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---' , ELSEV I ER Neuroblastoma in Two Siblings Supports the Role of lp36 Deletion in Tumor Development Crocifissa Lo Cunsolo, Achille Iolascon, Andrea Cavazzana, Roberto Cusano, Paolo Strigini, Katia Mazzocco, Lucia Giordani, Luisa Massimo, Bruno De Bernardi, Massimo Conte, and Gian Paolo Tonini ABSTRAC'R Familial neuroblastoma occurs rarely. We studied a family with three children; one of them has a disseminated (stage 4) and another has a localized (stage 2) neuroblastoma. We observed subtelomeric locus D1Z2 ( 1 ~ 3 6d)eletion in both tumors by using double-color fluorescence in situ hybridization. The MYNC gene was found in single copy in both tumors. Loss of heterozygosity (LOH) and restriction fragment length polymorphism analyses were performed by using DNA from frozen tumor cells and from microdissected tumor areas excised from parafthembedded sections. We detected somatic LOH at locus DlS468 ( 2 ~ 3 6in) a tumor-cell population with a trisomy 1 of the stage-2 patient. Neuroblastoma cells of the stage-4 patient were diploid and showed allelic loss at the following loci:DlS172, DlS80, DlS94, DlS243. DlS468, DlS214, DlS241. and DlS164. Haplotype study showed that the siblings inherited the same paternal Ip36-pter chromosomeregion by homologous recombina- tion and that. in the two tumors, arm I p of different chromosomes of maternal origin was damaged. Our results sugest that the siblings inherited the predisposition to neuroblastoma associated with paternal Ip36 region and that tumors developed as a consequence of somatic loss of the maternal lp36 allele. B Elsevier Science Inc., 1999. All rights reserved. ;q< 1' -&pODUCTION Neuroblastoma (NB), a neural crest-derived neoplasia, usually occurs as a sporadic tumor, and families with recurrent neuroblastoma are rare. In 1945, Dodge and Benner 111reported a neuroblastoma in siblings and, after that, other observations on familial neuroblastoma were described (for review, see [2-4]). Tumor cells from patients with disseminated sporadic neuroblastoma very often show a nonrandom deletion of the chromosome arm 1p [5]. Data from loss of heterozygosity (LOH) studies indicate that a putative NB suppressor gene should be located at the lp36 region f6-81. re- cently, Kaghad et al. [9] identified the p73 gene as a strong candidate for the NB suppressor gene. On the other hand, Maris et al. [IO]excluded an NB predisposition gene being located at lp36 by linkage analysis in familial neuroblas- toma. Although several cytogenetic and molecular studies on sporadic neuroblastoma have been carried out, there are very few data on familial cases. The study of Ip dele- tion in familial neuroblastoma can help to better under- stand the role of this chromosomal abnormality and to identify the gene(s) involved in the tumor development. In the present paper, we report the cytogenetic and mo- lecular studies of chromosome arm I p in a family with re- From the Unit of Solid Tumor Biology, Advanced Biotechnol- current neuroblastoma. ogy Center, (C. L. C., K. M., G. P. T.), Genoa, Italy; the Laboratory of Genetics Population (G. P. T.]and the Session of Genetics Epidemiology (P.S.), National Institute for Cancer Research, Genoa, PATIENTS AM)METHODS Ital-v: the Laboratory of Molecular Genetics (R.C.), the Laboratory of Oncology (C. L. C., K. M.1, and the Department of Hematology/ Oncology (L. iM.,B. D. B., M. C.), G. Gaslini Children's Hospital, Genoa. Italy: the Department of Biomedicine of the Evolution Age A 5-year-old boy (11-1)was admitted to the G. Gaslini Children's Hospital with a month-long history of fever, abdominal pain, and anemia. A large solid abdominal mass was (A. I.. L. G.), University of Bari, Bari, Italy: and Anatomy Pathol- histologically diagnosed as an unfavorable neuroblastoma ogy. Ospedale S. Camillo Forlanini (A. C.),Rome, Italv. Address reprint requests to: Gian Paolo Tonini, Ph.D.. Unit of Solid Tumor Biology Advanced Biotechnology Center, Largo R. [Ill. The patient was staged as 4 [12] and.after a high-dose chemotherapy cycle, died from toxicity. Three months later. t Benzi 10. I61 32 Genoa. Italy. his 3-year-old sister (11-2) was admitted to the Children's Received December 26, 1997: accepted June 25,1998. Hospital with a diagnosis of histologically favorable neu- t I Cancer Genet Cytogenet 109:126-130 (1999) @ Elsevier Science Inc.. 1999. All rights reserved. 