Original Paper

International Journal of Cell Cloning 8:409-424 (1990)

Establishment and Characterization of a Primitive Neuroectodermal Tumor of Bone Continuous Cell Line (LAP-35)

Gian Paolo Bagnam". ', Massimo Serra". : Marco Giovannini"' ', Manuela Bdiali", Mario Stella: Anna Monrald: Donatella Gmchi", Rwlo Paolucci", Paola Rocchit Annalisa Pession: Pier0 Picci", : Guido Paolucci", Mario Campanacci". Lino Tessarollo', Andrea 0. Cavazzanaf

"G. Prodi Interdepartmental Center for Cancer Research, binstitUte of Histology and Embryology, University of Bologna, Bologna, Italy; 'Laboratory of Oncological Research, Istituti Ortopedici Rizzoli, Bologna, Italy; dDepartment of Human Genet- ics, San Bortolo Hospital, Vicenza, Italy; 'Institute of Experimental Oncology, 'In- stitute of Pathology, University of Padova, Padova, Italy

Key Words. Primitive neuroectodermal tumor of bone Ewing's sarcoma differentiation Cytogenetics * Molecular genetics

Neural

Abstract. A continuous tumor cell line (LAP-35) was established from a primitive neuroectodermal tumor of bone from the right tibia of a 12-year-old female. The neural character of the cell line was documented by the spontaneous growth of neurites and by the presence of several neural markers, including neuron-specific enolase (NSE), $100 protein, neurofilaments, chromogranin A, synaptophysin and positivity to monoclonal anti- bodies UJl27.11, UJ13A, UJ181.4. Cell-sorter analysis showed a high expression of nerve growth factor receptor (NGFr) and major histocompatibility complex class I-related molecules. A unique cytogenetic profile was observed, including a reciprocal chromosomal translocation (rct) 11:22 (q24;ql2), typically associated with Ewing's sarcoma and neuro- epithelioma, and deletion of the short arm of chromosome 1 (lp-), otherwise a feature of neuroblastoma. N-myc proto-oncogene was neither amplified nor expressed, whereas the expression of c-myc was documented by northern blot analysis. These features distinguish this new cell line from previously reported neuroectodermal cell lines, identifying LAP-35 as a unique model of a group of neural bone tumors that share characteristics of neuro- blastoma as well as neuroepithelioma.

Correspondence: Dr. Marco Giovannini, Istituto di Istologia ed Embr. Gen., Via Bel-

Received April 30, 1990; provisionally accepted June 21, 1990; accepted for publica-

0737-1454190/$2.00/0 @AlphaMed Press

meloro 8, 40126 Bologna, Italy.

tion July 27, 1990.

Bagnara et al. 410

Introduction

Small round cell tumors of bone (RCTB) comprise a heterogeneous group of lesions which range from undifferentiated variants, such as Ewing’s sarcoma (ES), to more differentiated neoplasms such as small cell osteosarcoma. Recently, a new class of RCIB was determined to be neural in origin, and the name of primi- tive neuroectodermal tumor of bone (PNETB) has been proposed [I, 21. These rare tumors are often confused with ES due to their similar anatomo-clinical fea- tures [3,4]. In fact, both tumors affect the long bones of young persons and share an undifferentiated small round cell morphology. Currently, the presence of rosettes and/or pseudorosettes, together with positivity for neural markers [5 ] , are the two most reliable features in differentiating PNETB from ES. The comparative rar- ity of PNETB and the lack of tumor cell lines derived from these tumors makes it difficult to define whether these tumors actually belong to the spectrum of ES rather than represent a distinct entity. LAP-35 is, to our knowledge, the first con- tinuous tumor cell line derived from a PNETB, offering new insight into the bio- logical characteristics of peripheral primitive neuroectodermal tumors.

Materials and Methods

Tumor and Cell Culture In December 1987, a 12-year-old girl was admitted to the “Istituti Ortopedici Rizzoli”

in Bologna for pain and swelling of the right knee. X-rays and CT scan evaluation of the limb showed an aggressive osteolytic lesion of the right proximal tibia involving the soft tissues. Radiographic examination of the lungs showed no sign of metastatic disease. Inci- sional biopsy revealed a proliferation of undifferentiated small round cells consistent with Ewing’s sarcoma. Therefore, the patient’s leg was amputated above the knee. Histological examination of the surgical specimen revealed a tumor composed of a uniform population of small round cells arranged in a vague lobular pattern. Homer-Wright-like rosettes were occasionally detected as well as numerous pseudorosettes (Fig. 1). Immunocytochemical analysis showed an intense positivity to neuron-specific enolase (NSE) and S-100 protein. On the basis of the histological features and the immunocytochemical results, the diagno- sis of PNETB was made. At the time of writing, the patient was alive and well and had no evidence of metastatic disease.

