Proc. Natl. Acad. Sci. USA Vol. 93, pp. 9172-9176, August 1996 Medical Sciences
Microsatellite instability in childhood rhabdomyosarcoma is locus specific and correlates with fractional allelic loss MIKE 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. Box 22700, 1100 DZ, Amsterdam, The Netherlands; §Department of Genetics, Leiden University, P.O. Box 9503, 2300 RA Leiden, The Netherlands; and 1Department of 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)
24). FAL, the fraction of chromosomal arms that undergo allelic loss, can be used as a molecular measure for chromosomal imbalance. Sporadic colorectal tumors demonstrating RERs and a low FAL have a better prognosis than their counterparts with high FAL and low RER (1). Thus, the identification of microsatellite instability and FAL in a particular tumor type may be of clinical importance. The inverse correlation between FAL and RERs in colorectal cancers suggests that extensive loss of heterozygosity (LOH) or the presence of RERs might be used to differentiate between the two different mechanisms of tumorigenesis. In the case of RER-positive tumors, defective MMR could be the initial event, resulting in an increased mutation rate, which might be the major driving force in tumorigenesis. In tumors with high FAL, 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 childhood cancers. Alveolar (ARMS) and embryonal RMS (ERMS) are the two main types and account for 19% and 57% of RMS, respectively (25-27). ARMS is characterized by a translocation t(2;13)(q35;q14), resulting in a chimeric transcript and fusion protein of the intact PAX3 DNA binding domain and the distal half of the forkhead (FKHR) gene (28, 29). No specific translocations have been identified in ERMS yet. Two putative tumor suppressor genes have been mapped in ERMS. EMRS1 is located on chromosome lip15.5 (30-34) and ERMS2 has been mapped to chromosome llq (34). In this study, we have analyzed the occurrence of RERs in human RMS, and the relation of RERs to allelic loss.
Replication errors (RERs) were initially ABSTRACT identified in hereditary nonpolyposis colon cancer and other tumors of Lynch syndrome II. Mutations in genes involved in mismatch repair give rise to a mutator phenotype, resulting in RERs. The mutator phenotype is thought to predispose to malignant transformation. Here we show that in the embryonal form of childhood rhabdomyosarcoma, RERs also occur, but in contrast to hereditary nonpolyposis colon cancer, only a subset of the microsatellite loci analyzed show RERs. The occurrence of RERs is strongly correlated with increased fractional allelic loss (P < 0.001), suggesting that the occurrence of RERs is a secondary phenomenon in rhabdomyosarcoma. Coincidental loss of genes involved in mismatch repair, possibly due to their proximity to tumor suppressor genes involved in tumor progression of embryonal form of childhood rhabdomyosarcoma, could explain the observed phenomenon.
Genomic instability at mnicrosatellite loci, which is manifested as the occurrence of replication errors (RERs), is a hallmark of 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 other extracolonic tumors not described in Lynch syndrome II (10-12). The genomic instability seems to be due to a mutator phenotype and could be a general mechanism for genetic alterations in several types of human cancer (13, 14). In HNPCC and some cases of sporadic colorectal cancer the mutator phenotype is associated with a defect in the mismatch repair (MMR) system (reviewed in ref. 15). At least five human genes, hMSH2, hMLH1, hPMS1, hPMS2, and GTBP, located on chromosomes 2pl6, 3p2l-23, 2q31-33, 7p22, and 2p16, respectively, are involved in the MMR system (16-19). Germline mutations in four of these genes have been identified in HNPCC kindreds (4, 16) and are thought to be responsible for the accumulation of RERs. Somatic mutations of MMR genes were also identified in sporadic colorectal cancers and cell lines demonstrating RERs (16, 19, 20). The observation of microsatellite instability in premalignant colorectal lesions of HNPCC tumors is compatible with the hypothesis that the mutator phenotype is responsible for the neoplastic transformation (1, 13, 21). However, RERs were identified recently in nonneoplastic tissue of patients with germ-line mutations in MMR genes (22) and transgenic mice homozygous for a disrupted MutS gene develop normally (23). This suggests that a defective MMR phenotype is compatible with normal development and that additional alterations are necessary for malignant transformation. In colorectal tumors, fractional allelic loss (FAL) and microsatellite instability are of prognostic importance (2, 3,
MATERIALS AND METHODS Microdissection of Normal and Tumor Tissue. Tumor tissue was obtained from formalin-fixed, paraffin-embedded tissue. Tumors obtained from the archieval collection of the Emma Kinderziekenhuis were from the period 1968-1990. Tumors were histologically classified by at least two independent pathologists (27). If no constitutional tissue was available in the form of peripheral blood, normal tissue was also obtained from the paraffin blocks or from bone marrow smears. Histological sections (7 ,um) from the paraffin blocks were hematoxylin/ eosin stained, and microdissection was performed to separate normal and tumor tissue. Every 10th section was hematoxylin/ eosin stained and analyzed for the presence of tumor and normal tissue. Abbreviations: RER, replication error; MMR, mismatch repair; HNPCC, hereditary nonpolyposis colon cancer; FAL, fractional allelic loss; LOH, loss of heterozygosity; RMS, rhabdomyosarcoma; ARMS, alveolar RMS; ERMS, embryonal RMS. 'To whom reprint requests should be addressed at: Neurozintuigen Laboratory, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. e-mail:
[email protected].
