Primitive neuroectodermal tumors of the central nervous system express neuroendocrine markers and may express all classes of intermediate filaments

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Primitive Neuroectodermal Tumors of the Central Nervous System Express Neuroendocrine Markers and May Express All Classes of Intermediate Filaments VICTOR E. GOULD, MD, LUCY B. RORKE, MD, DESIREE SoJANSSON, BS, WILLEMINA M. MOLENAAR, MD, PHD, JOHN Q, TROJANOWSKI, MD, PHD, VlRGlNlA M. Y. LEE, PHD, ROGER J. PACKER, MD, AND WERNER W. FRANKE, PHD THE CENTRAL NERVOUS SYSTEM PRIMITIVE NEUROECTODERMAL TUMOR CONCEPT: HISTORICAL BACKGROUND AND CONTROVERSY

ently based on a misunderstanding of Schaper’s earlier embryologic concept of “indifferent cells”4 which occurred throughout the developing central nervous system (CNS), and had the capability to evolve into neurons or glial elements. The confusion was compounded by Kershman, who suggested that indifferent cells were unique to the cerebellum and corresponded to granular cells, and thus justified the notion that medulloblastomas were exclusively cerebellar tumors.5 The latter idea contradicted observations by Cushing,6 and by Bailey’ who noted that medulloblastomas were most common in the cerebellum of children but also found that histologically indistinguishable tumors occurred in the cerebrum and spinal cord. Bailey even underlined the histologic similarities between medulloblastoma and retinoblastoma. Evident contradictions notwithstanding, it became customary to designate as medulloblastomas those primitive-appearing tumors arising in the cerebellum, while histologically similar tumors arising in other CNS sites received diverse designations including neuroblastomas, ependymoblastomas, and pineoblastomas, among many others.g-15* for overviewsee l6 In 1973, Hart and Earle described a group of 23 poorly differentiated cerebral tumors in children and young adults which they designated as “primitive neuroectodermal tumors” (PNETs) since they did not fit into the defined groups of other “primitive” neoplasms, ie, medulloepitheliomas, neuroblastomas, polar spongioblastomas, ependymoblastomas, or medulloblastomas’O; they noted that some of those tumors showed glial and/or neuronal features, andtall had a “mesenchymal” component. Subsequently, other observers reported similar tumors in other CNS sites, and stressed the “primitive neuroectodermal” designation. 17*18 At present, most neuropathologists follow the tradition of calling medulloblastomas those PNETs arising in the cerebellum, while applying different diagnostic designations to similar tumors arising elsewhere in the CNS. The logic of this approach has been challenged; one of us (L.B.R.) argued that

In 1925, Bailey and Cushing described a group of childhood cerebellar tumors comprised of primitive-appearing cells ‘; they initially thought that those tumors “derived” from glial progenitors, ie, spongioblasts, and named them “spongioblastoma cerebelli”. However, Globus and Strauss had concomitantly applied that designation to another tumor type, currently known as glioblastoma multiforme.2 Bailey and Cushing therefore adopted a new diagnostic term: “medulloblastoma cerebelli”‘; they also abandoned the concept of spongioblastic origin in favor of derivation from a hypothetical, multipotential cell, ie, the medulloblast, which they thought was capable of differentiating into astrocytes, oligodendrocytes, and neurons.3 Bailey and Cushing attempted to define their neoplastic offspring by the then-prevalent histogenetic approach; accordingly, they suggested that medulloblasts were one of five stem cells populating the primitive neural tube.3 This notion was apparFrom the Department of Pathology, Rush Medical College, Chicago, IL; the Departments of Pathology and Neurology, The Children’s Hospital, Philadelphia, PA; the Department of Pathology, Swedish University of Agricultural Sciences, Uppsala, Sweden; the Department of Pathology, University of Pennsylvania, Philadelphia, PA; and the Institute of Cell and Tumor Biology, German Cancer Research Center, Heidelberg, Federal Republic of Germany. Supported in part by the Otho S. A. Sprague Memorial Fund (to V.E.G.), and by NIH grant CA-36245 from the National Cancer Institute (to J.Q.T.) Dr Molenaar was a Fulbright Scholar on leave from the University of Groningen, Groningen, The Netherlands. Key words: central nervous system tumors, immunocytochemistry, cytoskeleton, intermediate filaments. Address correspondence and reprint requests to Victor E. Gould, MD, Department of Pathology, Rush Medical College, Chicago, IL 60612. 01990 by W.B. Saunders Company. 0046-S 17719012 103-0002$5.00/O