655 Avenue of the Americas. New York. NY 10010 0165-P4n60S80/9196/5$-4se6e08f(r9o8n)t0m01t5tcQx Metion ,f lp36 in Siblings with Neuroblastoma 127 &lastoma. The postsurgical evaluation of metastasis was foundto be negative. and the patient was staged as 2B. So fa,after standard-dose chemotherapy. she is in complete remission. In the course of the daughter's disease, the mother m e pregnant. Ultrasonographies were performed e\.erq' 2 months throughout the pregnancy and 3 months after birth: the results were negative. Two vears after birth. !he girl (11-3) is living without any sign of neoplastic dis+:*e. The pedegree of the familv is shown in Figure 3. To detect the lp36 deletion in tumors. we performed double-color fluorescence in situ hybridization (FISH) on interphase nuclei on tumor touch preparations from patients 11-1and 11-2 as described elsewhere [13].The probes that we used were the p l - 7 9 for subtelomeric locus D1ZZ and the chromosome 1 a-satellite for the centromeric region. Fluorescent signals were observed by an Ol\-mpus BH-2 microscope. We also carried out an LOH study by using restriction fragment length polymorphisms (RFLPs) and mini- and microsatellite analyses for chromosome arm 1p loci. We extracted DNA from tumor tissues of both patients and h m peripheral blood leukocytes of the mother. the father. and the sisters (11-2.11-3)according tQ the standard method !14]. A blood sample from patient 11-1 was not available. High-molecular-weight DNAs (10 p,g) purified from the tumors of patients 11-1 and 11-2 and from the leukocytes of patients II-2,II-3, the mother, and the father were digested with appropriate restriction enzymes. The resu!ting fragments were separated by agarose gel !0.8/~e)lectrophore- sis and blotted onto a nylon membrane filter, according to Southern blot techniques [Is]. The filter was hvbridized i With randomly primer [d2P]dCTPlabeled probes, washed. and exposed to x-ray film at -80C as described elsewhere 1141. RFLP analysis was done with the following restrichprobes (locus) and enzymes: CEB15 ( D l S l 7 2 ) / 7 a q I . . PI-24 (DlS94)lSphI,pl- 31 ( D I S I12)/Sspl.and perlecan (HSPG2)lBamHI.A polymerase chain reaction (PCR)study fOr locus Dl S80 was carried out according to Martine et al. 'j IW. The resulting amplified fragments were separated by 1.2% agarose gel electrophoresis stained with ethidium bramide. To further study the extension of the chromo%mearm 1p deletion. we performed PCR by using a series of microsatellites: tumor DNA of patient 11-1 and tumor md constitutiona! DN.4 of patient 11-2 were used as b p l a t e s for PCR amplification for the following loci: DIs243, DlS468. DIs214. D1SI60. D l s 2 4 4 . DlS170, D1s241,D1S201. DlS164. D l S 2 1 1 . D1S1.97. DIS200. and *1s220. The PCRs were carried out according to the conations described by Genome Data Base (Johns Hopkins Universitv. Baltimore. MD. LlS.4. 1990). Finally. to stud!; w t i t u t i o n a l DNA from patient 11-1 and to determine i *cb parental chromosome 1 of patient 11-2 had been de- -b,we obtained tumor and normal cells from 5-w paraf- h-mbedded tissue sections. Briefly. tumoral cells were -dissected in such a way that DN.4 was prepared only areas containing more than 9c% of neoplastic cells. The cells from the uninvolved area of the tissue provided' somatic DN.4. which was compared with the tuDNA. Thr sections were slight!! counterstained with %'s Haematoxlin ior 3 3 se: and then microdissected under light microscopy control as previousl?7 described [171. The microdissected areas were digested overnight at 37C in a TE (Tris-HCI 100 mM, 1 mM EDTA, pH 8.0) buffer containing 200 mg/mL of proteinase K (Boehringer. Germany). DNA ( l / l O of the final volume) was used as template for the PCR reaction. RESULTS AND DISCUSSION Double-target FISH showed that the tumor from patient 11-1was composed of disomic cells for chromosome 1 that displayed loss of a green telomeric signal and retained two centromeric red signals (Fig. 1A). The neuroblastoma of the sister (11-2) shows two cell populations-one disomic and