, were co-expressed in mouse NIH3T3 cells, human malignant cells and human testis tissue, but not in human normal fibroblasts. Stable transfection of an SYT/SSX4 expression vector into human and murine cell lines correlated with a down-regulation of SYT transcripts. This was also observed in a synovial sarcoma tumor expressing SYT/SSX4. This suggests that the SYT/SSX fusion gene may regulate SYT expression from the normal allele and as such alter the normal function of SYT.

Association of HLA class I and class II antigen expression and mortality in uveal melanoma.

PURPOSE: Malignant transformation of cells is frequently associated with abnormalities in human leukocyte antigen (HLA) expression. These abnormalities may play a role in the clinical course of the disease, because HLA antigens mediate interactions of tumor cells with T cells and NK cells. Uveal melanoma is a highly malignant tumor of the eye and is characterized by a hematogenic spread to the liver. Little is known about the role of HLA expression in progression of this malignant disease. METHODS: In the present study HLA class I antigen, beta(2)-microglobulin (beta(2)-m), and HLA class II antigen expression was analyzed in primary uveal melanoma lesions by immunoperoxidase staining with monoclonal antibodies of 65 archival clinical samples. The results were correlated with the clinical course of the disease. RESULTS: HLA class I antigen expression and beta(2)-m expression were downregulated in 40 and 35 lesions, respectively. HLA class II antigens were expressed in 30 lesions. Patients with high HLA class I, including beta(2)-m, and HLA class II antigen expression in their primary melanoma lesions had a significantly decreased survival (P = 0.009, P < 0.001, and P = 0.006, respectively). CONCLUSIONS: The findings argue against a major role of cytotoxic T-lymphocyte (CTL)-mediated control of tumor growth in the clinical course of uveal melanoma and are compatible with a potential role of NK-cell-mediated control of hematogenic metastatic spread.

Murine monoclonal antibody elicited with antibiotic-exposed Escherichia coli exerts protective capacity in experimental bacterial infections.

Escherichia coli pre-exposed to a sub-minimal inhibitory concentration (sub-MIC) of several antibiotics elicits an enhanced humoral response which is protective against challenges with untreated homologous and heterologous bacteria. To characterise the specificity of this response we produced murine monoclonal antibodies (MAbs) to aztreonam-treated E. coli O6:K-. This resulted in the identification of MAb MT 1F, of isotype IgG1, that recognised a 12-kDa protein component of the untreated bacterial cells. After passive transfer, the MAb displayed protective activity in mice infected with lethal doses of live E. coli O6:K- and E. coli O111:B4. In ELISA experiments the MAb cross-reacted with structures located on whole cells of E. coli O6:K-, E. coli O111:B4, E. coli J5 and Salmonella minnesota Re595 and it also exerted a bactericidal activity against live E. coli O6:K-. The modifications induced by antibiotic treatment may unmask bacterial epitopes that may elicit the production of MAbs endowed with protective capacity.

CD44s adhesive function spontaneous and PMA-inducible CD44 cleavage are regulated at post-translational level in cells of melanocytic lineage.

Adhesion between the CD44s receptor and hyaluronic acid plays an important role in cell migration, tumour growth and progression. Although the alternative splicing of CD44 variant exons represents the principal regulatory mechanism of CD44-mediated functions, CD44v spliced variants are scantily expressed in melanoma cells. For this reason, we have investigated the possibility that post-translational modifications of the CD44 standard receptor could play a pivotal role in regulating CD44-mediated functions in melanoma. Using metabolic inhibitors of N- and O-glycosylation, as well as melanoma transfectants expressing CD44s O-glycosylation site-specific mutants, we performed structural and functional analysis of N- and O-deglycosylated CD44s molecules expressed in melanoma cells. We discovered that complete N- and O-glycosylation is not required by CD44s to be correctly expressed on the melanoma cell surface. Indeed, variably glycosylated and functionally different CD44s molecules were constitutively expressed in primary and metastatic lesions. Furthermore, we observed that changes in N- and O-glycosylation of CD44s could modulate its cleavage. In fact, spontaneous CD44s shedding was dependent on the presence of partial or complete O-glycosylation of four serine-glycine motifs localized in the membrane-proximal CD44 ectodomain. Mutation of these serine residues, as well as an extensive metabolic O-deglycosylation, strongly impaired spontaneous CD44 shedding. Furthermore, an O-glycosylation-independent mechanism of CD44 cleavage has been identified. This alternative mechanism of receptor cleavage is phorbol 12-myristate-13-acetate (PMA) inducible, mediated by metalloproteinase and requires the presence of N-linked sugar residues. Our findings demonstrate that the post-translational modification of CD44s represents the principal regulatory mechanism of CD44s-mediated functions in melanoma.

