Acta Path. Jap. 29(2): 289-301, 1979
PRIMITIVE NEUROECTODERMAL TUMOR (NEUROEPITHELIOMA) OF SPINAL NERVE ROOT
- Report of a n Adult Case and Establishment of a Cell Line Shinobu ISHIKAWA*, Yoshihiko OHSHIMA**, Toshimitsu SUZUKI*, and (late) Shoichi OBOSHI* First Department of Pathology*, and Department of Orthopedics**, Niigata University, School of Medicine, Niigata (Received on July 15, 1978)
A case of primitive neuroectodermal tumor arising in the cervical nerve root of a 28-year-old m a n is presented. Histologically, the tumor was characterised by proliferation of primitive neuroectodermal cells and formation of numerous Homer-Wright type rosettes. A cell line (Nagai line) was established from the tumor. Electron microscopic examination of Nagai cells revealed numerous microrosette formation with microvilli-like cytoplasmic processes projecting into the central lumina. Neurosecretory granules appeared in the cytoplasmic processes when Nagai cells were treated with dibutyryl cyclic AMP. Primitive satellite cells which completely surrounded other tumor cells with their tongue-like slender cytoplasmic processes were also found. Histogenesis of this unique tumor was discussed comparing with the neuroblastoma of sympathetic nervous system, medulloblastoma of the central nervous system, and with the tumors induced by Adenovirus type 12 in animals. It was concluded that the tumor was neuroepithelioma derived from a primitive stem cell of neural crest origin which possesses the bipotency t o differentiate toward either neuroblastic or neurilemmal line. ACTA PATH. JAP. 29: 289-301, 1979.
Introduction
Primitive neuroectodermal tumor of peripheral nerves simulating medulloblastoma or medulloepithelioma of the central nervous system have been rarely reported and designated as neuroepithelioma or m e d u l l ~ e p i t h e l i o m a ? Their ~ ~ ~ ~ origin ~ ~ ~ have been attributed to persistent or aberrant neural crest cells but the evidence has been lacking
until now. In this paper a case of primitive neuroectodermal tumor arising in the cervical nerve root and the characteristics of a cultured cell line derived from the tumor will be reported, and the discussion on the histogenesis of this unique tumor will be made.
Zl'l Ev i'ig SB, &*8J% (%k)A&! S1 Asahimachi-dori, Niigata, 951, Japan. 289
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Case Report A 28-year-old man felt the sensory deficit of the ulnar site of the left forearm and weakness of flexor muscles of the 4th and 5th finger (root sign of 8th cervical nerve) in 1971. Then a mass appeared in the left supraclavicular fossa and grew gradually, but he had left it without consulting a doctor. On January 9, 1974, he was admitted to the University of Niigata Hospital with left Homer’s sign and slight pyramidal tract sign of bilateral lower extremities. X-ray examinations revealed a mass in the left supraclavicular fossa and enlargement of the left 8th cervical neural foramen. Myelography revealed extradural impingement upon the cord which was shown by blockage of flow of radio-opaque dye in the subarachnoid space. At surgery on January 23, 1974, the mass in the supraclavicular fossa was found within the epineural sheath of the left 8th cervical nerve root (Fig. 1). Only the tumor mass was enucleated with slight difficulty due to the adhesion to the epineurium. One week later, the tumor of the epidural space was also resected. The tumor was confined in the epidural space and the spinal cord was not infiltrated by the tumor. Following surgery, the patient was treated with radiation and received a total of 4500 rads with use of the telecobalt unit. He was in good health for 2 years until February, 1976, when a mass reappeared in the left supraclavicular fossa. Partial resection of the tumor was performed. Vincristine (total, 5 g) and Endoxan (total, 500 mg) were administered postoperatively, but the flaccid palsy of extremities and left phrenic palsy occurred. He was put in an iron lung since July 9, due to the paralysis of the respiratory muscles, but the patient’s general condition became worse and death came on August 8. The urinary excretion of vanillylmandelic acid (VMA) was within normal limit.
Pathological Findings The tumor mass excised at surgery from the supraclavicular fossa was about 6x3 cm in size and the tumor of the epidural space was removed in fragments. The tumor was relatively firm and tan to gray-white in color. Sectioning revealed occasional necrosis with small cystic cavities. Microscopic examination of the tumor revealed nests or sheets of primitive small round cells with scant cytoplasm and poorly defined cell borders. The nuclei of these tumor cells were round to oval and contained dense chromatin nets. Formation of rosettes was not prominent in the surgical specimens. Fibrillar material was hardly seen around the tumor cells nor neurites were demonstrated with Bodian’s method for silver impregnation. There was no evidence of maturation toward ganglion cells. Relatively intact residual nerve bundles and delicate connective Fig. 1. Supraclavicular tumor a t the first operation. The tumor is present within the epineural sheath of left 8th cervical nerve root. Fig. 2. Main tumor mass a t autopsy. The tumor involves the epidural space and compresses the spinal cord. Subdural space is not infiltrated by the tumor. Destruction of the spinal cord is artifact a t autopsy. Fig. 3. Histologic appearance of the tumor a t autopsy. Note numerous Homer-Wright type rosettes. H.E. x 100. Fig. 4. Higher magnification of rosettes. Fine fibrillar material is present in the center of rosettes but little fibrillar material outside the rosettes. H.E. x 300.
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tissue septa carrying small blood vessels were present among the tumor tissue. The tumor was diagnosed as undifferentiated neuroblastoma. Examination of the epidural tumor and the recurrent tumor at the supraclavicular fossa revealed the same histologic appearance. The autopsy revealed large, irregularly-shaped, and partially necrotic tumor mass in the left supraclavicular fossa. The tumor was in continuity via the left 8th cervical neural foramen with the mass occupying the epidural space. I n the spinal canal, tumor masses projected from the thoracic and lumbar vertebrae into the epidural space, and compressed the spinal cord a t various levels. The subdural space was not infiltrated by the tumor (Fig. 2). The tumor also involved the left cervical paravertebral sympathetic ganglia, mediastinal lymph nodes and upper lobe of the left lung. Metastases of the tumor were found a t the pericapsular region of thoracic paravertebral sympathetic ganglia, in paraortal lymph nodes, each lobe of both lungs, systemic bones, and in hypophysis. Metastasis in the hypophysis was in continuity with the metastasis in the sphenoidal bone. Tumor was found neither in the central nervous system nor in bilateral adernals. Microscopic appearance of the tumor of autopsy material was similar to that of surgical material except for the presence of numerous Homer-Wright type rosettes in autopsy materials (Pig. 3). These rosettes added the striking characteristics to this tumor. At the center of rosettes some fine fibrillar material was faintly visible with H & E stain (Fig. 4),but the fibrils were not demonstrated by Bodian’s method. Though the preservation of the structures was not so good, electron microscopic examination of the tumor a t autopsy revealed the presence of arrays of rough-surfaced endoplasmic reticulum (rER), clusters of glycogen particles, and oligocilia in some tumor cells. Other findings a t autopsy were moderate lobular pneumonia of both lungs, extramedullary hematopoiesis in the liver and spleen, and bilateral pyelitis.