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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Medical Sciences: Visser et al. Isolation of DNA. To dissolve the paraffin, three sections were treated twice with xylene and washed with 100% ethanol. Sections were dried and incubated in 200 gl 10 mM Tris (pH 8.3), 1 mM EDTA, and 0.5% Tween 20 containing 10 mg-ml-' of proteinase K and incubated overnight at 37°C. The DNA obtained was extracted with phenol/chloroform and precipitated with ethanol according to standard procedures. For the hematoxylin/eosin-stained bone marrow smears, cells were scraped from the slides and DNA was extracted as described above. DNA quality was independent of the age of the tissue blocks. PCR Amplification. Primer sequences for the amplification of the microsatellites were obtained from the Genome Data Base (see Table 1). Standard PCRs were performed in a volume 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 0.8 unit of Taq polymerase (GIBCO/BRL). The MgCl2 concentration was adjusted for each primer pair for optimal amplification and ranged from 0.5 to 2.0 mM. Conditions are available upon request. PCR was performed in a multiwell thermocycler (MJ Research, Waltham, MA) for 35 cycles of 1 min 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 one primer was end-labeled with fluorescein isothiocyanate. The PCR products were denaturated and separated in a denaturing 6% polyacrylamide gel and exposed to an x-ray film or in the case the fluorescein isothiocyanate-labeled primer, the PCR products were detected on an automated sequencer (Millipore). Data Analysis. LOH and microsatellite instability were scored visually. Statistical analysis was performed with the two sample t test. All P values are two-sided. All data were collected blind by two independent investigators. Loci were scored RER positive when in two independent experiments addition alleles were detected. FAL is defined as the fraction of chromosome arms that undergo allelic loss. For each chromosome arm at least one marker was used (see Table 1). FAL is the ratio of informative loci that show LOH dived by all informative loci tested in a specific tumor.
RESULTS Allelic Loss. Thirty two primary RMS were analyzed (26 EMRS, 6 ARMS). Eight relapses and three metastases of 10 primary ERMS and one relapse and six metastases of three primary ARMS were also included in the analyses. In total, 57 microsatellite markers, covering all autosomes, were used in this study (Table 1). At least one microsatellite was tested for each nonacrocentric chromosome arm, and larger chromosome arms were analyzed by using multiple microsatellites. The data obtained in the allelic loss analysis were used to calculate the FAL. The observed LOH per chromosome arm was divided by the total number of informative chromosome arms. ARMS had a mean FAL of 0.06. ERMS showed a Gaussian distribution for the FAL with a mean of 0.142. Microsatellite Instability. Twenty-two of the 57 microsatellites tested demonstrated instability (Fig. 1.). All forms of RER were found-i.e., single new alleles (Fig. 1A, T1), multiple new alleles (Fig. 1A, T2 and 1C, T1) and even replacement of both normal alleles (Fig. 1B, T1). Every microsatellite repeat was tested in at least 30 samples. Some loci were frequently affected (e.g., CFTR, D9S66, D12S62, D14S51, CYP19) (Table 1). This suggests that in childhood RMS some loci are more prone to somatic instability than others. The microsatellite locus most frequently affected by instability was CFTR. The repeat of this marker is complex. It consists of two repeat units; a polymorphic TA dinucleotide repeat and a nonpolymorphic CA dinucleotide repeat (35). Other microsatellites frequently affected by instability are dinucleotide (D9S66, D12S62, D14S51) or tetranucleotide