The LAP-35 cell line was established from the amputation specimen. Under sterile conditions, the surgical material was collected, placed in Iscove’s modified Dulbecco’s medium (IMDM; GIBCO, Wley , Scotland) and immediately processed. Tissue was minced and incubated at 37°C in collagenase type I (Sigma, St. Louis, MO) at a concentration of 2 m g / d in phosphate-buffered saline (PBS) for 30 min. Three cycles of enzymatic diges- tion were performed for a total incubation time of 90 min. After each cycle, cell suspen- sions were washed, seeded in 25 cmz or 75 cm2 flasks (Coming, Stone Staffordshire, England) with IMDM supplemented either with 10% fetal calf serum (FCS; Biological Industries, Kibbutz Beth Haemek, Israel) or with 30 nM selenium dioxide, 5 p g / d insu- lin and 5 p g / d transferrin (all from Sigma). Both culture media were supplemented with penicillin (100 U / d ; GIBCO), streptomicin (100 pg/ml; GIBCO), and cells were incu- bated at 37°C in a humidified atmosphere of 5% C02. The medium was changed every 3 days, and the culture was grown for more than 50 passages at the split ratio of 1 5 every 7 days.

Primitive Neuroectodermal Tumor of Bone Cell Line 411

Fig. 1. This picture shows the original biopsy of the tumor. The tumor was composed of a uniform population of small round cells destroying bone trabecolae; a preliminary diagnosis of ES was made. (Paraffin-embedded tissue section stained with hematoxylin- eosin, ~ 2 0 0 . )

Growth In Mrro Doubling time and saturation density were determined by seeding 2.5 X los cells into

a 25 cm2 flask. To determine the LAP-35 growth curve and doubling time, adherent cells were counted in duplicate at 24 h intervals. Cell doubling time was calculated during the logarithmic phase of growth (from 48 to 96 h after seeding). Once growth had stopped, the medium was changed daily and saturation density w a s calculated by counting adherent cells every 2 days. Growth in semisolid medium was determined by seeding 1 X lo3 to 1 X los cells into 60 mm culture dishes; colonies were scored after 14 days. Growth in plastic was determined by limiting dilution assay (up to 100 cells/well). Proliferation rate was determined by 'H-thymidine ('H-Tdr; hersham, Buckinghamshire, England) uptake. Cells were seeded in quadruplicate at concentrations of 0.5, 1, 10, 100, 200 (all X 1031ml) in 16 mm wells in IMDM + 10% FCS. After 72 h of culture, the cells were labeled with 'H-Tdr (1 pCi/well) for 4 h, and assayed for incorporated radioactivity by using a multiple cell harvester (Skatron, Oslo, Norway) and a Beta-counter (LKB, Uppsala, Sweden).

Electron Microscopy Glutataldehyde (2.5%) was gently added at mom temperature to cells grown in 25 cmz

flasks. After 30 min, cells were scraped from the bottom and collected by centrifugation.

Bagnara et al. 412

The final pellet was post-fixed in osmium tetraoxide (0.1%), dehydrated and embedded in Epon 812 by routine techniques. Ultrathin sections were stained with uranyl acetate and lead cytrate and scoped with a Siemens Elmiskop 101 electron microscope.

Growrh in Nude Mice Injections of 30 x lo7 cells suspended in 0.5 ml of PBS were administered in the right

legs of 4-week-old female nude mice (strain: Crl.nu/nu(CD-1)BR). Animals were observed for a detectable tumor mass every 2 days for 12 weeks and then sacrificed.

Mycoplasma Test The cell line was considered free of mycoplasma contamination with the following

methods: colony formation in agar-broth selective medium (Mycotrim; Hana Media Inc., Berkeley, CA), fluorescent Hoechst 33258 stain (Calbiochem, La Jolla, CA) [6] and 3H-Tdr incorporation by culture supernatant [7].