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teins (NFP).23,26,30-35 I n a retrospective immunocytochemical study based on paraffin sections of 46 PNETs, GFP was shown in 38 cases and vimentin in 15, while NFP was noted in only three instances.31 In a subsequent prospective study of six cases, GFP was noted in paraffin sections in all cases while vimentin and NFP were not observed in any of them. However, when adjacent freshly frozen samples of the identical cases were analyzed by immunoblot, GPF, vimentin and NFP were recognized in six, three, and four instances, respectively. 32 A recent study of cerebellar PNETs emphasizing the classical neuroblastic rosettes and the “pale islands” or “follicles” revealed GFP, NSE and tubulin immunoreactivity as well as neural features by electron microscopy; yet no immunostaining was obtained with an antibody said to recognize the 150/200 kd subunits of NFP.23 Most of the aforementioned IF studies on PNETs were carried out on variably fixed and paraffin-embedded tissue samples. Therefore, the fact that GFP was the most consistently demonstrated IF protein should not be surprising, considering that a number of available GFP antibodies bind to epitopes that are not inactivated by aldehyde fixation and routine tissue processing. Conversely, epitopes of vimentin, NFP, desmin and certain cytokeratin polypeptides are far more sensitive, and are either destroyed, masked or otherwise rendered unavailable for binding by such treatment, particularly if the level of expression is low. These phenomena may explain many falsely negative reactions noted in CNS PNETs as described above.

the “primitive” cells of those CNS tumors were indeed their dominant characteristic, and proposed that all tumors with those histologic features be classified as PNETs regardless of location in the CNS.“j This concept has gained support, and was incorporated into the 1985 World Health Organization classification scheme of pediatric CNS tumors.lg Yet serious scholars have reserved judgment,*O or have maintained that cerebellar medulloblastomas, cerebral neuroblastomas, ependymoblastomas, et al, are distinct entities, and have advanced conjectural histogenetic notions in support of their contention,21-23 despite the fact that our knowledge of the normal development of the CNS is incomplete, and that the very idea of classifying tumors based on restrictive histogenetic assumptions has been seriously questioned.24s25 Thus, the controversy goes on. CYTOLOGIC AND IMMUNOHISTOCHEMICAL FEATURES OF CENTRAL NERVOUS SYSTEM PRlMlTlVE NEUROECTODERMAL TUMORS The “primitive neuroepithelial cells” typical of CNS PNETs have been extensively described.“j By light microscopy, they have been said to be small to medium sized, round to oval with basophilic nuclei, and with or without detectable nucleoli; cytoplasm is scanty. By electron microscopy, their complement of organelles is variable but is generally scanty; conspicuous cytoplasmic processes, small neurosecretory granules, arrays of microtubules, synaptic-type structures, and other features consistent with neuronal differentiation have been noted in some studies but not in others; these inconsistencies are not surprising given the evident heterogeneity shown by many PNETs at the light microscopic level. By immunocytochemistry, neuron-specific enolase (NSE) has been demonstrated in PNETs.‘~ In this context, this finding may indeed be significant; however, we should recall that the gamma-subunit of NSE may be expressed by non-neuronal, and nonneuroendocrine (NE) cells. Therefore, NSE can no longer be regarded as a specific neural or NE marker.*(j A PNET-medulloblastoma cell line and its xenografts were shown to immunostain with the “pan-neuroectodermal” monoclonal antibody (Mab) UJ13A. *’ Notably, while the precise characteristics of the antigen recognized by Mab UJ 13A have not been defined, it was noted that it also reacted with pulmonary oat cell carcinomas 28 and, whereas the latter cannot be currently said to be neuroectodermal tumors, they are indeed NE tumors.2g The complement of intermediate filament (IF) proteins expressed by PNET-medulloblastomas and other CNS PNETs has been given relatively little attention; a few small series have been studied and the findings have been variable and apparently contradictory. The IF class most frequently described in PNETs has been glial filament protein (GFP) which was found either alone or variably coexpressed with vimentin and/or, less often, with neurofilament pro-

