Identification of Three Regions on Chromosome 17Q in Primary Human Breast Carcinomas Which Are Frequently Deleted I

[CANCER RESEARCH 53, 5617-5619, December 1, 1993] Advances in Brief

Identification of Three Regions on Chromosome 17q in Primary Human Breast Carcinomas Which Are Frequently Deleted I

Craig S. Cropp, Marie-Helene Champeme, Rosette Lidereau, and Robert Callahan 2 Laboratory of Tumor Immunology and Biology, National Cancer Institute, NIH, Bethesda, Maryland 20892 [C. C., R. C.], and INSERM, Centre Rene Huguenin, 92211 Saint Cloud, France [M. H. C., R. L.]

Abstract Materials and Methods

We have examined the long arm of chromosome 17 in sporadic breast Primary breast carcinomas and matching peripheral lymphocytes were col- carcinomas for the loss of heterozygosity (LOH) at 18 polymorphic loci. At lected at the Centre Rene Huguenin in Saint Cloud, France, from patients who least three distinct regions could be identified by the frequency of LOH had received no prior therapy. The distribution of clinical and pathological and confirmed by high density deletion maps of individual tumor DNAs. parameters associated with the tumors and patients has been published (14). A proximal region affected by LOH is located in a 22-cM region defined Genomic DNA was extracted, and 10/xg was digested with the restriction by D17S73 and NME1 and thus is similar in location to the region thought enzyme of choice. The digested DNA was fractionated by agarose gel elec- to contain the BRCA1 gene associated with familial breast and breast/ trophoresis, transferred to Genatran 45 nylon membranes (Plasco, Woburn, ovarian cancer. The central region affected by LOH is bordered by the MA), and baked for 2-3 h at 80~ The membranes were prehybridized and D17S86 and D17S21 loci and is estimated to be 28 cM in size. The third then hybridized with 32p-labeled DNA probes made by the nick-translation or region is bordered by the DI 7S20 and 1917S77 loci which are II cM apart. random-primer system. The DNA probes and the polymorphic restriction sites These results define three independent regions of chromosome 17q which used are listed in Table 1 in their published chromosomal order (15, 16). After are likely to contain tumor suppressor genes relevant to the etiology of hybridization, the membranes were washed under stringent conditions (15 mM sporadic breast carcinoma. NaCI-I.5 mM sodium citrate, pH 7, at 65~ for 20 min) and autoradiographed.