Localization of the alpha 3 beta 1 integrin in some common epithelial tumors of the ovary and in normal equivalents.

Monoclonal antibodies (mAbs) against cultured human ovary carcinoma cells were produced. We obtained 7 mAbs which reacted diffusely with carcinoma of the ovary but only weakly with vessels and the surface epithelial layer of normal ovary. Biochemical characterization of these mAbs indicated that 3 out of 7 were specific for the alpha 3 chain of the Vla-3 integrin, a receptor for fibronectin, collagen and laminin. Using one of these mAbs, we have studied, by immunohistochemical methods, the distribution of alpha 3 beta 1 integrin in mucinous, serous and endometrioid cystoadenocarcinoma of the ovary and in their normal equivalent: endocervical, tubal and endometrial epithelia. The results show that alpha 3 beta 1 is present in cell-cell contact areas and more abundantly at the junction between epithelial cells and basement membrane in endocervical, tubal epithelia, in epithelium lining the cavity of the uterus and in surface epithelium of the ovary. However, endometrial glands showed only weak and fragmented positivity at the basal pole of the cells. 26 out of 31 ovarian cancers studied, expressed the alpha 3 beta 1 integrin. However, basal localization, typical of normal epithelia, is not prominent or disappears in tumors and is replaced by a more diffuse reaction with variable immunohistochemical staining of the neoplastic cells. Furthermore, a comparative analysis of the expression of alpha 3 beta 1 and its ligands, laminin (LM), fibronectin (FN) and collagen IV (Coll IV), demonstrated that basal polarization of Vla-3 was always correlated with the presence of laminin and Coll IV, intrinsic components of the basement membranes.

Cytogenetic, molecular and phenotypic characterization of the newly established renal carcinoma cell line KJ29. Evidence of translocations for chromosomes 1 and 3.

The established non papillary human renal carcinoma cell line (RCC) KJ29 was submitted to a multiparametric characterization to evaluate its potential use for in vitro and in vivo studies. The cell line grows in vitro as monolayer as well as cell suspension. Cytogenetic analysis has shown a modal chromosome number of 50 with some marker chromosomes, including rearrangements of chromosomes 1 and 3. The antigenic phenotype is characterized by co-expression of cytokeratin and vimentin, as well as expression of urothelium differentiation antigens, low levels of class II MHC antigens and no class I antigens. A differential expression of the VLA-3 integrin heterodimer has been detected between the adherent and non adherent cell population. The cell line which is highly tumorigenic in athymic mice displays expression of erb B-2 and c-met oncogenes and high expression of cell-cycle related and Ha-ras 1 genes.

Vla-3 distribution in normal and neoplastic non-lymphoid human tissues.

Using monoclonal antibody (mAb) M-Kid 2 to the alpha 3 beta 1 heterodimer, we have evaluated immunohistochemically the in vivo expression of the Vla-3 integrin in normal and transformed non-lymphoid human tissues. In normal tissues the alpha 3 beta 1 complex displays a polarized distribution at the baso-lateral aspect of most keratinizing and glandular epithelia. In addition the integrin is detected in perineurium, basal lamina of smooth muscular fibers, vascular media, podocytes and Bowman's capsule, myoepithelial cells of the parotid and breast, and in pulmonary alveoli. Neoplastic transformation is associated with qualitative and quantitative changes in expression of this integrin. The loss of polarized distribution often occurs in various malignancies. Furthermore, a significant decrease in expression occurs in 13% of the colon-rectum carcinomas, 75% of the ductal invasive, and 40% of the lobular invasive breast carcinomas. Among the lung malignancies tested, the small cell lung carcinomas (SCLC) were found to be consistently unreactive with mAb M-Kid 2. Analysis of Vla-3 expression in established tumor cell lines demonstrated that the integrin is almost invariably expressed by the plastic adherent cell subpopulations.

