Breast Cancer Cytogenetics: Clues to Genetic Complexity of the
Marilyn L. Slovak, Ph.D.* and Sandra R. Wolrnant Department of Cytogenetics, City of Hope National Medical Center,
Duarte, California and tOncor, 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 alter- ations that contribute to tumor development and progres-
sion in neoplasia. Cytogenetic analysis has spearheaded the localization, identification, and cloning of genes crit- ical to the development of hematopoietic 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 Cutu- log of Chromosome Aberrations in Cancer (1) (the ma- 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.
Characteristics inherent in breast cancers (e.g., heter- ogeneity, slow growth) have delayed identification of breast-cancer-specific cytogenetic associations. Other features that have hampered recognition of common cy- togenetic alterations include tumor necrosis, low mitotic activity, the outgrowth of stromal elements, relatively few good quality metaphases, and highiy complex rear- rangements in tumor cells. The published cytogenetic
Address correspondence and reprint requests to: Marilyn L. Slovak, Ph.D., Department of Cytogenetics, City of Hope National Medical Center, Northwest Building, Room 2255, 1500 E. Duarte Road, Duarte, CA 91010- 3000, U.S.A.
0 1996 Blackwell Science Inc., 107S-l22Xl96/$10.SOlO The Breast Journal, Volume 2, Number 2, 1996 124-140
studies of breast cancer before 1985 often examined tu- mor cells at advanced stages of disease or were based on data gleaned from cell lines established from pleural ef- fusions. Few clinicopathological correlations were avail- able. These studies described numerous and complex karyotypic aberrations defining multiple related clones and provided little evidence pointing toward the possi- ble primary (initiation) or secondary (progression) ge- netic events in breast cancer pathogenesis. The extraor- dinary diversity of chromosomal aberrations with high intra- and intertumor variability made interpretation of their clinical and biological significance in breast tumors difficult.
Additionally, diploid primary breast tumors that re- tained their invasive capacity after a week in culture have been reported, suggesting that at least some pri- mary breast tumors are characterized by apparently dip- loid karyotypes. Differences in the results of breast can- cer cytogenetic analyses (i.e., cytogenetically normal diploid tumors versus aneuploid tumors) were often at- tributed to problems of methodology. Caution had to be exercised in interpretation of chromosome changes de- rived from tumor tissue maintained in vitro. Cytogenetic studies of benign tumors and early breast cancers, which are needed to establish the primary karyotypic events re- lated to the disease, were essentially nonexistent prior to 1985 because of procedural limitations. Not only the technical limitations, but also difficulties in interpreta- tion of results, and the uncertainty of the role of the chromosome/genetic alterations in breast disease were major considerations. Technical improvements account
Breast Cancer Cytogenetics 125
for many of the recent data that have increased our un- derstanding of the genetics of breast cancer.
Even though the pre-1985 cytogenetic studies were of limited value, they raised some very important biologic questions: (a) Do the multiple clones observed in vitro also occur in vivo? (b) What are the short-term versus long-term effects of cell culture on the results of chro- mosome studies? (c) Will methodological advances re- flect the true genetic changes in breast cancer? (d) Do cy- togenetically normal diploid breast tumors exist? and (e) Do the nonrandom break points observed in advanced breast cancers localize to sites in the genome that iden- tify genes relevant to mechanisms of origin, progression, and clinical behavior of breast tumors? These questions acknowledge that breast cancer is a complex, polygenic disease (2) that will require merging of information from many disciplines to permit a broad overview of the tumor, incorporating data on the genetic makeup of in- dividual tumor cells, specific gene alterations, clonal evolution of disease, intratumor heterogeneity, and in- tertumor heterogeneity. Thus, the early breast cancer cy- togenetic studies essentially outlined the need and, thus, laid the foundation for a systematic, multiparameter ap- proach to our current studies of the disease.
