The prognosis of patients with advanced neuroblastoma remains poor despite recent progress in chemo/radiotherapy. Therapeutic trials on the induction of differentiation of neuroblastoma by chemical and biological agents have been attempted to improve patients' prognosis. Recently a new synthetic polyprenoic acid, E5166, having retinoic acid properties, has been described. In this study two human neuroblastoma cell lines, KP-N-RT(LN) and SK-N-DZ, were treated in vitro by E5166. Morphological differentiation of KP-N-RT(LN) and SK-N-DZ cells could be induced by E5166 in the presence of 1.7 X 10(-5) M E5166 for 10 days in culture. Levels of catecholamines (dopamine, adrenaline, and noradrenaline) were not elevated in the E5166-differentiated cells. E5166-induced differentiation may not be cyclic AMP dependent, since levels of cyclic AMP did not increase after exposure of cells to this agent. No significant increase in neuron-specific enolase levels could be demonstrated in E5166-treated neuroblastoma as compared to control untreated cells. E5166 treatment of KP-N-RT(LN) and SK-N-DZ cells was found to inhibit colony formation in soft agar in a dose-dependent manner. Colonies of KP-N-RT(LN) cells in the presence of E5166 showed morphological differentiation as defined by the expression of long neurite processes. E5166 is a less toxic reagent than the retinoic acids used for the induction of differentiation, it can be administered to patients p.o., and the concentration of E5166 which induces the morphological differentiation in vitro can be achievable in vivo. Therefore our study suggests that E5166 could be a useful therapeutic agent in advanced neuroblastoma to differentiate residual anaplastic tumor cells to a benign form (ganglioneuroma) after surgery and chemotherapy.

Cell Differentiation↗

PubMed1987

Novel non-isotopic in situ hybridization technique detects small (1 Kb) unique sequences in routinely G-banded human chromosomes: fine mapping of N-myc and beta-NGF genes.

A novel in situ hybridization technique is described. This non-radioactive technique combines, for the first time, the high spacial resolution and rapid signal development of the non-isotopic approach with the previously unrivalled sensitivity of autoradiography. The procedure, which employs biotin labelled DNA probes and a streptavidin-alkaline phosphatase based detection system, is compatible with pre G-banding and can be performed on archival material. Unique sequences as small as 1 Kb are detectable. Using this technique, we have mapped the N-myc oncogene and the gene for beta-Nerve Growth Factor to 2p24 and 1p13 respectively.

Alkaline Phosphatase↗

PubMed1987

Cell surface membrane antigen present on neuroblastoma cells but not fetal neuroblasts recognized by a monoclonal antibody (KP-NAC8).

Neuroblastoma (NB) arises from primitive sympathetic neuroblasts in the adrenal gland or the sympathetic ganglion. NB in situ, sometimes observed in the adrenal glands of autopsied infants, is considered to be a premalignant lesion that may develop into NB. Little is understood about the morphological and biochemical changes that accompany this malignant progression. In this study, a unique monoclonal antibody, KP-NAC8, raised against a human NB cell line is described. This binds to NB cells but not to fetal neuroblasts. The antibody recognizes a Mr 200,000 surface protein on NB cells. KP-NAC8 binds to 15 of 17 human NB cell lines and all 26 fresh NB samples either from tumor tissues or from marrow aspirates involved with tumor. The antibody was found to cross-react with some other tumor cell lines, namely, Ewing's sarcoma (1 of 2), melanoma (1 of 4), lung cancer (3 of 3), and leukemia (2 of 14) cell lines. However, KP-NAC8 did not bind to any rhabdomyosarcoma (0 of 4), Wilms' tumor (0 of 4), retinoblastoma (0 of 2), glioma (0 of 4), and gastric cancer (0 of 2) cell lines examined. Among fetal tissues, KP-NAC8 did not react with normal neuroblasts in the adrenal glands of 5 fetuses. In a further study, the membrane phenotype of fetal adrenal neuroblasts was analyzed by a panel of 12 monoclonal antibodies including KP-NAC8. A comparison of the binding of the same panel of antibodies to fresh NB revealed that antibodies UJ13A, UJ127:11, PI153/3, anti-Thy-1, A2B5, BA-1, BA-2, HSAN1.2, and Leu-7 bound to both fetal adrenal neuroblasts and NB cells. Monoclonal antibodies OKIa-1 and J5 did not bind to either tissues. The only antibody that could distinguish fetal adrenal neuroblasts from NB cells was KP-NAC8. KP-NAC8 may, therefore, define a differentiation-related antigen that may prove helpful in understanding the biological nature of NB and NB in situ.

Adrenal Glands↗

PubMed1987

The present role of monoclonal antibodies in the diagnosis and therapy of brain tumours.

