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Tenascin is an extracellular matrix molecule with structural similarity to fibronectin. An increase in extracellular matrix content of both tenascin and fibronectin is associated with early wound healing and with various skin fibroses. However, the relationship of tenascin and fibronectin expression during scar remodeling and the formation of pathologic scars such as keloids is unknown. Expression of tenascin in normal and abnormal human scars was examined and compared with that of fibronectin by immunohistochemistry and in situ hybridization. Tenascin and fibronectin protein and messenger RNA contents were elevated in normal, mature scars relative to quiescent skin, similar to the situation during earlier stages of healing. Tenascin and fibronectin expression was further enhanced in keloids relative to normal skin and scar, and, as has been shown for fibronectin, tenascin expression in uninjured skin adjacent to keloids was indistinguishable from that in quiescent skin from unaffected individuals. These data suggest that tenascin and fibronectin gene expression are coordinated during later stages of normal wound healing and that a defect involving common regulatory elements for these genes is associated with the formation of keloids.

The human F8/G253 antigen, B-CAM, is a cell surface glycoprotein that is expressed with restricted distribution pattern in normal fetal and adult tissues, and is up-regulated following malignant transformation in some cell types. We have isolated a complementary DNA for B-CAM using an expression cloning technique. The complementary DNA (EMBL accession number X80026) encodes a 588-amino acid protein which is a novel member of the immunoglobulin superfamily with a characteristic V-V-C2-C2-C2 immunoglobulin domain structure. This structure has been described previously for the human MUC18 melanoma antigen (31% amino acid identity) and chicken and rat versions of a neural adhesion molecule referred to as SC1/DM-GRASP/BEN or KG-CAM, respectively (26% amino acid identity). This homology is suggestive of a role for B-CAM in cell-cell or cell-matrix adhesion. The gene for B-CAM has been mapped by fluorescence in situ hybridization to chromosome 19q13.2-13.3.

Background: The vascular endothelium participates in diverse physiologic and pathologic processes, ranging from blood clotting to leukocyte trafficking and tumor metastasis. Many of these functions are mediated by endothelial surface molecules, and monoclonal antibodies (mAb) have been used extensively to define endothelial cell surface structures in normal and lesional tissues., Experimental Design: MAbs raised against human umbilical vein endothelial cells were tested on a panel of cultured cell types representing diverse cell lineages. In vivo antigen expression was examined through immunohistochemical tests with normal and lesional tissues. Immunochemical methods were employed to characterize mAb-defined antigens in endothelial cell extracts., Results: The newly-derived mAbH572 reacts with a high molecular weight glycoprotein complex of cultured endothelial cells comprised of disulfide-bonded subunits of molecular weight 190,000, 140,000, 125,000, and 110,000. This antigen, designated endoGlyx-1, is expressed on the surface of cultured endothelium, with no changes in expression after cytokine-stimulation. Immunohistochemical assays detect endoGlyx-1 in endothelial cells of large, medium-sized, and small blood vessels, including capillaries, in all human organs tested; only hepatic and splenic sinusoids lack the molecule. EndoGlyx-1 is not found in any of the normal fetal and adult nonendothelial cell types tested. In a study of > 100 tumors, endoGlyx-1 immunostaining was consistently found in tumor capillaries, including "hot spots" of neoangiogenesis in certain tumors. EndoGlyx-1 is also expressed in hemangiomas and angiosarcomas., Conclusions: The highly restricted expression of endoGlyx-1 in normal and tumor blood vessels suggests a role for this molecule unique to endothelial cell physiology. In fact, endoGlyx-1 appears to be the only endothelial lineage-specific cell surface glycoprotein identified to date. The availability of endoGlyx-1 antibodies may aid in the characterization and quantification of the angiogenic phase of solid tumors and facilitate the search for endothelial precursors in bone marrow or peripheral blood.

The human fibroblast activation protein (FAP) defined by monoclonal antibody (MAb) F19 is a cell surface antigen expressed in reactive stromal fibroblasts of breast, colorectal, lung and other epithelial cancers. In contrast to its stroma-specific localization in epithelial neoplasms, FAP is expressed in the malignant mesenchymal cells of bone and soft tissue sarcomas. FAP is transiently expressed in some fetal mesenchymal tissues but is absent or expressed at low levels in most adult tissues. FAP is induced in cultured fibroblasts and, in these cells, consists of a M(r) 95,000 subunit (FAP alpha) carrying the F19 epitope and a non-covalently bound M(r) 105,000 subunit (FAP beta) lacking the F19 epitope. Using MAb F19 and 5 newly derived MAbs, we identify 3 distinct epitopes on FAP alpha and tentatively assign one epitope to FAP beta. Analysis of detergent extracts of a FAP alpha high beta- sarcoma cell line by size exclusion-high performance liquid chromatography (HPLC) revealed that FAP alpha does not elute as a M(r) 95,000 species but as part of a high-molecular weight complex (M(r) > 400,000) that dissociates into M(r) 95,000 subunits in SDS gels. Immunoaffinity purification of FAP alpha followed by tryptic digestion, reversed-phase HPLC and microsequencing identified 3 unique FAP alpha peptides, with 2 showing sequence similarity (23/38 identical amino acids) to segments of CD26, a T-cell activation antigen. CD26 is a membrane-bound enzyme (dipeptidyl aminopeptidase IV), but immunopurified FAP alpha has little if any dipeptidase activity with typical CD26 substrates. Finally, studies with FAPlow leptomeningeal fibroblasts revealed that transforming growth factor-beta, 12-O-tetradecanoyl phorbol-13-acetate and retinoids can upregulate FAP expression, whereas serum and several other factors had no or little effect on FAP levels. FAP and CD26 may belong to a family of structurally related but functionally distinct activation proteins that are expressed on different cell types and show unique modes of regulation in normal and malignant cells.

