[The immunological cellular phenotype and the prognosis in neuroblastoma in children]. / Immunologicheskii fenotip kletok i prognoz pri neiroblastome u detei.

Ganglioside profile was evaluated in 19 samples of tumor tissue obtained from 13 surgical patients with various morphological patterns of neuroblastoma. In six of those cases, two samples from each proving most different in terms of cell maturity were selected for examination. The relative content of GD2 gangliosides was 27.0-37.6% in sympathoblastoma and as low as 5.1-14.8% in ganglioneuroblastoma. Ganglioneuroblastomas showed fairly high levels of GM1 and GD1a gangliosides which were almost completely absent in sympathoblastomas. Ganglioside profile variations seen within each tumor type were incomparable with differences in profile established between morphological patterns of neuroblastoma studied.

Synaptophysin: a sensitive and specific marker for ganglion cells in central nervous system neoplasms.

Synaptophysin, a 38-kilodalton glycoprotein found in synaptic vesicle membranes, has been shown to be a sensitive marker of neuroendocrine differentiation in non-central nervous system (CNS) tumors. We analyzed the patterns of synaptophysin immunoreactivity in CNS neoplasms in comparison with various normal CNS sites in biopsies. Normal gray matter structures all showed a diffuse punctate granular pattern of neuropil staining without staining of neuronal cell bodies. In contrast, neoplastic ganglion cells in 18 of 18 gangliogliomas/gangliocytomas showed intense immunoreactivity outlinging the borders of the cell bodies. Focal staining was also seen in five of 16 primitive neuroectodermal tumors and in one of three central neurocytomas, but these tumors had a finely granular neuropil pattern of immunoreactivity more like that of normal gray matter than like that of the gangliogliomas. All 35 examples of pure gliomas of various types showed no immunoreactivity. Our data highlight synaptophysin as a sensitive and specific marker of both neuronal lineage and neoplastic character in gangliogliomas.

Expression of pan-neuroendocrine proteins in 53 neuroblastic tumors. An immunohistochemical study with neuron-specific enolase, chromogranin, and synaptophysin.

The presence and distribution of pan-neuroendocrine markers such as neuron-specific enolase (NSE), chromogranin (CG), and synaptophysin (SYP) were investigated by immunohistochemistry in 53 cases of neuroblastic tumors, including three cases of ganglioneuromas, 17 ganglioneuroblastomas, and 33 neuroblastomas. In ganglioneuromas, all three markers were observed both in ganglion cells and in neurofibrils. All cases of ganglioneuroblastoma were positive for these markers, however, some variability of staining intensity was noted. Of the 33 cases of neuroblastomas, all were positive for NSE, 23 (70%) for CG, and 31 (94%) for SYP. Neuron-specific enolase was detected not only in the majority of the neuroblasts showing signs of differentiation, but also in some undifferentiated neuroblasts. Chromogranin was found mainly in differentiated neuroblasts with enlarged cytoplasm and nuclei, but was scarcely found in undifferentiated cells. Synaptophysin was detected in some undifferentiated neuroblasts, as well as in differentiated neuroblasts. Two cases without SYP-positive cells were also negative for CG. Our observations conclude that antibodies against NSE and SYP are helpful as a diagnostic aid for neuroblastic tumors.

Endogenous sugar receptor pattern in human glioblastomas and gangliocytomas studied by histochemical application of biotinylated (neo)glycoproteins and affinity chromatography.

Biotinylation of chemically glycosylated bovine serum albumin, yielding a panel of neoglycoproteins, and of desialylated, naturally occurring glycoproteins allowed to systematically evaluate presence and distribution of various types of endogenous sugar receptors in the sections of human glioblastomas and gangliocytomas by a routine histochemical procedure. Pronounced cytoplasmic staining with markers, carrying constituents of natural glycoconjugates, e.g. for beta-galactoside-specific receptors, contrasted with the different intensities, noticed for alpha- and beta-glucoside-specific receptors. Significant qualitative differences between the two tumor types were detected with N-acetyl-D-galactosamine- and sialic acid-carrying probes. Nuclear staining with only a part of the applied panel underscored the specificity of the protein-carbohydrate interaction. Fine structural features of the synthetic neoglycoproteins, e.g. the mode of coupling of the carbohydrate moiety to the protein, were found to exert a significant influence on their suitability as histochemical markers. On the basis of the histochemical results, exemplary biochemical analysis of certain classes of endogenous sugar receptors by affinity chromatography and subsequent gel electrophoresis, namely of beta-galactoside-, alpha-fucoside-, alpha-mannoside- and alpha-glucoside-specific proteins, revealed presence and characteristics of respective sugar receptors that can contribute to the histochemical staining. Similar extent of histochemical staining with the respective probes notwithstanding, the different tumor types exhibited qualitative differences in the expression of individual endogenous sugar receptors. The combined histochemical and biochemical analysis is supposed to be of conspicuous value for biological and clinical investigations on endogenous sugar receptors.