Monoclonal antibodies already have an established role as tumour markers for use in histological and cytological diagnosis of tumours, such as to distinguish between astrocytomas, oligodendrogliomas and ependymomas. Most recently monoclonal antibodies are employed in combination with radioisotopes or cytostatic agents for radioimmuno-localisation and antibody-targeted therapy. In a preliminary study involving 12 patients with malignant gliomas the distribution of the antibody UJ = 13A labelled with J-131 has been examined. The ratio of radiolabel in normal to malignant tissue ranged from 3.1 to 12.8, however the absolute amount of labelled antibody reaching the tumour was around 0.001% of the injected dose. A phase I trial is presently conducted with an intrathecal radio-antibody therapy in cases of leptomeningeal malignancies resulting from various types of neoplasia. A clinical improvement was seen in 4 out of 5 cases, without serious side-effects. Such an antibody-guided irradiation would be most suitable for radiosensitive targets such as medulloblastomas, neuroblastomas and some tumours of the pineal region. The rapid advances that are currently made in our understanding of immunoregulation and T-cell function, as well as new technologies to gain human monoclonal antibodies by B-cell immortalisation and recombinant DNA technology and our preliminary results justify the great hope we have in immunotherapy.

Antibodies, Monoclonal↗

PubMed1987

A monoclonal antibody (FMG25) that can differentiate neuroblastoma from other small round-cell tumours of childhood.

The monoclonal antibody (MAb), FMG25, raised following immunization of mice with the human T cell line HUT 78, binds to human neuroblastoma but not to other small round-cell tumours of childhood (rhabdomyosarcoma and Ewing's sarcoma). The specificity of the reagent is paralleled on normal tissues binding to brain but not frozen sections of thymus, tonsil, lymph node and spleen. Of the 15 T-cell malignancies examined, only 3, belonging to the large T-cell type, were positive. This represents only 50% of this type examined. Only one early case of pre-B ALL was found to be positive for FMG25 binding out of 27 non-T-cell malignancies. The pattern of reactivity of FMG25 makes the MAb useful as a diagnostic reagent in paediatric pathology. In addition, the antibody may be useful as one of a panel of reagents applied to detect and remove tumour cells from bone marrow harvested for autologous transplantation.

Antibodies, Monoclonal↗

PubMed1987

The use of a panel of monoclonal antibodies in pediatric oncology.

A panel of monoclonal antibodies including antibodies against neuroectodermal antigens, (UJ13A, UJ127.11, UJ181.4), leukocytes (2D1), intermediate filament antigens cytokeratin (LE61), Vimentin, Desmin (Labsystems, Helsinki, Finland), myoglobin, and neurofilament (155) antigens were assessed for their use as an adjunct to light microscopy in pediatric pathology, with particular emphasis on the "small round cell" tumors. One hundred thirty-four tumors were studied using immunofluorescence and immunoperoxidase techniques. The differentiation of neuroblastoma from lymphoma proved to have a clear-cut immunologic profile, as did the rhabdomyosarcomas, which showed consistent positivity with neuroectodermal antibody UJ13A and in positive binding with antidesmin. Ewing's sarcoma did not give a clear immunohistologic pattern with these antibodies. This panel was shown to have been a valuable aid to diagnosis in 12% of cases studied. The future use of such a panel for routine diagnostic use is discussed, but it is emphasized that the binding pattern of these tumors is often heterogeneous, and examination in conjunction with conventional histology is essential if the correct conclusions are to be made.

Antibodies, Monoclonal↗

PubMed1987

Comparative localization of glioma-reactive monoclonal antibodies in vivo in an athymic mouse human glioma xenograft model.

Radioiodinated murine monoclonal antibodies (Mabs) 81C6, Me 1-14, C12, D12, and E9, made against or reactive with human gliomas but not normal brain, and Mab UJ13A, a pan-neuroectodermal Mab reactive with normal human glial and neural cells, were evaluated in paired label studies in the D-54 MG subcutaneous human glioma xenograft model system in nude mice. Following intravenous injection in the tail vein of mice bearing 200-400 mm3 tumors, specific localization of Mabs to tumor over time (6 h-9 days) was evaluated by tissue counting; each Mab demonstrated a unique localization profile. The comparison of localization indices (LI), determined as a ratio of tissue level of Mab to control immunoglobulin with simultaneous correction for blood levels of each, showed Mabs 81C6 and Me 1-14 to steadily accumulate in glioma xenografts, maintaining LI from 5-20 at 7-9 days after Mab injection. Mab UJ13A peaked at day 1, maintaining this level through day 2, and declining thereafter. Mabs D12 and C12 peaked at days 3 and 4, respectively, and E9 maintained an LI of greater than 3 from days 3-9. Percent injected dose localized/g of tumor varied from a peak high of 16% (81C6) to a low of 5% (Me 1-14 and UJ13A). Immunoperoxidase histochemistry, performed with each Mab on a battery of primary human brain neoplasms, revealed that Mabs 81C6 and E9, which demonstrated the highest levels of percent injected dose localized/g of tumor over time, reacted with antigens expressed in the extracellular matrix. This finding suggests that extracellular matrix localization of antigen represents a biologically significant factor affecting localization and/or binding in the xenograft model used. The demonstration of significant localization, varied kinetics and patterns of localization of this localizing Mab panel warrants their continued investigation as potential imaging and therapeutic agents for human trials.

Animals↗

PubMed1987

Immunohistochemical performance testing of monoclonal antibodies to neuroblastoma cells on normal adrenals, spinal and sympathetic ganglia, and neural crest tumours.

