Loss heterozygosity the short arm ofchromosome1 in ... · .Sy*-__ __ __ Case 1 0 T N ~-. *A* 40 "Wq ... Restriction enzymesknownto showRFLPwith the various probes wereusedto digest

Proc. Natl. Acad. Sci. USAVol. 86, pp. 3753-3757, May 1989Genetics

Loss of heterozygosity for the short arm of chromosome 1 in humanneuroblastomas: Correlation with N-myc amplification

(suppressor gene/oncogene/restriction fragment length polymorphism/neural-crest tumors)

CHIN-To FONG*, NICHOLAS C. DRACOPOLIt, PETER S. WHITE*, PAULINE T. MERRILLROGERS C. GRIFFITH*t, DAVID E. HOUSMANt, AND GARRETT M. BRODEUR*§¶*Departments of Pediatrics and Genetics, Washington University School of Medicine, Saint Louis, MO 63110; §Pediatric Oncology Group, Saint Louis, MO63110; tCenter for Cancer Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139; and tDepartment ofPathology, The Miriam Hospital, Providence, RI 02906

Communicated by Donald C. Shreffler, February 21, 1989

ABSTRACT Partial monosomy of the short arm of chro-mosome 1 is the most consistent cytogenetic abnormality foundin human neuroblastomas, but its overall frequency and sig-nificance are unclear. Using a panel of chromosome-1-specificDNA probes that identify restriction fragment length polymor-phisms, we demonstrate that 13 of 47 human neuroblastomas(28%) have somatic loss of heterozygosity (LOH) at one ormore loci on the distal short arm of chromosome 1. Thechromosomal region that shows LOH most consistently isbetween lp36.1 and lp36.3; loss ofa gene or genes in this regionmay be critical for the development or progression of neuro-blastomas. The region of LOH in human neuroblastoma mayresemble that described for pheochromocytoma, medullarythyroid carcinoma, and melanoma, which are also tumors ofneural-crest origin. Although LOH for distal chromosome lpcan occur in early stages of neuroblastoma, the loss usuallyoccurs in tumors of advanced clinical stages. LOH for the shortarm of chromosome 1 correlates significantly with N-mycamplification, suggesting that these two genetic events arerelated. Indeed, these two lesions appear to characterize agenetically distinct subset of particularly aggressive neuroblas-tomas.

Cytogenetic and molecular studies of human cancer cellshave revealed characteristic genetic lesions, usually consist-ing of deletions, translocations, and gene amplification (1, 2).Although the latter two abnormalities have been associatedprimarily with oncogene activation, deletions are thought toidentify the location of putative cancer suppressor genes or"antioncogenes," the loss or inactivation of which may playa role in tumor development or progression. The locations fora number of these putative suppressor genes have beenidentified, based on consistent chromosome deletions orallelic loss in specific malignant diseases (1, 2). However,only one candidate member of this class of cancer-relatedgenes has been cloned-the retinoblastoma gene located atchromosomal region 13q14 (3-5).

Cytogenetic analysis of near-diploid neuroblastomas andtumor-derived cell lines has revealed a consistent deletion ofthe distal short arm of chromosome 1 in >70% of cases (6-8). Indeed, this finding is confirmed by a recent statisticalanalysis of 60 near-diploid neuroblastoma karyotypes, whichindicates that deletion of chromosome lp is the only numer-ical or structural abnormality that occurs with increasedfrequency (P < 0.001) (9). Unfortunately, most tumors ana-lyzed have come from patients with advanced stages ofdisease or from established neuroblastoma cell lines. Inaddition, cytogenetic analysis of primary-tumor tissue is notalways successful and may be difficult to interpret.

Therefore, we have taken a molecular approach by exam-ining restriction fragment length polymorphisms (RFLPs) inboth normal and tumor tissues from individual patients toidentify loss of heterozygosity (LOH) on chromosome 1 in alarge number of unselected neuroblastomas. We intended todetermine the frequency of this lesion in an unselected seriesof neuroblastomas, to further define the region that is con-sistently deleted in these tumors, and to determine therelationship of this abnormality to N-myc amplification andclinical variables.