Flow cytometric DNA analysis of neuroblastoma and ganglioneuroma. A 10-year retrospective study.

Retrospective quantitative DNA analysis was done on 147 samples from 89 patients with neuroblastoma and ganglioneuroma using flow cytometry. In the neuroblastoma patients, nuclear DNA content was found to be a stable tumor marker irrespective of site (primary versus metastatic) and despite changes with time in tumor progression, maturation, or therapy. The occurrence of DNA aneuploidy, which was detected in 60% of the neuroblastoma patients, paralleled other favorable indicators and was highly associated with survival (P less than 0.001). Of clinical stage, age, primary site, sex, and DNA content, only stage and DNA content correlated with survival. Those patients with favorable stage and DNA aneuploidy had higher survival rates. Further, favorable stage and the presence of DNA aneuploidy were independent prognostic indicators. Abnormal DNA content was also detected in samples from ganglioneuromas in which significant numbers of ganglion cell nuclei were recovered. These results indicate a striking difference between neuroblastoma and adult tumors in which DNA aneuploidy is generally a poor prognostic sign and provide a molecular link between ganglioneuromas and their malignant counterparts.

Flow cytometric DNA analysis of neuroblastoma: correlation with prognostic variables and survival of patients.

The DNA ploidy of tumor tissues obtained from 41 patients with neuroblastoma, or ganglioneuroblastoma, which is the histologic variant of neuroblastoma, was determined by flow cytometry. The DNA ploidy was diploid in 7 tumors (5 neuroblastomas and 2 ganglioneuroblastomas) and aneuploid in 34 tumors (20 neuroblastomas and 14 ganglioneuroblastomas). The DNA ploidy of tumor cells did not correlate with the survival of patients and there was no correlation between the DNA ploidy of tumor cells and such prognostic variables, histology, primary site, staging of tumors and operative curability.

[Immunohistochemical detection of growth hormone-releasing factor (GRF) in neuroendocrine tumors]. / Immunhistochemischer Nachweis des Growth hormone-releasing factor (GRF) in neuroendokrinen Tumoren.

We examined formalin-fixed, paraffin-embedded human tumors (42) from autopsies and surgical specimens for the presence of human pancreatic GRF-40 using the unlabeled antibody peroxidase-antiperoxidase method to assess the prevalence of tumors containing GRF, to define their primary sites and cellular derivations, and to correlate clinical and pathological features. Two paragangliomas, 1 pancreatic endocrine tumor, 1 bronchial carcinoid and 1 ganglioneuroma were immunoreactive for GRF. Of the GRF-containing tumors, only one bronchial carcinoid and one paraganglioma were associated with acromegaly.

The morphology and neuroendocrine profile of pancreatic epithelial VIPomas and extrapancreatic, VIP-producing, neurogenic tumors.

The histology, histochemistry, and ultrastructure of 43 VIP-producing tumors (34 from the pancreas, one jejunal, six retroperitoneal and two mediastinic), 37 of which were associated with the WDHA syndrome, have been investigated on paraffin sections of primary or metastatic tumor tissue. The pancreatic and jejunal tumors showed all structural and secretory patterns of epithelial endocrine tumors, including expression of cytokeratin, neuroendocrine markers like neuron-specific enolase, chromogranins and synaptophysin, peptides like VIP, PHM, GRH, PP, insulin, neurotensin, glucagon, somatostatin and enkephalin, secretory granules, small clear vesicles, peculiar osmiophilic bodies, and occasional formation of tubules or microacini with specialized luminal surfaces. All the remaining tumors were neurogenic, showing either neurons and nerve fibers together with Schwann cells (ganglioneuromas and ganglioneuroblastomas) or endocrine cells (pheochromocytomas) reacting with VIP, PHM, NPY, enkephalin, somatostatin, neuron-specific enolase, synaptophysin, and MAP2 (but not cytokeratin, PP, or GRH) antibodies. A possible origin of pancreatic VIPomas from transformed pancreatic PP cells or ductular stem cells partially committed to differentiation along the PP cell line is suggested.

An immunocytochemical demonstration of calcineurin in human nerve cell tumors. A comparison with neuron-specific enolase and glial fibrillary acidic protein.

