[CANCER RESEARCH 39, 519-526, February 1979)0008-5472/79/0039-0000$02.00

Genetic Basis of Susceptibility for Neuroblastoma followingTreatmentwith N-Methyl-Nnitrosourea and X-Rays inXiphophoru&

Manfred Schwab,2 Gerhard Kollinger, Joachim Haas, Mulkh R. Ahuja, Safia Abdo,3 Anneross And•rs,andFritz Anders

Genetisches Institut der Justus-Liebig-Universitaet Giessen, Heinrich-Buff-Ring 58-62, D-6300 Giessen, Federal Republic of Germany

ABSTRACT INTRODUCTION

In 65 genotypes of Xiphophorus (nonhybnids and interpopulational and interspecific F and backcnoss hybrids),the susceptibility to neuroblastoma following treatmentwith carcinogenic-mutagenic agents was tested. The fishwere exposed to an aqueous solution of N-methyl-N-n itrosounea (MNU) or exposed to X-rays. Neunoblastoma wasinduced by MNU in 64 of about 3500 treated fish and by Xnays in 4 of about 5500 treated fish; in the control fish, aswell as in the vast number of fish routinely bred in our fishlaboratory during the last 20 years, neunoblastoma was notobserved. Sixty of the 64 MNU-induced neuroblastomasand all 4 X-ray-induced neuroblastomas developed in the605 and 859 treated fish, respectively, of a group of backcross genotypes carrying the “lineatus―chromosome.These fish are derived from Xiphophorus variatus x Xiphophorus helleri hybrids with X. helleri as the recurrent parent.The remaining four cases of neuroblastoma induced byMNU were distributed at random throughout fish of backcross genotypes derived from other species; within these, arelationship to the genotype is not detectable. In both X.variatus and the F (X. variatus x X. helleri), althoughcarrying the same lineatus chromosome, as well as in X.helleri lacking this chromosome and in the lineatus-lackingsegnegants, from each of which a comparable number offish was treated, neuroblastoma could not be induced. Theresults are interpreted to imply that the neuroblastoma istriggered by somatic mutation of regulating genes suppressing another gene that favors neoplastic transformationand that is located on the lineatus chromosome. Thedifferential susceptibility of the fish carrying this chromosome (X. variatus, F , and backcross hybrids) might dependon the number of regulating genes. Accordingly, insusceptibility, as in the case of X. variatus, would be due to apolygenic regulating system, which, in practice, cannot beimpaired in one cell. Susceptibility, as in the case of thelineatus backcross segnegants, would be due to a monogenic regulating system, which can easily be impaired byone single mutation, and this monogenic state of regulationmay be achieved by elimination of regulating genes in thecourse of selective backcrossing.

, This work was supported by Deutsche Forschungsgemeinschaft through

Sonderforschungsbereich 103 ‘Zellenergetikund Zelldifferenzierung' (Projects C 11 and C 12), Marburg; and by Land Hessen through Justus-LiebigUniversitaet Giessen. This work is dedicated to Peter Karlson on the occasionof his 60th birthday. This paper contains parts of the dissertations of GerhardKollinger, Joachim Haas, and Safia Abdo.

2 To wham requests for reprints should be addressed.3OnleavefromtheUniversityofAlexandria;supportedbytheEgyptianMinistry of Education.

Received August 11, 1978; accepted October 23. 1978.

Certain interpopulational and interspecific hybrids between the platyfish (Xiphophorus maculatus) and theswordtail (Xiphophorus helleri) spontaneously develop melanomas (20, 21, 24, 36), thyroid tumors (41), or oculartumors (22). Subsequent analysis of melanomas as well asother neoplasms of Xiphophorus has led to the suggestionthat development of a neoplasm may result from a misguided expression of an endogenous entity capable oftransforming cells from the normal to the neoplastic state(4-6). This entity was called Tu4 (7). Tu appears to bepresent in all populations and species of Xiphophorus andmay exist in an individual in multiple form distributed ovenseveral chromosomes. Although the nature and the biological function of Tu are still largely obscure, some speculations on these aspects of Tu have been presented recently(2-4).