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Table 1. Microsatellite loci, LOH, and RER in childhood RMS Chromosome Marker Locus Location LOH* RERt FGR FGR lp 1 lp36.2-p36.1 0/11 MFD60 D1S322 lp31 1/17 0 lq MFD96 DlSl75 lp21-ql2 0 0/12 MLOWl 1 DlS158 lq32-q41 0/14 MFD52 DlSlO2 lq42-q43 1/8 0 2p MYCN MYCN 2p24 0 0/12 AFMO92xh D2S123 2plS-pl6 3 2/14 2q HGBC 1 HGBC 2q34-q35 2/16 E41-31.1 3p D3S11 3p2l-p4l 3 2/21 3q GLUT2 GLUT2 3q26-q26.3 0/9 0 4p MFD59 D4S174 4p14 1/8 0 GABRB1 GABRB1 4pl3-pl2 4/24 3 4q MFD22 D4S171 4q33-q35 0 2/16 5p MFD88 0 D5S208 Spl5.3-15.1 1/24 5q MFD27 D5S1O7 5qll.2-ql3.3 0 1/6 IL-9 IL-9 5q22.3-q31.3 0 3/22 6p Fl3Al 5/22 F13A1 6p25-p24 1 1717/171 D6S89 6p24-p23 4/13 2 6q MFD131 3/27 D6S251 6ql3-21.1 2 7p MFD172 D7S472 7p2l-22 0 0/18 7q CFTR CFTR 7q31-p32 -/22f 11 7q COS43 D7S466 7q32 0 0/20 8p MFD199 0 2/16 D8S201 8p23 8q MFD159 0 0/21 D8S166 8ql1-ql2 9p MFD121 2/14 D9S104 9p2l 0 9q MFD94 D9S51 9q3 0 0/12 5964 D9S66 9q34.1-q34.3 5 4/26 lop MFD28 DlOS89 lOpter-pl 1.2 0 0/13 10q MFD150 0 1/21 DlOS109 lOqll.2-qter llp HRAS1 HRAS1 llpl5.5 0 6/10 TH 1 lpl5.5 TH 21/24 2 MFD166 D11S875 llpl5.5-15.4 1 12/22 38811 13/24 0 D11S554 llpl2-11.2 llq MFD127 0 1/3 DllS873 llql3-q23 TYR TYRSIN 1lq14-q21 0 1/2 ph2-22 DllS35 llq22 3/7 0 2 MFD161 D11S874 1 1q23-qter 13/21 MFD129 12p D12S62 12pter-pl2 1/20 7 12q MFD109 D12S60 12qter 6/28 2 13q MFD44 D13S71 13q31-q33 0 0/15 2E12B 14q 0 0/8 D14S43 14q24.3 7 MFD165 5/20 D14S51 14q31.l-qter 15q CYPl9 6 CYP19 15q21.l 3/19 HBAP1 16p HBAP1 l6pl3.3 0 1/17 1 16q 16AC1.15 D16S305 16q24.3 5/17 17p 12G6 0 D17S513 l7pl13 2/14 17q HGF HGF 1 17q22-q24 1/17 2 18p AFM178 D18S59 18pter 6/25 0 MFD80 MFD80 18pl 1.32-11.31 5/17 MBP MBP 0 18q 18q22-qter 0/12 l9p MFD120 D19S177 19p13.3 0 1/20 4 19q MFD5 APOC2 19ql2-ql3.2 1/21 IP2017 0 20p 0/15 D20S59 20pl2 20q 0 AFM27xhl D20S120 20ql2-ql3 0/20 C21G-K23A D2113E 21qll.2 0 21q 2/12 0 MFD55 D21S156 21q22.3 0/29 CYP2D 0 22q CYP2D 22ql 1 0/12
*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 these markers was inherent to their structure, tumors derived from another tissue type were analyzed. Twenty-three breast cancers samples and matched normal DNAs were tested with the CFTR microsatellite. Only one sample demonstrated instability, whereas three samples demonstrated LOH. These breast
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A N T1 T2
B N Tl T2
C N
TI
T2
FIG. 1. Three different patients with normal tissue and tumors demonstrating RERs. N, normal tissue; Ti, primary tumor; T2, relapse or metastases. (A) Normal tissue is homozygous for D9S66. In Ti a new allele is detected, whereas in T2 even more RERs are found. (B) Normal tissue is heterozygous for D9S66. In Ti the normal alleles are replaced by two alleles of different size, whereas T2 shows the same alleles as detected in normal tissue. (C) Normal tissue is heterozygous for TH. In Ti two additional alleles occur whereas T2 shows the same heterozygous situation as present in normal DNA. PCR was performed with one fluorescein isothiocyanate-labeled primer and data were collected on a fluorescent imaging system (Millipore).