Establishment of Cultured Cell Line Materials and Methods Culture method: RPXI-1640 medium (Nissui) supplemented with 200 pg/ml of kanamycin and 20% fetal calf serum (Microbiol. Assoc.) was used. The tumor tissue obtained a t autopsy was minced finely with scissors. An aliquot of the tissue fragments was explanted i n Petri glass dishes. The remainder was suspended in culture medium after trypsinization, and cultured in Petri dishes. All the cultures were maintained a t 37°C in a humidified incubator with constant flow of 5% CO, in air. One-half the amount of medium was exchanged twice a week i n primary culture. After the cell line was established, serial passages were carried out every 15 days. Light microscopic examination: Cultured cells were observed with a phase contrast microscope. Paraffin sections of large cell clumps stained with H&E were examined with a light microscope. Electron microscopic examination: The floating cell clumps and adherent cells which were scraped off with rubber policeman were centrifuged a t 800 rpm for 5 min., respectively. The resulting pellets were fixed in 1.25% glutaraldehyde (0.1 M phosphate buffer solution, p H 7.4) for 10 min. in ice cooling, washed with cold phosphate buffer solution for one hour, post fixed i n 1% osmium tetroxide for 30 min., dehydrated in graded series of ethanol, and embedded in epoxy resin. The ultrathin sections were double-stained with lead citrate and uranyl acetate and examined under a Hitachi HS-9 electron microscope. Cell growth estimation: The cells in duplicate plates were counted a t 3 or 4 days-interval.
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Cell viability was determined by trypan blue dye exclusion test. Chromosome analysis: The culture was treated with colchicin (30 ,ug/ml) for 2 hours before harvest. Chromosome specimens were prepared by the air-drying method after hypotonic treatment and acetic-methanol fixation. The slides were stained with Giemsa solution. Dibutyryl cyclic A M P treatment : N6,0a'-dibutyryl adenosine 3' :5'-cyclic monophosphoric acid, sodium salt (dibutyryl CAMP, Sigma, grade 11) was dissolved in RPMI-1640 medium and freezestocked a t -2OOC until use. The cells were dispersed by trypsinization and seeded in Petri dishes of 35 mm in diameter. Each dish was adjusted to contain 5 x lo5 cells in 2 ml of medium. Five days after subculture, when some cells adhered to the glass surface and began to grow, the dishes were divided into three groups. I n the f i s t and second group the medium of the dishes was replaced with the medium containing 0.1 mM or 1.0 mM dibutyryl CAMP,respectively. The third group were cultured with the usual medium as a control. One-half the amount of medium was exchanged twice a week in each dish. The cells were observed continually with a phase contrast microscope. The cells cultured for 2 weeks with dibutyryl CAMP containing medium were prepared for electron microscopic examination as well as the control cells.
Characteristics of the Cell Line About a week after initiation of culture, fibroblast-like cells were found t o be adherent to the glass surface in both dishes of explantation and suspension culture. These cells proliferated and became confluent in a few days. The clumps of the small round tumor cells were found slightly adherent to the fibroblast-like cells. The tumor cells did not increase in number until two months later when they began to proliferate slowly. The tumor cells were detached from the fibroblast-like cells by trypsinization and transfered to the other dishes. Thereafter a constant cell growth was achieved and serial passages were carried out every 15 days. The cell line, reached 40th subcultivation in June, 1978 and maintained for 22 months in vitro, was designated as Nagai line after the patient's name. Microscopically the majority of tumor cells did not adhere to the surface of the culture dishes and formed the large cell clumps. Small proportion of cells was adherent to the glass surface. The shape of the adherent cells were oval, tear dropshaped, or polyhedral. They occasionally had short cytoplasmic processes (Pig. 5). Section of cell clumps after usual histological procedure revealed the appearance similar to that of the tumor a t autopsy with occasional formation of rosettes. Chromosome analysis, performed a t the 12th subcultivation revealed a modal number between 76 and 85 (Fig. 6). No marker chromosome was identified (Pig. 7). Population-doubling time determined at the 17th subcultivation in the exponential phase of growth was 130 hours. Ultrastructure of the Cultured Tumor Cells In a low power view, rosette-like arrangement of the tumor cells was frequently observed. The general configuration of the tumor cells was round, oval, or carrot-shaped since their apical cytoplasm faced the central lumen and the nuclei situated a t the pole away from the lumen. Numerous microvilli-like cytoplasmic processes were noted and projected in the space of the rosette (Pig. 8). The nuclei were round, oval, or irregular with occasional cytoplasmic invaginations. Chromatin substance was diffusely dispersed and one or two nucleoli were present. The cytoplasm of the main
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Fig. 5. Adherent tumor cells of Nagai line with short cytoplasmic processes. Phase contrast micrograph, x 100.
mnn 160
l3-u 50
Chromosome Number
Fig. 6. Distribution of chromosome number in Nagai line a t the 12th subcultivation.
tumor cells contained abundant free ribosomes and polysomes, a few rER, and moderate number of mitochondria. Many cells were abundant in glycogen particles which occasionally formed a large aggregate. Some cells contained well-developed Golgi apparatus and arrays of rER (Fig. 8). Occasionally large osmiophilic granules which resembled the lipofuscin were present. The finding to be noted was the presence of single or oligocilia not infrequently observed in some tumor cells. Cross section of the cilia revealed the 9+0 type structure (Fig. 9). Arrays of microtubules were seen though rarely at the root of cytoplasmic projections (Pig. lo), and also rarely bundles of microfilaments with a thickness of about 10 nm were present in the cytoplasmic processes (Fig. 11). Between the neighboring tumor cells, special types of cell junctions such as gap junctions or adhesion plaques were occasionally seen (Fig. 12).
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Pig. 7. Representative karyotype of Nagai line a t the 12th subcultivation.
Fig. 8. (Figures 8-16: electron micrograph of Nagai line) Main tumor cell forming the rosette. Microvilli-like cytoplasmic processes are noted projecting in the space of the rosette (right). Note also arrays of rER, Golgi apparatus and a large aggregate of glycogen particles. x 5,000.