Structural alterations of p53 and overexpression of the p53 protein are found in a large proportion of human cancers. In this study, we examined the frequency of p53 mutations and p53 overexpression in squamous cell carcinomas (SQCC) of head and neck. Expression of p53 was detected by immunochemistry (IHC) with monoclonal antibodies defining three distinct epitopes: PAb421 (species cross-reactive epitope on normal and mutated p53), PAb1801 (epitope on normal and mutated human p53), and PAb240 (conformational epitope of mutated p53 and denatured normal p53). Genetic alterations of p53 were identified by single-strand conformational polymorphism (SSCP) analysis and DNA sequencing in selected cases. IHC assays revealed nuclear p53 immunostaining in 53% of cases (32 of 60) with PAb1801, 38% (23 of 60) with PAb421, and 32% (19 of 60) with PAb240. Cases positive with PAb421 or PAb240 were also positive with PAb1801, whereas PAb421 and PAb240 identified overlapping but distinct tumor subsets. Areas of carcinoma in situ present in the tumor specimens showed nuclear p53 immunostaining in 11 of 26 cases. SSCP analysis for exons 5-9, the most common sites of p53 abnormalities, revealed mutations in 26% (15 of 58) of the evaluable cases. Comparison of the SSCP results with the IHC results for PAb1801 identified 11 cases that were positive by both methods, 4 cases with p53 mutations that were negative by IHC, 20 cases positive by IHC but without detectable p53 mutations, and 23 cases negative by both methods. IHC with PAb240, which is thought to be specific for mutated p53, was positive in 9 cases with demonstrable p53 mutations and in 9 cases with no detectable mutations. DNA sequence analysis of nine tumors identified point mutations, nonsense mutations, and frame-shift mutations. In conclusion, our study shows that p53 overexpression in SQCC of head and neck as detected by IHC is a frequent finding, and that overexpression is associated with common types of p53 mutations in some cases. However, IHC identifies p53 overexpression in some tumors with apparently normal SSCP patterns and, conversely, tumors with nonsense or frameshift mutations may not express any p53 detectable by IHC.

The translocation t(11;22)(q24;q12) can be identified in its classical or variant form in approximately 90% of cases of Ewing's sarcoma (ES) and peripheral neuroectodermal tumor (PNET). In this tumor group in which the histopathologic diagnosis is often one of exclusion, the cytogenetic demonstration of this translocation has become an invaluable positive diagnostic marker. With the recent cloning of the breakpoint regions of the t(11;22), molecular genetic approaches to the detection of this translocation have become possible. By Southern blotting, the position of the breakpoints on chromosome 22 has been found to be tightly clustered within a 7-kilobase (kb) fragment of the genomic DNA, within a gene designated EWS. In the present study, we examined the efficacy of an EWS complementary DNA (cDNA) probe in detecting the t(11;22) in Southern blots of EcoRI- or HindIII-digested DNA extracted from cases of ES and PNET. We also compared the results of the molecular and cytogenetic analysis with the expression of the ES cell surface antigen MIC2, as demonstrated by immunoperoxidase staining with the monoclonal antibodies O13 and HBA71. Twenty-three specimens were studied, including 18 ES and five PNET. Of 16 cases with clonally abnormal karyotypes, 14 (88%) showed a typical or variant t(11;22). Rearrangements were demonstrated within the EWS gene with the EWS cDNA probe in 20 of 23 specimens (87%), including all of the 14 cases, as well as in one case that displayed clonal numerical chromosome abnormalities only. The MIC2 antigen was expressed in 19 of 20 cases (95%), including all three cases lacking EWS rearrangement.(ABSTRACT TRUNCATED AT 250 WORDS)

The human fibroblast activation protein (FAP), defined by monoclonal antibody F19, is expressed in vivo in reactive stromal fibroblasts of epithelial cancers, subsets of bone and soft tissue sarcomas, and granulation tissue of healing wounds. FAP is generally absent from the stroma of benign epithelial tumors and normal adult tissues. In vitro FAP induction is observed in proliferating cultured fibroblasts and in melanocytes grown with fibroblast growth factor and phorbol ester. In the present study, we show that fibroblast and melanocyte FAP is a cell surface protein comprising noncovalently linked M(r) 95,000 (p95) and M(r) 105,000 (p105) subunits. In contrast, cultured sarcoma and melanoma cell lines express only p95 or are FAP negative. Immunoblot experiments show that p95, but not p105, carries the epitope defined by monoclonal antibody F19. Furthermore, peptide maps of purified p95 and p105 differ, suggesting that they may be distinct gene products. Loss of FAP or a change from p95/p105 to p95 expression accompanies the acquisition of growth factor independence and tumorigenicity in several in vitro test systems, including simian virus 40 transformation of normal fibroblasts, Ha-ras transformation of normal melanocytes, supertransformation of osteosarcoma cells, and enhanced N-MYC expression in variant neuroblastoma cells, whereas serum-starved normal fibroblasts continue to express p95/p105. Thus, fAP expression appears to be linked to the growth factor-dependent proliferative capacity of normal cells and is not merely a secondary event in proliferating cells; furthermore, FAP expression is inversely correlated with growth factor independence and tumorigenicity in transformed cell lines. This distribution pattern is consistent with a role for p95/p105 in mediating extrinsic, growth regulatory signals in normal cells, possibly as a heteromeric cell surface receptor. Such a physiological function may be obviated when oncogenes with cytoplasmic or nuclear sites of action are activated, reducing extrinsic growth factor dependence and permitting down-regulation of FAP in certain transformed cells.