cancer samples were previously analyzed for RERs with another set of microsatellite markers (10). In that study, five samples showed microsatellite instability. The tumor sample that showed instability at the CFTR locus did not show RERs in the previous study (10). Microsatellite instability was also evaluated in the 18 relapses and metastases of the primary tumors analyzed. In contrast to the allelotype analysis, where only minor differences were found in the LOH pattern between the primary tumor and the relapse or metastasis of the same patient (data not shown), many differences were found with respect to microsatellite instability in the different tumors of the patients (Fig. 1). In two of the three patients presented in Fig. 1, there were differences between the RER pattern of the primary tumor and the relapse. In Fig. 1A, the RER at locus D9S66 is only found in the relaps, so it had to be generated after the occurrance of the metastasis from the primary site. In Fig. 1 B and C, however, the RER is only present in the the primary tumor (T1) and had to be generated after the metastases (T2) were formed (Fig. 1 B and C). Thus microsatellite instability is not a prerequisite for the development of RMS nor metastases. This suggests that the development of RER in RMS is a late phenomenon and that the RERs observed are generated
constantly. Microsatellite Instability Is Correlated with FAL. In the study of Aaltonen et al. (1), the FAL of RER positive (+) sporadic colorectal cancer was 6 times lower than the FAL of RER negative (-) sporadic colorectal cancers (0.039 versus 0.254). To test whether such a correlation also exists in RMS, we classified the RMS into RER+ and RER- group. Tumors were scored as RER+ when at least 1 locus was affected. In ERMS, 14 of 26 tumors were RER+, whereas in ARMS, only one of 6 tumors was RER+. We found a strong correlation between FAL and RER (Table 2). The difference in the mean FAL in the RER+ (0.1907 ± 0.063) and RER- (0.085 ± 0.057) ERMS was statistically significant (P < 0.001, two sample t test). When the group of RMS (ERMS and ARMS) was analyzed as a whole, the difference in FAL between the RER+
Table 2. RER and FAL in ERMS Markers Tumor tested RER+ 20 30 19 29 34 38 41 35 35 30 26 33 17 37 33 19 24 33 27 37 6 55 32 42 S 52 29 47 RER2 53 18 18 17 29 16 34 7 34 3 49 1 47 11 48 13 46 9 52 36 15 8 50
(1996)
RER
FAL
9 7 7 S 4 4 4 3 3 2 2 2 2 1
0.13 0.05 0.32 0.20 0.13 0.18 0.27 0.18 0.22 0.17 0.18 0.23 0.17 0.24
0 0 0 0 0 0 0 0 0 0 0 0
0.14 0.06 0.07 0.05
0 0.19 0 0.08 0.14 0.14 0.04 0.14
and RER- group was also significant (P < 0.001, two sample t test). The cutoff value of 1 or more loci with RER for the inclusion of a tumor in the RER+ group is arbitrary. If a cutoff value of two or more RERs per tumor for inclusion in the RER+ group is used, the correlation between FAL and RER was still significant (P < 0.01). In view of the high frequency of RER at the CFTR locus, we also calculated the significance of the correlation between RER and FAL if we ommitted the highly RER prone CFTR locus. In that case the correlation was also highly significant, P < 0.001. In view of the correlation between FAL and RERs, we
analyzed whether the chromosomal position of the currently known genes involved in mismatch repair frequently showed LOH in the RER+ group. Microsatellite markers on chromosome 2pl5-16 (D2S123), 2q35-36 (HGBC), 3p2l (D3S11), and 7p22 (D7S472) were tested. No significant difference was found between the LOH pattern in the RER+ and RERgroup. Only two RER+ tumors showed LOH at any of the tested loci (LOH of D3S11 was found in tumors 23 and 29, tumor 29 also showed LOH of D2S123). Only one of the RERtumors (11) showed LOH at any of the four loci tested (HGBC).