Some cytoplasmic processes intervened between the neighboring tumor cells and piled doubly or triply (Fig. 13). There were some cells which have crescent-shaped cell body and nucleus. They projected narrow cytoplasmic expansions completely surrounding the other tumor cell (Fig. 14). These features resembled the ultrastructure of the
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Fig. 9. Cilia projecting from a tumor cell ( x 5,000) with 9+0 type microtubular structure (inlet, x 20,000). Fig. 10. Bundle of microtubules seen a t the base of a cytoplasmic process. x 10,000. Fig. 11. Bundle of microfilaments i n a cytoplasmic process. (Scale, 100 nm). Fig. 12. A gap junction (long arrow) and adhesion plaques (short arrows) seen between the cell membrane of adjacent tumor cells. x 7,000.
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Fig. 13. Slender cytoplasmic processes intervene between the neighboring tumor cells. x 7,000. Fig. 14. A cell which considered to be the primitive satellite cell. Slender cytoplasmic process completely embraces the other tumor cell. x 3,000. Fig. 15. Cells treated with 0.1 mM dibutyryl CAMP for 2 weeks. Note marked elongation of microvilli-like cytoplasmic processes projecting into the central space of a rosette (Compare with Fig. 8). x 3,000. Fig. 16. Cells treated with 0.1 mM dibutyryl CAMPfor 2 weeks. Note dense core vesicles and clear vesicles seen in the bulgings of microvilli-like cytoplasmic processes (Scale, 200 nm).
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satellite cells. Cells with transitional figures from the crescent-shaped cells to the main tumor cells were also present.
Morphological Changes Induced by Dibutyryl Cyclic AMP Treatment Under phase contrast microscope, the tumor cells treated with 0.1 mM dibutyryl CAMP showed no apparent morphological changes even after two weeks of treatment. The cells treated with 1.0 mM dibutyryl CAMPreduced their cytoplasmic processes and became rather plump, probably due to the toxic effect of the dibutyryl CAMP. Electron microscopic examination, however, revealed striking changes in the cells treated with 0.1 mM dibutyryl CAMP. The microvilli-like cytoplasmic processes which projected into the central lumen of the rosettes were markedly elongated with occasional bulgings (Fig. 15). There appeared many dense core vesicles of 80 to 150 nm in diameter and clear round or oval vesicles of 50 to 100 nm in these processes (Fig. 16). These vesicles closely resembled the neurosecretory granules or synaptic vesicles. No apparent synaptic complexes were observed.
Discussion The tumor reported here generally simulated the neuroblastoma of the sympathetic nervous system. The histologic appearance of this tumor, however, seemed to be rather identical with rare primitive neuroectodermal tumor of peripheral since this tumor was composed of nerves reported by STOUT^^ and by NESBITT~~, primitive neuroectodermal cells which formed numerous Homer-Wright type rosettes without argyrophile fibers or neurites but with some fine fibrillar material in their centers, and did not show any differentiation tendency to ganglion cells. These histological features were different from those of neuroblastoma. The case reported by STOUT^^ was a 42-year-old man with a tumor in the ulnar nerve. Histological features of the tumor were identical with those of the tumor of our case. He suggested that the tumor might have arisen from embryonally misplaced neuroepithelium of the neural crest origin and called the tumor as “neuroepithelioma”. NE S BI T Talso ~~ reported a case of “pimitive neuroectodermal tumor (neuroblastoma) arising in sciatic nerve of a child’’ and described that the histologic pattern of the tumor was similar to that of STOUT’Scase. He also reviewed 15 cases of primitive neuroectodermal tumor of peripheral nerves in the literature. They have been reported under various names such as medulloepithelioma, neuroepithelioma, medulloblastoma or neuroblastoma. The nomenclature of this kind of tumor is controversial even now. Some differences exist between the tumor and neuroblastoma in histological features as above-mentioned. Moreover, Nagai-cell, cultured cell line derived from the tumor, was apparently different from the cultured neuroblastoma cells derived from children’s neuroblastoma of sympathetic nervous system. I n morphology in vitro, Nagai-cell mainly formed large aggregates with partial adherence to the glass surface, extended short cytoplasmic processes, and the processes remained short even after dibutyryl CAMP treatment and negative for Bodian’s staining. On the other hand, cultured neuro-
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blastoma cells comprised of small spiny cells or large epithelioid cells with neurites of various length which could be markedly elongated by dibutyryl CAMP treatment and stained positively with Bodian's staining4. Population doubling time of Nagai-cell was about 130 hours and that of cultured neuroblastoma cells about 20 to 60 hOUIS.4~9*20 The chromosome number of Nagai-cell was the lower tetraploid and that of cultured neuroblastoma cell near the d i p l ~ i d . ~ ~ ~ ~ Ultrastructure of Nagai-cell and cultured neuroblastoma cells was also different. Nagai-cell showed numerous microrosettes with microvilli-like projections into the central lumen and oligocilia, 9 +0 type, were observed occasionally. The neurosecretory granules were not found under the ordinary culture condition, but when treated with dibutyryl CAMP, the dense core vesicles appeared in the varicosity of microvilli-like cell processes. The varicosity closely resembled the growth cone of sympathetic neurons in culture2 and the dense core vesicles observed in Nagai-cell were also found not only in adrenergic neurons but also in various central and peripheral neurons.16 A more characteristic finding was the presence of satellite-like cells which embraced other tumor cells with narrow cytoplasmic processes without neurotubules nor neurofilaments. This was similar to the primitive satellite cells observed in the posterior spinal ganglia of chick embryo14, or sympathetic ganglia of human fetus15 and newborn ~ ~ Oin biopsy rats. On the other hand, neuroblastoma cells of both in c ~ l t u r e ~and material618 were mainly composed of undifferentiated neuroblastic cells but some had Nissl substance, bundles of neurofilaments and microtubules within cytoplasm and neurites, neurosecretory granules, and synaptic structures. When treated with dibutyryl CAMP, cultured neuroblastoma cells showed marked differentiation ability to either ganglionic and paraganglionic line as shown by marked increase in both number and size of the neurosecretory granules.4 From these results, it is suggested that Nagai-cell has different differentiation