The present investigation is part of a broad-scale expeniment in which a large number of defined genotypes weretested for their susceptibility to develop neoplasms following treatment with mutagenic-cancinogenic agents (1, 9, 10,23, 35, 47-51 ). We have tested the susceptibility with 2basically different types of agents, namely, a chemicalagent, MNU, which is a direct-acting mutagen-carcinogennot requiring metabolic activation (37, 40, 42), and a physical agent, X-nays. Both exert their carcinogenic effect mostlikely via mutation (for MNU, see Refs. 15 and 28; for X-nays,see Refs. 13 and 17). This experiment has shown thus fan,that, following exposure to both agents, various neoplasmscan be induced, including melanoma, neunoblastoma,squamous cell carcinoma, epithelioma, carcinoma, adenocarcinoma, papilloma, hepatoma, fibrosarcoma, rhabdomyosarcoma, and lymphosancoma; the susceptibility to 5everal of these neoplasms appears to be confined to particularchromosomes (49, 51). In a previous paper, we have pnesented data on the susceptibility to both the fibrosancomaand rhabdomyosarcoma (49). This report deals with thesusceptibility to neuroblastoma.

MATERIALS AND METHODS

Fish Genotypes

Sixty-five different genotypes from X. maculatus, Xiphophorus xiphidium, Xiphophorus variatus, Xiphophorus

4 The abbreviations used are: Tu, tumor gene; MNU, N-methyl-N-nitrosou

rea; BC, backcross; PBS, phosphate-buffered saline, containing (per literI1@O)5.82 g NaCI, 0.145 g KCI, 1.05 g Na@HPO,'2I4@O,0.145 g K142P0,,0.073g CaCL@,0.073 g MgCL'6H,O; Li, lineatus chromosome; A-gene, regulatinggene.

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montezumae cortezi, and Xiphophorus helleri guentheri, aswell as F1and BC hybrids, were used in this study (Table 1).The species and subspecies were originally obtained fromMyron Gordon in 1959 and from Curt Kosswig in 1960.Gordon had collected these fish between 1939 and 1951.The fish have been maintained since their collection inclosed stocks. The approximate number of generationssince then is between 40 and 60 for the different species(for further descriptions of these fish and for the localitieswhere the original material was collected, see Refs. 29, 30,58, and 59).

The different genotypes exhibit, on lack, specific melanophore spot patterns, which are due to the expression ofspecific genes. Each is located on a particular chromosome. All these genes are in any genotype coexpressed(codominant); this makes it possible to recognize the presence onabsence of specific chromosomes in any genotype.

Treatment

From each of the genotypes, at least 50 individuals (forsome genotypes, up to 700) were treated, all being between6 weeks and 6 months old. About 3500 fish were treatedwith MNU, and about 5500 fish were treated with X-nays.The same number of animals served as controls; they werehandled like the treated ones.

MNU Treatment. Animals were exposed in aquariums tofreshly prepared aqueous solutions of MNU (1 mM; pH 6.0to 6.5; 27°).Four 1-hr treatments were given in 2-weekintervals.

X-lrradlation. Animalswere whole-body-irradiatedwith1000 A for 45 mm (dose nate, 22 A/mm; 150 kV, 12 ma); thisdose was administered 3 times at 6-week intervals (the totaldose was 3000 R/fish; for details, see Ref. 46).

Genotypes treated with MNU and X-nays

Species,populations, and hybridsNonhybrids

Xiphophorus maculatus (Rio Jamapa)x.maculatus(RioSt.Pedro,Usumacintasystem)x.xiphidium(RioPurification,RioSotoIaMarina)x. variatus(RioPanuco)x. montezumaecortezi(RioAxtla-RiaPanucasystem)x.helleriguentheri(BelizeRiver)x. helleriguentheri(RioLancetilla)

Hybridsx. maculatus(RioJamapa)x X.helleri(RioLancetilla)without

markerF1BC@(n = 1, 3, 5, 7); BC parent X. he/len

x. maculatus(RioJamapa)x X.montezumaeFBC@(n = 2, 3, 5); BC parentX.montezumae

x. maculatus(RioJamapa)x X.xiphidiumFIWF

x. vaniatusx X.he/len(RioLancetilla)withoutmarkerF,BC@(n = 1, 4, 15); BC parent X. he/lenBC@(n = 1, 4, 15); BC parent X. helleni albino

Melanophorespat pattern markerfor different chromosomes

Sd,@'Sd' Sp, Sn,Sn',Dt, Cn,Sh, Os,Sd°',Sn°'Sh, Os; without markerF!, Ct, FIDILi, PuScDb,; without markerDb2; without marker