At the cellular level, classic cytogenetic studies and DNA content by flow cytometry or image analysis are
Table 1. Comparisons of Genetic Techniques
the methods of choice to determine overall genetic changes, whereas individual gene mutations, deletions, and amplifications are best investigated by molecular strategies (Table 1). Each aspect of genetic analysis has its advantages and limitations. The chief advantage of classic cytogenetic analysis is that it is currently the only genetic method that provides an overview of the com- plexity of the genetic changes in individual tumor cells, and best illustrates intratumor heterogeneity and clonal evolution. Although cytogenetic alterations do not ex- plain what is happening at the genetic level, they focus attention on areas where critical genes may be found and thus lead to the development of molecular genetic assays.
Molecular testing is not the procedure of choice to describe events in individual tumor cells. Although mo- lecular tests are highly specific, the information gleaned is limited to the selected genetic aberration being tested and reveals only the composition of an idealized average tumor cell. After a recurring karyotypic aberration is de- fined within a tumor, restriction fragment length poly- morphism (RFLPs) analysis using a set of polymorphic markers for that targeted chromosomal region is per- formed. Tumor DNA is compared to the constitutional genotype and an allelotype describing the loss of het- erozygosity (LOH) is generated. This approach, how-
Techniaues Strenaths Weaknesses Resolution
Molecular Defines selected genetic aberrations in cell population
Sensitive/specific Evaluation of minimal residual disease for
individual gene mutations, deletions, and amplification
~l~~~~~~~~~~ in situ Defines complex rearrangements ID abnormalities in interphase Associate genetic alteration with morphology/or
May use archived tissues
Detects DNA amplified sequences within tumor
No need for specific probes No need of prior knowledge of aberrations
Comparative genomic hybridization (CGH) genome
Cytogenetics Overview of genetic changes in individual cells ID intratumor heterogeneity ID clonality Defines target regions
Estimate DNA content and proliferative (5- phase)
Aneuploid detection in paraffin-embedded, fresh,
Flow Cytometry fraction
frozen, and formalin-fixed
Limited to specific probe (gene) alterations 0.2-50 Kb No evaluation of intratumor cell heterogeneity or
May need polymorphic (informative) markers Tumor clone 325% Need consitutional DNA
Limited probe availability >2.5 Kb
?>2 Mb Little data regarding intratumor heterogeneity Requires >5 t o 7-fold amplification for detection Compromised by normal cell contamination No data regarding point mutations, transcriptional
activation, or chromosomal translocation
Limited genic level data 2-20 Mb Requires mitotic cells Selection due to in vitro culturing
No specific genetic alterations defined Low sensitivity Loss or gain of small chromosomes not detectable False aneuploidy due t o fixation, stain variations, or
Chromosome number 2 2
126 S L O V A K AND WOLMAN
ever, may be hampered by stromal cells and infiltrating lymphocytes thus obscuring the extent of allele loss. The most sensitive category of testing is that based on the polymerase chain reaction (PCR), which repeatedly am- plifies short specific segments of DNA so that even a rare molecule can be detected and analyzed. Even though it is possible to isolate single cells for PCR test- ing, this procedure is generally not applicable to the evaluation of intratumor cell heterogeneity or clonal evolution. PCR and in situ hybridization (ISH) proce- dures are, however, the most sensitive tests for questions concerning residual disease as identified by specific ge- netic aberrations.
Fluorescence in situ hybridization (FISH) studies and comparative genomic hybridization (CGH) are the re- sult of a marriage between molecular and cytogenetic in- vestigations. FISH using single short probes for repeti- tive DNA, multiple probes for whole chromosomes, or cosmid probes for regional localization, allows for the detection of chromosome aberrations in interphase nu- clei as well as metaphase cells. Denaturation of DNA to a single-stranded configuration, followed by incubation with specific labeled DNA (hybridization) will result in appearance of label at the normal chromosomal or in- tranuclear location where that particular DNA resides. FISH probes in metaphase preparations are useful for resolution of components of rearranged chromosomes and for detection of microdeletions. In interphase, the repetitive and cosmid probes are helpful in detection of abnormal chromosome copy number (aneusomy) and of clonal aberrations within a tumor population. Cosmid probes for specific oncogenes or tumor suppressor genes, such as ERBB2, N or CMYC, or TP53, can be used to detect gene amplification or deletion. Most im- portant, these probes are applicable to interphase tumor cells without loss of details of cellular and tissue mor- phology, so that one can associate the genetic alteration with specific cell types or areas within a tumor. FISH has been used successfully to determine the genetic alter- ations in archived paraffin-embedded tumor and freshly prepared touch preparations or fine-needle aspirations, thus allowing for confirmation of the breast tumor cyto- genetic results after in vitro cell culture (3-5). In this in- stance, FISH analysis holds promise for identifying ge- netically defined subgroups that may predict tumor behavior.