RECENT IMMUNOHISTOCHEMICAL WESTIGATIONS ON FROZEN SAMPLES OF CENTRAL NERVOUS SYSTEM PRIMITM NEUROECTODERMAL TUMORS We studied by immunocytochemistry the patterns of expression of synaptophysin,26T36*37 all classes of IF proteins, and desmoplakins I and 113* in frozen samples of 22 PNETs. 3g Eighteen tumors were primary in the posterior fossa, three in the cerebrum, and one in the spinal cord. Antibodies to all classes of IF proteins recognizing diverse epitopes were used. In the case of cytokeratins, we applied broad spectrum antibodies (AEl-AE3, Lu-5), and antibodies specific for cytokeratin 18. All cases were studied by the avidin-biotin-peroxidase complex method; 11 cases were also studied by indirect fluorescence microscopy, and several cases were studied with doubleand triple-label immunofluorescence microscopy for various possible antigenic coexpressions. A subgroup of 10 cases was investigated for the expression of four selected neuropeptides: bombesin, somatostatin, substance P, and vasoactive intestinal polypeptide. Selected samples were studied by two-dimensional gel electrophoresis and immunoblot analysis of IF proteins. Preliminary reports on these studies have been recently published.3gr40 246

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We found that all cases expressed synaptophysin. The expression was invariably extensive; in those cases with insular architecture, synaptophysinexpressing cells were found within and outside the islands. Notably, staining was observed predominantly in small “undifferentiated” cells but was also seen in large, stellate cells with coarse processes. The immunoreaction showed the well-described finely punctate patterns (Fig 1). In the 10 cases studied, variable expression of at least two neuropeptides was found; the materials most often noted were somatostatin and substance P (Fig 2). Twenty-one of 22 PNETs were not immunostained with desmoplakin antibodies; in a single case, focal and questionable reactivity was noted. All 22 PNETs showed variable but often extensive areas comprised of cells that reacted with vimentin antibodies. Twenty-one of 22 cases were immunostained for GFP, and 16 of 22 expressed one or more of the subunits of NFP (Figs 3-6). Vimentin and GFP reactions were characteristically coarse, whereas NFP staining was noted either in fine neuropils or in coarse globular and annular profiles as observed in some carcinoids and NE carcinomas.41 All these IF proteins were noted within and outside the islands, and, whereas reactivity predominated in undifferentiated-appearing small cells, staining was also noted in the large astrocyte-like cells. Four of 22 PNETs had either isolated cells or occasional cell clusters that expressed desmin (Fig 7). While the desminimmunoreactive cells were indeed convincing, the actual amounts of the antigen present in those tumor regions were quite limited. Thus, desmin was not detected on two-dimensional gel electrophoresis by Coomassie blue or Ponceau S staining, but it was shown in immunoblots with ‘251-labelled secondary antibodies. Three of 22 PNETs had isolated cells that immunostained with broad spectrum cytokeratin antibodies as well as with antibodies specific for cyto-

FIGURE 2. PNET immunostained with substance P antiserum: aggregate of tumor cells and some delicate and coarse processes are strongly reactive. (Avidin-biotin-peroxidase complex hematoxylin-eosin counterstain, magnification x 420.)

keratin 18 (Fig 8). Interestingly, the staining patterns of cytokeratin-positive cells were not reticular or finely linear or ftbrillar but rather coarse and uneven as described in Ewing’s sarcomas.42 Double- and triple-labeled immunofluorescence microscopy demonstrated various antigens coexpressed in individual cells; these included synaptophysin with vimentin, GFP, NFP (Fig 9) and desmin, vimentin-GFP, vimentin-NFP, vimentin-desmin, vimentin-cytokeratin and NFP-desmin (Fig 10). Coexpression of vimentin-GFP-NFP, vimentinGFP-desmin, and vimentin-GFP-cytokeratin was also noted.

