Genome Instability in Secondary Solid Tumors Developing After Radiotherapy of Bilateral Retinoblastoma

Genome instability in secondary solid tumors developing after radiotherapy of bilateral retinoblastoma

Sandrine-HeÂ leÁ ne LefeÁ vre1, Nicolas Vogt1, Anne-Marie Dutrillaux1, Laurent Chauveinc2, Dominique Stoppa-Lyonnet3, FrancËois Doz4, Laurence Desjardins5, Bernard Dutrillaux1,6, Sylvie Chevillard6 and Bernard Malfoy*,1

1Institut Curie ± CNRS UMR 147, 26 rue d'Ulm 75248 Paris Cedex 05, France; 2Institut Curie, Service de RadiotheÂrapie, 26 rue d'Ulm 75248 Paris Cedex 05, France; 3Institut Curie, Service de GeÂneÂtique Oncologique, 26 rue d'Ulm 75248 Paris Cedex 05, France; 4Institut Curie, Service de PeÂdiatrie, 26 rue d'Ulm 75248 Paris Cedex 05, France; 5Institut Curie, Service d'Ophtalmologie, 26 rue d'Ulm 75248 Paris Cedex 05, France; 6CEA, DSV DRR, 60 avenue du GeÂneÂral Leclerc 92265 Fontenay-aux-Roses, France