The LK26 antigen is a cell surface glycoprotein (M(r)35,000 to 40,000) of normal placenta and gestational choriocarcinomas that shows highly restricted distribution in normal tissues, being expressed primarily in a subset of simple epithelia. In this study, immunohistochemical methods were used to examine LK26 expression in 78 ovarian tumors and > 400 tumors of other histological types. Ovarian carcinomas derived from coelomic epithelium showed the most consistent and strongest immunostaining for LK26, with 52 of 56 cases being LK26+. Ovarian tumors of sex cord, germ cell, and stromal origin were generally LK26-. LK26 was not found in normal fetal or adult ovary; however, it was present in the lining epithelia of some benign ovarian cysts. Mesotheliomas, which share a common mesothelial origin with LK26+ ovarian tumors, expressed no or only low levels of LK26. Other epithelial cancers expressed LK26 in subsets of cases and generally showed heterogeneous or weak immunostaining; this group of LK26+ tumors includes endometrial (10 of 11 cases tested), colorectal (six of 27), breast (11 of 53), lung (six of 18), and renal cell (nine of 18) carcinomas. Four of five brain metastases derived from epithelial cancers and three of 21 neuroendocrine carcinomas showed prominent LK26 immunoreactivity. Only rare neuroectodermal tumors (two of 70) and none of the sarcomas (none of 58) or lymphomas tested (none of 21) were LK26+. Tests with cultured cells showed that the LK26 proteins expressed in choriocarcinoma and ovarian cancer cells are biochemically similar, and transfection experiments identified LK26 as an adult-type, high-affinity folate-binding protein. The present study provides the first detailed specificity and sensitivity analysis for folate-binding protein/LK26 in human tumors and defines a role for folate-binding protein/LK26 in immunobiological studies of ovarian cancers and other LK26+ neoplasms.

Hybridoma technology, immunohistochemistry and molecular genetic techniques have paved the way for a general serological mapping of molecules expressed on the surface of normal and neoplastic cells. The emerging findings challenge cell lineage-based models of differentiation and point to new, modular concepts to explain complex tumor phenotypes. In parallel studies, the repertoire of T-cell recognized tumor antigens--still mysterious but probably quite distinct from the antibody-defined repertoire--has also become accessible to molecular analysis.

Two extracellular matrix proteins of brain tissue, neuronectin (NEC1) and cytotactin (CT), are disulfide-bonded multimers of M(r) 180,000-250,000 subunits. The previously known distribution of these molecules is, however, very different. Human NEC1 is found throughout the white matter of rostral segments of the adult central nervous system (CNS) but not in rostral gray matter or in caudal CNS segments, including the cerebellum. In contrast, CT is absent or expressed at a low level in the adult chicken cerebrum but highly expressed in the cerebellum. Despite these differences in distribution, results obtained with antibodies that recognize NEC1 and CT in several vertebrate species indicate that these molecules are identical or at least closely related: (1) alpha NEC1 antibodies recognize proteins affinity-purified with CT-binding proteoglycan; (2) proteins recognized by alpha NEC1 and alpha CT antibodies in cells constitutively expressing the molecules, cells in which expression is induced by growth factors and phorbol ester and cells treated with tunicamycin (to block glycosylation) are identical in subunit composition and mobility on SDS gels; (3) the removal of NEC1 from culture supernatants by immunoprecipitation removes all molecules reactive with alpha CT antibodies and vice versa; (4) immunoblots of brain extracts with alpha NEC1 and alpha CT antibodies yield identical results. Having demonstrated the structural similarity between NEC1 and CT, we reexamined their distribution in the CNS. Surprisingly, the temporal and spatial distribution pattern of NEC1/CT varied greatly among species. Immunohistochemical and immunoblot experiments with adult human CNS tissues revealed significant levels of NEC1/CT in rostral but not caudal segments. In contrast, in cows and pigs the molecule is found throughout the CNS. Adult rat and mouse brains show regionally restricted expression of NEC1/CT in several areas of the cerebrum--distinct from those showing NEC1/CT in the human--and in the molecular layer of the cerebellum. Tests with fetal and newborn tissues revealed that CNS development in humans, cows and pigs is not accompanied by the marked decline in NEC1/CT levels or the changes in subunit composition found in the chicken CNS. The marked species diversity in temporospatial expression patterns suggests that intrinsic and/or extrinsic elements controlling the expression of NEC1/CT have diverged during vertebrate evolution.

The HBA71 antigen is an M(r) 30,000/32,000 cell surface glycoprotein (p30/32MIC2), encoded by the pseudoautosomal MIC2 gene on chromosomes X and Y, that is expressed in Ewing's sarcomas. Immunohistochemical studies demonstrate a striking specificity for HBA71 among neoplasms of diverse histologic types. In the present study, 43 cases of Ewing's sarcoma of bone were tested for HBA71 expression and six additional immunohistochemical markers regularly used in the differential diagnosis of small round-cell tumors of childhood and adolescence (neuron-specific enolase, vimentin, leukocyte common antigen, cytokeratins, muscle-specific actin, desmin). The study design included (a) random selection of Ewing's sarcoma cases from the files of Memorial Hospital beginning in 1968, (b) blind review of the original histopathologic diagnoses of ES, (c) side-by-side immunohistochemical study of recut histologic specimens, and (d) statistical analysis of immunohistochemical findings in view of clinical outcome. Of the seven antigens studied, only HBA71, neuron-specific enolase and vimentin were expressed in a significant proportion of cases. Forty-one of the 43 cases were HBA71+ (95% sensitivity); of these, 21 were neuron-specific enolase+, 29 were vimentin+, and 15 were both neuron-specific enolase+ and vimentin+. One tumor lacked all antigens, and one was vimentin+ only. Comparison of tumor tissues in five patients obtained before and after cytostatic chemotherapy showed no change in HBA71 expression or in the other antigens tested. Product-limit survival analysis (median disease-free survival was 27.3 months for the study cohort) revealed no significance of neuron-specific enolase or vimentin marker status. These results raise doubts about the usefulness of neuron-specific enolase and vimentin immunohistochemistry to distinguish Ewing's sarcoma from other small round-cell tumors of childhood and adolescence or as prognostic indicators in Ewing's sarcoma. The positive identification of Ewing's sarcoma of bone now becomes a reality using HBA71 immunohistochemistry, either as a sole method or in combination with chromosomal breakpoint analysis. This may result in achieving uniform diagnostic criteria for evaluating the biologic, therapeutic, and prognostic aspects of Ewing's sarcoma and related neoplasms.