Introduction Results and Discussion Several studies confirm the importance of genetic alterations to the The frequencies of LOH detected at 18 different loci on chromo- development of breast carcinoma (I). We and others have been search- some 17q in our panel of sporadic breast carcinoma DNAs is sum- ing for tumor suppressor genes relevant to breast cancer by screening marized in Table 1. Based on the frequency of LOH at individual loci, primary breast tumor DNA samples for LOH. 3 According to Knud- chromosome 17q may be divided into at least three independent son's "two-hit" hypothesis for the inactivation of a tumor suppressor regions which are affected by interstitial deletions. The borders of gene, one allele contains a point mutation or a small deletion, while these regions can be defined by the genotypes of individual tumor the second allele is lost by an interstitial deletion, chromosome loss, DNAs. Fig. 1 shows examples of Southern analysis that depict partial or an aberrant mitotic recombinational event (2). Chromosome 17 deletions of chromosome 17q. Panel A shows examples of interstitial contains several genes important to the development and/or progres- deletions in the proximal region. Tumor DNAs 132 and 134 are sion of human breast cancer. Already identified are the TP53 tumor informative and unaffected at D17S73 (LEW207) but have deletions suppressor gene on chromosome 17p13.1 (3, 4) and the ERBB2 gene of the lower allele for D17S41 (LEW102) and NME1, respectively. on 17q (5, 6). We have previously shown a high frequency of LOH on Tumor 132 is informative at D17S86 (CMM86) while tumor 134 is chromosome 17q that was associated with estrogen receptor-negative not. Tumor DNA 303 lost the upper allele at EPB3 but retained both tumors (7). Recently, several groups have published evidence for a alleles of the NME1 and D17S41 (LEW102) loci. Tumor 307 is gene on 17q linked to familial breast and breast/ovarian cancers (8- deleted for the lower allele of NME1 but informative for D17S86 10). This gene, named BRCA1, maps to 17q21. An initial candidate (CMM86) and uninformative for D17S73 (LEW207). Based on these tumor suppressor gene in this region was NM23 or NME1 (11). We data and data summarized in Fig. 2, we conclude that a target gene(s) found the NME1 locus deleted in 64% of sporadic breast cancers (12). for LOH is located in the 22-cM region defined by D17S73 and NME1 However, no mutations were found in the coding sequence and thus (15, 16). It is pertinent that recent genetic linkage studies also indicate excluded it as the BRCA1 gene.4 Others (13) have also excluded that the familial breast and breast/ovarian cancer locus (BRCA1) is NME1 as a candidate for BRCA1. We have continued to further define located centromeric to NME1 on chromosome 17q21, raising the the regions of chromosome 17q which might contain tumor suppressor possibility that this locus is also affected in sporadic breast cancer genes relevant to breast cancer by screening tumor DNA samples for (13). LOH. Here we describe our results with 18 polymorphic loci on A second region of chromosome 17 that is affected by LOH is chromosome 17q. A high density deletion map has been constructed, centered around the D17S40 locus. Fig. 1B illustrates two examples of and the results are consistent with at least three independent regions interstitial deletions in this region (tumor DNAs 385 and 600). Both which are affected by LOH. tumors are informative at D17S86 (CMM86) and TK1. Tumor 385 is deleted for the lower alleles of both D17S40 (LEW101) and D17S21 Received 9/7/93; accepted 10/20/93. (c63), whereas tumor 600 is deleted for the lower allele at GH1 and The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with the upper allele of D17S40 (LEW101). Taken together, these deletions 18 U.S.C. Section 1734 solely to indicate this fact. point to the presence of a target gene within the 28-cM region lying 1 Supported by the Ligue Nationale de Lutte Contre le Cancer; the Comites Regionaux between D17S21 and D17S86 (Refs. 15 and 16; summarized in Fig. des Hauts de Seine, du Val d'Oise et des Yvelines; and Association pour la Recherche sur le Cancer. 2). 2 To whom requests for reprints should be addressed, at National Cancer Institute, In Fig. 1C are selections of interstitial deletions in the third or distal Building 10, Room 5B50, Bethesda, MD 20892. 3 The abbreviation used is: LOH, loss of heterozygosity. region of chromosome 17q. Tumors 70 and 106 each have LOH for 4 Unpublished results. the lower allele at D17S4 (THH59) but are informative and unaffected 5617

Table 1 Frequencies of LOH detected at 18 loci on chromosome I7q in sporadic breast carcinoma DNAs Probe name Locussymbol Enzyme Total no.tumors Allelic ]oss/informative case (%) VAW212 D17S117 PSTI 49 6/15 (40) LEW207 Dt 7S73 BGLtl, HINDIII 82 10/27 (37) PPY PPY MSPI 35 5/20 (25) EPB3 EPB3 PSTI 60 6/15 (40) NME1 NME1 BGLII, ECORI 140 25/55 (45) LEW102 D17S41 PSTI, TAQI 70 21/43 (49) CMM86 D17S86 MSPI, BGLII 93 9/44 (20) GH1 GH1 BGLII 79 13/59 (22) LEW101 D17S40 MSPI 38 10/23 (43) c63 D17S21 MSPI 38 7/20 (35) TK1 TK1 TAQI 23 1/21 (5) C1-26 D17S20 TAQI, GBLII, ECORI 92 5/72 (7) THH59 D17S4 TAQI, PSTI, ECORI 83 24/63 (38) p 128E1 D17S77 TAQ1 54 11/30 (37) TH17.26 D17S773 TAQI 20 2/9 (22) EFD52 D17S26 PSTI 12 0/9 (0) AC256 D17S79 PSTI 21 2/9 (22) RMU3 D17S24 TAQI, BAMHI, MSPI 69 27/59 (46)

B C 132 303 307 134 385 600 70 271 106 T L T L T L T L T L TL TL T L TL 7 ......

