Proc. Natl. Acad. Sci. USAVol. 93, pp. 9172-9176, August 1996Medical Sciences

Microsatellite instability in childhood rhabdomyosarcoma is locusspecific and correlates with fractional allelic lossMIKE VISSER*t, JOHANNES BRASS, CARIN SIJMONS*, PETER DEVILEE§, LILIANE C. D. WIJNAENDTST,J. C. VAN DER LINDENS, P. A. VOUTEt, AND FRANK BAAs*II*Neurozintuigen Laboratory, tDepartment of Pathology, and tDepartment of Pediatric Oncology, Emma Kinderziekenhuis, Academic Medical Center, P.O. Box22700, 1100 DZ, Amsterdam, The Netherlands; §Department of Genetics, Leiden University, P.O. Box 9503, 2300 RA Leiden, The Netherlands; and 1Departmentof Pathology, Free University, P.O. Box 7057, 1007 MB Amsterdam, The Netherlands

Communicated by David. E. Housman, Massachusetts Institute of Technology, Cambridge, MA, May 30, 1996 (received for review December 9,1995)

ABSTRACT Replication errors (RERs) were initiallyidentified in hereditary nonpolyposis colon cancer and othertumors of Lynch syndrome II. Mutations in genes involved inmismatch repair give rise to a mutator phenotype, resulting inRERs. The mutator phenotype is thought to predispose tomalignant transformation. Here we show that in the embry-onal form of childhood rhabdomyosarcoma, RERs also occur,but in contrast to hereditary nonpolyposis colon cancer, onlya subset of the microsatellite loci analyzed show RERs. Theoccurrence of RERs is strongly correlated with increasedfractional allelic loss (P < 0.001), suggesting that the occur-rence of RERs is a secondary phenomenon in rhabdomyosar-coma. Coincidental loss of genes involved in mismatch repair,possibly due to their proximity to tumor suppressor genesinvolved in tumor progression ofembryonal form ofchildhoodrhabdomyosarcoma, could explain the observed phenomenon.

Genomic instability at mnicrosatellite loci, which is manifestedas the occurrence of replication errors (RERs), is a hallmarkof hereditary nonpolyposis colorectal cancer (HNPCC) (1-5)and other tumors of Lynch syndrome II (6-9). Occasionally,RERs are found in sporadic colorectal cancers (1) and otherextracolonic tumors not described in Lynch syndrome II(10-12). The genomic instability seems to be due to a mutatorphenotype and could be a general mechanism for geneticalterations in several types of human cancer (13, 14). InHNPCC and some cases of sporadic colorectal cancer themutator phenotype is associated with a defect in the mismatchrepair (MMR) system (reviewed in ref. 15). At least five humangenes, hMSH2, hMLH1, hPMS1, hPMS2, and GTBP, locatedon chromosomes 2pl6, 3p2l-23, 2q31-33, 7p22, and 2p16,respectively, are involved in the MMR system (16-19). Germ-line mutations in four of these genes have been identified inHNPCC kindreds (4, 16) and are thought to be responsible forthe accumulation of RERs. Somatic mutations ofMMR geneswere also identified in sporadic colorectal cancers and cell linesdemonstrating RERs (16, 19, 20). The observation of micro-satellite instability in premalignant colorectal lesions ofHNPCC tumors is compatible with the hypothesis that themutator phenotype is responsible for the neoplastic transfor-mation (1, 13, 21). However, RERs were identified recently innonneoplastic tissue of patients with germ-line mutations inMMR genes (22) and transgenic mice homozygous for adisrupted MutS gene develop normally (23). This suggests thata defective MMR phenotype is compatible with normal de-velopment and that additional alterations are necessary formalignant transformation.

In colorectal tumors, fractional allelic loss (FAL) andmicrosatellite instability are of prognostic importance (2, 3,

24). FAL, the fraction of chromosomal arms that undergoallelic loss, can be used as a molecular measure for chromo-somal imbalance. Sporadic colorectal tumors demonstratingRERs and a low FAL have a better prognosis than theircounterparts with high FAL and low RER (1). Thus, theidentification of microsatellite instability and FAL in a par-ticular tumor type may be of clinical importance. The inversecorrelation between FAL and RERs in colorectal cancerssuggests that extensive loss of heterozygosity (LOH) or thepresence of RERs might be used to differentiate between thetwo different mechanisms of tumorigenesis. In the case ofRER-positive tumors, defective MMR could be the initialevent, resulting in an increased mutation rate, which might bethe major driving force in tumorigenesis. In tumors with highFAL, loss of tumor suppressor genes plays an important role.Rhabdomyosarcoma (RMS) is the most common pediatric