CENTRAL NERVOUS SYSTEM PRIMITIVE NEUROECTODERMALTUMORS AS NEUROENDOCRINENEOPIASMS: COMMENT AND OVERVIEW The

consistent expression of synaptoand of neuropeptides by CNS PNETs indicate that they share very significant NE markers phyc&26,36.37

FIWRE 1. PNET; immunofluorescence microscopy with synaptophysin antibody. Strong and diffuse staining in an “island” is evident; note also staining in cells outside the island, in segments of large stellate cells (arrows), and the punctate pattern of the reaction, All photos are from frozen sections that were air-dried and then briefly fixed in cold acetone. (Avidin-biotin-peroxidase complex hematoxvkn-eosin counterstain, magnification x 340.)

FIGURE 3. PNET immunostained wlth vimentin antim, strong reactivity is noted In perikarya, delicate and coarse processes, and In stellate cells. (Avidin-biotin-peroxidase complex hematoxyllneosin counterstain, magnification x 420.)

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FIGURE 4. PNET immunostained with GFP antibody; intense and diffuse reactiiity is noted in perikarya of small cells and in processes of variable thickness. (Avidin-biottn-peroxidase complex hematoxylin-eosin counterstain, magnification x 420.)

FIGURE 6. PNET immunostained with antibody to the 68 kd subunit of NFP; reactivity is noted in some perlkarya, and in delicate and coarse processes. (Avidin-biotin-peroxidase complex hematoxylin-eosin counterstain, magnification x 420.)

with classical peripheral neural NE neoplasms (eg, neuroblastomas and pheochromocytomas-paragangliomas), and with epithelial NE neoplasms (eg, hypophyseal and islet-cell adenomas, medullary thyroid carcinomas, and carcinoids and NE carcinomas from diverse primary sites). 26,2g,36*41Yet our finding that CNS PNETs frequently express three classes of IF protein (vimentin-GFP-NFP), and occasionally may even add desmin and cytokeratins to their IF complement, indicates that they differ substantially in their cytoskeletal profile from peripheral NE tumors. We should recall that neural NE tumors express NFP for the most part exclusively,26 and under exceptional conditions coexpress NFP-cytokeratins.43 On the other hand, epithelial NE tumors express either cytokeratins with desmoplakins exclusively, or coexpress them with NFP, and rarely with vimentin.26,44,45 Thus, whereas PNETs show significant antigenic homologies with peripheral NE tumors, their complex IF complement would place them in a distinct category of NE neoplasms. The IF protein complement of CNS PNETs is exceedingly complex. Interestingly, embryonic glial

cells express vimentin,46 and embryonic neurons may express vimentin alone or with NFp46-51; desmin in developing neurons has also been shown.51 Yet it remains that the IF protein complement of CNS PNETs is dominated by vimentin-GFP-NFP while desmin and cytokeratins do not appear to be “constitutive” proteins but seem to represent options only sporadically and focally exercised. 52 Notable, however, is that large numbers of small, apparently undifferentiated cells may express features of mesenchymal, neuronal, glial, myoid, and epithelial differentiation routes while coexpressing NE antigens. Some of these peculiar coexpression patterns were also noted in the astrocyte-like cells (see below). The cytoskeletal profile of PNETs is distinct from that of other CNS tumors such as meningiomas which coexpress vimentin-desmoplakins,53 and gliomas and ependymomas which mostly, though variably, express GFP-vimentin54; moreover, none of these tumors seem to express synaptophysin.26 Predictably, the rather rare gangliogliomas were shown to express synaptophysin, but their IF protein profile appears restricted to NFP-GFP-vimentin. Notable

FIQURE 5. PNET immunostalned with GFP antibody: in this field strong reactivity is evident in large stellate cells. (Avidinbiotln-peroxtdase complex hematoxylin-eosin counterstain, magnification x 540.)