potentiality from that of neuroblastoma cells. It must be noted that the tumor reported here bears a close resemblance in microscopic appearance to the medulloblastoma of the cerebellum. Although in many cases no indication for neuronal or glial differentiation of medulloblastoma cells were found, some cases showed formation of cytoplasmic processes, rudimentary synaptic structure or glial filaments indicating either neuronal or glial differentiation.l]l7 Oligocilia and microrosettes with many cytoplasmic processes in the center were observed in some cases.7~18 From these observations, it is suggested that the medulloblastoma arises from the undifferentiated cell (medulloblast) seen in developing encephalon which later differentiates into neuroblastic or glial ~ e l l s . ~ P Primitive neuroectodermal tumors have been induced in central and peripheral nervous system of the various rodents by the inoculation of Adenovirus type l2.lo~l2~l3 Histological and ultrastructural figures were the same regardless of the animal species and the site where the tumor has developed. Histology of the tumor was very similar to that of Primitive neuroectodermal tumor, and the tumor referred to as medullab1astoma13 Or medul1oepithelioma.lo The ultrastructure of the tumor revealed to that of the tumor cells reported here with occasional 9 + 0 type oligoc~ia.lO,ls
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MUKAI~Odescribed that oligocilia have been observed in sensory neurons and their precursors. They were also observed in undifferentiated neural crest cells, neuroblastic cells and in immature satellite cells of posterior ganglia of chick embryo.14 O G A W A ~ ~ ~ ~ ~ observed glial or neurilemmal differentiation, and MUKAI~Oobserved neuronal differentiation in Adenovirus type 12-induced tumors. Because of the morphological similarity between Adenovirus type 12-induced tumors and that of our case, Adenovirus type 12-specific surface antigen and tumor antigen of the cultured tumor cells were examined by fluorescent antibody technique, and tumor antigen of primary tumor by complement fixation technique (HAMADA and MAEDA3). Each test, however, was negative. From the studies on the medulloblastoma and Adenovirus type 12-induced tumors, it might be suggested that there might exist similar, if not exactly identical, undifferentiated stem cells in both central and peripheral nervous systems, which have bipotency to differentiate toward either neuroblastic or glial line and neuroectodermal tumors might originate from the provisional stem cells in the central or peripheral nervous system. In conclusion, the tumor reported here was different from sympathetic neuroblastoma in many respects but identical to neuroepithelioma derived from primitive stem cells of neural crest origin. The tumor cells possessed bioptency to differentiate along both neuroblastic and neurilemmal line. Therefore it was regarded as homologous histogenetically to the medulloblastoma of the central nervous system and to the primitive neuroectodermal tumors of rodents induced by Adenovirus type 12. Acknowledgement: The authors are grateful t o Prof. T. HAMADA and Dr. H. MAEDA,Department of Virology, Niigata University, School of Medicine, for the examination of Adenovirus type 12specific antigens of the tumor cells. This study was supported in part by a Grant-in Aid for Cancer Research from the Ministry of Education, Science and Culture, and Ministry of Health and Welfare.
References 1. BAILEY,P. and CUSHINC,H. : Tumors of the glioma group. Lippincott, Philadelphia, 1926. 2. BUNCE,M.B.: Fine structure of nerve fibers and growth cones of isolated sympathetic neurons in culture. J. Cell Biol. 56: 713-735, 1973. 3. HAMADA, T. and MAEDA,H.: Personal communication. 4. ISHIKAWA, S. : Differentiation of human neuroblastoma cells in vitro. Morphological changes induced by dibutyryl cyclic AMP. Acta Path. Jap. 27: 697-711, 1977. 5. KANERVA,L. : Ultrastructure of sympathetic ganglion cells and granule-containing cells in the paracervical (Frankenhauser) ganglion of the newborn rat. Z. Zellforsch. 126: 2 5 4 0 ,
1972. 6. MACKAY,B., LUNA,M.A., and BUTLER,J.J.: Adult neuroblastoma. Electron microscopic observations in nine cases. Cancer 37: 1334-1351, 1976. J., and GULLOTTA, F. : The ultrastructure of medulloblasto7. MATAKAS,F., CERVBS-NAVARRO, mas. Acta neuropath. (Berl.) 16: 271-284. 1970. 8. MISUGI, K., MISUCI, N., and NEWTON,W.A.: Fine structural study of neuroblastoma, ganglioneuroblastoma, and pheochromocytoma. Arch Path. 86: 160-170, 1968. S., and INUI, A.: Comparison between the cells i n v i m g. MIYAI(E, s., OKABE, H., IMASHIJKU, and i n vitro, concerning catecholamine metabolism. Soshiki-baiyo 1: 23-27, 1975 (in Japanese). 10. MUKAI,N. and KOBAYASHI, S. : Human adenovirus-induced medulloepitheliomatous neoplasms
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in Sprague-Dawley rats. Am. J. Pathol. 73: 671-690, 1973. 11. NESBITT, K.A. and VIDONE,R.A. : Primitive neuroectodermal tumor (neuroblastoma) arising in sciatic nerve of a child. Cancer 37: 1562-1570, 1976. 12. OUAWA, K., TSUTSUMI, A., IWATA, K., FUJII,Y., OHMORI,M., TAUUCHI, K., and YABE,Y.: Histogenesis of malignant neoplasm induced by adenovirus type 12. Gann 57: 43-52, 1966. 13. OGAWA, K., HAMAYA, K., FUJII,Y., MATSUURA,K., and ENDO,T.: Tumor induction by adenovirus type 12 and its target cells in the central nervous system. Gann 60: 383-392,1969. 14. PANNESE, E.: in BRODAL, A., HILD, W., VAN LIMBORGH, J., ORTMANN, R., SCHIEBLER,T.H., and TONDURY,G. (Eds). Advances in Anatomy, Embryology and Cell Biology: The histogenesis of the spinal ganglia, Vol. 47, Fasc. 5, Springer-Verlag, Berlin, 1974. 15. PICK,J., GERDIN,C., and DELEMOS, C.: An electron microscopical study of developing sympathetic neurons in man. Z. Zellforsch. 62: 402-415, 1964. 16. POTTER, L.T.: Storage of norepinephrine in sympathetic nerves. Phamacol. Rev. 18: 439451, 1966. 17. RUSSELL, D.S. and RUBINSTEIN, L.J.: Pathology of tumours of the nervous system. 4th ed. Arnold, London, 1977. T. and KITAMURA, M.: in YAMADA, E., UCHIZONO, K., and WATANABE, Y. (Eds), 18. SAWADA, Fine Structure of Cells and Tissues, Vol. 6, 264-267, Igakushoin, Tokyo, 1971, (in Japanese). 19. SEIMURA,T .: Electron microscopic studies on advenovirus type 12-induced hamster tumors. Comparison among tumors in different locations. Niigata Medical J. 90: 229236, 1976 (in Japanese). H.R., GERSON,J.M., MOORHEAD, P.S., MAQUIRE, H., and HUMMELER, K.: 20. SCHLESINGER, Establishment and characterization of human neuroblastoma cell lines. Cancer Res. 36 : 3094-3100, 1976. G.F., and HAAQENSEN, C.D.: in ZABRISKIE,E.G., FRANTZ, A.M., and 21. STOUT,A.P., LAIDLAW, HARE,C. (Eds), Tumors of the Nervous System: Tumors of the peripheral nerves, 417-439, Williams & Wilkins, Baltimore, 1937.