Sd, Sd', Sp, Sn,Sn',Dt, Cr, Sh, Os,Sd°',SnDI50%segregantswith markeras in F , 50%without marker

Sd, Sn,Sc50%segregantswith markeras in F,, 50%without marker

Sn, Sd, Sp, Sd, Sd'@1

Li50% segregants with marker as in F, , 50% without marker50% segregants with marker as in F,, 50% without marker

a Cn, crescent; Ct,cutcrescent; Db, dabbed; Dt, dot; Fl, flecked; Fl°', deletion of flecked; Li, lineatus; Os, one spat; Pu, punctatus; Sc,

spotted caudal; Sd, spotted dorsal; Sd°', deletion of spotted dorsal; Sd' , spotted dorsal mutation; Sh, shoulder spat; Sp, spotted; Sn,striped, Sn' , striped mutation. Far further details, see Refs. 29, 30, 58, and 59.

520 CANCER RESEARCHVOL. 39

Examination

LightMicroscopyExamination.Wholefishwere fixed inBouin's solution, dehydrated, and then embedded in paraffin and sectioned. Sections were stained with hematoxylinand eosin and were selectively stained with periodic acidSchiff, Van Gieson's stain, and G@mOni'sstain.

Electron Microscopy Examination. Tissue samples werefixed in 2.5% glutanaldehyde in PBS. All specimens werepostfixed for 2 hr in 1% osmium tetroxide. After dehydration, they were embedded in ERL-4206 (Serva). Ultrathinsections were cut with a diamond knife using a ReichertOm U2 ultramicrotome. Sections were stained with uranylacetate and lead citrate and were examined in a Zeiss EM10 electron microscope.

RESULTS

Description of the Neuroblastoma

Morphology.Followinga latent periodof 3 to 7 monthsafter the last treatment, the first clinically observablechange in the area of the eye occurs. It consists of a slightprotrusion of the eye, apparently due to proliferation of theunderlying tissue. During the following 2 to 3 weeks, thetumor grows rapidly and may reach up to 7 mm in diameterwithin this period. In most cases, only one eye is affected(Fig. 1, a and b) and, exceptionally, both eyes show theprotrusion.

The eye itself apparently remains functional in most ofthe fish as judged from their response to stimuli.

Histology.Sagittalsectionsrevealedthat the neuroblastoma is in most cases located exterior to the sclena in theretrobulban space within the area of the onbita (Fig. lb).Only in advanced stages are the tumor cells also found

Table 1

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Susceptibility ofX. vaniatus (Li/Li), X. he/len (—I—),their F, (Lii—),and backcnoss segregants (Li/—and —/—)frombackcnossesof Lii—animals to X. helleni (Fig. 2) to develop neunoblastoma following treatment with MNU andX-naysAnimals

NeoplasmaTreated

Survived

Genotype MNU X-rays MNU %(1 X-rays %@ MNU %(@ X-rays%@x.

variatusLi/Li 286 698 271 94.7 635 91.0 0 0 00x.helleni —I— 198 68 182 91.9 59 86.8 0 0 00F,

Lii— 103 476 89 86.4 462 97.0 0 0 0 0BC, Lii— 226 684 212 93.8 661 96.6 17'@― 8.0 2b0.3—I—

201 352 198 98.5 335 95.2 0 0 00BC,Lii— 181 104 170 93.9 102 98.1 15@@ 8.8 l'@1.0—I-.

153 88 146 95.4 82 93.2 0 0 00BC,@Lii— 198 111 193 97.4 96 86.5 28'@' 14.5 1b@,0‘-I—

165 91 151 91.5 83 91.2 0 0 0 0

Neuroblastoma Induced in Xiphophorus

Table 2

(I Percentage of incidencebased on numberof survivors.

b The susceptibility of the Li segregants differs significantly from the insusceptibility of the group of othergenotypes, including the Li-lacking segregants, the F, , X. vaniatus, and X. he/len; p < 0.001.

(. The susceptibilities of the BC, and BC4 are not significantly different; p < 0.001.

d The incidences at BC, and BC4 to those at BC,@ fit a 1 :2 ratio; p < 0.05. p calculated by the @2method.

within the eye, invading the sclena and the retina as well asthe vitreous body and the area around the iris; the opticnerve is invaded in few cases. These findings would suggestthat the nidus of the neuroblastoma is outside the eye. Thetumor often shows extensive necrotic and hemorrhagicareas.