FIQURE 7. PNET immunostained with antibody of desmln; aggregate of small tumor cells, many of which are strongh/ reactive. (Avidin-biotin-peroxidase complex hematoxylin-eosin counterstain, magnification x 420.)

PRIMITIVE NEUROECTODERMAL TUMORS (Gould et al)

dermal” tumors55 remains to be clarified. However, at least in rare cases, bone tumors may display NE and IF profiles distinct from Ewing’s sarcomas but reminiscent of those of CNS PNETs.~~ These observations may prove useful in various differential diagnoses. The presence in most CNS PNETs of large stellate cells is well known. It has long been argued whether they represent “trapped” reactive astrocytes or neoplastic cells. Many scholars seem to have favored the former view. In agreement with earlier reports we found that those cells expressed vimentin and/or GFP; yet we also found that some of those cells reacted with antibodies to synaptophysin, NFP, desmin, and cytokeratins. The presence of these proteins argues for NE, neuronal, myoid, and epithelial differentiation pathways which may be seen as casting doubt on the idea that they are reactive astrocytes. Thus, a subpopulation of these cells may be neoplastic, as suggested by their presence in spinal seedings, and in true metastases of PNETs.~~ Still, this notion may be tempered by recent evidence indicating that astrocytes in culture may express neuropeptide genes.57 Doubts about the justification for classifying CNS PNETs as a group of tumors have been expressed.20-23 Unquestionably, while most of these tumors are dominated by apparently undifferentiated cells, some cases may exhibit marked diversity and may display variable admixtures of neuronal, glial, and

FIGURE 8. PNET immunostained with antibody to cvtokeratin 18; several cells show strong reacttvii in the form of coarse clumps (arrows]. [Avidin-biotin-peroxidase complex. hematoxylin-eosin counterstain, magnification x 420.)

also are the differences in IF protein complement and NE-antigen expression between CNS PNETs and certain pediatric peripheral small-cell cancers, particularly classical Ewing’s sarcomas. The latter were shown to coexpress vimentin-desmoplakins, have cytokeratin and NFP subpopulations, but do not express synaptophysin. 42 The extent, if any, of the homology of NE- and IF-protein expression between CNS PNETs and certain peripheral “neuroecto-

FIWRE 9. PNET; double labeled immunofluorescence microscopy with antibodies to svnaptophvsin [left) and NFP [right); more cells appear to express synaptophysin than NFP but the majoritv are positlve for both. Note the punctate pattern of svnaptophvsin and the coarse pattern of NFP; also, reacttvity involves small cells as well as a very large stellate cell in the right lower field. (Magnification x 620.)

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FIGURE IO. PNET; double label immunofluorescence microscopy with antibodies to NFP [left) and desmin (right); more cells and larger segments thereof react for NFP than for desmin; yet, a number of individual cells cc-express both IF proteins (arrows). Note also desmin reactivity in coarse cell processes. [Magnification x 620.)

omas,2g~63 designations

as vague as “small-cell cancer” or as incongruous as “undifferentiated neuroendocrine carcinoma” are still applied. We, and PNETs, hope for a kinder and gentler destiny.

ependymal features. Indeed, our immunophenotypic findings not only confirmed but widened the scope of that diversity; yet, our findings also underline a very significant homology-their NE differentiation. While not intending to exacerbate the polemic surrounding PNETs, we suggest that the unique overall antigenic profile of CNS PNETs would justify their consideration as a distinct group. Admittedly, PNETs comprise a heterogeneous clinicopathologic group, but, in this aspect, they do not differ from other broad groups of epithelial visceral neoplasms.58*5g Additionally, whether significant clinical correlations may be derived from the subsets of CNS PNETs defined by these parallel studies that showed variable expressions of NFP isoforms,60 is yet to be determined. CNS tumors currently termed PNETs were known by the turn of the century; they were said to lack differentiation, and were often classified as “sarcomas.“24 Today, the systematic application of molecular markers indicates that these tumors manifest a distinctive, albeit complex and variable phenotypic profile. Thus, regardless of taxonomic preferences, we can no longer view them as undifferentiated. A notable historical parallel is afforded by “small-cell lung cancers”; these were also regarded as “sarcomas” until the late 1920s.61 While they were shown to have neurosecretory granules in 1968,@ and were subsequently proven to be NE carcin-