PRIMITIVE NEUROECTODERMAL TUMOR (NEUROEPITHELIOMA) OF SPINAL NERVE ROOT
- Report of a n Adult Case and Establishment of a Cell Line Shinobu ISHIKAWA*, Yoshihiko OHSHIMA**, Toshimitsu SUZUKI*, and (late) Shoichi OBOSHI* First Department of Pathology*, and Department of Orthopedics**, Niigata University, School of Medicine, Niigata (Received on July 15, 1978)
A case of primitive neuroectodermal tumor arising in the cervical nerve root of a 28-year-old m a n is presented. Histologically, the tumor was characterised by proliferation of primitive neuroectodermal cells and formation of numerous Homer-Wright type rosettes. A cell line (Nagai line) was established from the tumor. Electron microscopic examination of Nagai cells revealed numerous microrosette formation with microvilli-like cytoplasmic processes projecting into the central lumina. Neurosecretory granules appeared in the cytoplasmic processes when Nagai cells were treated with dibutyryl cyclic AMP. Primitive satellite cells which completely surrounded other tumor cells with their tongue-like slender cytoplasmic processes were also found. Histogenesis of this unique tumor was discussed comparing with the neuroblastoma of sympathetic nervous system, medulloblastoma of the central nervous system, and with the tumors induced by Adenovirus type 12 in animals. It was concluded that the tumor was neuroepithelioma derived from a primitive stem cell of neural crest origin which possesses the bipotency t o differentiate toward either neuroblastic or neurilemmal line. ACTA PATH. JAP. 29: 289-301, 1979.
Introduction
Primitive neuroectodermal tumor of peripheral nerves simulating medulloblastoma or medulloepithelioma of the central nervous system have been rarely reported and designated as neuroepithelioma or m e d u l l ~ e p i t h e l i o m a ? Their ~ ~ ~ ~ origin ~ ~ ~ have been attributed to persistent or aberrant neural crest cells but the evidence has been lacking
until now. In this paper a case of primitive neuroectodermal tumor arising in the cervical nerve root and the characteristics of a cultured cell line derived from the tumor will be reported, and the discussion on the histogenesis of this unique tumor will be made.
Zl'l Ev i'ig SB, &*8J% (%k)A&! S1 Asahimachi-dori, Niigata, 951, Japan. 289
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Case Report A 28-year-old man felt the sensory deficit of the ulnar site of the left forearm and weakness of flexor muscles of the 4th and 5th finger (root sign of 8th cervical nerve) in 1971. Then a mass appeared in the left supraclavicular fossa and grew gradually, but he had left it without consulting a doctor. On January 9, 1974, he was admitted to the University of Niigata Hospital with left Homer’s sign and slight pyramidal tract sign of bilateral lower extremities. X-ray examinations revealed a mass in the left supraclavicular fossa and enlargement of the left 8th cervical neural foramen. Myelography revealed extradural impingement upon the cord which was shown by blockage of flow of radio-opaque dye in the subarachnoid space. At surgery on January 23, 1974, the mass in the supraclavicular fossa was found within the epineural sheath of the left 8th cervical nerve root (Fig. 1). Only the tumor mass was enucleated with slight difficulty due to the adhesion to the epineurium. One week later, the tumor of the epidural space was also resected. The tumor was confined in the epidural space and the spinal cord was not infiltrated by the tumor. Following surgery, the patient was treated with radiation and received a total of 4500 rads with use of the telecobalt unit. He was in good health for 2 years until February, 1976, when a mass reappeared in the left supraclavicular fossa. Partial resection of the tumor was performed. Vincristine (total, 5 g) and Endoxan (total, 500 mg) were administered postoperatively, but the flaccid palsy of extremities and left phrenic palsy occurred. He was put in an iron lung since July 9, due to the paralysis of the respiratory muscles, but the patient’s general condition became worse and death came on August 8. The urinary excretion of vanillylmandelic acid (VMA) was within normal limit.
Pathological Findings The tumor mass excised at surgery from the supraclavicular fossa was about 6x3 cm in size and the tumor of the epidural space was removed in fragments. The tumor was relatively firm and tan to gray-white in color. Sectioning revealed occasional necrosis with small cystic cavities. Microscopic examination of the tumor revealed nests or sheets of primitive small round cells with scant cytoplasm and poorly defined cell borders. The nuclei of these tumor cells were round to oval and contained dense chromatin nets. Formation of rosettes was not prominent in the surgical specimens. Fibrillar material was hardly seen around the tumor cells nor neurites were demonstrated with Bodian’s method for silver impregnation. There was no evidence of maturation toward ganglion cells. Relatively intact residual nerve bundles and delicate connective Fig. 1. Supraclavicular tumor a t the first operation. The tumor is present within the epineural sheath of left 8th cervical nerve root. Fig. 2. Main tumor mass a t autopsy. The tumor involves the epidural space and compresses the spinal cord. Subdural space is not infiltrated by the tumor. Destruction of the spinal cord is artifact a t autopsy. Fig. 3. Histologic appearance of the tumor a t autopsy. Note numerous Homer-Wright type rosettes. H.E. x 100. Fig. 4. Higher magnification of rosettes. Fine fibrillar material is present in the center of rosettes but little fibrillar material outside the rosettes. H.E. x 300.
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tissue septa carrying small blood vessels were present among the tumor tissue. The tumor was diagnosed as undifferentiated neuroblastoma. Examination of the epidural tumor and the recurrent tumor at the supraclavicular fossa revealed the same histologic appearance. The autopsy revealed large, irregularly-shaped, and partially necrotic tumor mass in the left supraclavicular fossa. The tumor was in continuity via the left 8th cervical neural foramen with the mass occupying the epidural space. I n the spinal canal, tumor masses projected from the thoracic and lumbar vertebrae into the epidural space, and compressed the spinal cord a t various levels. The subdural space was not infiltrated by the tumor (Fig. 2). The tumor also involved the left cervical paravertebral sympathetic ganglia, mediastinal lymph nodes and upper lobe of the left lung. Metastases of the tumor were found a t the pericapsular region of thoracic paravertebral sympathetic ganglia, in paraortal lymph nodes, each lobe of both lungs, systemic bones, and in hypophysis. Metastasis in the hypophysis was in continuity with the metastasis in the sphenoidal bone. Tumor was found neither in the central nervous system nor in bilateral adernals. Microscopic appearance of the tumor of autopsy material was similar to that of surgical material except for the presence of numerous Homer-Wright type rosettes in autopsy materials (Pig. 3). These rosettes added the striking characteristics to this tumor. At the center of rosettes some fine fibrillar material was faintly visible with H & E stain (Fig. 4),but the fibrils were not demonstrated by Bodian’s method. Though the preservation of the structures was not so good, electron microscopic examination of the tumor a t autopsy revealed the presence of arrays of rough-surfaced endoplasmic reticulum (rER), clusters of glycogen particles, and oligocilia in some tumor cells. Other findings a t autopsy were moderate lobular pneumonia of both lungs, extramedullary hematopoiesis in the liver and spleen, and bilateral pyelitis.