Monoclonal antibody (MAb) HBA71, which was raised against Ewing's sarcoma cells, recognizes a cell-surface glycoprotein, p30/32MIC2, that is encoded by the MIC2 gene in the pseudoautosomal region of human chromosomes X and Y. This immunohistochemical study evaluates the specificity and sensitivity of MAb HBA71 for tumor diagnosis. Frozen and paraffin-embedded tissues of more than 300 tumors of diverse histologic type, including more than 100 small round cell tumors of childhood and adolescence, were tested with this MAb by the avidin-biotin immunoperoxidase procedure. The authors found HBA71 immunoreactivity in 61 of 63 Ewing's sarcomas studied and 9 of 11 primitive neuroectodermal tumors and peripheral neuroepitheliomas. HBA71-negative tumors included neuroblastomas (0 of 24), melanomas (0 of 13), an esthesioneuroblastoma, small cell osteosarcomas (0 of 2), a malignant ectomesenchymoma, desmoplastic SRCT (0 of 5), and medulloblastomas (0 of 5). Heterogeneous expression of HBA71 immunostaining was found in some embryonal rhabdomyosarcomas (3 of 14) and astrocytomas (4 of 7), and in a few neuroendocrine tumors (4 of 26), carcinomas (3 of 94), and lymphomas (6 of 30). Because Ewing's sarcomas are consistently HBA71 positive, the authors searched for antigen-positive normal cells that may represent precursors for these tumors; however, no obvious candidate for the elusive cell of origin for Ewing's sarcoma was identified in the normal fetal tissues tested. Their findings indicate that HBA71 is a highly restricted cell-surface antigen of Ewing's sarcomas and primitive neuroectodermal tumors, and immunohistochemistry employing this antibody may be of value in the differential diagnosis of selected small round cell tumors in childhood and adolescence.

The neural cell adhesion molecule (NCAM) was discovered in a search for cell surface antigens of chicken neurons that contribute to cell adhesion and pattern formation during development. Homologous adhesion molecules have been identified in several species, including humans. In this immunohistochemical study, the authors examine the role of human NCAM in tumor diagnosis. The authors used a monoclonal antibody (MAb), 5.1H11, to examine NCAM immunoreactivity in frozen sections of more than 450 tumors, including more than 80 small round cell tumors (SRCT) of childhood and adolescence (neuroblastomas, Ewing's sarcomas [ES], peripheral neuroepitheliomas [PN], primitive neuroectodermal tumors [PNET], esthesioneuroblastomas, malignant ectomesenchymoma, medulloblastomas, small cell osteosarcomas, mesenchymal chondrosarcomas, embryonal rhabdomyosarcomas, and lymphomas). The authors show that 1) neuroblastomas and primary brain tumors are NCAM+; 2) ES, most PN/PNETs, and melanomas are NCAM-; 3) embryonal rhabdomyosarcomas and various other sarcomas are NCAM+; 4) neuroendocrine tumors are NCAM+; 5) subsets of carcinomas of kidney, ovary, lung and other organs are NCAM+; and 6) lymphoid tumors are NCAM-. Tests with normal fetal and adult tissues indicate that these findings reflect only in part the NCAM phenotypes of corresponding normal tissues. Notably the NCAM- phenotype of ES and PN/PNET is not explained by current histogenetic models for these tumors, which suggest a primitive neuroectodermal origin. Finally the authors show that NCAM expression among SRCT has an inverse relationship with the expression of p30/32MIC2, a cell surface antigen of ES and PN/PNET detected with MAb HBA71. These results suggest that immunohistochemical assays for NCAM and p30/32MIC2 expression may aid in the further characterization of SRCT of childhood and adolescence.

Monoclonal antibody HBA71 detects a cell surface antigen of human Ewing's sarcomas and peripheral neuroepitheliomas that distinguishes these tumors from other small round cell tumors of childhood and adolescence. In the present study, we show that monoclonal antibody HBA71 reacts with polypeptides of Mr 32,000 and 30,000 and that the HBA71-coding gene segregates with human chromosomes X and Y in rodent-human hybrids. Therefore, we compared HBA71 to the T-cell leukemia antigen 12E7, which is encoded by the pseudoautosomal region of chromosomes X and Y. We show that monoclonal antibodies HBA71 and 12E7 (a) detect polypeptides of identical size, (b) react with mouse cells transfected with complementary DNA corresponding to the 12E7-coding gene, MIC2, and (c) give similar patterns of reactivity with human tumor cell lines and small round cell tumor tissues. Thus, HBA71 and 12E7 are identical or closely related antigens and the available MIC2 probes will facilitate analysis of the molecular mechanisms that determine differential HBA71 expression in small round cell tumors of childhood and adolescence.

The A4 and AO10 (110 kd glycoprotein) cell-surface antigens are biochemically distinct markers of cultured human astrocytomas that are expressed by only a limited number of other cultured cell types. To further characterize these two antigens, the authors used immunohistochemical methods to determine their expression in normal human tissues, astrocytomas, and over 100 tumors of other histologic types. They found that A4 is expressed 1) throughout the central (CNS), but not peripheral nervous system (PNS); 2) in smooth muscle and a small number of epithelial tissues; and 3) in reactive glia and in astrocytomas, but not in most tumors of other histologic types. In contrast, the AO10 antigen is expressed 1) in a small subset of CNS neurons, but not in astrocytes, PNS neurons, or other normal tissues; 2) in astrocytomas and reactive glia; and 3) in some additional neuroectodermal tumors, but not melanomas, carcinomas, or sarcomas. These findings show that A4 and AO10 are restricted markers for human astrocytomas in vivo. Furthermore, the antigens show distinct patterns of expression in normal human CNS but appear to be coordinately expressed in astrocytomas and astrocytoma-derived cell lines.

Serologic analysis of rodent-human somatic cell hybrids has permitted the assignment of loci coding for cell surface differentiation antigens 5.1H11 (gene symbol MSK39) and F35/9 (MSK40) to human chromosomes 11q13-qter and 22, respectively. Both antigens are expressed in hybrids constructed with antigen-positive human cells and certain hybrids constructed with antigen-negative human cells, indicating that the coding genes are not irreversibly silenced in human nonexpressor cells. Antigens 5.1H11 and F35/9, and at least six additional cell surface antigens encoded by chromosomes 11 and 22, are expressed on human Ewing sarcoma and peripheral neuroepithelioma cells, providing selectable markers for isolating and characterizing the specific t(11;22)(q24;q12) marker chromosomes of these tumors in interspecies hybrids.