Genome alterations of seven secondary tumors (®ve these cancers and the diculty in collecting a series of osteosarcomas, one malignant peripheral sheath nerve cases in clearly de®ned context. tumor, one leiomyosarcoma) occurring in the ®eld of It is well established that therapeutic irradiation can irradiation of patients treated for bilateral retinoblasto- induce secondary malignancies within or at the margin ma have been studied. These patients were predisposed to of the radiation ®eld after a long latent period. These develop radiation-induced tumors because of the presence tumors have dierent histology from the primary of a germ line mutation in the retinoblastoma gene lesions, with sarcomas being common (Robinson et (RB1). Tumor cells were characterized by a high al., 1988). Well documented data are available on chromosome instability whereas microsatellites and radiation-induced thyroid tumors developing after minisatellites were found to be stable. In all tumors, radiotherapy or accidental irradiation. A key role of the normal RB1 allele was lost with the corresponding the RET protooncogene was demonstrated in radia- chromosome 13, whereas the germ line mutated allele tion-induced as well as in spontaneous thyroid tumors, was retained. The two alleles of TP53 were inactivated, but both the frequency and the speci®city of RET one by deletion of the short arm of chromosome 17, the rearrangements seemed to be higher in radiation- other by mutation. As compared with non-radiation- induced tumors (Bongarzone et al., 1997; Bounacer et induced tumors, the observed panel of TP53 mutations al., 1997; Klugbauer et al., 1995; Nikiforov et al., 1997, was uncommon with sites not recurrently found otherwise 1999). Data is also available for other cancers and a high rate of deletions (3/7). In these predisposed developing after radiotherapy. Cytogenetic data in- patients, the loss of the single normal allele of RB1 is dicated that these radiation-induced cancers generally rather due to the radiation-induced chromosome in- had complex karyotypes with multiple deletions and stability than a direct eect of ionizing radiation. chromosome losses (Chauveinc et al., 1999; Mertens et Oncogene (2001) 20, 8092 ± 8099. al., 2000). This suggests that complex oncogenic processes are involved. The molecular analysis of two Keywords: ionizing radiation; secondary cancers; reti- series of radiation-induced tumors of the central noblastoma; chromosome instability; TP53; predisposi- nervous system has been published. No signi®cant tion dierence in the pattern of gene alterations was observed between radiation-induced and spontaneous astrocytomas (Brat et al., 1999). In contrast, for Introduction meningioma, it was concluded that radiation-induced forms displayed a distinct pattern of molecular genetic Despite extensive epidemiological studies and numer- alterations from that observed in spontaneous tumors ous case reports of ionizing radiation-induced human (Shoshan et al., 2000). De®nitive conclusions cannot be solid tumors, little data is available on the genetic drawn about secondary lung and breast cancers alterations present in these cancers. In particular, it is developing after radiotherapy for Hodgkin's disease. not currently known if speci®c pathways of cancer- In a series of lung cancers (De Benedetti et al., 1996), ogenesis are involved compared to spontaneous cancers radiation-induced characteristic mutations of TP53 of identical histological origin. Such studies are dicult gene were assumed to be present, whereas this was to carry out because of the heterogeneous origin of not con®rmed in more recent work (Behrens et al., 2000). In the latter study, an increase of microsatellite instability was noticed, as compared with sporadic cases, in therapy-related tumors post-Hodgkin's dis- *Correspondence: B Malfoy; E-mail: [email protected] Received 5 July 2001; revised 17 September 2001; accepted 1 ease. However, the role of smoking in pulmonary October 2001 neoplasia and the recurrent combination of chemother- Genome instability in radiation-induced tumors S-H LefeÁvre et al 8093 apy and radiotherapy complicates the interpretation of Radiotherapy was administered by photon or by these data. electron. The total radiation dose was 45 Gy. After a The paucity of available data does not allow an latent period of 7 ± 22 years, a secondary tumor overview of the speci®c eects of ionizing radiation in developed in the radiation ®eld. These tumors (®ve the induction of human tumors. In particular, the role osteosarcomas, one malignant peripheral sheath nerve and the consequences of genetic predispositions are tumor, one leiomyosarcoma) were considered to be poorly understood. Data on second cancers occurring radiation-induced according to the Cahan criteria after childhood cancers indicate that genetic predis- (Cahan et al., 1948). positions may also predispose to radiotherapy-related cancers (de Vathaire et al., 1992). Patients carrying a RB1 and TP53 analysis germ line mutation of the retinoblastoma gene (RB1) usually develop bilateral retinoblastoma (Draper et al., LOH analyses were performed on the seven radiation- 1992; Vogel, 1979). In addition to having a high risk of induced tumors (Table 1), using microsatellite se- developing other spontaneous cancers, these patients quences distributed along the whole of chromosomes also have a high frequency of secondary tumors, in 13 and 17 where the RB1 and TP53 genes are localized particular, osteosarcomas, in the ®eld of irradiation. on bands 13q14.1 and 17p13.1, respectively. Twenty- Cumulative incidence of a secondary cancer at 50 years one loci were analysed on chromosome 13, including after diagnosis was 51% for patients with predisposi- three in the RB1 gene, and 16 on chromosome 17, tion and 5% for patients which developed non- including one in the TP53 gene (Figure 1). For the hereditary retinoblastoma (Wong et al., 1997). three intragenic loci of RB1, LOH were observed in all These radiation-induced cancers, developing in a informative microsatellites indicating the loss of one known genetic context, may provide an opportunity to allele in the seven studied cases. For cases 1, 3, 4, 5 and determine the pathways underlying the radiation- 7, data were in favor of a whole chromosome 13 loss. induced eects. We have studied seven secondary For case 2, several loci in the distal and proximal parts tumors developing in the ®eld of irradiation in patients of the chromosome 13 were not informative and it was treated for bilateral retinoblastoma. We show that, in not possible to establish the boundaries of the deletion. these tumors characterized by high chromosome Heterozygosity was maintained only in the distal part instability, the normal allele of RB1 was lost with of chromosome 13 for case 6, suggesting that a large deletion of the chromosome 13 (where the RB1 gene is deletion occurred. The TP53 intragenic microsatellite localized) and not by inactivation due to an intragenic indicated a LOH in all of the six informative cases. In mutation attributable to the ionizing radiation. More- case 1, the loss of one copy of TP53 could be expected over, the uncommon panel of mutations observed on since TP53 was surrounded by loci which underwent TP53 suggests the presence of mutational mechanisms LOH (see also below). For cases 2, 3 and 4, a whole which could be speci®c to, or particularly active in, chromosome 17 short arm was deleted. In contrast, for radiation-induced tumors. cases 1, 5 and 6, the proximal part of the arm was not lost. All chromosome 17 was lost in case 7, no LOH being observed in the six other cases on the 17q arm. Results

Patients Seven children with bilateral retinoblastoma underwent therapeutic irradiation between 4 ± 14 months of age (Table 1). Case 2 was a low penetrancy familial case. No retinoblastoma was reported in other families.