Acknowledgment. The gathering and excellent preservation of the tissue samples were achieved thanks to the consistent cooperation of Drs. Luis Shut, Leslie Sutton and Arno Fried, and of Kathy Bonner, RN. Thanks are also due to C. Kuhn and S. Winter for outstanding technical work, for secretarial support.

and to Anne-Marie

Fornabaro

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NEUROECTODERMAL TUMORS (Gould et al) tern tumors using monoclonal antibodies against neurofilaments and glial filaments. HUM PATHOL 15:248, 1984 32. Tremblay GF, Lee VMY, Trojanowski JQ: Expression of vimentin, glial filament and neurofilament proteins in primitive childhood brain tumors. A comparative immunoblot and immunoperoxidase study. Acta Neuropathol (Berl) 68:239, 1985 33. Trojanowski JQ: Cytoskeletal proteins and neuronal tumors, in Colvin RB, Bhan AK, McCluskey RT (eds): Diagnostic Immunopathology. New York, NY, Raven, 1988, pp 225-243 34. Trojanowski JQ: Neurofilament proteins and neuroectodermal brain tumors, in Osborn M, Weber K (eds): Cytoskeletal Proteins in Tumor Diagnosis. Cold Spring Harbor, NY, Cold Spring Harbor Laboratory, 1989, .gp 11-18 35. Dehner LP, Abenoza P, SI ey RK: Prtmary cerebral neuroectodermal tumors: Neuroblastoma, differentiated neuroblastoma, and composite neuroectodermal tumor. Ultrastruct Path01 12:79, 1988 36. Gould VE, Lee I, Wiedenmann B, et al: Synaptophysin: A novel marker for neurons, certain neuroendocrine cells and their neoplasms. HUM PATHOL 17:979, 1986 37. Schwechheimer K, Wiedenmann B, Franke WW: Synaptophysin: A reliable marker for medulloblastomas. Virchows Arch [A] 411:53, 1987 38. Cowin P, Kapprell H-P, Franke WW: The complement of desmosomal plaque proteins in different cell types. J Cell Biol 101:1442, 1985 39. Gould VE, Jansson D, Molenaar WM. et al: Immunohistochemical profile of primitive neuroectodermal tumors. XVth World Conf Anat Clin Pathol, Florence, Italy, 1989, p 15 (abstr) 40. Molenaar WM, Jansson D, Gould VE, et al: The immunophenotype of central primitive neuroectodermal tumors (PNETs). J Neuropathol Exp Neurol 48:359, 1989 (abstr) 41. Gould VE, Moll R, Moll I, et al: Neuroendocrine (Merkel) cells of the skin. Hyperplasias, dysplasias and neoplasms. Lab Invest 52:334, 1985 42. Moll R, Lee I, Gould VE, et al: Immunocytochemical analysis of Ewing’s tumors. Patterns of expression of intermediate Iilaments and desmosomal proteins indicate cell type heterogeneity and pluripotential differentiation. Am J Path01 127:288, 1987 43. Franke WW, Gould C, Achstaetter T: Co-expression of cytokeratins and neurofilament proteins in a permanent cell line: Cultured rat PC12 cells combine neuronal and epithelial features. J Cell Biol 103:1933, 1986 44. Dockhorn-Dwornizcak B, Franke WW, Schroeder B, et al: Patterns of expression of cytoskeletal proteins in human thyroid gland and thyroid carcinomas. Differentiation 35:53, 1987 45. Gould VE, Jansson D: Neuroendocrine and nerve sheath neoplasms, in Osborn M, Weber K (eds): Cytoskeletal Proteins in Tumor Diagnosis. Cold Spring Harbor, NY, Cold Spring Harbor Laboratory, 1989, pp 23-29 46. Bignami A, Raju T, Dahl D: Localization of vimentin, the nonspecific intermediate filament protein in embryonal glial and in early differentiating neurons. Dev Biol 91:286, 1982 47. Draeger UC: Coexistence of neurofilaments and vimentin in neurons of adult mouse retina. Nature 303:169, 1983 48. Shaw G, Osborn M, Weber K: An immunofluorescence microscopical study of the neurofilament triplet protein, vimentin and glial Iibrillary acidic protein within the adult brain. Eur J Cell Biol26:68, 1981 49. Shaw G, Weber K: The structure and development of the rat retina: An immunofluorescence microscopical study using antibodies specific for intermediate filament proteins. Eur J Cell Biol 30:219, 1983 50. Tapscott SJ, Bennett GS, Holtzer H: Neuronal precursor cells in the chick neural tube express neurofilament proteins. Nature 292:836, 1981 5 1. Ziller C, Dupin E, Brazeau P, et al: Early segregation of a neuronal precursor cell line in the neural crest as revealed by culture in a chemically defined medium. Cell 32:627, 1983 52. Franke WW, Jahn L, Knapp AC: Cytokeratins and desmosomal proteins in certain epithelioid and non-epithelial cell, in Osborn M, Weber K (eds): Cytoskeletal Proteins in Tumor Diagnosis. Cold Spring Harbor, NY, Cold Spring Harbor Laboratory, 1989, pp 151-172