another trisomic for chromosome 1: the latter accounted for about 30%. Loss of a green signal was observed only in the nuclei with trisomy 1(Fig. 1B). Figure 1 The figure IS representative of double-color FISH anaivsis performed on interphase nuclei of tumor 11-1 (A) and tumor II-: (B) bi using ar a-satpllite probe (red' speufic for the centromere of chromosome 1 and a probe for subtelomeric lp36 locus 0122 (green).h [Aj twc red signals for locus QC and onl\ one green signal for locus 012.2 at each nucleus are visrble In (B) the nuclei sho- three red and two green signals I128 c. Lo c u m o l o ~ ~ ALLELIC DISTRIBUTIONAT LOCUS DlS80 -II M I-? 1 1-2 23 ll-2t I t - 2 ~ 11-3 m- - 517 - 394 Figure 2 PCR for locus DIS80. Two PCR products are generated: one at 394 bp and the other at 517 bp. The mother (1-1)is homozygous for the 517-bp allele, whereas the father (1-2) is heterozygous. The tumor of patient (11-It) shows only the paternal allele at 394 bp; the constitutional DNA of the patient was not available. The tumor (II-2t) and the constitutional (11-2c)DNA of patient U-2 retains the two alleles, and no LOH was observed. The constitutional DNA (II-3) of the youngest sister was homozygous for the 517-bp allele. poor outcome of patients [la]. In our experience, at stage 2, whose tumor shows chromosome arm 1p damr6, have a high chance of relapsing (of 28 stage-2 NBs, bow of three have lp36 deletion and relapsed, persona hr uaThis fact suggests that the lp36 deletion characte- gressive behavior of neuroblastoma cells in l o c w PhEg mors, too. In this regard, it is notable that patient II-2 stage-2 NB and showed a tumor-cell clone with a some lp36 deletion. PCR for locus DlS80 shows two ucts, a 394-bp and a 517-bp band (Fig. 2). The mother wa homozygous for the 517-bp allele, and the father wm her. erozygous (394 and 517). The presence of the 394-bp in the tumor cells of patient 11-1 indicates that the &le deleted was the 517-bp allele of maternal origin. In &e tu. mor of II-2, minisatellite analysis failed to show LOR for locus D1S172 was observed in the tumor of patient &I, whereas the loci D1S94, HSPGZ,and D l S l 1 2 results wm uninformative. Heterozygosity for D1s 9 4 , HSPGZ a d D l S l 12 was retained in the neuroblastoma cells of patient 11-2 (Table 1). Because the peripheral blood of patient a-1 was not available, loci D1S160,DlS244, D1S170, D ~ s 1 g 7 , and D1.5220 were uninformative. In contrast, allele sqp. gation in the kindred allowed us to easily assign LON D1S468, D1S214, D1S241, and D1S164 at the mat- Chromosome 1 [Table 1). The microsatellite study of t\lmor paraffin-embedded tissue confirmed that the maternal cbto. mosome A was damaged in patient 11-1. A comparison be- --- 7, - .I Nonrandom chromosome lp36 deletion has often been detected in metastatic sporadic neuroblastoma (stage4), as it was in the tumor of the patient 11-1, rarely in localized tumors (stages 1 and 2), and it has been associated with a Table 1 LOH for chromosome arm l p in the siblings Locus" DlS172 DlS80 DlS94 DlS243' DlS468' DlS214* DlS160 DlS244 DlS170 HSPG2 DlSll2 DlS241 DlS201 DlS164 DlS211 DlS197 DlS200 D1S220 Map location 1p36.3 1p36.3 1~36.33 1~36.33 1p36.3 11336.22-21 1~36.2 1p36.11-12 1~36.11-12 1~36.1~35 lp36.11 lp35 lp35 lp35 lp32.3 11332.2 1p32.2-3 lp31-p32 Patient 11-1 AC W1 2 01 W1 W3 84 11 d 31 31 c1 il 1 W3 -- 2 31 c7 1 12 21 Patient 11-2 B D,,C 31 12 12 2 83 23 23 12 11 12 11 12 31 32 21 11 12 11 ".4lignment of loci for chromosome arm l p according to Genome Data Base (JohnsHopkins University, Baltimore, MD. USA. 1990). Symbols: W. deleted; 0.not informative; -, not done. %OH was studied by using DNA from microssected paraffin-embedded tissue. Table 2 LOCUS" Chromosome l genotype of mother, father, and daughters 11-2 and 11-3 Map ~1-1 -1-2 I f 4 - II-3 location A B cDB SA GD DlS172 D1S8O DlS94 DlS243 DlS468 DlS214 DlS160 DlS244 DlS170 HSPGZ DlS112 DlS241 D1S201 DlS164 D1S211 DlS197 DlS200 D1S220 CRP ,4PO-A2 DlS104 F13B DlS306 DlS249 1~36.3 2 3 113 1p36.3 1 1 211 1~36.33 2 1121 1~36.33 1 2 122 1p36.3 2 1331 1~36.22-21 1 2 4 3 2 1~36.2 1 2 132 1~36.11-12 1 1 1 2 1 1p36.11-12 1 1 1 1 1 1 ~ 3 6 . 