Human malignant melanoma cells express specific chondroitin sulfate proteoglycans (mel-CSPG) on the surface, both in vivo and in vitro. Melanocytes in normal skin show no detectable mel-CSPG but can be induced to express the antigen when cultured in the presence of cholera toxin and the tumor promoter 12-O-tetradecanoylphorbol-13-acetate. Most other cell types do not express mel-CSPG either in vivo or in vitro. A study was designed to examine regulatory signals controlling mel-CSPG expression. The gene encoding mel-CSPG was mapped to human chromosome 15, and this chromosome was introduced into rodent cells derived from distinct differentiation lineages. Three types of mel-CSPG--expressing hybrids were found: (i) hybrids derived from human melanomas; (ii) hybrids derived from human cells that do not express mel-CSPG; and (iii) hybrids derived from human cells expressing mel-CSPG that are antigen-negative but that are induced to express mel-CSPG when cultured on extracellular matrix instead of plastic surfaces. Thus, mel-CSPG expression can be controlled both through intrinsic signals, provided by the differentiation program of the rodent fusion partner, and through extrinsic signals, provided by specific cell-matrix interactions.

Three mouse monoclonal antibodies to distinct cell surface antigens were derived from immunizations with cells of Tera-1, a human teratocarcinoma cell line, and a membrane preparation of placental tissue. The distribution of the antigens on 165 cultured lines of various human tumors and normal cells was determined by mixed hemadsorption assays and on fresh tissues by immunofluorescence staining. K4 antigen is expressed on cell lines derived from teratocarcinomas but not on any other cultured cell tested. Normal adult colonic epithelium, some fetal tissues, and specimens of testicular teratocarcinoma were also K4 positive. K21 antigen was detected on teratocarcinoma cell lines and, at more than 100-fold lower levels, on cultures of normal and malignant kidney epithelium but not on other cultured cells. K21 expression in normal tissues is restricted to the epithelium of fetal intestine and bronchus. Other fetal tissues and all adult normal tissues tested lacked K21. A subset of teratocarcinoma specimens (5 of 8) was reactive with antibody K21. P12 antigen is represented on a wide range of cell lines and tissues, including a subset of teratocarcinomas. AbK4, AbK21, and AbP12 react with carbohydrate sequences present on high-molecular-weight glycoproteins. AbK21 and AbP12 recognize the lacto-N-tetraose and lacto-N-fucopentaose III (X-hapten) structures, respectively, whereas AbK4 reacts with a neuraminidase-sensitive determinant.

Mouse monoclonal antibodies have been used to study the distribution of the epidermal growth factor receptor in human cultured cells and tissues. As expected, most epithelial cells expressed epidermal growth factor receptor (EGFr), whereas cells of hematopoietic origin were EGFr-. However, EGFr was found to be differentially expressed on cultured cells of neuroectodermal origin. Normal melanocytes and a proportion of melanomas are EGFr-, whereas a distinct subset of other melanomas, astrocytomas, and neuroblastomas is EGFr+. Expression of the EGFr in melanomas was closely related to the expression of phenotypic traits of differentiation. Less differentiated melanomas have an epithelioid morphology and are nonpigmented, Ia+, and EGFr+; in contrast, more differentiated melanomas have a dendritic morphology and are pigmented, Ia-, and EGFr-. In the melanoma cell panel used, expression of EGFr did not correlate with rate of proliferation. Expression of EGFr in tissues also showed a lack of correlation with the proliferative state of cells. Our findings indicate that expression of EGFr is related to cell lineage and specific stages of cellular differentiation, rather than only to cell proliferation. The data suggest that receptor content may be elevated in a large number of tumor cell lines.

Monoclonal antibodies AbAJ9 and AbT87 define two distinct human cell-surface antigen systems (1, 2). Both antibodies react with a wide variety of cultured human cell types but not with rodent cell lines. AbAJ9 identifies a glycoprotein of 140,000 mol wt and AbT87 identifies a glycoprotein of 60,000 mol wt. The genetic control of antigen expression was studied in rodent-human somatic cell hybrids containing different subsets of the entire human chromosome complement, using an immune rosetting assay for serological analysis and hybrid selection. The presence of human chromosome 1 was found to be both necessary and sufficient for expression of antigens AJ9 and T87 in hybrid cells. Two independent gene loci on chromosome 1, designated MSK -1 for AJ9 and MSK -2 for T87 , control the expression of these antigens. We have assigned MSK -1 to region 1 cen-p22 and MSK -2 to region 1q32 -qter, using hybrids containing only fragments of human chromosome 1.

CD14 is a myelomonocytic differentiation antigen expressed by monocytes, macrophages, and activated granulocytes and is detectable with the monoclonal antibodies MO2, MY4, and LeuM3. Analyses of complementary DNA and genomic clones of CD14 show that it has a novel structure and that it maps to chromosome 5 within a region containing other genes encoding growth factors and receptors; it may therefore represent a new receptor important for myeloid differentiation. In addition, the CD14 gene is included in the "critical" region that is frequently deleted in certain myeloid leukemias.

We have previously assigned several genes controlling expression of cell-surface antigens to human chromosomes 1 and 12. In the present study, we characterize three additional cell-surface antigens determined by these chromosomes. Two monoclonal antibodies, AbSR75 and AbMG6, define antigens expressed on a wide range of cultured human cells. In contrast, AbK66 defines an antigen with a more restricted distribution which is expressed on normal and malignant human epithelial cells but not on neuroectoderm-derived cells or hematopoietic cells. Normal adult fibroblasts are K66-, whereas fetal fibroblasts are K66+. Serological analysis of rodent-human hybrid cells permitted the assignment of MSK31, controlling SR75, and MSK32, controlling K66, to chromosome 1, whereas MSK27, controlling MG6, maps to chromosome 12. Analysis of hybrids containing only deleted copies of chromosome 1 or chromosome 12 established regional assignments for MSK31, MSK32, and MSK27 and also for the previously defined genes MSK1, MSK4, and MSK7. The MSK1 gene, coding for a 140,000 mol wt cell surface glycoprotein, was found to map to the same band (1p22) as the NRAS protooncogene and the gene encoding the beta-subunit of nerve growth factor (NGFB).