Human central and peripheral nerve cell tumors were examined in detail using antibodies to calcineurin, glial fibrillary acidic protein (GFAP) and neuron-specific enolase (NSE). Forty-eight formalin-fixed and paraffin-embedded specimens of human neuronal tumors, including 27 medulloblastomas, were examined. Calcineurin-positive cells were found in all peripheral nerve cell tumors and the two gangliogliomas, whereas 20 of the 27 medulloblastomas and one of the two cerebral neuroblastomas did not contain calcineurin-positive cells. Differentiation of cells along the neuronal lines was positively correlated with calcineurin immunoreactivity. NSE-positive cells were found in all of the tumors with the exception of the one cerebral neuroblastoma. NSE immunoreactivity was not invariably consistent with calcineurin immunoreactivity and non-neuronal cells were often positive. Calcineurin-positive cells were all devoid of GFAP, but NSE-positive cells expressed GFAP in some tumors. GFAP-immunoreactive cells were found only in central nerve cell tumors, and not in peripheral tumors. In addition, GFAP-positive cells in some tumors such as retinoblastoma and medulloblastoma morphologically revealed not only neoplastic but also reactive astrocytic features.

Synaptophysin: a reliable marker for medulloblastomas.

Synaptophysin is an acidic, integral membrane glycoprotein (Mr 38,000) of presynaptic vesicles in various neurons and neuroendocrine cells, and in tumours derived from such cells. By indirect immunofluorescence microscopy of cryostat sections, using the monoclonal antibody SY 38 to synaptophysin, a consistent positive immunoreactivity was observed in all medulloblastomas (n = 6) and neuroblastomas (n = 3) as well as a ganglioneuroma and a glioneuronal hamartoma. The presence of synaptophysin in medulloblastomas was confirmed biochemically by immunoblotting experiments. For purpose of comparison, the expression of intermediate-sized filament (IF) proteins was also examined. While neurofilament proteins were consistently expressed in the neuroblastomas (3/3), the ganglioneuroma and the glioneuronal hamartoma, IF distribution in medulloblastomas was variable. A neurofilament-positive type of tumour (1/6) could be distinguished from vimentin-expressing neoplasms (4/6) by immunocytochemistry. These data indicate that synaptophysin is a reliable marker for medulloblastomas as well as other differentiated and undifferentiated neuronal tumours and in this respect is superior to the more heterogeneous expression patterns of IF proteins in these tumours.

Aspiration cytology and immunohistochemistry of a pheochromocytoma-ganglioneuroma of the adrenal gland.

The results of fine needle aspiration (FNA) cytology, immunohistochemical staining and electron microscopy of combined pheochromocytoma-ganglioneuroma are presented. The cytologic findings included clusters of neoplastic cells with features of a neuroendocrine tumor and aggregates of spindle-shaped cells with interspersed ganglionlike cells. Aspirated material was tested for a panel of hormones by the peroxidase-antiperoxidase method. The neoplastic cells showed positive staining for neuron-specific enolase (NSE) and vasoactive intestinal polypeptide (VIP). Electron microscopy demonstrated granules similar to those of a pancreatic VIPoma in the nerve processes of the ganglioneuroma component. This appears to be the first description of the FNA cytology of a combined pheochromocytoma-ganglioneuroma. The value of immunohistochemical staining in the study of FNA material for making a specific diagnosis and the clinical implications of a preoperative diagnosis in the management of this lesion are discussed.

Gangliorhabdomyosarcoma: a histopathological and immunohistochemical study of three cases.

A histopathological and immunoperoxidase study on three cases of genitourinary gangliorhabdomyosarcoma using a spectrum of conventional staining methods and antibodies against myoglobin, neuron-specific enolase and S-100 protein is presented. The results of the study have shown that differentiated myoblasts, ganglion cells and Schwann cells reacted positively with the particular antisera, but the majority of undifferentiated cells were negative. From the immunopathology results it was not possible to determine whether the undifferentiated cells were precursors of neural cells or myoblasts; the histological appearance resembled that of mesenchymal cells commonly seen in rhabdomyosarcomas. Theories concerning the origin of these tumours from neural crest ectomesenchyme or from neural crest and somitic mesenchyme are considered. Further study is needed to establish their histogenesis.

S-100 protein expression in satellite and Schwann cells in neuroblastoma. An immunohistochemical and ultrastructural study.

Immunohistochemical evidence has recently been provided that in the normal adrenal medulla as well as in autonomic ganglia, satellite cells and Schwann cells react with S-100 protein antiserum. In the light of these data, we investigated primary peripheral neuroblastoma and ganglioneuroblastoma to determine firstly whether both cell populations actually exist in the malignancies, using the definite criteria of electron microscopy for their identification, and secondly whether they express S-100 protein using on immunohistochemical technique and light microscopy. The results indicate that in both neuroblastoma variants, satellite and Schwann cells are present and specifically express the S-100 antigen.

