Breast Cancer Cytogenetics: Clues to Genetic Complexity
of the Disease
Marilyn L. Slovak, Ph.D.: and Sandra R. Wolman, M.D.t Department of Cytogenetics, City of Hope National Medical Center, Duarte, California,
Oncor, Inc., Gaithersburg, Maryland
enetic aberrations in cancer have become a pri- G mary focus for understanding the pathogenesis of neoplasia. In this respect, cancer cytogenetic observa- tions have been pivotal to studies of specific genetic alterations that contribute to tumor development and progression in the hematopoietic disorders and small round-cell tumors of childhood. Today, cytogenetic in- vestigations are yielding insights into epithelial tumor initiation and progression as well. In fact, 6% of the solid tumor cytogenetic studies listed in the 1994 Cata- log of Chromosome Aberrations iii Cancer (l), the nia- jor data bank for neoplastic cytogenetic abnormalities, are derived from breast tumors, a figure very different from the fewer than 70 tumors in the 1988 edition.
The cytogenetic studies of breast cancer published be- fore 1985 often examined tumor cells at advanced stages of disease or were based on data gleaned from cell lines established from pleural effusions. These earlier studies provided little information regarding primary (initiation) or secondary (tumor progression) alter- ations; however, they did recognize that breast cancer is a complex, polygenic disease (2) that will require merg- ing of information from many disciplines to permit a broad overview of the tumor, that is, incorporating data on the genetic make-up of individual tumor cells, spe- cific gene alterations, clonal evolution of disease, intra-
Address correspondence to: Marilyn L. Slovak, Dept. of Cytogenetics, City of Hope National Medical Center, 1500 E. Duarte Road, Duarte, CA 91010-3000, U.S.A.
d 1996 Blackwell Science Inc., ~ 0 7 5 - 1 2 2 ~ / 9 ~ / $ 1 0 . 5 0 / ~ The Breast Journal, Volume 2, Number 1, 1996 27-30
tumor heterogeneity, and intertumor heterogeneity. The early breast cancer cytogenetic studies essentially out- lined the need and, thus, laid the foundation for a sys- tematic, multi-parameter approach to our current stud- ies of the disease.
Because the etiology of breast cancer is complicated by disease heterogeneity accompanied by numerous ge- netic changes, the most productive route to detect genes that are causal of or contributory to cancer is the recog- nition of frequent and specific chromosome aberrations associated with particular tumors or tumor-prone indi- viduals. Although most breast cancer cases are sporadic, familial clustering is observed in -20% of breast tumors and at least 5-10% of cases appear to be due to the in- heritance of an autosomal dominant gene (3-5). The ex- act number and distribution of predisposing genes is currently unknown. The underlying etiology of breast cancer is poorly understood, and only recently have sev- eral genetic-based mechanisms emerged (6, 7). This lack of essential genetic information is a major limitation to the development of clinical applications in breast cancer.
Five genetic approaches (cytogenetics, fluorescence in situ hybridization [FISH], comparative genomic hybrid- ization [CGH], flow cytometry, and molecular genetic studies) will be discussed. Cytogenetic studies and DNA content are the methods of choice to determine overall genetic changes, whereas individual gene mutations, de- letions, and amplifications are best investigated by mo- lecular strategies.
Recurring karyotypic aberrations are found in pri- mary breast cancers utilizing either direct or short-term cultures; these chromosomal regions may house genes
28 SLOVAK A N D WOLMAN
critical to the basic pathobiology of the disease. For the sake of brevity and clarity, this review will focus on five recent studies that include a total of 231 primary breast tumors (8-1 7). Chromosomal banding studies of these primary breast cancers indicate the most common nu- merical aberrations include gains of chromosomes 7, 8, 18, and 20 and losses of X, 8, 9, 13, 14, 17, and 22. Structural aberrations have defined nonrandom gains of lq, 3q, 6p and 8q and losses of lp , 4p, 6q, 8p, 9p, l l p , l l q , 15p, 16q, 17p, 19p, and 19q. The heterogeneity of karyotypic findings in breast cancer may reflect the many different mechanisms or routes associated with breast cancer tumorigenesis. Taken together, these data correspond with intratumor heterogeneity, variable phe- notypes, and perhaps polyclonality in breast cancer. Po- sitional cloning studies are needed to identify the candi- date tumor suppressor and oncogene genes in these specific chromosomal regions. Correlations of these crit- ical genes or regions of interest to the clinico-pathologi- cal features should define genetically based prognostic subgroups.
In general, cytogenetic alterations are expected to support loss of heterozygosity (LOH) reported for many chromosomal arms ( lp , lq, Sp, 6q, 7q, Sp, 9q, l l p , 13q, 14q, 15q, 16q, 17p, 17q, 18q, 22q, and Xp) (for review of allelotype studies see 6,7) . Correspondence of cytogenetic and molecular data for chromosomes 1, 3, 6 ,7 , 8, 11, 16, and 17 will be emphasized. Briefly, early genetic alterations appear to involve losses on 3p, 7q31, l lq13 , 16q22-24, 17~13.1, or gain of lq . Clonal evo- lution of breast cancer shows preferential association with lp36, 6q24-27 or 11q22-23 aberrations. A poor prognosis has been reported for mutations of TP.53, loss within bands 7q31 and l l q l 3 or amplification of 11q13. Breast cancer susceptibility loci have been mapped to 13q12-13, 17q21, 17~13.1, and perhaps 11~15.5 and 1 lq23. Of course, the potential interactions of environ- mental influences of this multifactorial (polygenic) dis- ease remain to be determined. Rearrangements of 1 ~ 3 6 , 17~13.1 , and perhaps other alterations of DNA repair genes appear to have an effect on intra- and intertumor heterogeneity resulting from genetic instability. These data are the infrastructure for a genetic dissection strat- egy which combines screening genetic alterations in many tumors to determine the smallest chromosomal re- gion of overlap (aka smallest common deleted region) with molecular assays in precursor or in situ lesions to determine alterations that are critical in the early stages of breast tumorigenesis.