Table 1 Localizations and histological types of the radiation- induced tumors Case Delaya Localization Type Xenograftb

1 7 Maxilla bone right osteosarcoma 7 2 9 Maxilla bone left osteosarcoma 7 3 22 Orbital bone right Malignant peripheral + sheath nerve tumor 4 20 Nasal fossa left Leiomyosarcoma + 5 11 Temporal bone left osteosarcoma + 6 20 Nasal fossa left osteosarcoma 7 7 13 Maxilla bone right osteosarcoma 7 Figure 1 LOH pattern of radiation-induced tumors on chromo- aDelay in years between the irradiation of the retinoblastoma and the somes 13 (a) and 17 (b). Filled sequences: LOH; Empty sequence: development of the secondary tumor. bGrown (+) or not grown (7) no LOH; point: non-informative. In case 3, identical results were as xenograft obtained in fresh and xenografted tumors

Oncogene Genome instability in radiation-induced tumors S-H LefeÁvre et al 8094 To determine the status of the remaining allele of was found by cDNA sequencing and con®rmed by RB1 and TP53 in the tumors, the coding regions and multiprimer analysis of the DNA. For tumor seven, a parts of some introns were analysed by DGGE and/or Leu?Val missense mutation was found at codon 12 by by sequencing. sequencing the whole cDNA. This mutation was not With the exception of two cases for which RNA found in the screening of 200 DNA from healthy from normal tissue (case 3) or tumor (case 7) was not donors and thus is unlikely to be a polymorphism (not available, the full length coding sequence of RB1 was shown). In all the cases, the RB1 constitutionally ®rst studied by DGGE using RNA from both tumor mutant allele was detected in the normal tissue jointly and normal tissues. In cases 2, 4 and 6, one RT ± PCR with the wild type allele, whereas only the mutant allele fragment prepared for DGGE analysis was found to was detected in the tumors. In cases 1, 2, 3, 4 and 6, harbor a deletion. In these tumors, the other DGGE the germ line mutation induced the formation of a fragments did not display abnormal electrophoretic non-functional protein. The situation in case 7 was less behavior. The fragments with deletions were sequenced clear since the mutation observed corresponded to a and it was shown that the totality of exon 9 in case 2 Leu?Val change in the N-terminal region of the and of exon 12 in cases 4 and 6 were missing. Deletion protein, a region where few mutations have been of exon 9 maintained the open reading frame, whereas described (Lohmann, 1999) and whose function is not those of exon 12 induced a stop codon at position 380 well characterized (Kaelin, 1999). The consequence of (Table 2). In case 2, the deletion of exon 8 was the amino-acid change on the functionality of the associated with a T?C transition in the polypyrimi- protein is dicult to estimate, but several arguments dine tract of the 5' splicing site localized in intron 8. suggest that this mutation is actually the germ line This mutation strongly reduces the splicing eciency of mutation and is sucient to signi®cantly alter the exon 9 (unpublished data). In cases 4 and 6, the 3' activity of the protein: (1) no other mutation was splicing site of exon 12 was altered by a G?A found in the gene; (2) it is not a known polymorphism; transition (ac/GT?ac/AT). Only one abnormal elec- (3) the mutation was found to be heterozygous in the trophoretic fragment encompassing exons 17 and 23, in normal tissue and the other allele was lost in the cases 1 and 3 respectively, was demonstrated by tumor. Thus, in these seven tumors, the tumor DGGE. In exon 17, a C?T transition at codon 556 suppressor role of RB1 was altered. introduced a stop codon. In exon 23, the deletion of a Somatic mutations of the TP53 gene were analysed T at codon 777 introduced a stop codon in 809. All by cDNA sequencing in ®ve tumors (cases 1, 3 ± 6) and these mutations were con®rmed on genomic DNA the found mutations were con®rmed by studying the (Table 2). For tumor ®ve, deletion of exons 7, 8 and 9 corresponding genomic DNA. For cases 2 and 7, RNA