The major part of the neunoblastoma is packed denselywith round to ovoid, closely aggregated, regular hyperchromatic cells, measuring between 3 and 7 @min diameter.The cells possess a scanty, ill-defined cytoplasm (Fig. lc).The nuclei usually occupy an abnormally large portion ofthe cell; mitotic figures are abundant in some areas of theneuroblastoma. The tumor cells are frequently arranged inrosette-like structures (Fig. lc, arrows).

Ultrastructure. The nuclei are polymorphic, with pocketsand projections (Fig. ld), and the nuclear mass appearsfrequently bipartite, depending on the direction in whichthe nucleus had been sectioned. This nuclear appearancein neunoblastoma is similar to that found in earlier investigations in the melanoma of Xiphophorus (54, 55). Cytoplasmic organelles are scarce in most of the cases; they show afew abnormally large mitochondniaand small vesicles, fewendoplasmic neticula, and varying numbers of nibosomes.In other cases, however, large areas of distinct endoplasmic

neticula were observed. In many cells, characteristic solitarycilia were detectable which, without exception, consistedof 9 pairs of peripheral doublets of microtubules with nocentral axis (Fig. ld, inset). In addition, in some of theneoplasms, particles were detectable that resemble in sizeand structure C-type viruses5 (see Ref. 35).

Dependence of the SusceptibilIty on the Genotype.Within the 3500 fish of the 65 genotypes treated with MNU,

the neuroblastoma developed in 64 fish, and within the5500 treated with X-rays, neuroblastoma was found in 4. Inthe untreated controls, which were handled the same as thetreated fish, neuroblastoma did not develop. In addition, inthe vast number of fish of the same genotypes used in thisstudy that were bred and maintained over the last 20 yearsin our fish laboratory, neuroblastoma has not been ob

5Manuscriptinpreparation.

served. This would imply that all of the neunoblastomasobserved following treatment with MNU and X-rays in fishof these genotypes are due to the action of these agents.

In the case of the MNU-induced neuroblastoma, all but 4fish developed neuroblastomas in one group of backcnoss

segnegants carrying the Li chromosome (derived from X.variatus; its presence can be recognized by the Li spotpattern) (Fig. 2); the remaining 4 neunoblastomas weredistributed at random throughout backcnoss genotypesderived from other species, and within these a relationshipof the susceptibility to the genotype cannot be drawn. Inthe case of X-ray-induced neuroblastoma, all fish developedneuroblastomas also in the group of BC segregants carrying the Li chromosome.

The Li backcross segregants, in which the high susceptibility for neuroblastoma was observed, were obtained according to the crossing principles shown in Fig. 2. In thisstudy, according to availability, 2 earlier backcnosses, BC,and BC4, and one later backcross, BC,@,were used (Table1). MNU and X-nays induced the neuroblastoma in BC, in 17of 212 fish (8.0%) versus 2 of 661 fish (0.3%); in BC4 in 15 of170 fish (8.8%) versus 1 of 102 fish (1.0%); and in BC,5 in 28of 193 fish (14.5%) versus 1 of 96 fish (1.0%; Table 2). ForMNU, the incidences between BC, and BC4 are not significantly different (p < 0.001), and the mean value of theincidences at BC1 and BC4to that at BC,5 fit a 1:2 ratio (p <0.05). The incidence for X-irradiation is much lower, disallowing such a relationship between BC,, BC4, and BC,5.

As a result, only the Li segregants of the backcrosses aresusceptible to neuroblastoma, both following MNU treatment and X-irradiation. In contrast, the F, and the parentalspecies X. variatus, although carrying the same Li chromosome, are insusceptible, and the BC segregants lacking theLi chromosome, as well as the parental species X. helleri,which also lacks this chromosome, are also insusceptible(p <0.001).