Comparative (or competitive) genome hybridization (CGH) is a new technique for the detection and localiza- tion of DNA sequence copy number variation within the entire tumor genome. It is based on simultaneous in situ
hybridization of differentially labeled tumor- and nor- mal reference-DNA to normal metaphase chromo- somes. The labeled DNAs are detected using two differ- ent fluorochromes; the relative DNA sequence copy numbers of all regions in the tumor genome can then be quantitated by measuring the intensity ratios of the two fluorochromes along each human chromosome. The ad- vantage of CGH is that it provides an overview of copy- number alterations occurring in breast tumors without the use of specific probes or prior knowledge of aberra- tions. However, this technique provides little informa- tion regarding individual tumor cells and requires am- plification levels of >5- to 7-fold for detection. The sensitivity of CGH is compromised by normal cell con- tamination and intratumor heterogeneity. Furthermore, amplification is only one of the mechanisms by which gene expression may be elevated in tumor cells. Activa- tion of genetic alterations by point mutation, transcrip- tional activation, or chromosomal translocation would not be detected by this method.
Today, combined approaches using several of the above have begun to define specific genetic alterations associated with individual risk assessment and risk fac- tors in breast cancer. This review is by no means inclu- sive of the vast breast cancer literature, but attempts to describe the recent, major contributions of cytogenetics and molecular cytogenetics as they relate to the clinico- pathological features of breast cancer. We will outline the specific contributions and limitations of genetic test- ing in breast cancer with suggestions for a practical clin- ical-based understanding of these genetic changes. Fi- nally, we will suggest some ways in which these data may identify new avenues toward breast cancer treat- ment and prevention.
DIPLOID TUMORS IN BREAST CANCER
Diploid or cytogenetically normal primary breast tu- mors were reported by several investigators (6-8). Their findings were supported by monoclonal antibody test- ing for cytokeratins, invasion assays, growth in agar, and morphology (9). However, others believe they have failed to capture the tumor cell population (10,l l) . We must also recognize that a normal karyotype does not exclude the possibility of relatively small changes in DNA content that might not be visible by standard cyto- genetics at the 400-550 band levels of resolution. Con- versely, based on a survey of chromosome studies after direct preparation of malignant solid tumors, Atkin and Baker (12) estimated this frequency to be less than 1%. Bullerdiek et a1 (13) found a high proportion of diploid
Breast Cancer Cytogenetics 127
tumors (11/16), but attributed this result to culture con- ditions that favored fibroblast growth. Pandis et a1 (14) found normal karyotypes in 4/20 primary breast cancers and interpreted these findings either as subvisible ge- netic changes present in the tumors or as nondividing tu- mor cells in the presence of dividing normal epithelial cells. Slovak et a1 (15) observed both normal and ab- normal karyotypes more commonly in breast tumors with numerous infiltrating lymphocytes. Multiparame- ter pathogenetic testing, such as combined FISH and im- munohistochemistry, is needed to resolve the clinical significance of diploid karyotypes in breast tumors.
CYTOGENETIC CHANGES IN PRIMARY BREAST DISEASE
Recurring karyotypic aberrations are found in pri- mary breast cancers utilizing either direct or short-term cultures; these chromosomal regions may house genes 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. Their data confirm earlier reports as well as more recent anecdotal information. The cases cover a broad range in numbers, case selection, and focus of in- terpretation. One study evaluated 30 near-diploid or paradiploid cases (16), with the presumption that near- diploid cases were more likely to reveal primary chro- mosomal events. However, these paradiploid tumors were studied relatively late in their evolution, and the re- sults could equally well reflect tumor progression rather than initiation. In contrast, the study of Hainsworth et a1 (17) reported clonal cytogenetic anomalies in 24/26 (92%) breast tumors with many tumors in the triploid to tetraploid range. The third study described clonal cy- togenetic aberrations in 28 human breast cancers almost all derived from ductal carcinomas in situ (DCIS), pre- sumably an earlier stage of disease (11). A larger study that focused on methodological modifications for short- term culturing of breast tumors (14, 18-23) described clonal abnormalities in 100/122 breast tumors and nor- mal karyotypes in the remaining 22 carcinomas. Finally, data from 40 primary breast cancers analyzed in short- term culture studies ongoing in one of our laboratories ( 1.9, include 10 near-diploid, 7 near-triploid, 1 tetra- ploid, and 10 bimodal cytogenetically aberrant cases.