Table 2 Mutations in the RB1 and TP53 genes of the radiation-induced tumors RB1a TP53b Case RNA DNA RNA DNA

1 E17 Co556 E17 B78250 E7 Co237 E7 B14038 CGA?TGA C?TATG (Met) ATC (Ileu) G?C Stop codon missense 2 E9 total deletion I8 B61720 Mis E4 Co83 E4 B12171 deletion E9 inframe T?C deletion 2 bp deletion 2 bp (CG) polypyrimidine tract frameshift: stop Co148 3d E23 Co777 E23 B162209 E5 Col164-187 E5 ± I5 B3170-13303 CCT?CC ± deletion T deletion 72 bp deletion 134 bp deletion 1 bp insertion 16 bp from I5 frameshift: stop Co809 frameshift: stop Co216 4 E12 total deletion I12 B70330 E5 Co135 E5 B13083 frameshift: stop Co380 GT?AT TGC (Cys) ? TTC Phe) G?T splicing site missense 5 E7-8-9 total deletion E7-8-9 deletionc E6 Co216 E6 B13407 frameshift: stop Co204 GTG (Val) ? GAG (Glu) T?A missense 6 E12 total deletion I12 B70330 E7 Co258 E7 B14099 frameshift: stop Co380 GT?AT GAA (Glu) ? AAA (Lys) G?A splicing site missense 7 E7 Co220 E7 B56903 Mis E8 Co284-285 E8 B14520 ± 14523 CTA (Leu) ? GTA (Val) C?G deletion 4 bp deletion 4 bp (CAGA) missense frameshift: stop Co344

E: exon, I: intron, Co: codon (genebank accession number: RB1, NM00321; TP53, NM000546). B: base position in the genomic sequence (Genebank accession number: RB1, L11910; TP53, U94788). Mis: RNA from these tumors was missing, data in italics were deduced from experiments on genomic DNA. aAnalysis performed on DNA and RNA from peripheral lymphocytes and tumors except for cases 2 and 7 where RNA from tumor was missing. bAnalysis performed on tumoral DNA and RNA, the absence of mutation was veri®ed on peripheral lymphocytes. cDeletion found by multiprimer analysis, the extent of the deletion in 3' and 5' of the three exons was not precisely mapped. dIdentical results were obtained in fresh and xenografted tumors