soft tissue sarcoma and accounts for 5-15% of all childhoodcancers. Alveolar (ARMS) and embryonal RMS (ERMS) arethe two main types and account for 19% and 57% of RMS,respectively (25-27). ARMS is characterized by a translocationt(2;13)(q35;q14), resulting in a chimeric transcript and fusionprotein of the intact PAX3 DNA binding domain and the distalhalf of the forkhead (FKHR) gene (28, 29). No specifictranslocations have been identified in ERMS yet. Two putativetumor suppressor genes have been mapped in ERMS. EMRS1is located on chromosome lip15.5 (30-34) and ERMS2 hasbeen mapped to chromosome llq (34). In this study, we haveanalyzed the occurrence of RERs in human RMS, and therelation of RERs to allelic loss.

MATERIALS AND METHODSMicrodissection ofNormal and Tumor Tissue. Tumor tissue

was obtained from formalin-fixed, paraffin-embedded tissue.Tumors obtained from the archieval collection of the EmmaKinderziekenhuis were from the period 1968-1990. Tumorswere histologically classified by at least two independentpathologists (27). If no constitutional tissue was available in theform of peripheral blood, normal tissue was also obtained fromthe paraffin blocks or from bone marrow smears. Histologicalsections (7 ,um) from the paraffin blocks were hematoxylin/eosin stained, and microdissection was performed to separatenormal and tumor tissue. Every 10th section was hematoxylin/eosin stained and analyzed for the presence of tumor andnormal tissue.

Abbreviations: RER, replication error; MMR, mismatch repair;HNPCC, hereditary nonpolyposis colon cancer; FAL, fractional allelicloss; LOH, loss of heterozygosity; RMS, rhabdomyosarcoma; ARMS,alveolar RMS; ERMS, embryonal RMS.'To whom reprint requests should be addressed at: NeurozintuigenLaboratory, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands.e-mail: f.baas@amc.uva.nl.

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

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Isolation of DNA. To dissolve the paraffin, three sectionswere treated twice with xylene and washed with 100% ethanol.Sections were dried and incubated in 200 gl 10 mM Tris (pH8.3), 1 mM EDTA, and 0.5% Tween 20 containing 10 mg-ml-'of proteinase K and incubated overnight at 37°C. The DNAobtained was extracted with phenol/chloroform and precipi-tated with ethanol according to standard procedures. For thehematoxylin/eosin-stained bone marrow smears, cells werescraped from the slides and DNA was extracted as describedabove. DNA quality was independent of the age of the tissueblocks.PCR Amplification. Primer sequences for the amplification

of the microsatellites were obtained from the Genome DataBase (see Table 1). Standard PCRs were performed in avolume of 10 ,u with 10-80 ng DNA, 50 ng of each primer, 200,uM of each dNTP, 50 mM KCl, 10 mM Tris (pH 8.3), and 0.8unit of Taq polymerase (GIBCO/BRL). The MgCl2 concen-tration was adjusted for each primer pair for optimal ampli-fication and ranged from 0.5 to 2.0 mM. Conditions areavailable upon request. PCR was performed in a multiwellthermocycler (MJ Research, Waltham, MA) for 35 cycles of 1min each at 94°C, 55°C, and 72°C. For detection either 1 ,uCi(1 Ci = 37 GBq) of [a-32P]dATP (Amersham) was used or oneprimer was end-labeled with fluorescein isothiocyanate. ThePCR products were denaturated and separated in a denaturing6% polyacrylamide gel and exposed to an x-ray film or in thecase the fluorescein isothiocyanate-labeled primer, the PCRproducts were detected on an automated sequencer (Milli-pore).Data Analysis. LOH and microsatellite instability were

scored visually. Statistical analysis was performed with the twosample t test. All P values are two-sided. All data werecollected blind by two independent investigators. Loci werescored RER positive when in two independent experimentsaddition alleles were detected. FAL is defined as the fractionof chromosome arms that undergo allelic loss. For eachchromosome arm at least one marker was used (see Table 1).FAL is the ratio of informative loci that show LOH dived byall informative loci tested in a specific tumor.

RESULTSAllelic Loss. Thirty two primary RMS were analyzed (26

EMRS, 6 ARMS). Eight relapses and three metastases of 10primary ERMS and one relapse and six metastases of threeprimary ARMS were also included in the analyses. In total, 57microsatellite markers, covering all autosomes, were used inthis study (Table 1). At least one microsatellite was tested foreach nonacrocentric chromosome arm, and larger chromo-some arms were analyzed by using multiple microsatellites.The data obtained in the allelic loss analysis were used tocalculate the FAL. The observed LOH per chromosome armwas divided by the total number of informative chromosomearms. ARMS had a mean FAL of 0.06. ERMS showed aGaussian distribution for the FAL with a mean of 0.142.