Expression of neurofilament triplet proteins in human neural tumors. An immunohistochemical study of paraganglioma, ganglioneuroma, ganglioneuroblastoma, and neuroblastoma.

Intermediate filaments which are specific to neural cells, ie, neurofilaments, consist of three subcomponents--68, 150, and 200 kd. Thirty human neural tumors were examined for the presence of these three subcomponents by means of their monospecific antisera. All 8 paragangliomas contained cells that were positive for the 68-kd component, but only 5 of them had cells positive for the 150-kd and 200-kd components. All 4 ganglioneuromas and 11 ganglioneuroblastomas contained cells that reacted with antibodies to all three components. All 7 neuroblastomas had cells reacting with antibody to 68 kd, but only 3 of them had cells that reacted with antibodies to 150 kd and 200 kd. In each case, the number of positive cells depended on the antibody used. The largest number reacting with antibody to 68 kd and the smallest with antibody to 200 kd. Furthermore, it was possible to detect tumor cells in which the 68-kd subcomponent existed by itself, but no tumor cells in which the 150-kd or 200-kd subcomponent existed alone could be detected. These results seem to indicate that antibody to the 68-kd component is sufficiently discriminating to be applied diagnostically.

The isolation and sequence analysis of vasoactive intestinal peptide from a ganglioneuroblastoma.

A ganglioneuroblastoma was excised at surgery from a 1-yr-old girl with severe watery diarrhea. The tumor, weighing 1 g, was extracted in trifluoracetic acid and contained 8.3 nmol immunoreactive vasoactive intestinal peptide. The peptide was isolated by affinity chromatography and high pressure liquid chromatography and was found to be identical to porcine vasoactive intestinal peptide by amino acid analysis and microsequence analysis.

Immunohistochemical study on the distribution of alpha and beta subunits of S-100 protein in human neoplasm and normal tissues.

The immunohistochemical distribution and localization of the alpha and beta subunits of S-100 protein in human neoplasms and normal tissues were studied by the PAP method using monospecific rabbit antibodies against each subunit. Beta subunit immunoreactivity was detected in all S-100-positive cells and tumors reported previously. In contrast alpha subunit immunoreactivity was absent from Schwann cells, schwannomas, neurofibromas, granular cell myoblastomas, pituicytes of the neurohypophysis, Langerhans cells, interdigitating reticulum cells, and histiocytosis X cells. Interestingly, only the alpha subunit was detected in neurons of both central and peripheral nervous system, and in lymph node macrophages. Human S-100-positive cells are divided into three groups; the first is composed of cells containing only the beta subunit (probably S-100b; beta beta), the second consists of cells containing both the alpha and beta subunits, and the third is composed of cells containing only the alpha subunit (probably S- 100ao ; alpha alpha). The ontogentic relationships between S-100-positive cells and tumors are discussed in the light of these findings.

Concanavalin A binding and neuronal differentiation. A light microscopic study on neuronal tumours.

Concanavalin A (Con A) acceptors have been demonstrated in large differentiated neurons in a previous paper. In order to elucidate the correlation between Con A binding in normal and neoplastic neurons and lectin binding dependence upon the differentiation grade, 26 tumours of the neuronal series were examined using formalin fixed and paraffin embedded biopsy specimen. The neoplasms included 3 gangliocytomas, 7 gangliogliomas, 1 central neuroblastoma, 11 medulloblastomas, 2 retinoblastomas, and 2 sympathicoblastomas. Well differentiated neurons in gangliocytomas and gangliogliomas expressed a high intracytoplasmic Con A acceptor density comparable to the feature in large non-neoplastic neurons. Less differentiated neurons and neuroblasts showed a weak perinuclear fine granular binding or an absolute lack of binding molecules, respectively. Our results suggest that in a variety of tumours, Concanavalin A receptor density in neurons depends upon the degree of differentiation of the cell. Well differentiated cells have a higher density than poorly differentiated neoplastic neurons.

Neuropeptide Y in phaeochromocytomas and ganglioneuroblastomas.

Tumour tissue from nineteen patients with phaeochromocytomas and nine with ganglioneuroblastomas contained large numbers of neuropeptide Y (NPY) producing cells and extracts of these tumours had very high concentrations of immunoreactive NPY. Plasma NPY concentrations were also raised, averaging 460 pmol/l in patients with tumours of the sympathetic chain and 55 pmol/l in healthy controls. Since plasma NPY is straightforward to measure and relatively stable, its estimation may prove helpful as a screening tests for phaeochromocytoma.