Oncogene Genome instability in radiation-induced tumors S-H LefeÁvre et al 8095 was missing and mutations were searched for in tumor indicated that sequences from chromosome 13 were DNA only (Table 2). Missense mutations, localized in located on two derivatives (Figure 2a). Although exon 5, 6 or 7, were found in four cases. The three dierent, they had one arm in common, suggesting other mutations were deletions in exon 4 (2 bp), 8 they originated from a unique rearrangement, and thus (4 bp) and 5. In the latter, a genomic 134 bp deletion were likely to have the same parental origin. Sequences overlapping the 3' splicing site of exon 5 was observed. from chromosome 17 were distributed on 5 ± 6 The removal of this splicing site induced the non- chromosomes. Case 6 was hypotetraploid (mean 78, recognition of the partially deleted intron 5 (16 bp range 76 ± 80 chromosomes). After R-banding, only 10 remaining) and its insertion in the mRNA between chromosomes or less had a normal appearance. exon 5, deleted of 72 bp, and exon 6. All deletions Surprisingly there were three possible normal chromo- caused the formation of a downstream stop-codon. In some 13, but no normal chromosome 17. Four the tumors, only the TP53 mutated allele was found, chromosomes were completely labeled by the chromo- con®rming the LOH data. some 13 speci®c painting (Figure 2b). The three normal The three deletions induced the formation of a chromosomes observed after R-banding were not truncated protein, in the DNA binding domain in cases distinguishable from a fourth, rearranged chromosome. 2 and 3, and in the oligomerization domain in case 7. This rearranged chromosome likely contained the small Mutant cells with such truncations are known to be distal part of the chromosome 13, which did not defective for normal p53 function. For the four other undergo LOH (Figure 1). Sequences from chromosome cases, missense mutations were observed in the DNA 17 were present on 8 ± 12 derivatives of various binding domain. They can induce a loss of function by morphologies (Figure 2c). Painting of other chromo- disrupting the binding of the protein to DNA, but can somes also displayed a high level of chromosome also create a gain of function form of the protein rearrangements (not shown). Both the number and (reviewed in Sigal and Rotter, 2000). The mutation morphology of the rearranged chromosomes were Cys?Phe at codon 135 (case 4) produces a dominant highly variable from cell to cell: some of them were ± negative mutant (Brachmann et al., 1996). However, only found in one or a few cells, whereas others were in this case, the mutated protein is expressed in absence found in each analysed metaphase. At later passages in of the wild type form, a situation which may modify its athymic mice, the cells of the two tumors underwent biological function. The three other mutations have not endoreduplications (not shown). Although, these yet been studied and the activity of the mutated results con®rm the very strong chromosome instability proteins is currently unknown. Nevertheless, the four in these two tumors. missense mutations take place at codons coding amino acids evolutionarily conserved in monkey, mouse, rat, chicken, xenopus and trout (Soussi et al., 1990), a Discussion strong indication of their importance for the function of the protein. Changes in these amino acids would be In this series of patients treated by radiotherapy for a expected to profoundly alter protein function. bilateral retinoblastoma, ®ve developed an osteosarcoma, one a leiomyosarcoma and one a schwannoma in the radiation ®eld. As expected from clinical and/or Genome instability familial data, all patients had a germinal mutation of Genome instability was analysed using microsatellites, the RB1 gene, which we could detect in normal somatic minisatellites and when possible by cytogenetics. cells (Table 2). It is well established that such In addition to the 37 loci used for the LOH study of mutations not only predispose to retinoblastoma but chromosomes 13 and 17 (see Materials and methods also to other tumors, in particular to secondary and Figure 1), eight loci (BAT25, BAT26, BAT40, radiation-induced tumors (Wong et al., 1997). D2S123, D5S346, D10S137, D17S250, D18S60) con- We looked for possible alterations of the normal sidered as highly indicative of the presence of a RB1 allele in tumor cells. In all the cases, a LOH was microsatellite instability (Boland et al., 1998) were found. It involved not only the RB1 chromosome analysed. In the absence of LOH, none of these loci region but the whole chromosome 13 (six cases) or a displayed dierences in the banding pattern when large part of it (one case). In all cases, the remaining comparing tumor and normal tissues. Three dierent allele was that carrying the germ line mutation. Thus, minisatellite loci were analysed (D1S80, D17S30, H- although our series is limited, we conclude that the RAS1). In all cases, the pattern of ampli®cation was mechanism underlying the induction of second cancers similar in normal and tumor tissues (data not shown). by radiation in patients heterozygous for a RB1 Analysable metaphases were obtained for xeno- mutation is unlikely to involve the direct induction of grafted tumors from cases 3 and 6. They were studied mutations of the normal RB1 allele. The loss of a by classical cytogenetics and FISH using chromosome whole chromosome might be a consequence of speci®c painting probes. For case 3, most metaphases chromosome malsegregation, however, this has been were hypotriploid (mean 58 chromosomes, range: 49 ± extensively looked for and radiation does not seem to 61). After R-banding, no more than 25% of chromo- directly induce this (United Nations, 1993). Rather, the somes had a normal appearance. No normal chromo- data suggest the presence of a chromosome instability some 13 or 17 was observed. Chromosome painting in tumor cells.