MATERIALS AND METHODSTumors and Cell Lines. We studied pairs of human neuro-

blastoma DNA and constitutional DNA from 47 individualpatients. Of the 47 tumor samples, there were 45 primaryneuroblastomas and 2 tumor-derived cell lines. The 47 cor-responding constitutional DNA samples were derived fromEpstein-Barr-virus-transformed lymphoblastoid cell lines (34cases), untransformed leukocytes (8 cases), and other so-matic tissues (e.g., kidney, liver; 5 cases).DNA Probes. We used a panel of 19 chromosome-i-specific

DNA probes (10-22), and the order of these probes wasdetermined by genetic linkage in the Centre d'Etude duPolymorphisme Humain reference panel families (10). Ofthese probes, eight define known gene loci-proatriodilatin(PND) (11), a-fucosidase (FUCAl) (12), the protooncogenec-fgr (FGR) (13), the protooncogene L-myc (MYCL) (14),a-amylase (AMY1) (15), ,3 nerve growth factor (NGFB) (16),anti-thrombin III (AT3) (17), and renin (REN) (18); twoprobes define chromosome-i-specific repetitive sequences-D1Z2 (19) and D1S57 (20); seven probes were isolated froma flow-sorted chromosome 1 library (D1S15, D1S16, D1S17,D1S18, D1S19, D1S21, and D1S22) (10), and two probesdefine anonymous DNA sequences-CRI-L336 (D1S47) (21)and D1S2 (22). A locus is considered informative for aparticular patient when the constitutional DNA from thatpatient displays two different alleles (i.e., heterozygosity atthat locus).DNA Methods. DNA was isolated from tumor tissue and a

normal DNA source from each patient and quantitated usingfluorometric methods as described (23, 24). Five microgramsofDNA was digested with a restriction enzyme, electropho-resed on 0.8% agarose gels, and transferred to Zeta-probenylon membranes (Bio-Rad) according to a standard protocol(23, 24). Probes were labeled with dCT32P using the random-primer technique (25). Southern hybridization and autorad-

Abbreviations: LOH, loss of heterozygosity; RFLP, restrictionfragment length polymorphism.$To whom reprint requests should be addressed at: Department ofPediatrics, Washington University School of Medicine, 400 SouthKingshighway Boulevard, Saint Louis, MO 63110.

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

3753

Proc. Natl. Acad. Sci. USA 86 (1989)

iography were performed as described (23, 24). Thesetumor/normal DNA pairs were analyzed with probe andenzyme combinations known to show RFLPs in a substantialpercentage of cases.

RESULTSThe percentage of patients informative at any one locusranged from 18% to almost 100%. Of the 47 patients, all wereinformative at 3 or more loci (median 7 loci, range 3-11 loci)for a total of 311 informative loci in the 47 patients. Allpatients were informative for at least 2 loci at or distal to

Case 3T N

D1S57 at 1p32. LOH at two or more alleles on the distal shortarm of chromosome 1 was detected in 13 of the 47 cases(28%). Representative autoradiograms of selected loci areshown in Fig. 1.Twelve ofthe 13 tumors reveal a pattern ofLOH consistent

with terminal deletions of the short arm of chromosome 1(cases 1-4 and cases 6-13, Fig. 2), whereas the remainingtumor (case 5) may have an interstitial deletion. Thus, thecommon region of LOH in these 13 tumors lies between thedistal end of the DNA locus defined by probe FGR and theproximal end of the locus defined by probe D1Z2, corre-

Case 4T N

Case 5T N

20kb-

tooOWS oe.-

DI Z2(Taq I)

1.7kb- -

Dl S47(Rsa I)

FGR(EcoR I)

By.. _

.Sy*-_____

__

Case 1 0

T N

~-.