Microsatellite Instability. Twenty-two of the 57 microsat-ellites tested demonstrated instability (Fig. 1.). All forms ofRER were found-i.e., single new alleles (Fig. 1A, T1),multiple new alleles (Fig. 1A, T2 and 1C, T1) and evenreplacement of both normal alleles (Fig. 1B, T1). Everymicrosatellite repeat was tested in at least 30 samples. Someloci were frequently affected (e.g., CFTR, D9S66, D12S62,D14S51, CYP19) (Table 1). This suggests that in childhoodRMS some loci are more prone to somatic instability thanothers. The microsatellite locus most frequently affected byinstability was CFTR. The repeat of this marker is complex. Itconsists of two repeat units; a polymorphic TA dinucleotiderepeat and a nonpolymorphic CA dinucleotide repeat (35).Other microsatellites frequently affected by instability aredinucleotide (D9S66, D12S62, D14S51) or tetranucleotide

Table 1. Microsatellite loci, LOH, and RER in childhood RMS

Chromosome Marker Locus Location LOH* RERtlp FGR

MFD60lq MFD96

MLOWlMFD52

2p MYCNAFMO92xh

2q HGBC3p E41-31.13q GLUT24p MFD59

GABRB14q MFD225p MFD885q MFD27

IL-96p Fl3Al

1717/1716q MFD1317p MFD1727q CFTR7q COS438p MFD1998q MFD1599p MFD1219q MFD94

5964lop MFD2810q MFD150llp HRAS1

THMFD16638811

llq MFD127TYRph2-22MFD161

12p MFD12912q MFD10913q MFD4414q 2E12B

MFD16515q CYPl916p HBAP116q 16AC1.1517p 12G617q HGF18p AFM178

MFD8018q MBPl9p MFD12019q MFD520p IP201720q AFM27xhl21q C21G-K23A

MFD5522q CYP2D

FGRD1S322DlSl75DlS158DlSlO2MYCND2S123HGBCD3S11GLUT2D4S174

lp36.2-p36.1lp31lp21-ql2lq32-q41lq42-q432p242plS-pl62q34-q353p2l-p4l3q26-q26.34p14

GABRB1 4pl3-pl2D4S171D5S208D5S1O7IL-9F13A1D6S89D6S251D7S472CFTRD7S466D8S201D8S166D9S104D9S51D9S66DlOS89DlOS109HRAS1THD11S875D11S554DllS873TYRSINDllS35D11S874D12S62D12S60D13S71D14S43D14S51CYP19HBAP1D16S305D17S513HGFD18S59MFD80MBPD19S177APOC2D20S59D20S120

4q33-q35Spl5.3-15.15qll.2-ql3.35q22.3-q31.36p25-p246p24-p236ql3-21.17p2l-227q31-p327q328p238ql1-ql29p2l9q39q34.1-q34.3lOpter-pl 1.2lOqll.2-qterllpl5.51 lpl5.5llpl5.5-15.4llpl2-11.2llql3-q231lq14-q21llq2211q23-qter12pter-pl212qter13q31-q3314q24.314q31.l-qter15q21.ll6pl3.316q24.3l7pl1317q22-q2418pter18pl 1.32-11.3118q22-qter19p13.319ql2-ql3.220pl220ql2-ql3

D2113E 21qll.2D21S156 21q22.3CYP2D 22ql 1

0/111/170/120/141/8

0/122/142/162/210/91/8

10010031300

4/24 32/161/241/6

3/225/224/133/270/18-/22f0/202/160/212/140/124/260/131/216/10

21/2412/2213/24

1/31/23/7

13/211/206/280/150/85/203/191/175/172/141/176/255/170/121/201/210/150/202/120/290/12

00001220

11000005000210000272007601012000400000

*LOH/informative patient.tNumber of samples with RER.tCould not be scored due to high frequency of RER.