Oncogene Genome instability in radiation-induced tumors S-H LefeÁvre et al 8096

Figure 2 FISH with chromosome 13 speci®c probes (a,b) and 17 (c) on metaphase chromosomes from cases 3 (a) and 6 (b,c). (a) The segment shared by the two rearranged chromosomes labeled by chromosome 13 speci®c painting is indicated. In addition to a diuse background, few centromeres are labeled. (b) arrows indicate chromosomes labeled by chromosome 13 speci®c painting. (c) arrows indicate chromosomes labeled by chromosome 17 speci®c painting. Metaphases were obtained from xenografted tumor cells

It has been established in experimental systems that The pattern of alterations of TP53 observed in our one biological consequence of exposure to ionizing series of tumors is unusual in three respects. Firstly, we radiation is the induction of delayed genomic found three deletions among seven patients. This 43% instability characterized by gene mutations and rate is too high, compared with that of large series of chromosome aberrations (reviewed in Little, 2000; registered cases: deletions occurred in 9% of 11 100 Wright, 2000). These delayed responses have been cases, among which 4.2% involved a single base pair most eectively demonstrated as non-clonal muta- (Soussi et al., 2000). Thus, the excess of deletions is tions/aberrations in clonal descendants of irradiated highly signi®cant in our sample (w2=9.9, s=1, cells. Radiation of both high and low linear energy P=0.0016). The rates of two and four base pair transfer (LET) is able to induce genome instability but deletions were of 0.75% and 0.2% respectively in the high-LET (a particles, neutrons) seem to induce sample of 11 100 cases, that of deletions larger than 50 chromosome instability more eciently than low- base pairs was of 0.1%. When the analysis is restricted LET radiation (X-ray, g). The mechanisms at the to osteosarcomas and leiomyosarcomas, (data were not origin of the genome instability are currently un- available for schwannomas), deletions are found in 16 known. The implication of this delayed instability in of 120 published mutations (13%), among which 4% the development of cancer is presently not established, involve 1 bp and 0.8%, 4 bp. No deletion of 2 bp or but in mice a relationship has been found between longer than 24 bp was observed. Again, the excess of susceptibility to the induction of chromosome in- deletions is highly signi®cant in our series (w2=4.5, stability in mammary epithelial cells and predisposi- s=1, P=0.03). Thus, it was highly improbable that tion to radiation-induced mammary cancer (Ponnaiya these three deletions were observed by chance in our et al., 1997). Some characteristics of the radiation- small series. induced tumors have been analysed to establish if A second particularity concerns the type of base speci®cities could be found as compared with substitution in the missense mutations. In non- spontaneous tumors. radiation induced tumors, CpG dinucleotides are found mutated at a high rate (Pfeifer, 2000). It has been suggested that this type of alteration may be related to The panel of TP53 mutations is uncommon exposure to chemical mutagens or methylcytosine TP53 status has been described in several series of demethylation. None of our mutations involved a radiation-induced sarcomas and carcinomas (Behrens CpG dinucleotide. Bases in DNA can be oxidized by et al., 2000; Brachman et al., 1991; Brat et al., 1999; De hydroxyl radicals formed by water radiolysis (Wallace, Benedetti et al., 1996; Fogelfeld et al., 1996; Gamble et 1998). 8-oxoguanine (8-oxo-G) is one of the most al., 1999; Nakanishi et al., 1998; Nikiforov et al., 1996; damaging lesions among oxidized bases (Cadet et al., Tada et al., 1997). In these series, the rates of 1997; Le Page et al., 1999). The predominant resulting mutations ranged from 0 to 100%, which makes any mutation was G : C?T : A (Le Page et al., 1995; ®rm conclusion impossible. It must be stressed that Moriya, 1993), as a consequence of 8-oxo-G: Adeno- some studies were limited to exons known to be sine pairing (Kouchakdjian et al., 1991). Such a recurrently mutated in cancers and that rarer muta- mechanism could be at play in case 4, but for the tions may have escaped. other mutations involving a Guanine (cases 1 and 6), it

