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Cancer Genet Cytogenet 96:134–139 (1997)

Elsevier Science Inc., 1997655 Avenue of the Americas, New York, NY 10010

Loss of Heterozygosity at Chromosome 9p21 in Primary Neuroblastomas: Evidence for Two Deleted Regions

Brendan Marshall, Gloria Isidro, Antonio Gentil Martins,and Maria Guida Boavida

ABSTRACT:

The genes responsible for the development of neuroblastoma following in vivo deletion ormutation are largely unknown. We have performed loss of heterozygosity studies on a series of 24 Portu-guese primary neuroblastomas using 6 polymorphic markers located at chromosome 9p21 spanning thep16/MTS1/CDKN2, p15/MTS2/CDKN2B, and the interferon

a

and

b

genes. Loss of heterozygosity wasobserved in 4 of the 24 tumors (17%), a somewhat lower percentage than a previous study that identi-fied patients by a mass screening program. A correlation was also observed between 9p21 LOH and1p36 LOH in our group of tumors. Two distinct regions of 9p21 deletion were observed: one located inthe region adjacent to the markers D9S162 and D9S1747 and a second located centromerically of thep16 gene near the D9S171 marker. The latter region is exclusive of the p16 gene. This result suggests thepresence of at least one other tumor suppressor gene at 9p21, apart from the p16 and p15 genes, whichmay be of importance to the development of neuroblastoma. © Elsevier Science Inc., 1997

INTRODUCTION

Neuroblastoma is the most common solid tumor encoun-tered in young children [1]. A diverse range of geneticchanges have been described in primary neuroblastomasand cell lines and implicated in the process of tumourigene-sis. These include loss of heterozygosity (LOH) at chromo-some 1p36 [2–5], amplification of the N-MYC oncogene [6],mutations in the neurofibromatosis type 1 gene [7], muta-tions in the NM-23 gene [8], elevated telomerase activity[9], and frequent translocations involving the long arm ofchromosome 17 with a subsequent increase in the copynumber of this arm of the chromosome [10–12]. 1p36 LOH issignificantly associated with a poor prognosis and togetherwith N-MYC amplification is the most significant prognosticindicator so far identified, surpassing tumor stage, ploidy,and age at diagnosis [13–15]. Recently it has been reportedthat there are two distinct regions at 1p35-36 that aredeleted; a more distal region not associated with N-MYCamplification and a more proximal region that is associated

with N-MYC amplification [16–18]. The tumor suppressorgenes located in the 1p36 region await identification.

Despite the great interest in the identification of thegenes at 1p36, 1p36 LOH is detected in only 20 to 40% oftumors and N-MYC amplification in an even smaller per-centage [19]. Thus, the underlying genetic changes in themajority of neuroblastomas remain obscure. A distinguishingfeature of neuroblastoma is its heterogeneity in terms ofclinical symptoms, histology, its response to treatment, andeven its evolution (cases of spontaneous cure). One potentialcandidate gene that has so far received little attention isthe p16 gene located at chromosome 9p21. The gene issometimes also referred to as MTS1 (multiple tumor sup-pressor 1) or CDKN2. Originally identified and clonedbecause of its involvement in familial melanoma [20–21]the gene has since been implicated in the development ofa variety of neoplasms and mutations have been detectedin various tumors and tumor cell lines [22, 23]. One groupthat attempted to identify mutations in this gene in neuro-blastoma cell lines reported no success [24]. However, re-cently, Takita et al., [25] have reported the allelotype ofneuroblastoma that indicates that LOH at 9p21 is a rela-tively frequent event in neuroblastomas thus showing thatin this respect neuroblastomas are similar to a diversegroup of other malignancies such as leukemia [26], me-sothelioma [27], melanoma [28], brain glioma [29], andcarcinoma of the head and neck, lung, bladder, and oe-sophagus [30–38]. Using the IFN

b

1 probe, Takita et al. [25]observed LOH at 9p21 in 36% of the neuroblastomas stud-ied suggesting the involvement of one or more tumor sup-pressor genes at this locus. Although this might suggest

From the Departamento de Genetica Humana, Instituto Nacionalde Saude Dr Ricardo Jorge (B. M., G. I., M. G. B), Avenida PadreCruz, Codex, and the Serviço de Pediatria, Instituto Portugues deOncologia Francisco Gentil (A. G. M.), R. Prof. Lima Basto - Palhavã,1000 Lisboa, and Departamento de Cerurgiâ Pediatrica, Hospital D.Estafánia, Rua Jacinta Marto, Lisboa, Portugal.

Address reprint requests to: Dr. Brendan Marshall, Program inMolecular Immunology, Institute of Molecular Medicine and Genet-ics, Medical College of Georgia, Augusta, Georgia 30912-2600.

Received May 29, 1996; accepted August 19, 1996.