Among the genetic alterations described in primary
human breast carcinomas, gene amplification has re- ceived much attention, partly because of associations with poor prognosis in other tumors. Cytogenetic stud- ies on primary (untreated) breast cancers have indicated the presence of double minutes (dmins) (17, 18) or ho- mogeneously staining or abnormally banding regions (HSRs or ABRs) (13,19,20) in 4-60% of breast tumors studied. Although HSRs have been localized to many chromosome arms (6p, 8p, 9p, l l q , 15p, 16p, 17q, 19p, and 20q) (8, 16, 17, 19-23), there was little agreement on frequency, type of aberration (HSR vs. dmin), or lo- calization of amplification sites among the studies re- viewed.
Amplification of HER-Zlneu (ERBB2) is found in 15-60% of investigated breast tumors. In general, ERBB2 amplification correlates with high grade, nega- tive ER and PR status, and is an independent predictor of shorter disease-free survival in both node-negative and node-positive patients (24-27). ERBB2 alterations are present at all clinical stages with equivalent expres- sion in the non-invasive and invasive components of breast tumors. Such data are consistent with role of ERBBZ in the initiation of early progression of a subset of breast tumors (28).
An increased risk of breast cancer development has been associated with benign proliferative breast disor- ders (PBD), including diffuse epithelial hyperplasia with or without atypia, papillomas, and fibroadenomas (29-33). A recent study by Dietrich et a1 (34) described clonal cy- togenetic abnormalities in 16/30 cases of PBD. The re- current aberrations included del(l)(q12), de1(3)(p12- 14), r(9)(p24q34), and alterations of chromosomes 1 and 12, especially regions 12pll-13 and 12q13-15. Of interest, the cyclin D2 and CDK-4 genes map to 1 2 ~ 1 3 and 12q13, respectively, and may play a role in aberrant cell proliferation in PBD. Additional genetic alterations appear to be necessary for malignant transformation.
Lynch et a1 (35) were the first to recognize a domi- nant pattern of inheritance in breast cancer in a small subgroup ( - 4 4 % ) suggesting the existence of one or more breast cancer susceptibility genes. Recently, two breast cancer susceptibility genes have been localized, BRCA-2 to 17q21 and BRCA-2 to 13q12-13 (36, 37). Mutations of these genes confer a high risk of early on- set breast cancer. BRCA-1 confers an increased risk of ovarian cancer that is not associated with BRCA-2 (38). BRCA-2 mutations have been associated with male breast cancer, whereas no male breast cancers have been observed in BRCA-1 families (37). These genes confer risk, not 100% expression in a carrier, and loss of func-
Breust Cuncer Cytogenetics 29
tion of the BRCA-2 gene may be modulated by other risk factors and exposures. Finally, inherited (germline) mutations of TP53 in Li-Fraumeni syndrome, a familial autosomal dominant disorder, result in various types of cancer, predominately soft tissue sarcomas, brain tu- mors, and breast cancers (39).
Genetic information is being incorporated into thera- peutic strategies. In order to define genetic prognostic indicators in clinical breast cancer management, studies have been proposed to treat patients with multiple ge- netic ( 2 3 ) alterations as high risk for purposes of post- operative management and long-term outcome (regard- less whether lymph node status is negative or tumor is T1 stage) (40). Hormonal therapy appears advanta- geous in p53 negativelbcl-2 positive breast tumors (41). ERBB2 antisense oligonucleotides have been shown to inhibit the proliferation of ERBB2 amplified breast tu- mor cell lines, indicating potential use for new class of pharmacological agents in those breast tumors (-25%) overexpressing ERBB2 (42). Other gene therapy ap- proaches may exploit TSGs since it is conceivable that introduction of a functional tumor suppressor gene into a tumor cell may retard its growth.
Ten years ago, we understood little about the role of genetics in breast cancer. Today, the data gleaned collec- tively from cytogenetics, flow cytometry, FISH, compar- ative genomic hybridization, and molecular genetic test- ing approaches have identified multiple, recurring genetic alterations in breast tumors. However, no single common genetic alteration appears to be unique or com- mon to all breast cancers. Although ordering or deter- mining the number of genetic alterations in development and progression of specific solid tumors is desirable, many of the changes described in this review occur in 6- 30% of breast cancers. Thus, genetic events in breast cancer appear to involve a direrse range of genes in dif- ferent subsets of tumors.
We still lack information on genetic alterations in precursor lesions such as atypical hyperplasia or PBD and the later stages of ductal carcinoma in situ and lobu- lar carcinoma in situ. Associations between inherited predisposition and the increasing risk of breast cancer are still poorly understood but surely will provide criti- cal clues for the application of genetics to diagnosis and prevention of disease. Taken togther, the genetic clues summarized in this review underscore the need to create molecular diagnostic tools for early detection and de- velop ways to reverse the first steps of tumorigenesis (perhaps by gene therapy approaches) as an effective strategy of overcoming breast cancer.
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