Assignment of Common Alã-Eleloss In

[CANCER RESEARCH 49, 6247-6251. November 15. 1989] Assignment of Common AlÃeleLoss in Osteosarcoma to the Subregion 17pl3'

Junya Toguchida,2 Kanji Ishizaki,3 Yusuke Nakamura, Masao S. Sasaki, Mituo Ikenaga, Mitsuo Kato, Masayuki Sugimoto, Yoshihiko Kotoura, and Takao Yamamuro Radiation Biology Center [J. T., K. l., M. S. S., M. I., M. K.J and Department of Orthopaedic Surgery; Faculty of Medicine [J. T., M. S., Y. K., T. Y.], Kyoto University, Yoshida-konoecho, Sakyo-ku, Kyoto 606, Japan; and Howard Hughes Medical Institute and Department of Human Genetics, University of Utah Medical Center, Salt Lake City, Utah 84132 [Y. N.]

ABSTRACT MATERIALS AND METHODS Samples. Thirty-seven osteosarcoma tumors were analyzed, consist Human osteosarcomas frequently show loss of alÃeleson chromosome ing of 26 primary and 11 metastatic lesions. They were treated at the 17 as well as those on chromosome 13 that harbors the retinoblastoma Kyoto University Hospital or other medical centers in Japan, and tumor gene, indicating concerted operation of another tumor-suppressing gene tissues were frozen immediately after surgical removal and stored at on chromosome 17. To assign the affected gene to a defined region of â€”¿80Â°Cuntilisolation of DNA. Peripheral leukocytes of patients were chromosome 17, we performed mitotic recombination/deletion mapping isolated using Ficoll-Hypaque (Pharmacia) from heparinized blood by the use of 10 polymorphic loci on chromosome 17. Of 37 tumors which were collected before or after the surgical treatment. studied, 28 (75.7%) showed loss of heterozygosity on chromosome 17. DNA Isolation and Southern Blot Analysis. High molecular weight The affected regions varied among tumors, ranging in extent from a whole DNA was isolated from peripheral leukocytes and tumor tissues as chromosome to a distal segment of the short arm. However, alÃeleloss previously described (22). Restriction endonuclease digestion of these in one region, notably in 17pl3 between D17S1 and D17S30, was common samples, agarose gel electrophoresis, Southern hybridization, labeling to all 28 tumors, suggesting the presence of a tumor-suppressing gene in the probes by nick-translation, and autoradiography were also accom this defined region. plished in the same way as described previously (22). In some cases, we added human placenta! DNA to prehybridization and hybridization solution to eliminate background repetitive sequences (23). INTRODUCTION Polymorphic Probes. Zygotic changes at RFLP4 loci on chromosome 17 were analyzed with the following probes; pHHH152 is homologous Cancer may be regarded as the consequence of multiple to D17S32 and reveals alÃelesof 10.5 and 9.6 kilobases with Hindlll- genetic alterations (1). Some of these alterations are mutations digested DNA; pHHH202 to D17S33, 2.5 and 1.9 kilobases with /foal; pA 10-41 to D17S71, 2.4 and 1.9 kilobases with Mspl; p 10.5 to MYH2, that endow preexisting genes, protooncogenes, with oncogenic 5.3 and 4.9 kilobases with Hindlll; pHF12-l to D17S1, 2.9 and 2.1 potential (2). In contrast with these gain-of-function mutations, kilobases with Mspl; and pMCT35.1 to D17S31, 2.4 and 1.8 kilobases loss-of-function mutations of tumor-suppressing gene can also with Mspl. Probes pTHH59. pCMM86, and pYNZ22 are homologous occur (3, 4); this mechanism has been extensively studied in to D17S4, D17S74, and D17S30, respectively, and these three loci retinoblastoma (5-7). Such mutations are thought to be reces contain a variable number of tandem repeats (24) and exhibit alÃelesof sive at the cellular