Immunocytochemical Studies Cells were cultured on glass coverslips for 3 to 5 days, fixed in acetonemethanol(7:3)

at -20°C for 10 min and then tested by applying a wide panel of monoclonal antibodies (MoAbs) and specific antisera (Table I). Immunoenzymatic detection was achieved by the immunoalkaline phosphatase (APAAP) technique [8]. Staining of specific antisera was ob- tained with a 4-stage APAAP procedure [9]. MoAbs were diluted in IMDM with 10% FCS. PBS (0.05 M, pH 7.6) was employed to dilute both the antisera and APAAP complexes as well as for washing procedures. New fuchsin (Sigma) was utilized as chromogen. Nuclear counterstaining was performed with Gill’s hematoxylin (Sigma). Serum controls for each case consisted in the substitution of the specific antibody with non-immune mouse IgG.

Immunofluorescence and Flow Cytometry Adherent cells were removed with a tripsin-EmA solution (lox; GIBCO) diluted

1:lO in PBS and resuspended in culture medium for membrane immunofluorescence; ad- herent cells were fixed for 10 min in acetone-methanol (73) at -20°C for cytoplasmic im- munofluorescence. Cells were incubated for 30 min with the primary MoAbs, washed twice in PBS and incubated for 30 min with the fluorescein-conjugated anti-mouse F(ab’)2GaMIg (Technogenetics, Milan, Italy ) or rabbit anti-guinea pig (Miles Laboratories Inc., Elkart, IN). Incubations were performed at 4°C and at mom temperature for membrane and cytoplas- mic antigens, respectively. For flow cytometric analysis, cells were stained using MoAbs to nerve gravth factor receptor (NGFr; Amersham) and HLA-A, -B, -C [lo] (kindly provided by Dr. G. Damiani, Istituto di Chimica Biologica, Universita di Genova, Genova, Italy), followed by fluoresceinated rabbit anti-mouse Ig F(ab’)2 (Dakopatts, Glostrup, Denmark) as a second step. Green fluorescence was analyzed on an EPICS-C flow cytometer (Coulter Electronics, Luton Beds, UK). Debris was gated out by forward and 90” angle light scat- ter. Samples stained with the fluoresceinated secondary antibody alone were used as negative controls for background correction.

ffiryotype Analysis Cytogenetic analysis of the in vitro tumor cell line was performed at the 3rd, 15th and

30th passages. The technique used for the analysis is briefly summarized as follows. Cells were treated with 0.4 pglml colchicine for 1 h. KCI (0.075 M) was added, and cells were fixed with a 3:l mixture of methanol and glacial acetic acid. Fifty GIG-banded metaphases were examined to estimate the breakpoints, and CBG banding was used to evaluate the cen- tromeric heterochromatin. Karyotype was determined according to ISCN criteria [ll].

Primitive Neuroectodennal Tumor of Bone Cell Line 413

Table I. Immunocytochemical analysis of LAP-35 cells

Antibodies Specificity LAP-35- (source) positive

cells (%)

anti-NSE (Dakopatts) neuron-specific enolase 98 f 2 S-100 (Dakopatts) nervous cells 95 f 5 anti-NGF* (Amersham) NGF receptor 45 f 7

NR-4 (Boehringer) neurofdaments 68 kDa 0

NE-14 (Boehringer) neurofilaments 200 kDa 10 f 2 V9 (Dakopatts) vimentin 99 f 1 DE-B5 (Dakopans) desmin 0 CKl (LP34) (Dakopatts) citokratins 0 OK-DR (Ortho Diagnostic) Ags HLA class II W6/32 (Seralab) Ags HLA class I 99 f 1 A2BSb neuronal cells 0

neuroectoded-associated antigen 0

PHE5 (Ortho Diagnostic) chromogranin A 40 f 5 SY (Dakopatts) sy naptophysin 30 f 3

"-18 (Boehringer) neurofdaments 160 kDa 96 f 4

0

anti-Thy-lb Thy-1 antigen 30 f 5

0 UJ167b

30 f 3 UJ223-tIb

0 UJ127-llb UJ115-3b

20 f 2 15 f 1 UJ181-4b

Pil53-3' lymphoblastoid cells (LC), neuroblastoma (NB) 0 0

,I ,I II

I1 I1 I 1

w ,, I,

I , I, N

0 ,I ,I

UJUA~

CD 9 (BA-2) (Hybritech) CD24 (BA-1) (Hybritech) LC, NB, B lymphocyte, granulocyte 0 CD56 (NKHI) (Coulter) NK cells, N-CAM 0 CD57 (HNKI) (B & D) NK cells, neuroectodermal-associated antigen 0