Serological and immunopathological analysis of the expression of Lea, Leb, X, and Y blood group antigens on cell lines and tissues was performed using a panel of mouse monoclonal antibodies. The distribution of the antigens was determined on 155 malignant tumor cell lines of various types and 10 short term cultures of normal fibroblasts and kidney cells. Among colon cancers, all four blood group antigens were expressed on the majority of cell lines. On lung, breast, bladder, and ovarian cancer cell lines, X and Y antigens were the main specificities found, whereas few of the renal and hematopoietic tumor cell lines demonstrated any of the four blood group antigens. No blood group antigens could be detected on astrocytoma or melanoma cell lines. The expression of the antigens was also analyzed on frozen sections of colon carcinoma and adjacent normal colon tissue from 42 patients using the immunoperoxidase method. Lea and X were detected throughout the normal colon and on most colonic tumors. In poorly differentiated colon cancer and in metastatic cancer, decrease of Lea antigen was observed. Leb and Y expression was observed in only 20-45% of normal tissue samples but in almost all colonic carcinoma tissues. A selected number of tumor and normal specimens from patients whose secretor status was known were examined in more detail. Both the staining of the tissues and the reactivity of blood group glycolipids from the same specimens were determined. These studies confirmed the above findings and demonstrated the unexpected ability of tumors of nonsecretors to express Leb and/or Y antigens. In such individuals, in whom the expression of Leb and Y antigens in normal tissues is absent or minimal, these antigens provide possible targets for immunodiagnosis and therapy.

Cells of the normal colonic mucosa express several types of cytokeratins, the characteristic intermediate filament proteins of epithelial cells. An immunohistochemical study was designed to examine the expression of two distinct groups of cytokeratins, recognized by monoclonal antibodies AE1 and AE3, in the colonic mucosa and to compare the findings with those obtained with a large number of polypoid lesions (adenomatous and hyperplastic) and carcinomas of the colon. AE1 and AE3 immunostaining was found in the surface epithelium and upper portions of the crypts of Lieberkühn (functional zone) of normal colonic mucosa, whereas the lower portions of the crypts (proliferative compartment) were unreactive with both AE1 and AE3. Polypoid lesions of the colonic mucosa can be placed into two categories based on their patterns of cytokeratin expression. Solitary tubular adenomas and hyperplastic polyps are composed of AE1 and AE3 nonexpressing cells with only few, patchy areas of AE1 and AE3 expressing cells present within glands and in the surface epithelium. In contrast, villous adenomas show strong AE1 and AE3 reactivity throughout the glands. Furthermore, tubular and villous adenomas, and even histologically normal mucosa in patients with familial polyposis, show AE1/AE3 expression throughout the glands and surface epithelium. Colonic carcinomas show a predominance of AE1/AE3 expressing cells. Thus, cytokeratins recognized by monoclonal antibodies AE1 and AE3 represent molecular markers of cellular maturation in the normal colonic mucosa, that are expressed in colonic carcinomas and, in addition, serve as markers that distinguish colonic mucosa and adenomas with a high risk for development of cancers from those with a lower risk.

Neuronectin (NEC1) is a human extracellular matrix protein found throughout the white matter of rostral brain segments but not in most areas of the mesencephalon, in the pons, cerebellum, medulla, spinal cord, and peripheral nerves. This distribution may result from NEC1 expression by a unique subset of neural cells in the rostral brain or from expression by a more widely distributed cell type, conditional on induction of region-specific extrinsic factors. In this study, we show that two brain-derived polypeptides, acidic and basic fibroblast growth factor (FGF), induce NEC1 secretion in cultured neural cells. Induction is detected within five hours after addition of FGF, is reversible, and is blocked by inhibitors of RNA and protein synthesis. Other growth and differentiation factors do not induce NEC1, except for 12-O-tetradecanoyl phorbol-13-acetate, which acts through a different signalling pathway. Acidic and basic FGF may be members of a group of extrinsic factors that determine region-specific NEC1 expression in neural tissues.

Nerve growth factor (NGF) is a polypeptide hormone which plays a central role in the development and growth of sympathetic and sensory neurons. The effects of NGF on target cells are mediated by a specific cell surface structure, nerve growth factor receptor (NGFr), which has been identified in human cells as a 75,000-mol-wt glycoprotein. We have used a monoclonal antibody to human NGFr to study cell-surface expression of the receptor on a panel of mouse-human neuroblastoma hybrids, and the serological typing results permit assignment of the gene coding for NGFr (NGFR) to chromosome 17q21-qter. In addition to mouse-human neuroblastoma hybrids, human NGFr was also detected on hybrids derived from fusions between mouse L-cell fibroblasts and human neuroblastoma and melanoma cells. Furthermore, induction of human NGFr expression was observed in hybrids derived from NGFr- human kidney epithelial cells and mouse L cells, but not in hybrids derived from human kidney epithelial cells and mouse RAG kidney carcinoma cells. These results suggest that cell-surface expression of human NGFr is controlled by trans-acting regulatory signals.