*A* 40

"Wq

ammup

.P:

___W

4.6 kb -

4.0kb-

C-_,0 _

2 .6 kb-24kb-AN -"

D1 S57 5.3kb4.8 kb 4

(TaqI) 4.6kb m

3.5 kb-

a" 1*a

fta 6 40

4mm

D1 S21 1.6 kb-

(Taq I)1.3 kb-b

SimWWi,,_ _

FIG. 1. LOH on chromosome lp in human neuroblastomas. Odd lanes represent tumor DNA (T), and even lanes represent normal somaticDNA (N). Restriction enzymes known to show RFLP with the various probes were used to digest the DNA. A tumor/somatic DNA pair isuninformative when the somatic DNA shows only one of two (or more) possible RFLP alleles (e.g., case 3 for probes D1S47 and FGR, case5 for probes D1S57 and D1S21, and case 10 for probe FGR). The pair is informative when two RFLP alleles are seen in the somatic DNA. LOHin the informative cases is defined as loss of one of the two RFLP alleles in the tumor compared to normal somatic DNA (e.g., case 3 for probesD1Z2,* D1S57, and D1S21; case 4 for probes D1Z2, D1S47, FGR, and D1S57; case 5 for probes D1S47 and FGR; and case 10 for probes D1Z2and D1S47). There is no LOH in the informative cases when the two alleles seen in the somatic DNA are preserved in the tumor DNA (case4 for probe D1S21, case 5 for probe D1Z2, case 10 for probes D1S57 and DMS21).

*Buroker et al. (19) performed Southern hybridization studies on 27 unrelated individuals using the D1Z2 probe and Taq I. They found no twoindividuals with identical banding patterns due to the high degree ofpolymorphism. This observation is supported by our study of >50 unrelatedpatients (results not shown). Constitutional homozygosity at this locus is inferred to be extremely unlikely. Individuals showing no change inbanding patterns between tumor and normal DNA are considered to have shown no LOH; the possibility remains that LOH is not detectedin this case because of superimposition of bands.

3754 Genetics: Fong et al.

..am

Proc. Natl. Acad. Sci. USA 86 (1989) 3755

CASES INDIVIDUAL CASES4 5 6 7 6 3

Symbols Key

* Informative, LOH

o Informative, No LOH

1q23-254/ |^~T3 |11/37I | 0O0O| | °° 0°1 q23-25 R 21/36101|00100001 101

FIG. 2. Analysis for LOH at various chromosome 1 loci in human neuroblastomas. The probes used are listed at the left of the box, andthe order of the loci defined by the recombinant probes is established by Dracopoli et al. (10). The number of informative cases (INF) are listedover total (TOT) tested for each probe. To the right of totals are the specific results from the 13 cases in which LOH was detected. Methodsfor determining LOH are described in the legend for Fig. 1. Informative combinations are indicated by symbols (e = LOH; o= no LOH).Virtually all remaining probe and DNA pair combinations were noninformative. A few combinations (14/247) were not determined (especially9 for NGFB) due to technical reasons or because only a limited amount of DNA was available. The shaded area indicates the minimum area

presumed to be deleted, as defined by two or more probes showing LOH. The proximal breakpoints occur somewhere between the most proximalinformative probe showing LOH and the most distal informative probe showing no LOH, so afew cases may have larger deletions than indicated(e.g., cases 5 and 9). A dark bar is shown at left of probes, and it defines the region most consistently deleted, indicating that the smallest regionof overlap included in all deletions appears to be those gene loci probed between FGR and D1Z2.

sponding to a cytogenetic region between 1p36.1 and lp36.3.The possibility remains that LOH is not detected in case 5because of superimposition of bands (Fig. 2). Nevertheless,we postulate that loss or inactivation of a gene (or genes)within band 1p36 is critical for the development of neuro-blastoma, and so the putative neuroblastoma locus mostlikely lies within this region. Mutation in the critical region onone chromosome, followed by deletion of the same region onthe homologous chromosome (as manifested by LOH), maybe an important mechanism in the transformation or progres-sion of human neuroblastomas.Based on densitometric analysis of the intensity of auto-

radiographic signals (Fig. 1), the LOH for alleles at or distalto 1p22 appears to represent a loss of one allele withoutreduplication. Thus, the LOH most likely represents a simplehemizygous deletion of the short arm of chromosome 1, asopposed to mitotic recombination. This explanation is con-sistent with our previous cytogenetic studies on primaryneuroblastomas and tumor-derived cell lines that generallyshow simple deletions resulting in partial monosomy forchromosome ip (6, 7, 9). Because no LOH was detected atthe informative loci between 1p22 and lq (from P nervegrowth factor locus to renin locus) (Fig. 2), loss of an entirechromosome 1 or loss followed by reduplication of theremaining homolog was not observed.