DISCUSSION

The Relation of the Induced Neuroblastomato OcularDiseases in other Vertebrates. Protrusion of the eye,

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called exophthalmus or “pop-eye,―is a disease that istound frequently in Xiphophorus. It may be caused, forinstance, by infection with a variety of organisms (trematodes, bacteria, fungi, viruses, etc.), by hormonal influences, or by mechanical injury (see Ref. 19). The exophthalmus described in the present article could be identified byhistological examination as due to a neoplasm of theretrobulbar space of the eye (35). It has never been observed in our laboratory to occur spontaneously. The histological picture is that of a neuroblastoma which consistsof imperfectly differentiated cells of neural origin. Thisneuroblastoma is rather similar to the eye tumor of apparently neural origin described earlier in Xiphophorus (39). Itis, however, different from the neoplasms originating frompigment cell precursors also induced by MNU and by X-raysin various tissues, including the eye, in Xiphophorus.6 Thisneoplasm, furthermore, with respect to its histologicalstructure, is similar to the neoplasms induced by nitnosamines and nitrosamides in the nasal cavity and in the olfactorybulb of the rat (11) and also resembles the extnaoculanorbital tumors of apparently neural origin induced by human adenovirus type 12 in the hamster (45). The histologicalpicture of fish neunoblastoma is also similar to that of theintraocular netinoblastoma-like tumors induced by humanadenovirus in the rat (34). The neunoblastoma, however, tothe best of our knowledge was not induced thus far in fishby treatment with mutagens-carcinogens (for review oncarcinogenesis in aquarium fish, see Ref. 44).

Genetic Basis of the SusceptIbilityto Neuroblastoma.By fan, most of the neuroblastomas in this study wereinduced in fish of the Li genotype. This would suggest thatthe susceptibility to neuroblastoma following MNU treatment and X-irnadiation is determined by the genotype inXiphophorus.

The agents used in this susceptibility test, both the“radiomimetic― MNU (43) and the X-rays, are known to be

potent mutagens (for a comparison between MNU and Xrays, see Ref. 38). It has been proposed (12) that some typeof mutation is involved in the initiation step of the processleading to neoplasia (for more recent analyses, see Refs. 25and 26). Such a mutation could be a change in eitherchromosome number and/on chromosome structure leading to an imbalance between genes for “expression―or“suppression―of neoplastic transformation (14, 27, 52), orit could be a single gene mutation (15), involving a singlebase missense (28) leading to the impairment of genes thatare involved in the suppression of genes responsible forneoplastic transformation.

Speculating that the neuroblastoma induced in the present investigation might be due to somatic mutation, onehas to assume that the susceptibility in the Li segregantsdepends on a single gene. If 2 or more genes would beinvolved, the incidence should be extremely low (for relation between mutation rate, tumor incidence, and numberof genes controlling the susceptibility, see Ref. 4). On thisbasis, one might formally interpret the result that theparental species X. variatus and X. helleri, their F, and theLi-lacking segregants from backcnosses are insusceptible,whereas the Li segregants are susceptible to neuroblas

S ManUscript in preparation.

toma formation in the following way. X. variatus carries agenetic factor that favors neoplastic transformation of neurogenic cells (tentatively called Tu). Tu is controlled byother genetic factors (tentatively called A), one of which islinked to Tu whereas the others are nonlinked. Both Tu andR-genes are present in homozygous state. Because animpairment of several R-genes in the same cell by mutationis an extremely unlikely event, the animals, in practice,remain insusceptible. The same applies for the F,, whichalso carries both Tu and the linked and nonlinked R-genes,although only in hemizygous state. In the backcnoss generations, Tu is transmitted along with its linked R-gene andonly the nonlinked A-genes, due to new combinations ofchromosomes, are eliminated. Fish of this genotype arehighly susceptible. The increase of the susceptibility fromBC, to BC,@ (Table 2) to MNU from 8.8% to 14.5% can beexplained as due to the elimination of further nonlinked A-genes. The doubling of the susceptibility from BC, to BC,@mi@ht indicate that a single main nonlinked A-gene hadbeen eliminated. The existence of such main nonlinked A-genes was also shown to operate in the control of melanoma development (4, 8, 56).

Generalizationof the Results.The fact that, from genotypes insusceptible to neoplasia, other genotypes that aresusceptible can be constructed by selective matings givesfurther support to the idea that, in the origin of cancer, oneor several genetic changes are involved. In the developmentof neuroblastoma in Xiphophorus analyzed in this expeniment, apparently several genetic changes are involved,including new combination of chromosomes as well asmutational events. This would suggest that the genotypemay determine whether the organism does or does notrespond to the exposure to a carcinogen with the development of a neoplasm and that, in the analysis of the etiologyof neoplasia, the experimenter should be able to controlgenetic factors strictly.