(CYP19) repeats. To test whether the instability of thesemarkers was inherent to their structure, tumors derived fromanother tissue type were analyzed. Twenty-three breast can-cers samples and matched normal DNAs were tested with theCFTR microsatellite. Only one sample demonstrated instabil-ity, whereas three samples demonstrated LOH. These breast

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A B Table 2. RER and FAL in ERMSC

N T1 T2 N Tl T2 N TI T2

FIG. 1. Three different patients with normal tissue and tumorsdemonstrating RERs. N, normal tissue; Ti, primary tumor; T2, relapseor metastases. (A) Normal tissue is homozygous for D9S66. In Ti anew allele is detected, whereas in T2 even more RERs are found. (B)Normal tissue is heterozygous for D9S66. In Ti the normal alleles arereplaced by two alleles of different size, whereas T2 shows the samealleles as detected in normal tissue. (C) Normal tissue is heterozygousfor TH. In Ti two additional alleles occur whereas T2 shows the sameheterozygous situation as present in normal DNA. PCR was per-formed with one fluorescein isothiocyanate-labeled primer and datawere collected on a fluorescent imaging system (Millipore).

cancer samples were previously analyzed for RERs withanother set of microsatellite markers (10). In that study, fivesamples showed microsatellite instability. The tumor samplethat showed instability at the CFTR locus did not show RERsin the previous study (10).

Microsatellite instability was also evaluated in the 18 re-lapses and metastases of the primary tumors analyzed. Incontrast to the allelotype analysis, where only minor differ-ences were found in the LOH pattern between the primarytumor and the relapse or metastasis of the same patient (datanot shown), many differences were found with respect tomicrosatellite instability in the different tumors of the patients(Fig. 1). In two of the three patients presented in Fig. 1, therewere differences between the RER pattern of the primarytumor and the relapse. In Fig. 1A, the RER at locus D9S66 isonly found in the relaps, so it had to be generated after theoccurrance of the metastasis from the primary site. In Fig. 1B and C, however, the RER is only present in the the primarytumor (T1) and had to be generated after the metastases (T2)were formed (Fig. 1 B and C). Thus microsatellite instabilityis not a prerequisite for the development of RMS nor metas-tases. This suggests that the development of RER in RMS isa late phenomenon and that the RERs observed are generatedconstantly.

Microsatellite Instability Is Correlated with FAL. In thestudy of Aaltonen et al. (1), the FAL of RER positive (+)sporadic colorectal cancer was 6 times lower than the FAL ofRER negative (-) sporadic colorectal cancers (0.039 versus0.254). To test whether such a correlation also exists in RMS,we classified the RMS into RER+ and RER- group. Tumorswere scored as RER+ when at least 1 locus was affected. InERMS, 14 of 26 tumors were RER+, whereas in ARMS, onlyone of 6 tumors was RER+. We found a strong correlationbetween FAL and RER (Table 2). The difference in the meanFAL in the RER+ (0.1907 ± 0.063) and RER- (0.085 ± 0.057)ERMS was statistically significant (P < 0.001, two sample ttest). When the group of RMS (ERMS and ARMS) wasanalyzed as a whole, the difference in FAL between the RER+

Tumor

RER+201934353026373324276

32S

29RER-

2181716731

11139

158

Markerstested

3029384135331719333755425247

531829343449474846523650

RER FAL

977S4443322221

000000000000

0.130.050.320.200.130.180.270.180.220.170.180.230.170.24

0.140.060.070.0500.1900.080.140.140.040.14

and RER- group was also significant (P < 0.001, two samplet test). The cutoff value of 1 or more loci with RER for theinclusion of a tumor in the RER+ group is arbitrary. If a cutoffvalue of two or more RERs per tumor for inclusion in theRER+ group is used, the correlation between FAL and RERwas still significant (P < 0.01). In view of the high frequencyof RER at the CFTR locus, we also calculated the significanceof the correlation between RER and FAL if we ommitted thehighly RER prone CFTR locus. In that case the correlationwas also highly significant, P < 0.001.

In view of the correlation between FAL and RERs, weanalyzed whether the chromosomal position of the currentlyknown genes involved in mismatch repair frequently showedLOH in the RER+ group. Microsatellite markers on chromo-some 2pl5-16 (D2S123), 2q35-36 (HGBC), 3p2l (D3S11), and7p22 (D7S472) were tested. No significant difference wasfound between the LOH pattern in the RER+ and RER-group. Only two RER+ tumors showed LOH at any of thetested loci (LOH of D3S11 was found in tumors 23 and 29,tumor 29 also showed LOH of D2S123). Only one of the RER-tumors (11) showed LOH at any of the four loci tested(HGBC).