Expression of three distinct human cell surface antigens defined by monoclonal antibodies (mAbs) was examined in a series of rodent-human somatic cell hybrids retaining different subsets of human chromosomes. Cell surface reactivity with mAbs F8 and G253, detecting a 95 kilodalton (kD) glycoprotein (gp95); with mAbs F10 and A103, detecting a 50 kD glycoprotein (gp50); and with mAb S7 was found to cosegregate with human chromosome 19. However, differential antigen expression was observed with hybrids containing fragments of the 19 and hybrids constructed with different human cell types. Comparison of results from the serological typing with the presence of a number of chromosome 19 DNA markers in hybrid cells and cytogenetic analysis suggests that MSK20, the gene coding for the F10/A103 antigen gp50, is located in chromosome region 19pter----19p13.2. The genes coding for the F8/G253 antigen, gp95 (gene symbol MSK19) and the S7 antigen (MSK37) are located in region 19p13.2----19q13.2. Thus, the cell surface antigens described in this study may be used as selectable markers for specific portions of human chromosome 19.

We have identified and characterized an extracellular matrix (ECM) glycoprotein of cultured astrocytomas, NEC1, that is expressed in normal human brain parenchyma. Detailed immunohistochemical analysis reveals a region-specific NEC1 pattern along the rostrocaudal axis of the central nervous system (CNS), with strong expression throughout the white matter of telencephalon and diencephalon, scant expression in some areas of mesencephalon, and no expression in pons, cerebellum, medulla, spinal cord, and peripheral nervous system. NEC1 is not co-distributed with any known neural cell type, suggesting that expression of specific ECM proteins in the CNS is segmentally controlled.

Genomic DNA fragments encompassing the human Thy-1 or mouse Thy-1.1 gene have been microinjected into pronuclei of mouse embryos homozygous for the Thy-1.2 allele. In the resulting transgenic mice, the human gene is expressed in a pattern characteristic of normal human tissues, and is not influenced by the pattern of endogenous mouse Thy-1 expression. The mouse Thy-1.1 gene fragment is expressed in a pattern typical of mouse Thy-1, although it is more limited in its distribution. The results indicate the presence of multiple cis-acting regulators of Thy-1 gene expression that have changed in both their character and arrangement over the course of Thy-1 gene evolution.

The rapidly expanding list of monoclonal antibodies (MAbs) to human cell surface antigens provides reagents to probe the biology of malignant melanoma and to develop new diagnostic and therapeutic approaches to this disease. The criteria used to select MAb-defined antigens as targets for passive immunotherapy or immunolocalization of melanoma include: 1) consistent antigen expression in melanomas, 2) restricted antigen distribution in normal tissues and nonmelanocytic tumors, and 3) cytotoxic activity of the MAb or MAb conjugates. The present study examined the tissue distribution of three prototype melanoma cell surface antigens, the Mr 57,000 glycoprotein (gp57) recognized by MAb A42, the GD3 ganglioside, and the mel-CSPG chondroitin sulfate proteoglycan. The avidin-biotin immunoperoxidase method was used to examine a large panel of normal tissues and over 150 malignant tumors. It was found that A42 has a highly restricted distribution in normal tissues and is expressed in subsets of melanomas and nonmelanocytic tumors. It was also found that GD3 and mel-CSPG are more widely distributed in normal tissues and among tumors than was thought previously. These immunohistochemical patterns provide an essential data base to evaluate the ongoing clinical trials employing MAbs to GD3 and mel-CSPG for the therapy and immunolocalization of melanomas, and they identify gp57 as a potential marker for subsets of normal and transformed melanocytic cells.

Human neuroblastoma cells growing in culture offer a unique opportunity to study proliferating human cells with a neuronal phenotype. We have previously identified several neuroblastoma cell lines which show spontaneous conversion (N/S interconversion) between two morphologically distinct cell types: neuroblastic (N-type) cells and variant, substrate-adherent (S-type) cells resembling cultured glial or mesenchymal cells. In the present study, we have used molecular markers to confirm the neuronal phenotype of N-type cells and to demonstrate that S-type cells have a nonneuronal phenotype. Furthermore, we have used these markers, including a series of cell surface differentiation antigens, to compare S-type neuroblastoma cells with a wide range of cultured epithelial, mesenchymal, and neuroectodermal cells. The results suggest that N/S interconversion represents an ordered transition between two neuroectodermal differentiation programs rather than random phenotypic instability of cultured cells; S-type variant cells show a molecular phenotype most closely resembling the phenotype of cultured ectomesenchymal cells; and in vitro variant formation of human neuroblastomas may provide an experimental model for the observed in vivo transition of some malignant neuroblastomas into benign ganglioneuromas.

We have characterized five distinct cell surface antigens of human astrocytomas and correlated their expression with the expression of glial fibrillary acidic protein (GFAP) and four previously defined cell surface markers of astrocytomas. One of the newly studied antigens, A4, which was originally detected on rat central nervous system (but not peripheral nervous system) neurons, is expressed on GFAP+ human astrocytoma cells, but not on GFAP- astrocytomas or a wide range of other neuroectodermal, epithelial, and hematopoietic cells. Antigens F19 (Mr 140,000/90,000 glycoprotein) and F24 (Mr 90,000 glycoprotein) also show restricted distribution and are expressed on subsets of neuroectodermal and mesenchymal cells. Antigens G253 (Mr 95,000 glycoprotein) and S5 (Mr 120,000 glycoprotein) are more widely distributed on the cultured cell panel. The distribution of these antigens was determined on a series of 22 astrocytoma cell lines and in normal brain tissue and the results were compared with the distribution of 5 additional glial cell markers: GFAP and cell surface antigens A010 (Mr 110,000 glycoprotein); AJ8 (Mr 100,000 glycoprotein); LK26 (Mr 35,000 glycoprotein); and Thy-1. Distinct patterns of expression on cultured astrocytomas and in neural tissues were identified for all antigenic systems studied, and cell surface expression of antigen A4 was found to correlate closely with GFAP phenotype of cultured astrocytomas. The antigens described in this study provide new markers to study normal glial differentiation and to correlate the phenotypes and biological behavior of distinct subsets of astrocytomas.