level, and to be expressed when some various sizes in TaqI-digested DNA. The relative order and subchro- chromosomal mechanisms eliminate the wild-type alÃele(8). mosomal location of these polymorphic loci have been described by Y. This process has been identified as a loss of heterozygosity at Nakamura (25). In addition, we used pMCT35.2, which is homologous polymorphic loci on a specific chromosome (8). Although initial to the sequences located within 40 kilobases of the sequences recognized by pMCT35.1; this probe reveals two alÃelesof2.4 and 2.0 kilobases in studies of hereditary embryonic tumors emphasized alÃeleloss Mspl digests.5 on one specific chromosome (8, 9), recent studies have shown that loss of heterozygosity is not restricted to the specific chromosome that harbors the etiologically related gene, but RESULTS often occurs on several other chromosomes as well (10-18). The zygosity of each locus on chromosomes 17 was deter These observations imply that some tumor-suppressing genes mined by RFLP analysis in 37 paired samples of constitutional may play a role in the progression of tumor rather than an and tumor tissues. Of those, 28 tumors (75.7%) lost heterozy initiating role (19, 20). gosity on chromosome 17 (Table 1). Loss of alÃeleson chro We recently reported loss of heterozygosity on chromosome mosome 17 appeared to be more frequent in metastatic lesions 13 in osteosarcoma, which revealed a recessive mutation in the (81.8%) than in primary lesions (73.1%), although the differ retinoblastoma gene (21). We also investigated loss of hetero ence was not statistically significant. In four cases, we could zygosity on 10 chromosomes other than chromosome 13, and obtain both primary and metastatic tumors. In all cases, heter found that alÃelelossalso occurred on nine of 10 chromosomes, ozygosity on chromosome 17 were already lost in primary of which chromosome 17 showed the highest frequency, even lesions, and same alterations were observed in metastatic le higher than chromosome 13 (21). This observation suggests the sions (data not shown). presence of another tumor-suppressing gene on chromosome To identify the commonly involved region on chromosome 17. Assuming that this gene should be situated within a region 17, zygotic changes of each locus were analyzed in 28 cases of alÃeleloss common to different tumors, here we performed which lost heterozygosity at least at one locus of chromosome the mitotic recombination/deletion mapping in osteosarcoma 17. Results of Southern blot analysis in typical cases are shown by using 10 probes for polymorphic loci on chromosome 17. in Fig. 1, and the schematic representation of involved regions Received 4/24/89; revised 8/11/89; accepted 8/18/89. in corresponding cases are shown in Fig. 2. Of the 28 tumors The costs of publication of this article were defrayed in part by the payment which lost heterozygosity at loci on 17p, six had lost alÃelesat of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. all the informative loci on 17q as well, indicating loss of a 1Supported in part by grants from the Ministry of Education, Science, and whole chromosome (Figs, la and 2a). In the other 22 tumors, Culture, and the Ministry of Health and Welfare. Japan. 2 Present address: Massachusetts Eye and Ear Infirmary, Department of Oph 4The abbreviations used are: RFLP. restriction fragments length polymor thalmology, Harvard Medical School. 243 Charles Street. Boston. MA 02114. phism; SCCL, small cell carcinoma of the lung. 3To whom requests for reprints should be addressed. *Y. Nakamura, unpublished data. 6247