Tissue specimens of neural crest-derived structures like normal spinal and sympathetic ganglia and adrenals of adults, infants and fetuses were used as immunohistochemical test substrates for a panel of 10 monoclonal antibodies to neuroblastoma cells raised by immunization with a variety of immunogens (fetal brain, neuroblastoma cell lineages, chick retinal cells or purified Thy-1 antigen). Antigen expression varied among normal structures such as ganglion cells, satellite cells, various nerve fibres, and different cells in the adrenal cortex. This variability in immunoreactivity shown by different cell types and structures was comparable to that seen when the same panel of monoclonal antibodies was applied to neuroblastomas and to ganglioneuroblastomas of different degrees of maturation and differentiation. The neoplasms thus seemed to reflect the normal maturation in the neuroectodermally-derived structures. Also, the studies give indications as to the cell of origin of the undifferentiated neuroblastomas.

Adrenal Glands↗

PubMed1987

Immunohistochemical differentiation of neuroblastomas from other small round cell neoplasms of childhood using a panel of mono- and polyclonal antibodies.

A panel of monoclonal antibodies to neuroblastoma cells, leucocyte common antigen, vimentin and MHC class II antigens (HLA-DR) and a polyclonal antibody to epidermal keratin were used for immunohistochemistry on sections of ethanol fixed and paraffin embedded specimens from 40 undifferentiated small cell tumours and 10 neural crest neoplasms. With the exception of central nervous system neoplasms and two embryonal rhabdomyosarcomas, immunohistochemical examination discriminated between the neural crest neoplasms and the other small cell tumours. Moreover, the staining pattern of neoplastic cells and structures in the neural crest neoplasms obtained with antibodies to neuroblastoma cells seemed, in part, to reflect the degree of tumour differentiation.

Antibodies↗

PubMed1987

Diagnosis and therapy of neuroectodermally associated tumours using targeted radiation.

Monoclonal antibodies have been proposed as targeting agents for cytotoxic compounds in vivo. We have undertaken a phase 1 study of 131I UJ13A in patients with neuroblastoma as well as a pilot study for the intrathecal use of radiolabelled conjugates for diffuse malignant meningitis. Access to solid tumour deposits appears to be a major problem in targeting sufficient isotope to elicit a therapeutic response. This, however, is overcome in the intrathecal use of antibodies for diffuse disease. Problems have also been identified in changes in the pharmacokinetics of antibody handling that occur on repeated administration of conjugate to patient. This is presumably due to the generation of an anti-mouse Ig response. These studies indicate some of the limitations in using murine antibodies as targeting agents and point to areas where their use may be of maximal effect.

3-Iodobenzylguanidine↗

PubMed1987

High-resolution in situ hybridization technique using biotinylated NMYC oncogene probe reveals periodic structure of HSRs in human neuroblastoma.

Gene amplification in mammalian cells commonly manifests itself as homogeneously staining chromosomal regions (HSRs). These are frequently seen in neuroblastoma and have been shown to be the site of amplification of the NMYC oncogene. We have used a nonisotopic, high-resolution in situ hybridization technique to reveal a hitherto unrecognized periodic microstructure within HSRs of a human neuroblastoma cell line.

Biotin↗

PubMed1987

Comparison of pharmacokinetics of radiolabeled monoclonal antibody UJ13A in patients and animal models.

The antigen to which the monoclonal antibody UJ13A binds is expressed by normal primate brain and most tumors of neuroectodermal origin. The antibody has been used in radioimmunologic studies in patients with neuroblastoma and glioma, though the uptake of antibody into tumors is low. Animal models have been sought to allow definition of the parameters that influence tumor uptake. Animals include nude mice, in which xenografted human tumors can be studied, and marmosets, in which the primate response to exposure to murine antibodies can be investigated. This presentation reports the preliminary pharmacokinetic data from studies of mice and marmosets and compares the data with those obtained in patients.

Animals↗

PubMed1987

Neuroblastoma: new approaches to diagnosis.

Monoclonal antibodies can play an important part in distinguishing neuroblastoma from other small round cell tumours of childhood. In addition, they have been useful in the identification of metastatic tumour cells in bone marrow. Though these roles for antibodies are now well proven, other functions for these reagents such as radioimmunolocalization and targeted therapy need further evaluation. The problems in the diagnostic use of monoclonal antibodies are reviewed and an assessment given on the type of panels that can be constructed as diagnostic aids. Other new diagnostic approaches in vivo and in vitro are reviewed for comparison. The rapidly developing specialties of molecular biology and molecular cytogenetics will almost certainly have an impact on the diagnosis and prognosis of patients with neuroblastoma. Although these are currently research tools, in a short time they will be in use in routine diagnosis. With the increased basic research into neuroblastoma, it is hoped that it will not be too long before the overall survival rate for the disease is improved. In the meantime the new diagnostic approaches outlined in this paper will ensure that patients are treated with an appropriate chemotherapeutic regimen and will allow the selection of subgroups of individuals for new aggressive chemo/radiotherapeutic protocols.

Antibodies, Monoclonal↗