Angiomyofibroblastoma and aggressive angiomyxoma: two benign mesenchymal neoplasms of the female genital tract. An immunohistochemical study.

We describe a rare case of angiomyofibroblastoma (AMF) of the vulva and one case of aggressive angiomyxoma (AAM) of the pelvic region and, with the help of an extensive revision of the literature, we attempt to define their histogenesis and peculiar biological behaviour by an immunohistological evaluation. Our results indicate that AAM, which is characterized by the presence of a high content of glycosaminoglycans in the stroma, expresses uniformly vimentin and hyaluronate receptor CD44, and heterogeneously muscle specific actin (MSA) and desmin, while AMF displays a positive reaction for vimentin, desmin and laminin, and only a weak and heterogeneous positivity for CD44. Both AMF and AAM showed no immunohistochemical reactivity for alpha-smooth muscle actin (ASMA), myoglobin, cytokeratin, collagen type IV, CD68 and S-100. The stromal cells of AAM were negative for laminin. These findings support the suggestion of an origin of the two entities by a common myofibroblastic progenitor, which normally occurs in the lower female genital tract and subsequently undergoes a neoplastic transformation. The expression of CD44 by AAM, which has never been reported before, could be responsible for its more aggressive behaviour, because this receptor is able to mediate migration of neoplastic cells on a hyaluronate rich extracellular matrix. It is speculated that the neoplastic cell of the AAM and AMF of the vulva is a specific myofibroblast which probably arises from undifferentiated mesenchymal cells normally occurring in the lower female genital tract.

Malignant oncocytoma of major salivary glands. Report of a post-irradiation case.

The fourth case of malignant oncocytoma arising in the submandibular gland is here reported. This tumor arose in a 48-year-old man after radiation exposure, a finding never described before for malignant oncocytoma. In addition, several regional metastatic lymph nodes were found. The diagnosis was confirmed by histochemical and ultrastructural findings. The tumor cells showed easily recognizable mucus production and, ultrastructurally, abundant mitochondria, intracytoplasmic lumina lined by microvilli and lipid droplets. These last features have only seldom been described in malignant oncocytoma. Furthermore, the neoplastic cells were alpha-1-antitrypsin positive and S100, thyroglobulin, carcinoembryonic antigen, and smooth muscle actin negative. A thorough review of the literature is also presented.

Differential effect of human and murine polyclonal and monoclonal antisera on TNF-alpha production by human monocytes.

The capacity of human and murine polyclonal and monoclonal antibodies to inhibit lipopolysaccharide (LPS)-induced tumor necrosis factor-alpha (TNF-alpha) release from human monocytes was investigated. Human pooled immunoglobulin G (IVIG), human IgM monoclonal antibody (HA-1A) directed against the lipid A moiety of LPS, and murine IgG monoclonal antibody (MT-1F) raised in mice against antibiotic-treated Escherichia coli O6:K- were either added simultaneously with LPS to monocytes or preincubated for 1 h at 37 degrees C before being added to monocytes. TNF-alpha content in the monocyte supernatants was then tested. Simultaneous addition of increasing concentrations of IVIG (from 0.3 to 2.5 mg/ml) and 10 micrograms/ml of LPS to monocytes induced an enhanced release of TNF-alpha by monocytes in a dose dependent fashion. Preincubation of IVIG with LPS abolished the additive effect, but did not inhibit LPS-induced TNF-alpha release by monocytes. The simultaneous addition of LPS and HA-1A to monocytes had no additive effect nor did it inhibit TNF-alpha release. On the other hand, inhibition of TNF-alpha release was observed when HA-1A was preincubated with LPS before being added to monocytes. In all instances MT-1F inhibited TNF-alpha release when the monocytes were stimulated with smooth type LPS, but not with LPS isolated from rough mutants.