LC, NB, bone marrow mononuclear cells

'evaluated by FACS bkindly provided by J.T. Kemshead, Oncology Laboratory, Institute of Child Health,

'kindly provided by A.E. Evans, The Childrens Hospital, Philadelphia, PA, USA London, UK

Molecular Studies High molecular weight DNA was prepared from a frozen pellet of cultured cells by

sodium dodecyl sulfate-pmteinase-K lysis, phenol extraction and ethanol precipitation as pre- viously described [l2]. DNA (10 pg) was completely digested with EcoRI, analyzed by elec- trophoresis on 0.8% agarose gel and capillary blotted onto a nylon membrane (Hybond N; Amersham) in 1.5 M NaCl and 0.25 M NaOH. Overnight hybridization was carried out at 6YC, according to Church [13], with the probe for N-myc (pNBI EcoRUBamHI 1 kb size, 2nd exon) and c-myc (pGEM2 EcoRIIHindm, 3rd exon) and labeled according to the method developed by Feinberg and Volgestein [14]. Filters were washed as previously described [U]. Autoradiography was performed on X-ray fdm. Total RNA was prepared from a frozen pellet of cultured cells by homogenization in urea lysis buffer, phenol extrac-

Bagnara et al. 414

tion and ethanol precipitation as described [B]. Total RNA (20 pg) was run in 1% agarose formaldehyde gel and capillary blotted onto a nylon membrane in 0.05 M NaOH. Overnight hybridization and washing procedure was carried out as previously described [B]. Northern blot was hybridized with N-myc and c-myc probes, as well as labeled with b-actin probe (pBR322/PsrI) as previously described. DNA and RNA from a neuroblastoma cell line (IMR-32) and a Ewing’s sarcoma cell line (A-4575) were run in parallel as positive con- trols for N-myc and c-myc amplification and/or expression, respectively.

Results

Cell Morphology Adherent polygonal and/or triangular tumor cells were characterized by the

presence of long (more than twice of soma length) cytoplasmic processes. The latter displayed typical varicosities along their course, forming a delicate network between cellular soma (Fig. 2A). These cellular processes are reminiscent of neu- ritic extensions as typically observed in cultured neuroblastoma (NB) cells while ES cell lines show a primitive, uncommitted appearance (Fig. 2B).

Ultrastructurally, LAP-35 cells displayed a round to oval morphology with oval nuclei. Chromatin was finely distributed and small nucleoli were occasion- ally observed. The cytoplasm contained numerous free ribosomes, mitochondria and prominent Golgi complexes (Fig. 3A). Occasional dense core granules were observed at the periphery of the cytoplasm (Fig. 3B). Cellular processes were characterized by the presence of arrays of microtubules and intermediate filaments with few electron dense granules along their course.

lmmunocytochemistry and Flow Cytometry A wide panel of MoAbs was employed in order to confirm the neural nature

of the tumor cells (Table I). NSE, S-100 protein, chromogranin A and synapto- physin were highly expressed in the majority of LAP-35 cells. Among the MoAbs which recognize neuroectodermal epitopes [16, 171, UJ-181-4, UJ-l3A, UJ-l27:ll and anti-Thy-1 gave a positive reaction in about 30% of cells, whereas A2B5, UJ-167 and UJ-123 failed to react with LAP-35 cells. The cytoskeleton composition was then investigated and two types of intermediate filaments were readily identified with the immunofluorescence technique. Neurofilaments of 160 kDa and 200 kDa were co-expressed with vimentin. Desmin and keratin were absent. A strong positivity for NGF receptor and HLA-A, -B and -C epitopes was detected by cell sorter analysis.