DISCUSSIONFive human genes involved in mismatch repair have beenidentified to date and mutations in these genes have beenidentified in HNPCC and sporadic forms of colon cancers. Thepresence of germ-line mutations in four of these mismatchrepair genes in colorectal cancer strongly suggests that defec-tive mismatch repair is an initial event in progression towarda malignant phenotype. In this study we show that RERs alsooccur in RMS. Our analysis shows that RERs are found in 14of 26 childhood RMS. However, in contrast to HNPCC, where>80% of the loci analyzed are affected in each tumor (1), inRMS only a subset of microsatellite repeat loci is affected per

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tumor. The CFTR repeat is affected most frequently, suggest-ing that in RMS some loci are susceptible to replication errors,whereas others are not. Locus-specific RER is not found in alltypes of tumors: the CFTR marker was affected in only 1 of 23breast cancer samples.Why would some loci be more prone to RER? In view of the

coupling of transcription and replication to DNA repair, anincreased transcription rate or the presence of a marker in thevicinity of an origin of replication might affect the suscepti-bility of one locus compared with another. In yeast, increasedtranscription of the lys2 locus was associated with an increasedmutation frequency, suggesting that stimulation of transcrip-tion in yeast results in a higher mutation rate (36). On the otherhand, the structure of a locus could theoretically influence itsstability upon replication. A highly unstable minisatellite hasbeen described in mice (37). A comparison of the sequence ofthe microsatellites showing RERs does not show obvioussimilarities. The loci involved are simple dinucleotide repeats,(D9S66, D12S62, D14S51), complex dinucleotide repeats(CFTR), or even tetranucleotide repeats (CYP19). The highfrequency of RERs at the CFTR repeat is not an intrinsicproperty of the repeat, since (i) it is not unstable in over 100meioses we analyzed in linkage studies, and (ii) in the breastcancer samples analyzed, no locus-specific instability forCFTR was found. Our analysis of the breast cancer samplesalso shows that locus specific RER is not a general phenom-enon in all types of cancer. Sequence-specific replication errorshave been associated with mutations in GTBP. In tumors withmutations in GTBP, mononucleotide repeats are much moreunstable than dinucleotide repeats (19). We have also testedwhether poly(A) tracts are unstable in RER+ ERMS. Noinstability at locus BAT25 was found (data not shown). Weconsider it unlikely that the observed difference with HNPCCin the frequency of loci showing RER is due to bias introducedby the set of markers used since we also tested markers thatshowed RERs in the HNPCC studies (e.g. D2S123, D5S107).In this study, marker D2S123 showed RERs but D5S107 did not.A second difference with HNPCC is that in childhood RMS,

the occurrence of RERs is correlated with increased fractionalallelic loss. In HNPCC, a low FAL is found, suggesting thatdefective MMR is the primary mechanism for generatingmutations in the genome. In ERMS however, only the tumorswith a high FAL show RER, suggesting that RERs are asecondary phenomenon. It is conceivable that the RERs inhigh FAL RMS are due to loss of chromosomal regionscontaining genes involved in DNA repair. These genes couldbe located near thusfar unidentified tumor suppressor genesinvolved in RMS, and may be lost selectively in RMS. Ourallelotype analysis does not show a region that is lost in allRER+ tumors and is present in all RER- tumors (M.V., C.S.,J.B., M. Godfried, P.A.V., and F.B., unpublished data). How-ever, in view of the number of genes involved in MMR, loss ofa single chromosomal region is not a prerequisite. If ourhypothesis of coincidental loss of repair genes is true, RMS willrepresent a tumor that only after the initial steps of malignanttransformation has acquired a defective MMR system; we aretherefore analyzing tumor cells that have only recently startedto accumulate RERs. In contrast, inHNPCC a defectiveMMRsystem, resulting in RERs, is an initial event, and thereforeHNPCC cells represent cells that have gone through manyrounds of replication in an environment with defective MMR.Most likely, multiple mutations have to be introduced in thegenome of the cells with germ-line mutations in MMR genesbefore genes involved in cell cycle control are mutated in sucha way that a cell is transformed to the malignant state.

In conclusion, we propose that RERs in RMS are a sec-ondary phenomenon, possibly due to the loss of genes involvedin MMR. This assumption is based on the fact that RERs inERMS show a strong correlation with high FAL. This impliesthat ERMS with high FAL accumulate more mutations than

their counterparts with low FAL. The mutator phenotype willthus occur only in tumor cells that already show a high level ofgenome instability. In view of the recent finding of increasedhomologous recombination in MutS-deficient ES cells (23),defective MMR could theoretically also increase genomeinstability by illegitimate recombination. Whether a mutatorphenotype also occurs in other types of tumors with high FALand which factors are involved in the locus-specific instabilityremains to be established.

We thank Drs. A. Motley, H. te Riele, N. de Wind, and P. Borst forcomments on this manuscript. This work was supported by grants fromthe Dutch Cancer Society and the Foundation for Pediatric CancerResearch (to F.B.).

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