Allele Loss on Chromosome 16q24.2-qter Occurs Frequently in Breast Cancers Irrespectively of Differences in Phenotype and Extent of Spread

Loss of heterozygosity on chromosomal arm 16q has been shown to be a frequent event in sporadic breast cancer and is suggested to be involved in cancer development through inactivation of a tumor-suppressor gene. To specify the commonly deleted region in which the unknown tumorsuppressor gene is located, a deletion map of chromosome 16 was constructed for 78 breast cancers, using 27 polymorphic DNA markers. Loss of heterozygosity on chromosome 16q was detected in 38 of the tumors. From the deletion map, the incidence of the loss of heterozygosity was deduced to be ->36% in the region distal to 16q12 and was most frequent in the 16q24.2-qter region. Then, association of the loss of heterozygosity in the 16q24.2-qter region with clinicopathological parameters of the tumors was examined for a total of 234 tumors, to reveal its biological significance in breast cancer development. The total incidence of loss of heterozygosity in the 16q24.2-qter region was 52% (118 of 225), and loss of heterozygosity was frequent irrespectively of the presence of invasion and metastasis, differences in clinical stage, tumor size, histological grade, or type, or amounts of estrogen receptor. Inactivation of an unknown tumor-suppressor gene on 16q24.2-qter was thus suggested to be involved commonly in the genesis of sporadic breast cancer, irrespectively of the extent of tumor spread or grade of aggressiveness of the cancer cells. On the other hand, eight cases revealed loss of heterozygosity not at 16q24qter but in more proximal regions. Therefore, it appears that multiple tumor-suppressor genes are located on chromsome 16q. also carries mutations in sporadic breast cancer (10); another gene that causes familial breast cancer is still suggested to exist around 17q21 (11). Frequent LOH of chromosome 16q is reported not only in breast cancer (4, 5) but also in hepatocellular carcinoma (12) and prostatic cancer (13). We previously specified in hepatocellular carcinoma a commonly deleted region, where an unknown tumor-suppressor gene is considered to be located, on chromosome 16 between the HP locus on q22.1 and the CTRB locus on q23.2 (12). However, >60% of cases with the LOH were suggested to be due to monosomy of the entire chromosome 16. Sato et al. (5) also identified in breast cancer a commonly deleted region located between the HP and D16S157 loci on 16q24, using five polymorphic DNA markers. To further specify this region, a deletion map of a larger number of cases seemed to be necessary, employing a larger number of polymorphic DNA markers. Recently, physical and genetic chromosomal maps have been considerably refined using a larger number of novel polymorphic DNA markers (14-21). In the present study, we examined the incidence of LOH and tried to localize more accurately the region commonly deleted on chromosome 16, using polymorphic DNA markers at 27 different loci. Furthermore, association of the LOH with clinical and histological parameters was examined, to reveal its biological role in breast cancer development. I N T R O D U C T I O N In human breast cancer, LOH 3 on chromosomal arms has been detected frequently by RFLP analysis. LOH in a specific chromosomal region is generally considered to be involved in the genesis and progression of cancer, through inactivation of tumor-suppressor genes located there. Inactivation of tumor-suppressor genes occurs in a recessive manner; usually, one allele containing the gene is deleted, and the other allele contains point mutations or small deletions. Frequent LOH is reported for chromosomal arms lp, lq, 3p, 7q, l l p , 13q, 16q, 17p, 17q, and 18q in breast cancer (1-7). A gene associated with susceptibility to breast cancer is suggested to be located on chromosome 17q21 on the basis of linkage analysis of breast cancer families (8). Although it is impossible to apply such linkage to sporadic cases, several tumor-suppressor genes located on a commonly deleted chromosomal region in sporadic cancer have already been identified, e.g., p53 and prohibitin (9-10). For example, the p53 tumor-suppressor gene located on chromosome 17p13.1, which is a commonly deleted region in diverse types of human cancers, has also been shown to carry mutations frequently (9). The prohibitin gene mapped on 17q12-q21 Received 7/9/93; accepted 11/12/93. 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 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported in part by grants-in-aid from the Ministry of Education, Science, and Culture and from the Ministry of Health and Welfare of Japan and by a grant-in-aid from the Ministry of Health and Welfare of Japan for Comprehensive 10-Year Strategy for Cancer Control. 2 To whom requests for reprints should be addressed, at National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104, Japan. 3 The abbreviations used are: LOH, loss of heterozygosity; RFLP, restriction fragment length polymorphism. M A T E R I A L S AND M E T H O D S DNA Probes and Their Chromosomal Locations. The polymorphic DNA markers used, all localized on chromosome 16, are shown in Table 1. To specify the most frequently deleted region, the loci of these markers were assigned by referring to both genetic and physical maps already published by scientists involved in chromosome 16 mapping (14-21). The order of DNA markers on chromosome 16 according to the genetic map is as follows: pterHBA1 (JWlO1)-D16S83 (pEKMDA2.1)-D16S131 (VK45C6)-MT2-D16S151 (CJ52.209M1)-[D16S4 (ACH207), D16S38 (CRI-O2)]-HP-CTRB-D16S20 (r 8-9) -D16S157(CJ52.96)D16S43(CRI-O84)-[D16S7 (p79-2-23), APRT]-D16S44 (CRI-089)-qter (14-18). By the genetic map, the D16S155 (CJ52. 199) locus is determined to be between D16S43 and D16S7. 4 According to the physical map, the order is as follows: pter-(HBA1, D16S83)-D16S34 (16/55B)D16S32(16/118)-D16S131-cenD16S27(p16-5)-(MT2, CETP)-D16S151-(D16S4, D16S38)-D16S152 (CJ52.1)-(HP, TAT)-CTRB-D16S20(D16S157, D16S43)-(D16S7, APRT)-D16S44-qter (19). 4 D16S159 (CJ52. 94) and D16S23 (p16-1) are localized in the same physical interval in 16p12.2, and D16S137 (KKA22) maps proximal to D16S159 on the genetic map. 4 Westphal et al. (22) strongly suggested that the position of the TAT locus is proximal to CTRB but distal to liP from 16cen. When these data are combined, the most probable order of the DNA markers is as follows: pterHBAI-D16S83-D16S34-D16S32-D16S131-(D16S159, D16S23)-(D16S137, cen)-D16S27-(MT2, CETP)-D16S151-(D16S4, D16S38)-D16S152-HP-TATC TRB-D16S20-D16S15 7-( D16S4 3 , D16S15 5 )-( D16S T, APR T)-D16S44-qter. TheD16S35 (16/32) and D16S30 (p16--8) loci are physically mapped to the region pter-pl3 containing D16S32 and D16S131 and to the region q22.1-q24, respectively (14). Additional information on these two loci was not available. Tissue Samples, DNA Isolation, and Southern Blot Analysis. We collected 234 paired samples of breast cancer and noncancerous tissue from 225 patients with primary disease. In nine patients, bilateral primary lesions were 4 D. E Callen, unpublished observations.