NUMERICAL AND STRUCTURAL ABNORMALITIES IN BREAST CANCER
Chromosome 1 was the most frequently altered chro- mosome in the newer data, as in all previously reported
studies (1). Despite intercellular variability and unbal- anced rearrangements resulting in losses, gains of l q and 8q were the most frequent findings. The most recur- rent translocation, gain of l q and loss of 16q [i.e., der(1; 16) (qlO;plO)], was described frequently as a sole kary- otypic anomaly (1 1,16,20,22,23). Isochromosomes re- sulted in overrepresentation of lq , 3q, and 6p chromo- somal arms (11,22,23). Robertsonian translocations involving the loci for the ribosomal genes [e.g. t(14;15) ( p l l ; q l l ) ] were observed in two studies (15,16).
Many additional sites of loss or rearrangement were common to several studies, although their rank order varied. Their contribution to detection of specificity was limited, notably in the studies of Dutrilleux et a1 (16), because of imprecise localization (to whole chromosome arms rather than bands). Although involvement of cer- tain chromosomes was common to all studies, few were consistent in nature. For example, chromosome 6 aber- rations were represented by 6q losses (16), 6q22-27 breaks in triploid tumors (17), 6pll-13 breaks and 6p gains (ll), deletions of 6q21-22 (23), and 6p23 and 6q13 breaks (15).
Rearrangements of chromosome 6 were very com- mon (44% of cases) in the Thompson et a1 study ( f l ) , but less common in data from others (15-17,23). In- volvement of 19q13 was cited in three of the five studies (11,15,17). Cumulatively, more than 25 hot spots of breakage and rearrangement were identified but, other than those noted above, only a few (many different sites on chromosome 1, the region from 3pll-21, near 6q22, 11q21-25) were cited in more than one of these studies. It is clear that significant improvements in methodology and banding have resulted in new and more precise data on the cytogenetics of primary breast cancers. Neverthe- less, a substantial fraction of cases in these reports or cells in individual cases fail to demonstrate either clonal- ity or nondiploidy. Bimodal cases comprise up to 25% of cases in some series (15) and are absent from others (11). Other cases appear to be characterized by poly- clonality (14,23). Many cases with extensive complex rearrangements were associated with marked chromo- some instability, precluding precise modal characteriza- tion (15,16).
51 NG LE TRl SOM I ES
Trisomy as the sole aberration in breast cancer has been suggested as an early cytogenetic change, but was observed relatively infrequently in the studies summa- rized here. Trisomy 7 was the only recurrent, solitary numerical finding in 5/20 cases (14) and in two tumors
128 SLOVAK AND WOLMAN
reported by Thompson et a1 (11). Near-diploid clones were associated with simple numerical changes caused by nondisjunction resulting in monosomy 17, mono- somy 19, and trisomy 7 (11). However, trisomy 7 as the sole aberration in solid tumors has been challenged as a neoplasia-specific aberration, with evidence that it may occur in stromal elements, inflammatory cells, or tumor- infiltrating lymphocytes (24,25). Alternatively, trisomy may indicate a general tendency of diploid tumor stem cells to undergo mitotic nondisjunction (26).