Establishment of Cultured Cell Line Materials and Methods Culture method: RPXI-1640 medium (Nissui) supplemented with 200 pg/ml of kanamycin and 20% fetal calf serum (Microbiol. Assoc.) was used. The tumor tissue obtained a t autopsy was minced finely with scissors. An aliquot of the tissue fragments was explanted i n Petri glass dishes. The remainder was suspended in culture medium after trypsinization, and cultured in Petri dishes. All the cultures were maintained a t 37°C in a humidified incubator with constant flow of 5% CO, in air. One-half the amount of medium was exchanged twice a week i n primary culture. After the cell line was established, serial passages were carried out every 15 days. Light microscopic examination: Cultured cells were observed with a phase contrast microscope. Paraffin sections of large cell clumps stained with H&E were examined with a light microscope. Electron microscopic examination: The floating cell clumps and adherent cells which were scraped off with rubber policeman were centrifuged a t 800 rpm for 5 min., respectively. The resulting pellets were fixed in 1.25% glutaraldehyde (0.1 M phosphate buffer solution, p H 7.4) for 10 min. in ice cooling, washed with cold phosphate buffer solution for one hour, post fixed i n 1% osmium tetroxide for 30 min., dehydrated in graded series of ethanol, and embedded in epoxy resin. The ultrathin sections were double-stained with lead citrate and uranyl acetate and examined under a Hitachi HS-9 electron microscope. Cell growth estimation: The cells in duplicate plates were counted a t 3 or 4 days-interval.
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Cell viability was determined by trypan blue dye exclusion test. Chromosome analysis: The culture was treated with colchicin (30 ,ug/ml) for 2 hours before harvest. Chromosome specimens were prepared by the air-drying method after hypotonic treatment and acetic-methanol fixation. The slides were stained with Giemsa solution. Dibutyryl cyclic A M P treatment : N6,0a'-dibutyryl adenosine 3' :5'-cyclic monophosphoric acid, sodium salt (dibutyryl CAMP, Sigma, grade 11) was dissolved in RPMI-1640 medium and freezestocked a t -2OOC until use. The cells were dispersed by trypsinization and seeded in Petri dishes of 35 mm in diameter. Each dish was adjusted to contain 5 x lo5 cells in 2 ml of medium. Five days after subculture, when some cells adhered to the glass surface and began to grow, the dishes were divided into three groups. I n the f i s t and second group the medium of the dishes was replaced with the medium containing 0.1 mM or 1.0 mM dibutyryl CAMP,respectively. The third group were cultured with the usual medium as a control. One-half the amount of medium was exchanged twice a week in each dish. The cells were observed continually with a phase contrast microscope. The cells cultured for 2 weeks with dibutyryl CAMP containing medium were prepared for electron microscopic examination as well as the control cells.
Characteristics of the Cell Line About a week after initiation of culture, fibroblast-like cells were found t o be adherent to the glass surface in both dishes of explantation and suspension culture. These cells proliferated and became confluent in a few days. The clumps of the small round tumor cells were found slightly adherent to the fibroblast-like cells. The tumor cells did not increase in number until two months later when they began to proliferate slowly. The tumor cells were detached from the fibroblast-like cells by trypsinization and transfered to the other dishes. Thereafter a constant cell growth was achieved and serial passages were carried out every 15 days. The cell line, reached 40th subcultivation in June, 1978 and maintained for 22 months in vitro, was designated as Nagai line after the patient's name. Microscopically the majority of tumor cells did not adhere to the surface of the culture dishes and formed the large cell clumps. Small proportion of cells was adherent to the glass surface. The shape of the adherent cells were oval, tear dropshaped, or polyhedral. They occasionally had short cytoplasmic processes (Pig. 5). Section of cell clumps after usual histological procedure revealed the appearance similar to that of the tumor a t autopsy with occasional formation of rosettes. Chromosome analysis, performed a t the 12th subcultivation revealed a modal number between 76 and 85 (Fig. 6). No marker chromosome was identified (Pig. 7). Population-doubling time determined at the 17th subcultivation in the exponential phase of growth was 130 hours. Ultrastructure of the Cultured Tumor Cells In a low power view, rosette-like arrangement of the tumor cells was frequently observed. The general configuration of the tumor cells was round, oval, or carrot-shaped since their apical cytoplasm faced the central lumen and the nuclei situated a t the pole away from the lumen. Numerous microvilli-like cytoplasmic processes were noted and projected in the space of the rosette (Pig. 8). The nuclei were round, oval, or irregular with occasional cytoplasmic invaginations. Chromatin substance was diffusely dispersed and one or two nucleoli were present. The cytoplasm of the main
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Fig. 5. Adherent tumor cells of Nagai line with short cytoplasmic processes. Phase contrast micrograph, x 100.
mnn 160
l3-u 50
Chromosome Number
Fig. 6. Distribution of chromosome number in Nagai line a t the 12th subcultivation.
tumor cells contained abundant free ribosomes and polysomes, a few rER, and moderate number of mitochondria. Many cells were abundant in glycogen particles which occasionally formed a large aggregate. Some cells contained well-developed Golgi apparatus and arrays of rER (Fig. 8). Occasionally large osmiophilic granules which resembled the lipofuscin were present. The finding to be noted was the presence of single or oligocilia not infrequently observed in some tumor cells. Cross section of the cilia revealed the 9+0 type structure (Fig. 9). Arrays of microtubules were seen though rarely at the root of cytoplasmic projections (Pig. lo), and also rarely bundles of microfilaments with a thickness of about 10 nm were present in the cytoplasmic processes (Fig. 11). Between the neighboring tumor cells, special types of cell junctions such as gap junctions or adhesion plaques were occasionally seen (Fig. 12).
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Pig. 7. Representative karyotype of Nagai line a t the 12th subcultivation.
Fig. 8. (Figures 8-16: electron micrograph of Nagai line) Main tumor cell forming the rosette. Microvilli-like cytoplasmic processes are noted projecting in the space of the rosette (right). Note also arrays of rER, Golgi apparatus and a large aggregate of glycogen particles. x 5,000.
