[CANCER RESEARCH 60, 3884 –3892, July 15, 2000] Genome-wide Search for Loss of Heterozygosity Using Laser Capture Microdissected Tissue of Breast Carcinoma: An Implication for Mutator Phenotype and Breast Cancer Pathogenesis Chen-Yang Shen, 1 Jyh-Cherng Yu, Yen-Li Lo, Chia-Hui Kuo, Chung-Tai Yue, Yuh-Shan Jou, Chiun-Sheng Huang, Jia-Chyi Lung, and Cheng-Wen Wu Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan [C. Y. S., Y. L. L., C. H. K., J. C. L., C. W. W.]; Department of Surgery, Tri-Service General Hospital, Taipei 100, Taiwan [J. C. Y.]; Department of Pathology, Shin Kong Wu Ho-Su Memorial Hospital, Taipei 111, Taiwan [C. T. Y.]; National Health Research Institutes, Taipei 11529, Taiwan [Y. S. J., C. W. W.]; and Department of Surgery, National Taiwan University Hospital, Taipei 100, Taiwan [C. S. H.] ABSTRACT Breast cancer is considered to display a high degree of intratumor heterogeneity, without any obvious morphological and pathological steps to define sequential evolution, and its progression may vary among indi- vidual tumors. In an attempt to elucidate these etiological and phenotypic complexities, the present study, based on the fundamental concept that genomic instability is the engine of both tumor progression and tumor heterogeneity, was conducted to test the hypothesis that breast cancer pathogenesis is driven by double-strand break (DSB)-initiated chromo- some instability (CIN). The rationale underlying this hypothesis is derived from the clues provided by family breast cancer syndromes, in which susceptibility genes, including p53, ATM, BRCA1 and BRCA2, are in- volved within the common functional pathway of DSB-related checkpoint/ repair. Because genomic deletion caused by DSB is reflected in the genetic mechanism of loss of heterozygosity (LOH), this genome-wide LOH study was conducted, using 100 tumors and 400 microsatellite markers. To minimize the effect of heterogeneity within tumors, the experimental technique of laser capture microdissection was used to ensure that genetic and phenotypic examinations were based on the same tumor cells. Support for our hypothesis comes from the observations that: (a) the extent of DSB-initiated CIN in tumors significantly increased as tumors progressed to poorer grades or later stages; (b) in the sequential steps toward CIN, the loci of p53 and ATM, the key checkpoint genes against DSB, were lost at the earliest stage; and (c) many loci identified to be important in breast tumorigenesis were the genomic sites possibly harboring the genes in- volved in DSB-related checkpoint/repair (including RAD51, RAD52, and BRCA1) or CIN (including FA-A, FA-D, and WRN), and a higher number of these loci showing LOH was significantly associated with increased level of DSB-initiated CIN (P < 0.0001). Breast cancers are thus considered to be sequentially progressive with CIN. However, CIN might also cause genetic heterogeneity, which was revealed by the findings that LOH at some markers was observed only in the component of ductal carcinoma in situ but not in the invasive component of the same tumors. In addition, some markers were found to preferentially lose at specific tumor grades, implying their contribution to genetic heterogeneity during tumor devel- opment. Therefore, this study suggests that breast cancer progression is clonal with regard to CIN, but different breast cancers would present distinct molecular profiles resulting from genetic heterogeneity caused by CIN. INTRODUCTION Breast carcinoma is one of the most common cancers in women and is known to arise from a multifactorial process, the effect of repro- ductive risk factors strongly supporting a hormonal role in its etiology (reviewed in Ref. 1). Early menopause, for example, whether occur- ring naturally or as a result of oophorectomy, has been shown to significantly reduce the risk. However, these reproductive risk factors have been shown to only moderately increase the risk of developing breast cancer. More frustrating from a cancer prevention viewpoint is that two-thirds of women who develop this disease fit none of the currently identified at-risk groups (2). This has prompted scientists to search for clues at the molecular level that may help in understanding breast tumor pathogenesis. The greatest insights in our understanding of molecular tumorigen- esis have been obtained from the study of family cancer syndromes (3–5). In the case of breast cancer, an inherited component has been suspected for many years because of reports of families with large numbers of affected individuals. In the Li-Fraumeni syndrome, there is a high incidence of premenopausal breast cancer. As a result of the identification of the germline mutations in 50% of pedigrees, these syndromes were linked to the p53 tumor suppressor gene (6). The documentation of 1000 mutations of the p53, accounting for about 25–30% of all breast cancers tested, has clearly established its role in breast cancer development (7). The inheritance of breast cancer sus- ceptibility in families has also led to the localization of breast cancer susceptibility genes, including BRCA1 localized to chromosome arm 17q and BRCA2 to chromosome arm 13q (reviewed in Ref. 8). Furthermore, the mapping of homozygous deletions on human chro- mosome 10q23 has resulted in the isolation of a candidate TSG, 2 PTEN/MMAC1, that appears to show a considerable frequency of mutation in breast cancer and to be responsible for Cowden disease, an inherited breast and thyroid cancer syndrome (reviewed in Ref. 9). In addition to these highly penetrant genes, other genes, in which mutations might result in a more moderate increase in breast cancer risk, have been predicted; a well-known example is the ataxia telan- giectasis gene. It has been suggested that heterozygote carriers of defective forms of the gene predisposing to ataxia telangiectasia are at higher risk of developing breast cancer (reviewed in Refs. 10 and 11). The recent isolation of this gene, designated ATM, supports this hypothesis. To seek clues to the initiation of breast cancer development, we explored the suggested function of these genes linked to inherited breast cancer syndromes. It is intriguing to find that, except PTEN/ MMAC1, the products of the other four genes (p53, ATM, BRCA1, and BRCA2) are all directly involved in a common molecular pathway related to cellular responses against DSBs arisen in DNA (reviewed in Refs. 12 and 13). ATM is a component of the cell cycle checkpoint machinery that senses DNA damage and is activated after the forma- tion of DSBs induced by ionizing radiation or other DSB-inducing agents (reviewed in Ref. 11). p53, acting downstream of ATM, is then phosphorylated and consequently triggered to initiate a protective response either by blocking the cell cycle for DNA repair or by Received 12/17/99; accepted 5/16/00. 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 To whom requests for reprints should be addressed, at Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan. Fax: 886-2-2782-3047; E-mail: bmcys@ccvax.sinica.edu.tw. 2 The abbreviations used are: TSG, tumor suppressor gene; DSB, double-strand break; CIN, chromosome instability; LOH, loss of heterozygosity; LCM, laser capture micro- dissection; DCIS, ductal carcinoma in situ; IDC, invasive ductal carcinoma; CGH, comparative genomic hybridization; MIN, microsatellite instability; NIN, nucleotide instability; PLOH, the proportion of loci showing LOH in a tumor. 3884 Research. on December 31, 2015. © 2000 American Association for Cancer cancerres.aacrjournals.org Downloaded from