Oncogene Genome instability in radiation-induced tumors S-H LefeÁvre et al 8097 is unlikely that they were formed by oxidation damage and that ionizing radiation had a more general consecutive on irradiation. indirect eect. The mechanisms at the origin of A third peculiarity concerns the sites of mutations. minisatellite instability, which are presently poorly For the missense mutations of cases 1, 4 and 5, the understood (Jereys et al., 1997), seem infrequently transversions observed were rarely found at the same implicated in the development of the tumors of our position within the aected codons (Soussi et al., 2000): series. 3/114 for codon 237, 2/154 for codon 216 and 11/92 for codon 135. The last missense mutation G?A (case Tumors are characterized by a high chromosome 6) was more frequently observed: 29/63 for codon 258. instability In addition, these mutations are targeted on evolutionarily-conserved amino-acids (Soussi et al., 1990). This Cytogenetic studies were possible for two xenografted speci®city, as well as their localization in the DNA tumors at early passage. It has been shown that, in binding domain of the protein, probably indicate the these conditions, xenografts gave a relevant picture of eciency of the mutations in altering p53 function. For the cellular content of the original tumor (Lefrancois et the deletions, the deleted codons were also rarely found al., 1989). In the two cases studied by cytogenetics, to be involved in the whole data base, with one case very complex rearranged and unstable karyotypes were for codon 83, 37 cases for codon 164 and 12 cases for observed. Both for RB1 and TP53, the loss of one gene codon 284 (Soussi et al., 2000). In experimental copy was linked to a large deletion or a chromosome systems, it has been suggested that the delayed gene loss. The mechanisms inducing a delayed chromosome mutations could represent an increase in spontaneous instability are not known. However in senescent damages or a diminished ability to correct them (Little ®broblasts, it was shown that chromosome losses were et al., 1997). In our series of radiation-induced tumors, non-random and chromosome 13 was among those the uncommon panel of mutations found in TP53 recurrently involved (Martins et al., 1993). Thus, in suggests the preferential involvement of mechanisms of radiation-induced tumors, chromosome instability mutation induced by or associated with ionizing could be a long term consequence of exposure to radiation. radiation. This would ®t with the relatively long delay (14.5 years on average) that was observed between radiotherapy and the second tumor onset of our Microsatellites and minisatellites are stable patients. Whatever the mechanism of promotion of No microsatellite instability was observed in our series the delayed chromosome instability may be, in the of tumors. Published data on microsatellite instability tumors developed after radiotherapy of retinoblastoma, in radiation-induced tumors are con¯icting. A high rate RB1 and TP53 are likely to be implicated in the of microsatellite instability was described in a series of observed genome instability. In all cases, both RB1 and nine tumors secondary to irradiation of pediatric TP53 were biallelically altered. The absence of normal tumors (Gafanovich et al., 1999) and in two series of p53 may be at the origin of a genomic instability lung and breast cancers (19 each) after therapy of associated with the loss of control check points and Hodgkin's disease (radiotherapy plus chemotherapy) other functions of the protein. In addition, the tumor (Behrens et al., 2000). In contrast, data from post- suppressor role of RB1 is abolished in the tumors: RB1 Chernobyl pediatric thyroid carcinomas, suggest that regulates the expression of many genes whose products microsatellite instability is not a frequent alteration are required for DNA synthesis and cell cycle (Richter et al., 1999). A similar conclusion was drawn progression. Transcription activation of at least some for radiation-induced acute myeloid leukemia in the of these genes, such as cyclin E, is associated with the mouse (Richter et al., 1999). Cytogenetic data were loss of proliferation control as a consequence of RB1 missing in these studies and it is not possible to inactivation (Almasan et al., 1995; Herrera et al., 1996; establish if in these tumors, the microsatellite instability Hurford et al., 1997). However, such a loss of RB1 is associated with a low rate of rearranged chromo- function, in addition to unchecked proliferation, can somes. Currently, the origin of these discrepancies, also lead to p53-dependent apoptosis (Macleod et al., which could be related to the histological origin of the 1996; Morgenbesser et al., 1994). Accordingly, loss of cancers and/or with the genetic context, is unknown. RB1 functions may create a survival pressure for the The stability of microsatellites found in our series of cell to acquire mutations in the p53 pathway. Thus, tumors argues against a direct implication in the TP53 mutation eects reinforced by the loss of the development of the loss of function of genes involved tumor suppressor function of RB1 can explain, at least in the DNA mismatch repair (Jiricny and Nystrom- in part, the chromosome instability in these radiation- Lahti, 2000; Peltomaki and de la Chapelle, 1997). The induced tumors. These two genes are unlikely to be the three tested minisatellites were found to be stable in the only ones implicated in the development of the seven analysed tumors. Somatic minisatellite mutations radiation-induced tumors. Alterations, directly or have been detected in several models of radiation- indirectly induced by radiation of other genes which induced tumors (Fennelly et al., 1997 and references could promote genome instability are expected. The herein) and in post-Chernobyl tumors (Richter et al., multiple chromosome imbalances observed also suggest 1999). In these cases, it was concluded that mutations the involvement of other genes and other sites of were not caused directly by radiation-induced DNA recurrent deletions must be investigated.