6. Cushing H: Experiences with the cerebellar medulloblastoma. A critical review. Acta Path01 Microbial Immunol Stand [A] 7:1, 1930 7. Bailey P: Intracranial Tumors (ed 2). Springfield, IL, Thomas, 1948 8. Bailey P: Cellular types in primary tumors of the brain, in Penfield W (ed): Cytology and Cellular Pathology of the Nervous System, ~013. New York, NY, Hoeber, 1932, pp 905-931 9. Palmer JO, Kasselberg AC, Netsky MC: Differentiation of medulloblastoma. J Neurosurg 55:161, 1981 10. Hart MN, Earle KM: Primitive neuroectodermal tumors of the brain in children. Cancer 32:890, 1973 11. Hassoun J, Gambarelli D, Grissoli F, et al: Central neurocytoma. Acta Neuropathol (Berl) 56:151, 1982 12. Azzarelli B, Richards DE, Anton AH, et al: Central neuroblastoma-Electron microscopic observations and catecholamine determinations. J Neuropathol Exp Nemo1 36:384, 1977 13. Rubinstein LJ: Cytogenesis and differentiation of pineal neoplasms. HUM PATHOL 12:441, 1981 14. Ojeda VJ, Jacobsen PF, Papadimitriou JM: Primary cerebral neuroblastoma. Case report with light microscopy, tissue culture and electron microscopy study. Pathology 12:269, 1980 15. Horten BC, Rubinstein LJ: Primary cerebral neuroblastoma. Chnicopathologic study of 35 cases. Brain 99:735, 1976 16. Rorke LB: The cerebellar medulloblastoma and its relationship to primitive neuroectodermal tumors. J Neuropathol Exp Neurol 42:1, 1983 17. Kosnick EJ, Boesel CP, Bay J, et al: Primitive neuroectodermal tumors of the central nervous system in children. J Neurosurg 48:741, 1978 18. Boesel CP, Suhan JP, Bradel EJ: Ultrastructure of primitive neuroectodermal neoplasms of the central nervous system. Cancer 42: 194, 1978 19. Rorke LB, Gilles FH, Davis RL, et al: Revision of the World Health Organization Classification of brain tumors for childhood brain tumors. Cancer 56: 1869, 1985 (suppl) 20. Dehner LP: Peripheral and central primitive neuroectodermal tumors: A nosologic concept seeking a consensus. Arch Path01 Lab Med 110:997, 1986 2 1. Rubinstein LJ: A commentary on the proposed revision of the World Health Organization classification of brain tumors for childhood brain tumors. Cancer 56:1887, 1985 (suppl) 22. Rubinstein LJ: Embryonal central neuroepithelial tumors and their differentiating potential. A cytogenetic view of a complex neuro-oncological problem. J Neurosurg 62:795, 1985 23. Katsetos CD, Liu HM, Zacks SI: Immunohistochemical and ultrastructural observations on Homer Wright (neuroblastic) rosettes and the “pale islands” of human cerebellar medulloblastomas. HUM PATHOL 19:1219, 1988 24. Zuelch KL: Brain Tumors, Their Biology and Pathology (ed 2). New York, NY, Springer, 1965 25. Gould, VE: Histogenesis and differentiation. A reevaluation of these concepts as criteria for the classification of tumors. HUM PATHOL 17:212, 1986 26. Gould VE, Wiedenmann B, Lee I, et al: Synaptophysin expression in neuroendocrine neoplasms as determined by immunocytochemistry. Am J Path01 126243, 1987 27. Friedman HS, Burger PC, Bigner SH, et al: Phenotypic and genotypic analysis of a human medulloblastoma cell line and transplantable xenograft (D341 Med) demonstrating amplification of c-myc. Am J Path01 130:472, 1988 28. Allan PM, Garson JA, Harper EI, et al: Biological characterization and clinical applications of a monoclonal antibody recognizing an antigen restricted to neuroectodermal tissues. Int J Cancer 31:591, 1983 29. Gould VE, Linnoila RI, Memoli VA, et al: Neuroendocrine components of the bronchopulmonary tract. Hyperplasias, dysplasias and neoplasms. Lab Invest 49:519, 1983 30. Roessmann V, Velasco ME, Gambetti P, et al: Neuronal and astrocytic differentiation in human neuroepithelial neoplasms: An immunohistochemical study. J Neuropathol Exp Neurol 42:113, 1983 31. Trojanowski JQ, Lee VMY, Schalepfer WW: An immunohistochemical study of human central and peripheral nervous sys-