Antitumor activity of the proteinase inhibitor tetra-p-amidinophenoxyneopentane in a nude mouse model of human melanoma.

An aromatic poly-amidine (tetra-p-amidinophenoxyneopentane, TAPP-Br) exhibiting anti-proteinase activity and known to exert antitumor activity in vitro was analysed for its ability to inhibit the in vivo growth of a human melanoma cell line transplanted in nude mice. 5 X 10(6) melanoma cells were injected subcutaneously in groups of nude mice and treatment with TAPP-Br was performed (0.125-1 mg/0.2 ml injections, repeated three times at the beginning of the experiment and after 20 days). After 25 days tumors displaying a volume of 0.9-1.8 cm3 were detectable in control untreated mice. Mice treated with TAPP-Br on the other hand did not develop sizable tumors or consistently developed tumors of significantly smaller sizes. Despite these therapeutic effects, significant chronic toxicity of the compound was observed when administered at higher dosage (500-1000 micrograms). These side effects, which may hamper the therapeutic use of TAPP-Br, are likely to be circumvented by alternative routes of administration or by vehiculation into liposomes. These alternative strategies of treatment are currently investigated.

Generation and characterization of the murine monoclonal antibody M-KID 2 to VLA-3 integrin.

Through the analysis of the antigenic phenotype of a recently established human renal carcinoma cell line (KJ29), we have demonstrated that alpha 3 subunit of the integrin family is selectively expressed by the plastic adherent cell subpopulation. Because of the scanty availability of monoclonal antibodies to this adhesion molecule, we have used KJ29 cell line as immunogen to raise novel murine monoclonal antibodies. We isolated an hybridoma secreting the mAb M-KID 2 of the IGg1k isotype that immunoprecipitates from intrinsically [35S]-Methionine labeled KJ29 cells, an heterodimer of 130/130 and 110/150 Kd, in reducing and nonreducing conditions respectively. This reactivity was completely abolished by immunodepletion of the cell extract with a polyclonal anti alpha 3 chain antiserum. Treatment of M-KID 2 immunoprecipitates with various solutions of pH ranging from 2 to 10.5, to dissociate alpha 3 from beta 1 chains, showed a retention of both alpha 3 beta 1 chains thus indicating that the epitope identified by mAb M-Kid 2 is likely to be constituted by the alpha 3 beta 1 heterodimer. Furthermore immunohistochemical studies on selected frozen and paraffin embedded tissues with mAb M-Kid 2 have provided staining pattern indicating the recognition of Vla-3. These findings demonstrate that mAb M-KID 2 can represent a valuable reagent for the study of Vla-3 integrin in normal and pathologic conditions.

Monoclonal antibodies to a soluble metallic radioisotope chelator: development and characterization.

Monoclonal antibodies (MAbs) have been prepared with specificity for diethylenetriaminopentaacetic acid (DTPA) used to chelate metal radioisotopes to immunoglobulins for radioimmunoimaging and radioimmunotherapy. The use of fusion partners of lymph node-derived B cells resulted more frequently in the isolation of IgG secreting hybridomas than with splenocytes. All MAbs have been selected for simultaneous recognition of chelated and unchelated DTPA, and have been characterized in their biochemical, physico-chemical and immunochemical features. In view of the potential use in development of bifunctional MAb, these novel MAbs were also proven to lack detectable cross-reactivity with normal human tissues.

Extraskeletal osteosarcoma in a cat.

A 7-year-old spayed female domestic shorthair cat was referred for evaluation of a localized growing mass on the left flank. Cytologic and histologic findings suggested that the mass was an extraskeletal osteosarcoma. Radiography failed to reveal any association between the lesion and the axial or appendicular skeleton. Because of the large size of the tumor, the cat was treated with carboplatin prior to and after surgery (hemipelvectomy) to ensure that surgical margins were free of neoplastic cells and to prevent systemic dissemination of malignant cells. The tumor has not recurred during a 2-year follow-up period.