Growth Characteristics The kinetics of LAP-35 growth was investigated as described in the Materi-

als and Methods section. The doubling time turned out to be 49 h (Fig. 4A) with a saturation density of 60 x lo4 cells/cm2. Thymidine uptake (Fig. 4B) and cellu- lar concentration appeared to be inversely related. The concentration of 500 cells/ml showed the highest proliferative activity. No significant differences were

Primitive Neuroectodermal Tumor of Bone Cell Line 415

Fig. 2. Phase-contrast appearance of LAP-35 cell line. A) LAP-35 cells appeared uniformly small and pear-shaped with long cellular processes forming a delicate intercon- necting network. Note the presence of varicosities along the neuritic extensions (X200). B) Ewing’s Sarcoma cell line (A-4575): epithelioid-looking cells arranged in small clusters displaying a very primitive uncommitted appearance (~100) .

Bagnara et al. 416

Fig. 3. A) Ultrastructurally LAP-35 cells are characterized by the presence of round nuclei with finely dispersed chromatin and evident nucleoli (nc). The cytoplasm contained numerous large mitochondria, sparse lysosomes and evident Golgi’s complexes (Gc) ( ~ 1 2 , 2 0 0 ) . B) Occasional dense core granules at the periphery of the cytoplasm were also observed (x33,OOO).

Primitive Neuroectodermal Tumor of Bone Cell Line 417

Fig. 4. A) LAP-35 cell line growth curve. Data are the mean f SD of three different experiments performed in quadruplicate. B) 3H-Tdr uptake in LAP-35 cell line at vari- ous cellular concentrations. Data are from one representative experiment performed in quadruplicate.

noted in terms of growth conditions when LAP-35 cells were grown in serum- free IMDM containing insulin, transferrin and selenium dioxide. Plating efficiency was tested both in plastic and in soft agar. Plating efficiency was 100% in plastic and 5% in soft agar.

LAP-35 cells (3 x 10’) injected S.C. in nude mice did not lead to the develop- ment of a detectable tumor mass in 5 out of 5 treated Crl.nu/nu(CD-l)BR mice, neither in the injection site nor in the internal organs (brain, lung, liver, adrenal gland and kidney).

Cytogenetics The modal number of the tumor cells revealed a near diploid profile with

Bagnara et al. 418

Fig. 5. Representative karyotype of LAP-35 cell line. Formula: 48,XX,del(l)(p34), +8,t(ll;22)(q24;ql2),inv(14)(qll.2;q32), -16, +der(l6)t(1.16)(q11;qll.l), +20.

the mode at 48. GTG and CBG-banding revealed the major stem line as 48,XX, del(l)(p34), +8,t(ll;22)(q24;ql2),inv(14)(qll.2;q32), -16, +der(l6)t(l.l6)(qll;qll.l), +20 (Fig. 5) . Two major clusters of chromosome abnormality were identified. The first included several chromosomal abnormalities generally associated with ES and PN [18, 191, such as a reciprocal chromosome translocation (rct 11;22), trisomy of chromosome #8 and der(16)t(1;16). The second was characterized by the presence of a partial monosomy of short arm of chromosome #l (lp-)(p34) similar to the one often observed in neuroblastoma [20-221, while chromosome #14 showed an inversion of the long arm.

Oncogene Analysis The atypical neural trait characterized by the simultaneous expression of typi-

Primitive Neuroectodermal Tumor of Bone Cell Line 419

Fig. 6 Southern blot analysis of LAP-35 cell line digested with EcoRI and hybridized to N-myc (A) and c-myc (B) probes. a = neuroblastoma cell line (IMR-32); b = LAP-35 cell line; c = control DNA from lymphocytes.

cal markers of both neuroblastoma and neuroepithelioma required further inves- tigation at the molecular level. Southern blot analysis failed to reveal any amplifi- cation or expression of the N-myc or c-myc oncogenes (Fig. 6). As shown in Figure 7, analysis of oncogene expression revealed that the c-myc oncogene was expressed

Bagnara et al 420

Fig. 7. Expression of myc-related oncogenes (c-myc and N-wc). One Ewing’s sar- coma (A4575) and one neuroblastoma (IMR-32) cell line were selected as positive con- trols for c-myc and N-myc expression, respectively. The c-myc oncogene was expressed in the LAP-35 cell line (A) that failed to show any expression or amplification of the N- myc oncogene (B). pactin (C) expression was used as a quantitative mRNA standard. a = LAP-35 cell line; b = Ewing’s sarcoma cell line (A-4575); c = neuroblastoma cell line (IMR-32).

in LAP-35, while no N-myc mRNA was detected. In the LAP-35 cell line, no over-expression of c-myc was detected when compared with beta-actin expression.