DISCUSSION Five human genes involved in mismatch repair have been identified to date and mutations in these genes have been identified in HNPCC and sporadic forms of colon cancers. The presence of germ-line mutations in four of these mismatch repair genes in colorectal cancer strongly suggests that defective mismatch repair is an initial event in progression toward a malignant phenotype. In this study we show that RERs also occur in RMS. Our analysis shows that RERs are found in 14 of 26 childhood RMS. However, in contrast to HNPCC, where >80% of the loci analyzed are affected in each tumor (1), in RMS only a subset of microsatellite repeat loci is affected per
Medical Sciences: Visser et aL tumor. The CFTR repeat is affected most frequently, suggesting that in RMS some loci are susceptible to replication errors, whereas others are not. Locus-specific RER is not found in all types of tumors: the CFTR marker was affected in only 1 of 23 breast cancer samples. Why would some loci be more prone to RER? In view of the coupling of transcription and replication to DNA repair, an increased transcription rate or the presence of a marker in the vicinity of an origin of replication might affect the susceptibility of one locus compared with another. In yeast, increased transcription of the lys2 locus was associated with an increased mutation frequency, suggesting that stimulation of transcription in yeast results in a higher mutation rate (36). On the other hand, the structure of a locus could theoretically influence its stability upon replication. A highly unstable minisatellite has been described in mice (37). A comparison of the sequence of the microsatellites showing RERs does not show obvious similarities. The loci involved are simple dinucleotide repeats, (D9S66, D12S62, D14S51), complex dinucleotide repeats (CFTR), or even tetranucleotide repeats (CYP19). The high frequency of RERs at the CFTR repeat is not an intrinsic property of the repeat, since (i) it is not unstable in over 100 meioses we analyzed in linkage studies, and (ii) in the breast cancer samples analyzed, no locus-specific instability for CFTR was found. Our analysis of the breast cancer samples also shows that locus specific RER is not a general phenomenon in all types of cancer. Sequence-specific replication errors have been associated with mutations in GTBP. In tumors with mutations in GTBP, mononucleotide repeats are much more unstable than dinucleotide repeats (19). We have also tested whether poly(A) tracts are unstable in RER+ ERMS. No instability at locus BAT25 was found (data not shown). We consider it unlikely that the observed difference with HNPCC in the frequency of loci showing RER is due to bias introduced by the set of markers used since we also tested markers that showed 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 fractional allelic loss. In HNPCC, a low FAL is found, suggesting that defective MMR is the primary mechanism for generating mutations in the genome. In ERMS however, only the tumors with a high FAL show RER, suggesting that RERs are a secondary phenomenon. It is conceivable that the RERs in high FAL RMS are due to loss of chromosomal regions containing genes involved in DNA repair. These genes could be located near thusfar unidentified tumor suppressor genes involved in RMS, and may be lost selectively in RMS. Our allelotype analysis does not show a region that is lost in all RER+ tumors and is present in all RER- tumors (M.V., C.S., J.B., M. Godfried, P.A.V., and F.B., unpublished data). However, in view of the number of genes involved in MMR, loss of a single chromosomal region is not a prerequisite. If our hypothesis of coincidental loss of repair genes is true, RMS will represent a tumor that only after the initial steps of malignant transformation has acquired a defective MMR system; we are therefore analyzing tumor cells that have only recently started to accumulate RERs. In contrast, in HNPCC a defective MMR system, resulting in RERs, is an initial event, and therefore HNPCC cells represent cells that have gone through many rounds of replication in an environment with defective MMR. Most likely, multiple mutations have to be introduced in the genome of the cells with germ-line mutations in MMR genes before genes involved in cell cycle control are mutated in such a way that a cell is transformed to the malignant state. In conclusion, we propose that RERs in RMS are a secondary phenomenon, possibly due to the loss of genes involved in MMR. This assumption is based on the fact that RERs in ERMS show a strong correlation with high FAL. This implies that ERMS with high FAL accumulate more mutations than
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their counterparts with low FAL. The mutator phenotype will thus occur only in tumor cells that already show a high level of genome instability. In view of the recent finding of increased homologous recombination in MutS-deficient ES cells (23), defective MMR could theoretically also increase genome instability by illegitimate recombination. Whether a mutator phenotype also occurs in other types of tumors with high FAL and which factors are involved in the locus-specific instability remains to be established. We thank Drs. A. Motley, H. te Riele, N. de Wind, and P. Borst for comments on this manuscript. This work was supported by grants from
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