Oncogene Genome instability in radiation-induced tumors S-H LefeÁvre et al 8098 Predisposition to radiation-induced tumor Denaturing gradient gel electrophoresis (DGGE) The simple hypothesis proposing that patients hetero- RB1 mutations were screened by DGGE analysis of RT ± PCR zygous for a RB1 mutation are at risk of developing a ampli®ed materials as described (Chevillard et al., 1997, 1998). radiation induced cancer by a direct alteration of the Samples with altered DGGE pro®le were then sequenced. Study of the RB1 cDNA melting domain pro®le indicated that remaining allele by the ionizing radiation is obviously 14 independent RT ± PCR were mandatory to analyse nucleo- wrong, at least for those who developed their second tides 271 to 2962. Sequences of the primer used for RT ± PCR cancer after a long delay. Our data are in favor of a and experimental conditions are available on request. genome instability characterized by a particular panel of mutations associated with a high chromosome Sequencing instability. The discrimination between the mechanisms linked to predisposition and those linked to irradiation Overlapping fragments, prepared by RT ± PCR, covering the will need further investigation. full length of TP53 and several regions of RB1 cDNAs, were directly sequenced using the dideoxynucleotide method. Exons and parts of introns were sequenced by the same technique using genomic DNA and PCR ampli®cation. Primer sequences and experimental conditions are available Material and methods on request. Comparisons between pattern of mutations were performed using Yates w2. Biological material Tumors were collected at the Institut Curie. Informed Instability of microsatellites and minisatellites consent from patients or parents was obtained for all cases. Three tumors were grown as xenografts in athymic mouse In addition to the loci analysed for LOH of chromosomes 13 (cases 3, 4 and 6), while both fresh and xenografted tumors and 17, eight microsatellites known to be highly informative were available for case 3. Xenografted tumors were analysed were used: BAT25, BAT26, BAT40, D2S123, D5S346, at passage 3. Animal care and housing were in accordance D10S137, D17S250, D18S60 (Boland et al., 1998). The with institutional guidelines as put forth by the MinisteÁ re de stability of three minisatellites were studied: D1S80 (Sajantila l'Agriculture, Direction de la SanteÂ et de la Protection et al., 1992), D17S30 (Horn et al., 1989); H-RAS1 (Calvo et al., Animale. 1998) according to the conditions described by these authors.

DNA and RNA preparation Cytogenetic analysis DNA and RNA were extracted from frozen samples using Metaphase spreading, R-banding and ¯uorescence in situ Qiagen kits. Preparation integrity was checked by gel hybridization (FISH) using chromosome speci®c painting electrophoresis. Reverse transcription was performed using probes were performed as described (Bernardino et al., 1997; Superscript II (Life Technology) and random primers. Dutrillaux and Couturier, 1981).

Loss of heterozygosity Acknowledgments LOH was analysed by PCR ampli®cation of microsatellite This research was supported by grants from COGEMA DNA. Ten ng of template DNA was ampli®ed with 20 pmole (PIC D14), EDF (RB 2001 ± 26) the European Community of each primer in the presence of [32P]dCTP (Remvikos et al., (FIGH-CT-1999-00002) and the Institut Curie `Programme 1995). PCR products were separated through 6% acrylamide Incitatif et CoopeÂ ratif GeÂ notoxicologie'. S ± H LefeÁ vre was gel. LOH was visually scored on autoradiograms, the absence a fellow of the MinisteÁ re de l'Eduation Nationale et de la of signal of one allele in a tumor was recorded as LOH. Recherche.

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