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135

that p16 is the likely target of this LOH it is by no meanscertain as considerable debate has surrounded the exact in-volvement of p16 in tumorigenesis because of the relativeinfrequency of homozygous deletions in this gene in pri-mary tumors compared to cell lines [39] and the overallinfrequency of mutations detected in primary tumors exhib-iting 9p21 cytogenetic abnormalities [40]. Furthermore,Puig et al. [41] have reported that in a panel of primary spo-radic melanomas, the shortest region of overlap (SRO) forLOH at 9p21 was outside of the region containing the p16gene. A recent report has also detected the loss of thenearby p15/MTS2/CDKN2B gene rather than the p16 genein a sporadic melanoma [42]. Many of the studies citedabove centered around the IFN genes and reported the dele-tion of both the

a

and

b

genes in various tumors, indicatingthat these too may be likely candidate genes.

Therefore, to more precisely define the region affectedby LOH at 9p21 in neuroblastomas we have studied a groupof Portuguese neuroblastoma patients with a series of 6polymorphic microsatellite markers that are located withinthe 9p21 chromosomal region. The markers span the regionthat includes both the p16 and p15 genes and also the IFNgenes. We report evidence for two deleted regions in thesetumors, one of which is exclusive of the p16 and p15 genes.

MATERIALS AND METHODS

Tumor and Blood Specimens

Twenty-four primary neuroblastomas were obtained post-operatively, immediately following surgery at the Portu-guese Cancer Institute and at the Children’s Hospital D.Estefânia, Lisbon, during the period 1992–1995. Tumorswere immediately frozen in dry ice and maintained at

2

70

8

C until DNA extraction. Ten milliliters of peripheralblood in EDTA was also obtained from each patient.Tumors were graded according to Evans staging and con-sisted of 3 Stage I, 6 Stage II, 4 stage III, 7 Stage IV, and 4Stage IV.

Loss of Heterozygosity Analysis

High–molecular-weight DNA was isolated from both tumorsand leukocytes using the method of Laird et al. [43]. To ana-lyze LOH, we utilized 6 polymorphic microsatellite markers;D9S157, D9S162, and D9S171 from the Genethon map [44]and D9S1747, D9S1748, and D9S1753 as described by Cairnset al., [45]. The markers are located on chromosome 9p21and flank both the p16 and p15 genes (Fig. 1A). Markerswere PCR amplified in a volume of 50

m

l for a total of 35cycles. All PCR reactions involved in an initial denaturationstep at 94

8

C for 5 minutes followed by 35 cycles of 94

8

C,30 seconds, annealing at variable temperature for 1 minuteand extension at 72

8

C for 30 seconds using 1.5 units ofAmpliTaq DNA polymerase. The amplification was com-pleted by a final extension step at 72

8

C for 5 minutes.Annealing temperatures for each of the primers were:D9S157

2

58

8

C, D9S162

2

56

8

C, D9S171

2

60

8

C, D9S1747

2

60

8

C, D9S1748

2

58

8

C, and D9S1752

2

58

8

C. Amplifiedmicrosatellites were electrophoresed on 6% acrylamide

sequencing gels containing 33% formamide and visualizedby silver staining using a modification of the method ofBudowle et al., [46]. Samples showing LOH were reamplifiedand rerun to confirm them as true examples of LOH.

1p36 LOH was performed using both PCR based micro-satellite markers and Southern blotting. MicrosatellitesD1S214, D1S220, D1S243, D1S228, D1S201, D1S190, andD1S209 located in the region 1p31-36 were amplified andanalyzed as described by Schleiermacher et al., [17].Southern blotting of tumor and normal DNAs was per-formed using the CEB 15 probe following

HaeIII

digestionof the DNAs as described [47].

RESULTS

9p21 LOH was observed in four of the twenty-four tumorsanalyzed (17%) and two regions of deletion were identi-fied (Fig. 1A). The first region, (region 1) defined by tumor1832, is located in and around the D9S162 and D9S1747markers just telomeric of the p16 gene. Three tumors(2058, 1217, and 1832) exhibit LOH in this region butbecause of the noninformativeness of the D9S1747 markerin tumor 2058 we cannot draw any conclusions regardingthe possible involvement or noninvolvement of the p16gene in this region. In addition, a second region (region 2),defined by tumor 2058, located centromeric and exclusiveof the p16 gene near D9S171 was lost in 2 tumors (2058and 1711). Tumor 2058 has thus lost heterozygosity fortwo distinct regions. Amplification of parental DNAs fromthose patients showing 9p21 LOH indicated that bothtumors with LOH for region 2 had lost the maternal allelewhereas, of the three tumors showing LOH for region 1,one had lost the maternal allele whereas two (includingtumor 2058) had lost the paternal allele. Tumor 2058 thathas LOH for regions 1 and 2 has thus lost these regionsfrom different chromosomes.