Discussion

Neuroblastoma and peripheral neuroepithelioma (PN) represent two distinct prototypes of the adrenergic and cholinergic lineage of peripheral primitive neuro-

Primitive Neuroectodermd Tumor of Bone Cell Line 421

ectodermal tumors (PNETs), respectively. In fact, despite their shared neuroecto- dermal origin they dif i r in many respects, including biochemical, immunological, genetic and cytogenetic profiles. NB is a well known adrenergic tumor, associ- ated in most cases with abnormal catecholamine excretion [23]. On the other hand, PN is characterized by an exclusive cholinergic enzyme content [24]. Major histo- compatibility complexes are commonly expressed on PN cells [4], whereas they are generally absent in NB [25, 261. Furthermore, a different oncogenic profile characterizes NB and PN. N-myc oncogene amplification and/or expression is typically associated with NB [27-291, while the c-myc oncogene is commonly expressed in PN [30].

Neuroblastoma further differs from PN for consistent chromosomal abnor- malities. Deletion of the short arm of chromosome 1 (lp-), homogeneous stain- ing regions (HSRs) and double minutes (DMs) have been found in virtually all NB cell lines and in about one-third of primary tumors [31,32], while they have never been reported in PN. Moreover, an rct involving chromosomes 11 and 22 (q24;q12) is typically found in PN [33, 341. The rct 11:22 has also been found in another apparently unrelated small round cell tumor, namely Ewing’s sarcoma of bone [19]. This finding, together with the presence of neuroectodermal epi- topes [35] and in vitro-inducible neural differentiation [21], strongly supports a neuroectodermal origin for ES of bone. Indeed, ES is now proposed as an un- differentiated member of the PNETs family [3,36]. Nevertheless, primitive neuro- ectodermal tumors of bone, as distinct from ES, have also been recently described [l, 21. These tumors closely mimic ES, and although they are often microscopi- cally undistinguishable from the latter, they are recognizable on the basis of im- munocytochemical and ultrastructural findings. The expression of one or more neural antigens, such as NSE or S-100 protein, together with the presence of neuro- secretory granules at the ultrastructural level favors the diagnosis of PNETB rather than ES [6].

Very little is known about the biology of these rare tumors (PNETB), so the LAP-35 cell line represents a unique model for studying the biological charac- teristics of this new class of bone tumors and their relationship with ES on the one hand and NB and PN on the other.

LAP-35 showed a distinctive phase-contrast appearance under standard culture conditions characterized by the spontaneous growth of neurites. This in vitro behav- ior seems to reflect a degree of neural commitment and differentiation like con- ventional NB and PN and different from classical ES. Overt in vitro neural features are in fact only inducible in ES cell lines [21], whereas they are spontaneously observed in other neural tumors, such as neuroblastoma and neuroepithelioma.

The neural characteristics of the tumor cell line have been further documented by an intense positivity to a wide panel of neural markers such as UJ127.11, UJUA, UJ181.4, Thy-1, NSE, S-100 protein, NGFr and synaptophysin. LAP-35 cells also showed a strong cytoplasmatic positivity to anti-chromogranin A antibodies. This result is well correlated to the presence of dense core granules at the ultrastruc- tural level. Chromogranin A is in fact the major soluble protein in vesicles, in

Bagnara et al. 422

adrenal medulla [37] and in several neuroendocrine and nervous tissues [38]; its expression is commonly observed in NB cells [39], whereas it is generally ab- sent in ES and PN [40]. This finding, in association with the deletion of the short arm of chromosome #l, seems to suggest a closer histogenetic relationship with NB rather than PN or ES. However, LAP-35 cells were further characterized by the presence of an rct 11:22, expression of MHC-related molecules and absence of any detectable amplification and/or expression of the N-myc oncogene. The presence of chromosomal abnormalities that characterize both NB and PN/ES constitutes a unique combination in the family of neuroectodermal tumors, only previously described in two cases of olfactory neuroblastoma [41]. The significance of this finding is still obscure and further investigation is required to establish the role of these chromosomal markers in a given neural tumor phenotype.

The aforementioned features clearly distinguish the LAP-35 tumor cell line from any NB and PN as well as from ES cell lines previously reported, also provid- ing experimental evidence for the existence of an intermediate class of neural tumors which share both the characteristics of NB and PN/ES.

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