LEW207 ...... ~ el-26

.~.:! ... EPB3 G H 1 ~:-:;: Fig. 1. Examples of Southern blot analysis de- ol picting different regions of interstitial deletions.A, deletions of the proximal region affected by LOH. Tumor 132 is deleted for LEWI02; tumor 303 is i deleted for EPB3; and tumors 307 and 134 are deleted for NME1. B, deletions of the central region. Tumor 385 is deleted for LEWI01 and c63, LEW101 a. and tumor 600 is deleted for GH1 and LEWI01. C, NME1 deletions of the distal region. Tumors 70, 271, and : L 106 are deleted for THH59. i

uw1% i I: TK1 CMM86 11~ "1 I~ e!

at D17S20 (cl-26), D17S77 (p128E1), D17S79 (AC256), and tive. Of these 12 tumors, 11 had been tested for one or more markers D17S24 (RMU3). Tumor 271 lost the upper allele ofD17S4 (THH59) on chromosome 17p. All but 3 of these 11 tumors were also deleted for but retained heterozygosity at D17S24 (RMU3). These data and the the 17p probes, thus suggesting that if the long arm is deleted the data summarized in Fig. 2 suggest that a third potential target gene entire chromosome may be lost. could be located in the region between D17S20 and D17S77 which are Our findings of at least three independent regions on 17q affected estimated to be 11 cM a part (15, 16). by LOH in primary breast tumor DNA is consistent with other reports. Other more complex patterns of LOH were also observed in the Comparative genome fluorescent in situ hybridization shows a com- tumor DNAs such as tumors 540, 307, 385, and 448 in which more plex pattern of 17q deletions in breast tumors. 5 In addition, two groups than one region appears to be affected by LOH (Fig. 2). Moreover, it recently published evidence of separate regions on chromosome 17q is possible there is yet another target region of deletions more telo- affected by LOH in breast tumor DNAs. Cornelis et al. (17) examined meric located around RMU3 (tumor DNA 448; Fig. 2). However, most 11 loci on 17q and divided it into two regions with the border at the of the tumor DNAs deleted for D17S24 (RMU3) were also deleted for GH1 locus, which is in our central region of LOH. The difference D17S4 (THH59). Thus it is not clear whether some of these deletions between our results and theirs may be explained by their use of only are nonspecific. three markers distal to the growth hormone locus. Similarly, Saito et There were 12 tumors that may have lost the entire long arm of al. (18) have also shown two regions of LOH on 17q in breast cancers chromosome 17. An average of 5.5 (range, 3-10) 17q probes were tested on these 12 tumors, and all were either deleted or not informa- s E Waldman, personal communication. 5618

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,1., ,8, 8 8 : 8.~ .~ 8 |8 o COMMON ilol @ @ @ D D @ D D D D D D O REGION D D @ @ 0 D 0 IID D D @ D 22 D D D D 0 D 0 0 D D D D 0 26 O 0 O 0 0 D C) O 0 D 0 0 0 23 t59 D 0 D D D D 0 D D D D D D COMMON 8E1 D D D D D D C) C) O C) O D C) iREGION 24 7.26 D D D D D D 0 D 0 D D D D ,52 D D D D D D O D D D D D D 25 '-56 D D D D D D D D 0 D 0 D D J3 D D D D 0 D D 0 0 0 O D D 17q