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1989 American Association for Cancer Research. ALLELE LOSS AT 17p!3 IN OSTEOSARCOMA Table 1 Loss of hetero:ygosity on chromosome 17 a 10Â° o Origin of Frequency of alÃeleloss _J tumor DNA (tumor with loss/informative cases) Oi 1 Primary lesions 19/26(73.1%) < Metastatic lesions 9/11 (81.8%) Â£ Total 28/37 (75.7%) I 50- 6 222 ni 'S .-â€¢oj

KS-40 --0---0- O- -o-

KS-103 -Oâ€”X--O o oâ€” o o-

Polymorphic Loci on Chromosome 17

Fig. 3. Frequency of loss of heterozygosity at polymorphic loci on chromo KS-159--0-Â« some 17. Data for each locus are calculated from the results of 28 tumors which showed alÃelelossat least at one locus on chromosome 17.

Fig. 2. Schematic representation of the genotypes of tumors shown in Fig. 1. DISCUSSION â€¢¿,locuswhich retained constitutional heterozygosity; O. locus which showed alÃeleloss; - - - -, region assumed to lose the heterozygosity: x, putative breakpoint in each case. The present study revealed the involvement of alÃeleloss in a defined region of p 13, between loci D17S1 and D17S30, of chromosome 17 in the development of osteosarcoma. We know all informative loci on the long arm retained constitutional of at least three other types of tumor that show a high frequency heterozygosity. Tumor KS-159 (Figs, id and 2d) was the only of alÃeleloss on chromosome 17: SCCL (26), colorectal carci exception, in that the most distal locus (D17S4) on the long noma (16, 17), and breast cancer (27). In SCCL, loss of alÃeles arm lost heterozygosity whereas the proximal locus (D17S74) also occurs frequently on chromosomes 3 and 13 (26), and a did not. Some tumors lost heterozygosity at all informative loci predisposing gene for colorectal carcinoma has been located on on the short arm, including the most proximal (D17S71) and chromosome 5 (13, 14). These observations suggest that loss of the most distal (D17S30) (Figs, lÃ©and 2b). However, because a gene on chromosome 17 may play a cooperative role in the six tumors were differentially affected among neighboring loci development and/or progression of tumors in addition to mu (Figs. 1, c-e and 2, c-e), we were able to determine the relative tation of tumor-specific predisposing genes, such as those on position of the breakpoints between two markers. For example, chromosomes 3, 5, and 13 for SCCL, colon carcinoma, and in KS-42 (Figs, le and 2c), D17S71 remained heterozygous in osteosarcoma, respectively. From this standpoint, the relation tumor DNA, while the next distal locus, MYH2, had lost the ship of loss of heterozygosity on chromosomes 13 and 17 may longer alÃele;the proximal breakpoint in KS-42 appeared to be be intriguing. The osteosarcomas in the present study also situated between these two loci. In this way, the proximal showed loss of heterozygosity of chromosome 13 and structural breakpoint could be located between D17S71 and MYH2 in changes of the retinoblastoma gene and a part of them were three tumors (Figs. \c and 2c), between MYH2 and D17S1 in previously reported (21). Among 37 osteosarcomas 36 were two tumors (Figs. 1d and Id), and between D17S1 and D17S31 informative as to chromosome 13 and 24 showed loss of het in one tumor (Figs. \e and 2e). As for the distal breakpoints, erozygosity. Of 24 tumors losing heterozygosity on chromo all tumors except KS-81 lost heterozygosity even at the locus some 13, only one tumor retained heterozygosity on chromo nearest pter, D17S30. In KS-81, which had a proximal break some 17 and 23 tumors lost heterozygosity on both chromo point between D17S1 and D17S31, the distal breakpoint must some 13 and 17. Four tumors lost heterozygosity on be located between D17S31 and D17S30 because loss of het chromosome 17 but not on chromosome 13. Constitutional erozygosity was found at the former locus but not at the latter. heterozygosities on both chromosomes were kept in eight tu The frequencies of alÃeleloss were calculated in each locus mors (detailed data will be published elsewhere). Thus strong (Fig. 3). Fourteen individuals were informative for locus association of deletion of chromosome 13 and 17 was apparent. D17S4; 18 for D17S74; 10 for D17S32; nine for D17S33; 12 Vogelstein et a/. (19) have explicitly demonstrated that a gene forD17S71; 15forMYH2; 13forD17Sl; 15 for D17S31; and on the short arm of chromosome 17 may be associated with 17 for D17S30. Constitutional heterozygosities were lost with progression from a benign adenomatous polyp in the colon to similar frequency at each locus on the long arm, indicating that a malignant adenocarcinoma. Since no definite premalignant nearly 20% of tumors lost a whole one chromosome 17. On the lesion has been identified for osteosarcoma, we have not been contrary, the frequency of alÃeleloss on the short arm increased able to confirm such an association for this type of tumor. toward the telomere. AlÃeleloss was observed in 50 and 80% However, metastasis might be regarded as an advancement of of tumors at the locus on the region of pi 1 and p 12-13 respec tumors. Although the difference was not statistically significant, tively, indicating that many tumors lost heterozygosity in a the frequency of alÃeleloss on chromosome 17 in metastatic most part of short arm of chromosome 17. Although hetero lesions was higher than that in primary lesions, and our prelim zygosity was lost at three loci inbandl7pl3(D17Sl,D17S31, inary results in a follow-up study have indicated that primary and D17S30) in almost all tumors, only D17S31 was commonly lesions which show loss of heterozygosity on chromosome 17 involved. The locus homologous to pMCT35.2 also showed tend to metastasize earlier.6 loss of heterozygosity in all informative individuals (12 of 12 The results of recombination/deletion mapping showed that cases) (data not shown). This indicates that the target gene is present in a region between D17S1 and D17S30 in 17pl3. *J. Toguchida. Unpublished data. 6249

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Junya Toguchida, Kanji Ishizaki, Yusuke Nakamura, et al.

Cancer Res 1989;49:6247-6251.

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