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53. Kartenbeck J, Schwechheimer K, Moll R, et al: Attachment of vimentin filaments to desmosomal plaques in human meningioma cells and archnoidal tissue. J Cell Biol 98: 1072, 1984 54. Miettinen M, Lehto V-P, Virtanen I: Antibodies to intermediate filament proteins in the diagnosis and classification of human tumors. Ultrastruct Path01 7:83, 1984 55. Steiner CC, Graham S, Lewis MM: Malignant round tumor of bone with neuronal differentiation (neuroectodermal mor). Ultrastruct Path01 12:505, 1988 56. Ghobrial M, Velasco in metastatic medulloblastoma.

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58. Gould VE, Moll R, Chejfec G, et al: Cytoskeletal characteristics of epithelial neoplasms of the lung, in Rosen ST, Mulshine

JL, Cuttita F, et al (eds): Biology of Lung Cancer. New York, NY, Dekker, 1988, pp 121-154 59. Jansson D, Gould VE, Gooch CT, et al: Immunohistochemical analysis of colon carcinomas applying exocrine and neuroendocrine markers. Acta Path01 Microbial Immunol Stand 96: 1129, 1988 60. Molenaar WM, Jansson DS, Gould VE, et al: Molecular markers of primitive neuroectodermal tumors (PNETs) and other pediatric central nervous system tumors. Lab Invest 61:635, 1989 61. Barnard WC: The nature of the “oat celled sarcoma” of the mediastinum. J Path01 29:241, 1926 62. Bensch KG, Corrin B, Pariente R, et al: Oat-cell carcinoma of the lung: Its origin and relationship to bronchial carcinoid. Cancer 22:1163, 1968 63. Blobel G, Gould VE, Moll R, et al: Coexpression of neuroendocrine markers and epithelial cytoskeletal proteins in bronchopulmonary neuroendocrine neoplasms. Lab Invest 52:39, 1985

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