The monoclonal antibody AbO13 defines a cell-surface antigen that is expressed on most cultured human cells, but not on rodent cells. AbO13 precipitates glycoproteins of 25,000 and 30,000 mol. wt. from lysates of [3H]glucosamine-labeled human cells. Results of the serological typing of a panel of 25 rodent-human somatic cell hybrid clones show that reactivity with AbO13 segregates with the human X and Y chromosomes. The presence of either of these chromosomes is sufficient for O13 expression on the hybrid cell surface. Analysis of hybrid clones containing human X chromosomes with karyotypically defined deletions permitted the regional assignment of the X-linked gene locus controlling the expression of O13 to Xp22-pter. In addition, AbO13 is reactive with Chinese hamster-human hybrids derived from fibroblasts of a 49,XXXXX individual that contained only inactivated copies of the human X chromosome. These results suggest that the X-linked locus determining the expression of O13 is not subject to X-inactivation.

The gene for myotonic dystrophy (DM) is located on the proximal long arm of chromosome 19 along with at least 10 cloned genes and anonymous DNA segments. In order to refine the map of this region of the chromosome, we have constructed somatic cell hybrid lines from fibroblasts carrying a balanced translocation t(1, 19) with a breakpoint at 19q12. We have established that D19S7 is the most proximal of the available long-arm markers and confirmed that PEPD localizes to 19q, along with PVS, MSK19, and MSK37. We have also examined the segregation of markers from the proximal long-arm region of chromosome 19 in hybrids containing fragments of this chromosome.

A panel of 22 monoclonal antibodies (MAbs) recognizing 21 distinct human cell surface antigens was tested by mixed hemadsorption assays for reactivity with a large number of rodent-human somatic cell hybrids containing different subsets of the human chromosome complement. The serological typing results permit the assignment of six gene loci determining cell surface antigens to human chromosomes 3, 6, 11, 19, 20, and 22. In addition, analysis of hybrids retaining deleted copies (but no normal homologs) of specific human chromosomes provides regional assignments for 18 gene loci, located on seven different chromosomes. These findings extend and refine the genetic map for human cell surface antigens and identify new selectable markers for defined chromosome segments.

Nerve growth factor (NGF) is a protein which promotes the survival and differentiation of neuronal cells in vitro and plays an important role in neuronal development. In this study, we have examined the expression of the receptor for NGF (NGFR) in human neuronal and nonneuronal cells, both in tissue culture and in vivo. In addition to cell lines derived from neuroblastoma, astrocytoma, and melanoma, all of which share a common neuroectodermal origin, NGFR was detected in a number of cultured cells of mesenchymal, epithelial, and hematopoietic derivation. Immunohistochemical analysis showed that NGFR is expressed in several nonneural human tissues, and the cell types in which NGFR was found include derivatives from all three germ layers. Thus, our findings demonstrate that NGFR is much more widely expressed in human cells and tissues than was previously thought.

Six distinct cell surface antigens of human trophoblast and choriocarcinoma were defined with MAbs. The distribution of the antigens was determined by MHA assays on 150 tumor cell lines and normal cell cultures and by immunofluorescence tests with a wide range of normal adult and fetal tissues and a tumor panel. Antigen LK26 is expressed on all cultured choriocarcinoma, teratocarcinoma and renal cancer lines but is absent from most cell lines derived from other tumor types and from cultures of normal kidney epithelium and fibroblasts. LK26 expression in normal tissues is restricted to the trophoblast. No other adult or fetal tissue was found to express the antigen, but choriocarcinoma and teratocarcinoma tissues were LK26+. SV19 is expressed on cultured choriocarcinomas and teratocarcinomas and on subsets of breast and colon cancer lines, but not on 120 additional cultures tested. In tissues, SV19 is detected in normal placenta, mammary gland and colon epithelium as well as in tumors of breast, colon and lung. Two antibodies, AbSV63 and AbK8, react with PLAP and AbSV63 also reacts with the intestinal form of the enzyme. AbLK24 defines a heat-stable determinant present on choriocarcinoma and breast cancer cell lines but absent from most other cultured cells. It is expressed on a small range of normal and malignant epithelial tissues, including normal trophoblast, normal breast epithelium and urothelium and tumors derived from these tissues. One antigen, K66, showed a wide distribution on cultured epithelial cells but was not found in any normal or malignant tissue. Finally, S4, a previously described marker of normal and malignant kidney epithelial cells, was also expressed on the choriocarcinoma cell lines. Four of the antigens are glycoproteins that could be immunoprecipitated from radiolabelled extracts of choriocarcinoma cells: LK26 (Mr 35,000), SV19 (Mr 40,000), PLAP (Mr 68,000) and S4 (Mr 160,000). The highly restricted distribution of LK26, SV19, S4, and PLAP in normal tissues and their expression in tumors make these antigens potential diagnostic markers of gestational choriocarcinoma and germ-cell tumors and, possibly, targets for immunotherapy.

Monoclonal antibodies (MAbs) K21 and K4 define two carbohydrate determinants, lacto-N-tetraose (LNT) and sialylated LNT (NeuAc2----3LNT). respectively, which are expressed on the surface of cultured human teratocarcinoma cells, but not on a wide range of other epithelial and nonepithelial cells in culture. The present study used immunohistochemical methods to examine LNT and NeuAc2----3LNT expression in normal human tissues, 29 germ cell tumors, and over 200 tumors of other histologic types. In addition, their distribution was compared with that of Lex, a previously characterized cell surface carbohydrate antigen. Our results show that LNT and NeuAc2----3LNT are consistently expressed in germ cell tumors and that each has a distinct, highly restricted distribution in normal tissues. LNT and NeuAc2----3LNT expression in germ cell tumors is predominantly localized to embryonal carcinoma and yolk sac components, whereas Lex is found primarily in teratoma components with intestinal and bronchial differentiation. Areas of seminoma variably express NeuAc2----3LNT and LNT but are Lex-negative. Immunohistochemical results obtained with different fixation procedures suggest that some cell types in vivo express NeuAc2----3LNT, LNT, and Lex determinants only on glycolipids, whereas other express them on both glycolipids and glycoproteins or only on glycoproteins. Thus, the tissue distribution of these antigens may be controlled by differential expression of the requisite glycosyltransferases as well as the availability of different classes of carrier molecules.