D Deleted C) Informativeand retention of heterozygosity D Not informative or not done Fig. 2. Deletion map of representative tumors. The DNA probes are listed in the same chromosomal order as in Table 1 which is based on recent linkage analysis. Relevant tumors and their deletion patterns are shown which provide a summary of the three interstitial deletion regions. 9 retentionof heterozygosity (i.e., an informative tumor). using 11 markers. However, only two of the loci examined were distal 6. Slamon, D., Godolphin, W., Jones, L. A., et al. Studies of the HER2/neu protooncogene in human breast and ovarian cancer. Science (Washington DC), 244: 707-744, to D17S86 (CMM86), which is on the border between our proximal 1989. and central LOH-affected regions. In addition, most of the markers 7. Cropp, C., Lidereau, R., Campbell, G., Champeme, M-H., and Callahan, R. Loss of used in the latter study have not been mapped by linkage analysis, heterozygosity on chromosome 17 and 18 in breast carcinoma: two new regions identified. Proc. Natl. Acad. Sci. USA, 87: 7737-7741, 1990. making it difficult to compare results. 8. Bishop, D. T., and Easton, D. E Preface to the breast cancer linkage consortium The work described here expands our understanding of the genetic papers. Am. J. Hum. Genet., 52: 677, 1993. pathology of breast cancer by clearly defining the boundaries of three 9. Hall, J. M., Lee, M. K., Newman, B., Horrow, J. E., Anderson, L. A., Huey, B., and King, M. C. Linkage of early-onset familial breast cancer to chromosome 17q21. distinct regions on chromosome 17q. Because of the active research to Science (Washington DC), 250: 1684-1689, 1990. identify and clone the hereditary BRCA1 gene, it may soon be possible 10. Narod, S. A., Feunteun, J., Lynch, H. T., Watson, P., Conway, T., Lynch, J., and Lenoir, to determine whether or not it is the same target gene as in our G. M. Familial breast-ovarian cancer locus on chromosome 17q12-q23. Lancet, 338: 82-83, 1991. proximal region. However, the other two regions in the central and 11. Steeg, P. S., Bevilacqua, G., Kapper, L, Thorgersson, V. P., Talmadge, J. E., Liotta, distal parts of chromosome 17q are newly characterized. The identi- L. A., and Sobel, M. E. Evidence for a novel gene associated with low tumor fication of their boundaries to a 28- and ll-cM region, respectively, metastatic potential. J. Natl. Cancer Inst., 80: 200-204, 1988. 12. Leone, A., McBride, O. W., Westin, A., Wang, M., Anglard, P., Cropp, C. S., Linehan, enables the next step in their characterization. The availability of high M. W., Rees, R., Callahan, R., Harris, C., Liotta, L. A., and Steeg, P. S. Somatic allelic density polymorphic microsatellite loci on chromosome 17q should deletion of nm23 in human cancer. Cancer Res., 51: 2490-2493, 1991. help further localize and ultimately clone the putative target tumor 13. Easton, D. F., Bishop, D. T., Ford, D., Crockford, G. P., and Consortium, B. C. L. Genetic linkage analysis in familial breast and ovary cancer--results from 214 fami- suppressor genes. lies. Am. J. Hum. Genet., 52: 678--701, 1993. 14. Lidereau, R., Escot, C., Theillet, C., Champeme, M-H., Brunet, M., Gest, J., and References Callahan, R. High frequency of rare alleles of the human c-Ha-ras-1 protooncogene in breast cancer patients. J. Natl. Cancer Inst., 77: 697-701, 1986. 1. Callahan, R., Cropp, C. S., Merlo, G. R., Liscia, D. S., Cappa, A. P. M., and Lidereau, 15. O'Connell, P., Plaetke, R., Matsunami, N., Odelberg, S., Jorde, L., Chance, P., R. Somatic mutations and human breast cancer: a status report. Cancer (Phila.), 69: Leppert, M., Lalouel, J., and White, R. An extended genetic linkage map and an 1582-1588, 1992. "index" map for human chromosome 17. Genomics, 15: 38--47, 1993. 2. Knudson, A. G. Hereditary cancers: clue to mechanisms of carcinogenesis. Br. J. 16. NIH/CEPH Collaborative Mapping Group A comprehensive genetic linkage map of Cancer, 59: 661-666, 1989. the human genome. Science (Washington DC), 258: 67-86, 1992. 3. Hollstein, M., Sidransky, D., Vogelstein, B., and Harris, C. C. p53 mutations in human 17. Cornelis, R. S., Devilee, P., van Vliet, M., Kuipers-Dijkshoorn, N., Kersenmaeker, A., cancers. Science (Washington DC), 253: 49-53, 1991. Bardoel, A., Meera Khan, P., and Cornelisse, C. J. Allele loss patterns on chromosome 4. Osbourne, R. J., Merlo, G. R., Mitudomi, T., Venesio, T., Liscia, D. S., Cappa, A. P. 17q in 109 breast carcinomas indicate at least two distinct target regions. Oncogene, M., Chiba, I., Takahashi, T., Nau, M. M., Callahan, R., and Minna, J. D. Mutations in 8: 781-785, 1993. the p53 gene in primary human breast cancer. Cancer Res., 51: 6194--6198, 1991. 18. Saito, H., Inazawa, J., Saito, S., Kasumi, F., Koi, S., Sagae, S., Kudo, R., Saito, J., 5. Ali, I. U., Campbell, G., Lidereau, R., and Callahan, R. Lack of evidence for the Noda, K., and Nakamura, Y. Detailed deletion mapping of chromosome 17q in prognostic significance of c-erbB-2 amplification in human breast carcinoma. Onco- ovarian and breast cancers: 2-cM region on 17q21.3 often and commonly deleted in gene Res., 3: 139-146, 1988. tumors. Cancer Res., 53: 3382-3385, 1993.

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1993 American Association for Cancer Research. Identification of Three Regions on Chromosome 17q in Primary Human Breast Carcinomas Which Are Frequently Deleted

Craig S. Cropp, Marie-Helene Champeme, Rosette Lidereau, et al.

Cancer Res 1993;53:5617-5619.

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