Similar RFLP studies were done to look for LOH on otherchromosomes with a subset of the tumor/normal DNA pairsused in this study as well as 27 additional pairs from patientswith neuroblastoma (data not shown). The DNA pairs wereexamined for LOH with one to six probes per chromosome[median, two probes on at least 12 different chromosomes,including chromosomes 2-13, 15, 19, 20, and 22 (W. K.Cavenee and A. Koufos, personal communication)]. No

LOH has been detected to date on any of these otherchromosomes. These findings agree with the very low inci-dence of random chromosome deletion or loss for chromo-somes other than chromosome 1 seen in the cytogeneticanalysis of 60 near-diploid tumors (9). Thus, the backgroundofnonspecific LOH in neuroblastomas was extremely low forthe other chromosomes tested. Although not every chromo-some or chromosome arm was tested, our findings show ahighly significant and nonrandom involvement of chromo-some lp in human neuroblastomas.The other genetic lesion consistently associated with neu-

roblastoma is amplification of the protooncogene N-myc (23,24). The presence of N-myc amplification has been shown tocorrelate positively with advanced clinical stage and poorprognosis in human neuroblastomas (23, 24). Table 1 showsthe correlation between the clinical staging and either theLOH for chromosome lp or the degree of N-myc amplifica-tion (or both) for the 47 patients in this study. Although thereis a trend toward association with advanced stages of disease(stages 3 and 4), it is not yet statistically significant (y2 = 2.86;P > 0.05). However, there was a very strong correlationbetween N-myc amplification and chromosome lp LOH (x2= 17.24; P < 0.001): amplification was found in 8 of 13 cases(62%) with lp LOH, compared with only 1 of 34 (3%) withoutlp LOH. Moreover, both N-myc amplification and deletionof chromosome lp (as detected by cytogenetic analysis)appear strongly correlated with a poor clinical outcome (23,24, 26, 27).

DISCUSSIONMost previous cytogenetic studies of neuroblastomas sufferfrom the inherent bias that only =20% of the primary tumors

Genetics: Fong et al.

Proc. Natl. Acad. Sci. USA 86 (1989)

Table 1. Correlation of chromosome lp LOH and N-mycamplification with stage

Patients, no.

Both LOHLOH N-myc and N-myc

Stage* Total for ip amplification amplification

1 6 1 0 02 6 0 0 03 12 6 5 54 21 6 4 34S 2 0 0 0

Total 47 13 9 8

*Clinical stages are defined as follows. Stage 1, localized tumorconfined to the area of origin; complete gross excision with orwithout microscopic residual disease; identifiable ipsilateral andcontralateral lymph nodes negative microscopically. Stage 2, uni-lateral tumor with incomplete gross excision and/or with ipsilateralregional nodes positive for tumor; identifiable contralateral lymphnodes negative microscopically. Stage 3, tumor infiltrating acrossthe midline with or without regional lymph node involvement, orunilateral tumor with contralateral regional lymph node involve-ment, or midline tumor with bilateral regional lymph node involve-ment. Stage 4, dissemination oftumor to distant lymph nodes, bone,bone marrow, liver and/or other organs (except as defined in stage4S). Stage 4S, localized primary tumor as defined for stage 1 or 2with dissemination limited to liver, skin, and/or bone marrow(modified from ref. 57). There was an apparent correlation betweenLOH for chromosome ip and advanced stage, but it did not reachstatistical significance (two-by-two test, x2 = 2.86; P > 0.05).However, LOH for chromosome ip and N-myc amplification arehighly correlated (x2 = 17.24; P < 0.001).