Some cytoplasmic processes intervened between the neighboring tumor cells and piled doubly or triply (Fig. 13). There were some cells which have crescent-shaped cell body and nucleus. They projected narrow cytoplasmic expansions completely surrounding the other tumor cell (Fig. 14). These features resembled the ultrastructure of the
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Fig. 9. Cilia projecting from a tumor cell ( x 5,000) with 9+0 type microtubular structure (inlet, x 20,000). Fig. 10. Bundle of microtubules seen a t the base of a cytoplasmic process. x 10,000. Fig. 11. Bundle of microfilaments i n a cytoplasmic process. (Scale, 100 nm). Fig. 12. A gap junction (long arrow) and adhesion plaques (short arrows) seen between the cell membrane of adjacent tumor cells. x 7,000.
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Fig. 13. Slender cytoplasmic processes intervene between the neighboring tumor cells. x 7,000. Fig. 14. A cell which considered to be the primitive satellite cell. Slender cytoplasmic process completely embraces the other tumor cell. x 3,000. Fig. 15. Cells treated with 0.1 mM dibutyryl CAMP for 2 weeks. Note marked elongation of microvilli-like cytoplasmic processes projecting into the central space of a rosette (Compare with Fig. 8). x 3,000. Fig. 16. Cells treated with 0.1 mM dibutyryl CAMPfor 2 weeks. Note dense core vesicles and clear vesicles seen in the bulgings of microvilli-like cytoplasmic processes (Scale, 200 nm).
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satellite cells. Cells with transitional figures from the crescent-shaped cells to the main tumor cells were also present.
Morphological Changes Induced by Dibutyryl Cyclic AMP Treatment Under phase contrast microscope, the tumor cells treated with 0.1 mM dibutyryl CAMP showed no apparent morphological changes even after two weeks of treatment. The cells treated with 1.0 mM dibutyryl CAMPreduced their cytoplasmic processes and became rather plump, probably due to the toxic effect of the dibutyryl CAMP. Electron microscopic examination, however, revealed striking changes in the cells treated with 0.1 mM dibutyryl CAMP. The microvilli-like cytoplasmic processes which projected into the central lumen of the rosettes were markedly elongated with occasional bulgings (Fig. 15). There appeared many dense core vesicles of 80 to 150 nm in diameter and clear round or oval vesicles of 50 to 100 nm in these processes (Fig. 16). These vesicles closely resembled the neurosecretory granules or synaptic vesicles. No apparent synaptic complexes were observed.
Discussion The tumor reported here generally simulated the neuroblastoma of the sympathetic nervous system. The histologic appearance of this tumor, however, seemed to be rather identical with rare primitive neuroectodermal tumor of peripheral since this tumor was composed of nerves reported by STOUT^^ and by NESBITT~~, primitive neuroectodermal cells which formed numerous Homer-Wright type rosettes without argyrophile fibers or neurites but with some fine fibrillar material in their centers, and did not show any differentiation tendency to ganglion cells. These histological features were different from those of neuroblastoma. The case reported by STOUT^^ was a 42-year-old man with a tumor in the ulnar nerve. Histological features of the tumor were identical with those of the tumor of our case. He suggested that the tumor might have arisen from embryonally misplaced neuroepithelium of the neural crest origin and called the tumor as “neuroepithelioma”. NE S BI T Talso ~~ reported a case of “pimitive neuroectodermal tumor (neuroblastoma) arising in sciatic nerve of a child’’ and described that the histologic pattern of the tumor was similar to that of STOUT’Scase. He also reviewed 15 cases of primitive neuroectodermal tumor of peripheral nerves in the literature. They have been reported under various names such as medulloepithelioma, neuroepithelioma, medulloblastoma or neuroblastoma. The nomenclature of this kind of tumor is controversial even now. Some differences exist between the tumor and neuroblastoma in histological features as above-mentioned. Moreover, Nagai-cell, cultured cell line derived from the tumor, was apparently different from the cultured neuroblastoma cells derived from children’s neuroblastoma of sympathetic nervous system. I n morphology in vitro, Nagai-cell mainly formed large aggregates with partial adherence to the glass surface, extended short cytoplasmic processes, and the processes remained short even after dibutyryl CAMP treatment and negative for Bodian’s staining. On the other hand, cultured neuro-
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blastoma cells comprised of small spiny cells or large epithelioid cells with neurites of various length which could be markedly elongated by dibutyryl CAMP treatment and stained positively with Bodian's staining4. Population doubling time of Nagai-cell was about 130 hours and that of cultured neuroblastoma cells about 20 to 60 hOUIS.4~9*20 The chromosome number of Nagai-cell was the lower tetraploid and that of cultured neuroblastoma cell near the d i p l ~ i d . ~ ~ ~ ~ Ultrastructure of Nagai-cell and cultured neuroblastoma cells was also different. Nagai-cell showed numerous microrosettes with microvilli-like projections into the central lumen and oligocilia, 9 +0 type, were observed occasionally. The neurosecretory granules were not found under the ordinary culture condition, but when treated with dibutyryl CAMP, the dense core vesicles appeared in the varicosity of microvilli-like cell processes. The varicosity closely resembled the growth cone of sympathetic neurons in culture2 and the dense core vesicles observed in Nagai-cell were also found not only in adrenergic neurons but also in various central and peripheral neurons.16 A more characteristic finding was the presence of satellite-like cells which embraced other tumor cells with narrow cytoplasmic processes without neurotubules nor neurofilaments. This was similar to the primitive satellite cells observed in the posterior spinal ganglia of chick embryo14, or sympathetic ganglia of human fetus15 and newborn ~ ~ Oin biopsy rats. On the other hand, neuroblastoma cells of both in c ~ l t u r e ~and material618 were mainly composed of undifferentiated neuroblastic cells but some had Nissl substance, bundles of neurofilaments and microtubules within cytoplasm and neurites, neurosecretory granules, and synaptic structures. When treated with dibutyryl CAMP, cultured neuroblastoma cells showed marked differentiation ability to either ganglionic and paraganglionic line as shown by marked increase in both number and size of the neurosecretory granules.4 From these results, it is suggested that Nagai-cell has different differentiation