The fraction of tumors with LOH was substantiallylower than in the study of Takita et al. [25] that identified9p21 LOH in 36% of neuroblastomas. Therefore, to controlfor the possibility that our tumor samples contained suchamounts of normal cells as to make detection of LOH verydifficult under the PCR conditions used, we also investigated1p36 LOH in all tumors using both the PCR approach ofSchleiermacher et al. [17] and traditional Southern blot-ting using the CEB 15 probe. 1p36 LOH was identified infive of the twenty-four samples (21%) using the PCR ap-proach, a result that was confirmed by Southern blottingwith the CEB 15 probe (not shown). The results fromSouthern blotting were completely consistent with PCRand no 1p36 LOH was identified by Southern blotting thatcould not also be detected by PCR. We therefore concludethat the 17% of tumors showing 9p21 LOH is a realistic es-timate of the true LOH figure in these tumors.

No correlation was observed between 9p21 LOH andtumor stage or patient age in agreement with the results ofTakita et al., [25]. The four tumors showing 9p21 LOH con-sisted of one Stage II, two Stage III, and one Stage IV. Addi-tionally, all four patients were more than one-year-old atthe time of clinical presentation. However, when we com-pared the tumors showing 9p21 LOH with those showing

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B. Marshall et al.

Loss of Heterozygosity in Neuroblastomas

137

1p36 LOH we were surprised to observe that three of thefour tumors showing 9p21 LOH also showed 1p36 LOH(tumors 1217,1711, and 2058). The only exception was theStage II tumor (1832). Conversely, of the five tumors show-ing 1p36 LOH, three (60%) also showed 9p21 LOH,whereas in the nineteen tumors without 1p36 LOH onlyone (5%) showed 9p21 LOH (p

,

0.01

x

2

).

DISCUSSION

We have identified LOH at 9p21 in a small group of pri-mary neuroblastomas using six polymorphic microsatel-lite markers spanning the region that includes both thep16 and p15 genes as well as the genes for interferon

a

and

b

. The frequency of LOH is somewhat reduced comparedto that reported in a previous study that investigated 9p21LOH at the IFN

b

locus [25]. However, the patients used inthat study were identified by means of a mass screeningprogram and thus contained an increased proportion ofinfantile and early stage tumors, whereas our patientswere identified upon the presentation of clinical symp-toms at the Portuguese Cancer Institute and the Children’sHospital in Lisbon. It is possible that some of the earlystage tumors identified by mass screening may regressspontaneously and thus escape clinical detection. Thetumors used in the present study are distributed relativelyevenly among the various tumor stages suggesting that,though our sample size is relatively small, the tumors rep-resent a normal cross section of neuroblastomas. Alterna-tively, it is possible that the locus studied by Takita et al.[25] (IFN

b

), or another located close by, is the true targetof LOH in these tumors and that some of our patients haverelatively small regions of LOH at the IFN

b

locus that donot extend to the flanking markers that we have tested inthis study (D9S162 and D9S1747) and have thus goneundetected.

The frequency of 1p36 LOH that we find in our neuro-blastomas (21%) is within the range previously reportedalthough towards the lower limits of this range. However,in at least some of the previous studies there appears tohave existed a bias towards more serious, N-myc ampli-fied cases of neuroblastoma. Of particular interest in thisstudy, and in contrast to the results of Takita et al., [25] isthat we find a correlation between 9p21 LOH and 1p36LOH, albeit with small numbers of tumors. This pointtherefore warrants further investigation. As 1p36 LOH isone of the most significant prognostic indicators available,it would be of obvious interest to see if 9p21 LOH confersany prognostic significance over and above that conferredby 1p36 LOH. Takita et al., reported a statistically signifi-cant association between 9p21 LOH and short survivaltime. In view of the association between 9p21 LOH and

1p36 LOH reported here it is possible that this may be be-cause of 1p36 LOH although Takita et al., reported no as-sociation between these parameters.

With respect to the regions deleted in these tumors, re-gion 2 (Fig. 1A) is outside that which contains the p16 andp15 genes and would appear to be located in a similar po-sition to that reported by Puig et al. [41] as the SRO forLOH in a series of sporadic melanomas. The region is lo-cated centromeric of both the p16 and p15 genes andwould appear to contain at least one previously unidenti-fied tumor suppressor gene of importance for the develop-ment of both melanoma and neuroblastoma. The questionof p16 involvement in neuroblastoma is not definitivelyanswered by this study as we cannot exclude the possibil-ity that region 1 (Fig. 1A) extends as far as the p16 gene.The deleted region does include the interferon genes,however, and these may warrant further consideration asmentioned above. Previous studies have reported that oneof the preferred mechanisms of p16 inactivation in a vari-ety of tumors and tumor cell lines is homozygous deletion[45, 48]. However, we could find no evidence for such amechanism in this study. An additional means of p16 in-activation has also been reported by Merlo et al., [49] in-volving transcriptional silencing of the gene because ofCpG island methylation, suggesting that studies involvingLOH or homozygous deletion may not tell the whole story.Thus it is possible that the number of neuroblastomaswith chromosome 9 involvement may be an underestimateif a similar mechanism was operational in these tumors. Acloser investigation of the role of this and other 9p21genes in tumorigenesis is therefore warranted by thepresent study.

This work was supported by JNICT grant PECS/C/SAU/258/95 toB.M. and a grant from the Portuguese League Against Cancer(Southern Branch) to MGB.

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