can be karyotyped. Thus, the findings seen from thesecytogenetic studies may not be representative of all neuro-blastomas, but rather of a select subset. On the other hand,most molecular studies to date present only information onN-myc amplification and do not address LOH on chromo-some ip in the same patients. Therefore, our study of thesetwo important genetic lesions in a large series of unselectedpatients provided the most accurate genotypic analysis ofneuroblastomas to date.Our studies also provide a more precise localization of the

putative neuroblastoma locus on chromosome 1 to the region1p36.1-1p36.3. Our finding of LOH for chromosome lp in-:=30% of patients contrasts with the frequency of >70% seenin an analysis of near-diploid tumors and cell lines (9).However, if one includes only primary tumors and does notexclude those with a hyperdiploid or triploid karyotype (>57chromosomes), the frequency of lp deletions drops to =40o(9), which agrees more closely with our studies.Thus, several explanations are possible for why LOH was

not seen in a higher proportion of neuroblastomas: (i) mostcytogenetic analyses are derived from advanced-stage tu-mors with near-diploid karyotypes, in which the incidence oflarge chromosome lp deletions may be more common; (ii)mutational events at the critical region in some neuroblasto-mas may be too small (e.g., point mutations or small dele-tions) to be detectable by LOH analyses with currentlyavailable probes; (iii) LOH for chromosome ip may play arole in malignant transformation in only a subset of neuro-blastomas (such as those with advanced stages of disease);and (iv) LOH for chromosome ip may be a secondary eventthat occurs in some tumors during the course of clonalevolution. Although we did find LOH for chromosome ip inan early-stage neuroblastoma, our data predominantlyshowed LOH for lp in advanced disease stages (see below)and thus supports the latter two possibilities.LOH at specific chromosomal regions has been seen in a

variety of other human neoplasms (3-5, 28-51). In retino-

blastoma, deletion of a specific gene on chromosome 13,thought to be critical in tumorigenesis, has been identified (3-5, 51), and its RNA and protein product have been initiallycharacterized (52-54). However, even in this prototypicexample of a recessive cancer gene and even with probes thatare within the actual gene that is frequently deleted, abnor-malities of the gene structure and/or expression are in therange of 12-40% of the cases (3-5, 51), similar to our findings.

In some melanomas, medullary thyroid carcinomas, andpheochromocytomas, deletion or somatic loss of heterozy-gosity in the tumor tissue has been demonstrated at loci ondistal chromosome ip (28, 55). Because these three neo-plasms also are embryologically derived from neural-crestcells, our studies of neuroblastomas raise the possibility thata common mechanism may underlie the formation or pro-gression of these embryologically related tumors.

In summary, at least two genetic events in the course oftumor evolution in neuroblastoma have been identified-lossof a critical region on the short arm of chromosome 1 andactivation (usually by amplification) of the N-myc protoon-cogene. Our studies suggest that the two genetic events arerelated and that one may precede or predispose to the other.The functional relationship between the loss of a gene orgenes from this critical region of chromosome lp and othergenetic events related to neuroblastoma development orprogression remains to be determined. However, these data,as well as the consistency with which normal or increasedN-myc copy number is seen in tumors from individualpatients over time (56), suggest that deletion of chromosomelp as well as N-myc amplification are features ofa geneticallydistinct subset of particularly aggressive neuroblastomas.

We are very grateful to Drs. Webster K. Cavenee and Alex Koufosfor sharing their data on LOH for other chromosomes in their seriesof neuroblastomas. We thank the following individuals or institutionsfor providing us with recombinant probes used in the present study:M. Litt for D1Z2, C. Helms (Collaborative Research) for CRI-L336or D1S47, P. Frossard for PND, J. S. O'Brien for FUCA1, R. Whitefor D1S57, J. Minna for MYCL, P. Pearson for D1S2, A. Ullrich(Genentech) for NGFB, K. Ishizaki for AMY1, E. Prochownik forAT3, and J. Chirgwin for REN. We appreciate the technical assis-tance of J. Wasson, D. Norman, and J. Gordon throughout the courseof the study. Patient and tumor samples were kindly provided bymembers of the Pediatric Oncology Group. This work was supportedby grants from the National Institutes of Health-CA39771 andCA01027 (G.M.B), CA44176 (N.C.D.), CA40842 (D.E.H); the Na-tional Cancer Center (C.-t.F.); the American Cancer Society-IN-36-29-4 (C.-t.F.); the Children's United Research Effort (G.M.B.);the Joshua Macy, Jr. Foundation (P.S.W.); and the Fern WaldmanMemorial Fund for Cancer Research.

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Genetics: Fong et al.