potentiality from that of neuroblastoma cells. It must be noted that the tumor reported here bears a close resemblance in microscopic appearance to the medulloblastoma of the cerebellum. Although in many cases no indication for neuronal or glial differentiation of medulloblastoma cells were found, some cases showed formation of cytoplasmic processes, rudimentary synaptic structure or glial filaments indicating either neuronal or glial differentiation.l]l7 Oligocilia and microrosettes with many cytoplasmic processes in the center were observed in some cases.7~18 From these observations, it is suggested that the medulloblastoma arises from the undifferentiated cell (medulloblast) seen in developing encephalon which later differentiates into neuroblastic or glial ~ e l l s . ~ P Primitive neuroectodermal tumors have been induced in central and peripheral nervous system of the various rodents by the inoculation of Adenovirus type l2.lo~l2~l3 Histological and ultrastructural figures were the same regardless of the animal species and the site where the tumor has developed. Histology of the tumor was very similar to that of Primitive neuroectodermal tumor, and the tumor referred to as medullab1astoma13 Or medul1oepithelioma.lo The ultrastructure of the tumor revealed to that of the tumor cells reported here with occasional 9 + 0 type oligoc~ia.lO,ls
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MUKAI~Odescribed that oligocilia have been observed in sensory neurons and their precursors. They were also observed in undifferentiated neural crest cells, neuroblastic cells and in immature satellite cells of posterior ganglia of chick embryo.14 O G A W A ~ ~ ~ ~ ~ observed glial or neurilemmal differentiation, and MUKAI~Oobserved neuronal differentiation in Adenovirus type 12-induced tumors. Because of the morphological similarity between Adenovirus type 12-induced tumors and that of our case, Adenovirus type 12-specific surface antigen and tumor antigen of the cultured tumor cells were examined by fluorescent antibody technique, and tumor antigen of primary tumor by complement fixation technique (HAMADA and MAEDA3). Each test, however, was negative. From the studies on the medulloblastoma and Adenovirus type 12-induced tumors, it might be suggested that there might exist similar, if not exactly identical, undifferentiated stem cells in both central and peripheral nervous systems, which have bipotency to differentiate toward either neuroblastic or glial line and neuroectodermal tumors might originate from the provisional stem cells in the central or peripheral nervous system. In conclusion, the tumor reported here was different from sympathetic neuroblastoma in many respects but identical to neuroepithelioma derived from primitive stem cells of neural crest origin. The tumor cells possessed bioptency to differentiate along both neuroblastic and neurilemmal line. Therefore it was regarded as homologous histogenetically to the medulloblastoma of the central nervous system and to the primitive neuroectodermal tumors of rodents induced by Adenovirus type 12. Acknowledgement: The authors are grateful t o Prof. T. HAMADA and Dr. H. MAEDA,Department of Virology, Niigata University, School of Medicine, for the examination of Adenovirus type 12specific antigens of the tumor cells. This study was supported in part by a Grant-in Aid for Cancer Research from the Ministry of Education, Science and Culture, and Ministry of Health and Welfare.
References 1. BAILEY,P. and CUSHINC,H. : Tumors of the glioma group. Lippincott, Philadelphia, 1926. 2. BUNCE,M.B.: Fine structure of nerve fibers and growth cones of isolated sympathetic neurons in culture. J. Cell Biol. 56: 713-735, 1973. 3. HAMADA, T. and MAEDA,H.: Personal communication. 4. ISHIKAWA, S. : Differentiation of human neuroblastoma cells in vitro. Morphological changes induced by dibutyryl cyclic AMP. Acta Path. Jap. 27: 697-711, 1977. 5. KANERVA,L. : Ultrastructure of sympathetic ganglion cells and granule-containing cells in the paracervical (Frankenhauser) ganglion of the newborn rat. Z. Zellforsch. 126: 2 5 4 0 ,
1972. 6. MACKAY,B., LUNA,M.A., and BUTLER,J.J.: Adult neuroblastoma. Electron microscopic observations in nine cases. Cancer 37: 1334-1351, 1976. J., and GULLOTTA, F. : The ultrastructure of medulloblasto7. MATAKAS,F., CERVBS-NAVARRO, mas. Acta neuropath. (Berl.) 16: 271-284. 1970. 8. MISUGI, K., MISUCI, N., and NEWTON,W.A.: Fine structural study of neuroblastoma, ganglioneuroblastoma, and pheochromocytoma. Arch Path. 86: 160-170, 1968. S., and INUI, A.: Comparison between the cells i n v i m g. MIYAI(E, s., OKABE, H., IMASHIJKU, and i n vitro, concerning catecholamine metabolism. Soshiki-baiyo 1: 23-27, 1975 (in Japanese). 10. MUKAI,N. and KOBAYASHI, S. : Human adenovirus-induced medulloepitheliomatous neoplasms
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in Sprague-Dawley rats. Am. J. Pathol. 73: 671-690, 1973. 11. NESBITT, K.A. and VIDONE,R.A. : Primitive neuroectodermal tumor (neuroblastoma) arising in sciatic nerve of a child. Cancer 37: 1562-1570, 1976. 12. OUAWA, K., TSUTSUMI, A., IWATA, K., FUJII,Y., OHMORI,M., TAUUCHI, K., and YABE,Y.: Histogenesis of malignant neoplasm induced by adenovirus type 12. Gann 57: 43-52, 1966. 13. OGAWA, K., HAMAYA, K., FUJII,Y., MATSUURA,K., and ENDO,T.: Tumor induction by adenovirus type 12 and its target cells in the central nervous system. Gann 60: 383-392,1969. 14. PANNESE, E.: in BRODAL, A., HILD, W., VAN LIMBORGH, J., ORTMANN, R., SCHIEBLER,T.H., and TONDURY,G. (Eds). Advances in Anatomy, Embryology and Cell Biology: The histogenesis of the spinal ganglia, Vol. 47, Fasc. 5, Springer-Verlag, Berlin, 1974. 15. PICK,J., GERDIN,C., and DELEMOS, C.: An electron microscopical study of developing sympathetic neurons in man. Z. Zellforsch. 62: 402-415, 1964. 16. POTTER, L.T.: Storage of norepinephrine in sympathetic nerves. Phamacol. Rev. 18: 439451, 1966. 17. RUSSELL, D.S. and RUBINSTEIN, L.J.: Pathology of tumours of the nervous system. 4th ed. Arnold, London, 1977. T. and KITAMURA, M.: in YAMADA, E., UCHIZONO, K., and WATANABE, Y. (Eds), 18. SAWADA, Fine Structure of Cells and Tissues, Vol. 6, 264-267, Igakushoin, Tokyo, 1971, (in Japanese). 19. SEIMURA,T .: Electron microscopic studies on advenovirus type 12-induced hamster tumors. Comparison among tumors in different locations. Niigata Medical J. 90: 229236, 1976 (in Japanese). H.R., GERSON,J.M., MOORHEAD, P.S., MAQUIRE, H., and HUMMELER, K.: 20. SCHLESINGER, Establishment and characterization of human neuroblastoma cell lines. Cancer Res. 36 : 3094-3100, 1976. G.F., and HAAQENSEN, C.D.: in ZABRISKIE,E.G., FRANTZ, A.M., and 21. STOUT,A.P., LAIDLAW, HARE,C. (Eds), Tumors of the Nervous System: Tumors of the peripheral nerves, 417-439, Williams & Wilkins, Baltimore, 1937.