1 ~ 3 5 ND ND 1 2 1 lp36.11 NDND111 lp35 2 1321 lp35 13213 lp35 1 3 223 11332.3 3 2 112 1p32.2 1 1 111 1~32.2-3 1 1 221 lp31-p32 1 1 111 lq21323 2 2 112 lq21-23 3 2 3 12 lqZl-qZ3 1 2 12 2 lq31332.1 1 1 1 2 1 lq32.1 2 2 122 lq32.1 1 1 321 1 2 2 2 3 3 3 2 1 2 1 2 1 2 1 1 2 1 1 1 2 2 2 2 21 11 ND ND 12 23 13 13 12 11 ND ND ND ND ND ND 12 12 21 11 12 11 ND 23 21 11 21 13 Abbreviation: ND. not done. `Alignment of loci for chromosome 1 according to Genome Data (JohnsHopkins University. Baltimore. LMD.USA, 1990) ,% *n.* of lp36 in Siblings with Neuroblastoma 0 1 23 AC BD BC rigt~3 ~S~chematic representation of genotype distribution. chromosomal recombination in the family members. and chromom e 1p deletion in tumor-cell patients. The genotype of patient U-1 was studied by using tumor DNA. For chromosomes C. B, and D. the loci upstream and downstream of the crossing-over are w e d . Loci D1S21 J and DlS468,downstream at the deletion kakpoints of chromosomes A and B. are respectively reported. normal and tumor DNA of patient 11-2 showed LOH h locus D1S468 of maternal chromosome B (Table 1). NMYC gene amplification was evaluated by the dot blot -que [14]and extra copies of the NMYC gene were nat detected either in the tumor of patient 11-1 or that of n-2(data not shown). The genotypes of individuals I-l,I-2,II-2,and 11-3 were -died by using the microsatellites already reported and &a b h w i n g ones for the l q region: CRP.APO-AZ.Dl 9 0 4 , h3BBD,1S306, and Dl 9 4 9 . Chromosome segregation and -wm m b i n a t i o n observed in the family are shown in Table 2 and represented in Figure 3. The genotype of patient 11-1 studied by using the tumor DNA; it showed that the w e n t inherited the maternal chromosome A and the paC (not shown in Table 2). The maternal chromo- B was transmitted to patient 11-2, and the paternal h o s o m e presents a crossing-over between loci Dl S80 aDlS94. As a result, patients II-1 and 11-2 inherited the epaternal distal region of chromosome C The healthy In-3) has the recombinant maternal chromosome .4/B mdthe recombinant paternal chromosome C/D (Fig. 3). k u s e no hTifl%amplification was detected in either n-2 tumors, extra copies of the gene appeared not to bc required for the tumor development that should de- -& bona fida. on chromosome arm ~p deletion. How- m,Weiss et al. I391 provided evidence that the NMYC contributes to the genesis of neuroblastoma in a model. In sporadic tumors. we found that NMYC %cation and 1p LOH are significantly associated A majority of tumors with NMYC gene amplification 129 have chromosome arm 1p deletion, too, but not the contrary. Data from sporadic tumors again suggest that lp36 deletion is probably involved in neuroblastoma development than is NMI'C gene amplification [20]. Knudson and Meadows [21] proposed the two-hit model for neuroblastoma, and the lp36 damage detectable in the two patients is consistent with this hypothesis. The frequent lp36 deletion observed in neuroblastoma,as in this family, indeed suggests the presence of a tumor-suppressor gene. We could assume that both siblings, 11-1and 11-2, inherited the predisposition to neuroblastoma associated with paternal telomeric chromosome C and that tumors developed as a consequence of the somatic loss of maternal lp36 allele(s). This appears in contrast with data reported by Maris et al. [ l o ] and the possibility that other genes 122-241, located at different chromosomes. participate in neuroblastoma development cannot be excluded. The discrepancy between our data and that of Maris could be due to diverse reasons. One is that Maris et al. [lo] in- cluded in their study neuroblastoma, ganglioneuroblastoma, and ganglioneuroma, the last two rarely showing chromosomal abnormalities, whereas our study included neuroblastoma only. This work was supported by AIRC (Associazione Italiana Ricerca SUI Cancro). CNR-ACXO pro+& No. 94.01259/PF39, and Associazione ltaliana per la h t t e ai Neuroblastoma. We thank Dr. Hubert Caron, for his suggestions, and Miss Alessandra Zanotta. for her assistance in editing the manuscript. We are grateful io surgeons, clinicians. and pathologists of the Italian Cooperative Group for Neuroblastoma and to Associazione Italiana Ematologia e Oncologia Pediatrica (AIEOP) for providing tumor samples. REFERENCES 1. 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