This view is supported by several results including thoseobtained in higher vertebrates.

For instance, in man in the etiology of various humanneoplasms, including retinoblastoma, Wilms' tumor of kidney, or pheochnomocytoma, apparently 2 mutational eventsare involved, the first being a germ line mutation and thesecond being a somatic mutation (26, 31-33, 53). Thissituation may, in principle, be comparable to the neunoblastoma induced in Xiphophorus by MNU and by X-rays. Theonly difference might be that in Xiphophorus the susceptibility is achieved by new combinations of chromosomesinstead of the germ line mutation.

The role of genetic factors is also apparent in certaindomesticated animals. For instance, Boxer dogs have asignificantly higher incidence for spontaneous neoplasiathan do other breeds (16), although the genetic basis forthe high incidence has not been elucidated thus far; theyare also extremely susceptible to induction of neoplasiafollowing treatment of MNU (18). Further examples for apredisposition for cancer are certain breeds of horse, fowl,and other domesticated animals. In addition, certain breedsof mouse and rat have a high incidence for spontaneousneoplasms; in contrast, neoplasms are rare in natural populations of both the mouse and the rat (57). These findingsare in parallel to those obtained in Xiphophorus. While

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nonhybnids of Xiphophorus, which stem from natural populations, are apparently insusceptible, certain backcrossgenotypes develop neoplasms following the treatment ofvarious organs or tissues (see “Introduction' ‘; see alsoRefs. 47 to 51). On the basis of the findings in Xiphophorus,one might speculate on a more general mechanism for thed ifferential susceptibility of wild-type and domesticatedanimals. According to these findings, insusceptibility of thewild-type animals might be due to a polygenically controlledbalance between genes favoring neoplastic transformationand genes suppressing neoplastic transformation, and thisbalance might have been built up during the evolution ofthe populations in genetic isolates. In the course of crossing and subsequent backcrossing, as in the case of Xiphophorus in the breeding of the Li segregants, or in the caseof other animals during domestication, genes suppressingneoplastic transformation may be eliminated. If all suchgenes are eliminated, neoplasms develop spontaneouslywithout any further treatment. The most well analyzedexample is the spontaneously developing melanoma ofcertain breeds of Xiphophorus (4-8). If all of these genesare not eliminated, depending on the importance of theremaining genes, a more or less susceptible animal isobtained that may develop neoplasia following a furthergenetic event induced by mutagenic-carcinogenic agents;a typical example is the neuroblastoma described in thispaper (for further neoplasms, see Refs. 49 to 51). Theseapparent parallels in the etiology of cancer between Xiphophorus and higher vertebrates imply that this model mightbe suitable for analyzing further the genetic basis forneoplasia in general (for broader discussion, see Refs. 2 to4).

ACKNOWLEDGMENTS

We are indebted to Dr. H. D. Mennel, Pathologisches Institut der Universitaet Freiburg im Breisgau, for his help in the identification of the neoplasm.We thank Kaete Klinke for her help in the breeding of the fish, and JuttaSiegers for excellent technical assistance.

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524 CANCER RESEARCH VOL. 39

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Neuroblastoma Induced in Xiphophorus

Fig. 1. a, Li-carrying segregant of the BC, (X. variatus x X. helleri) x X. helleni exhibiting an MNU-induced neurablastama in the area of the left eye. x 3,b, sagittal section showing the tumor mass causing protrusion of the eye. H & E, x 6. c, neuroblastama cells. Note rosette-like arrangement (arrows). H & E,x 1400. d, electron micrograph of the neuroblastoma cells. Note nuclear packets (P), bipartite nuclear mass (N), abnormally large mitachondria (M), andsmall vesicles (V). x 14,000. Inset, cross-section of a cilium exhibiting the 9 + 0 pattern of microtubUles. x 44,000.

la

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FEBRUARY1979 525

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Further backcrosses were obtained by selective backcrossing of the Li segregants to X. helleni. Only the Li segregants were susceptible to neuroblastomafollowing MNU treatment and x-irradiation.

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1979;39:519-526. Cancer Res   Manfred Schwab, Gerhard Kollinger, Joachim Haas, et al.   Xiphophorus

-nitrosourea and X-Rays in N-Methyl-NTreatment with Genetic Basis of Susceptibility for Neuroblastoma following

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