In a study of 18.5 primary breast carcinomas, trisomy 8 was the most frequent clonal chromosomal gain (10 invasive ductal carcinomas and 1 invasive lobular carci- noma) (26). Because trisomy 8 occurs in benign as well as malignant tumors it may endow the cells with a growth advantage, contributing to the phenotype but not directly responsible for malignancy, which in turn may depend on other cytogenetically visible or invisible mutations. Genes on chromosome 8 include the CMYC oncogene (8q24) which has been associated with prolif- eration and unfavorable prognosis (27,28), the FGF re- ceptor gene FGFRIFLG (8p12), which is amplified in -15% of breast cancers and has been associated with small, low-grade, estrogen-positive tumors (29). These and other unknown genes could affect selection for tri- somy 8 as either a primary or a secondary cytogenetic aberration in breast cancer.
Trisomy 18 as the primary or sole cytogenetic aberra- tion has been described in breast tumors of varied mor- phology (8,14,23,26,30). Genes such as BCL-2 (18q21) an inhibitor of apoptosis (31) or DCC (deleted in colon cancer) (18q21) are potential sources of growth advan- tage in the development of breast cancer (32).
Over 90% of the cases reported by Thompson et a1 (ll), Hainsworth et a1 (17), and Slovak et a1 (15) were clonal. However, two independent reports by Pandis et a1 (14,23) described the presence of unrelated clones in 27/79 (34%) tumors suggesting a polyclonal origin in a significant subset (one-third) of breast tumors. This sup- position is exciting and perhaps supported by the vast tumor heterogeneity observed in these tumors. How- ever, environmental influences and chromosome insta- bility mechanisms may also have an underlying detri- mental effect on mitosis and segregation, leading to apparent cytogenetic polyclonality. Because most com- plex breast tumor karyotypes are described as compos- ite karyotypes, the question of unicellular versus multi- clonal origin needs to be addressed using a combined
FISWimmunohistochemistry (also known as FICTION) approach to determine whether karyotypically unre- lated clones contain common genic alterations in patho- logically defined breast tumor cells.
Discordance between flow cytometric and cytoge- netic studies was associated with either selective growth of a mitotic 46,XX population in an aneuploid tumor population, or a bimodal distribution (near-diploid/ near-tetraploid) of mitotic cells found by cytogenetics with a diploid DNA index, or cytogenetically aberrant and near-diploid, DNA-diploid tumors (15).
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) (15,33) or ho- mogeneously staining or abnormally banding regions (HSRs or ABRs) (20,34,3.5) in 4-60% of breast tumors studied. Although HSRs have been localized to many chromosome arms (6p, 8p, 9p, l l q , lSp, 16p, 17q, 19p, and 20q) (15,16,23,34,35-381, there was little agreement on frequency, type of aberration (HSR vs. dmin), or locali- zation of amplification sites among the studies reviewed.
Differences in amplification frequency range from re- ports of HSRs in 60% of 84 primary breast cancers (35) to average of < l o % (14,17,33,39) to absent (11). How- ever, the unrestrictive definition of an HSR used by Zaf- rani et a1 (35) may be questioned: They defined an HSR as a chromosome segment larger than the largest homo- geneously staining segment in the normal karyotype that did not result from a simple chromosomal rearrange- ment. This definition may have led to an inflated fre- quency of HSRs in cases with complex karyotypes, poor or suboptimal banding, or the presence of numerous marker chromosomes. Furthermore, the frequency dis- crepancy could result from different selection biases de- pendent on cell culturing methods (1 8). The latter prob- lem is substantial, as evident from the number of cases (n = 17) in which the HSR-derivative chromosome was unidentifiable because of extreme karyotypic complex- ity (3.5). Few correlates with diagnostic or prognostic factors in breast cancer were presented [HSRs correlated with young age ( a 0 years) (P < 0.05) but did not cor- relate significantly with clinicopathoIogica1 features of the disease (tumor size, histologic grade, metastatic axil- lary nodes, or loss of hormonal receptors] (35).
The genes most frequently amplified in breast cancer
Breast Cancer Cytogenetics 129
include CMYC, ERBB2, CCNDI, and INT2IFGF3 (re- viewed in 40 and 41), but these molecular amplifica- tions have correlated poorly if at all with cytogenetic ev- idence. Southern blot analyses of 16 proto-oncogenes failed to reveal association with the HSRs, even though low-level amplified sequences of HST and INT2 ( ~ 3 ) , CMYC (X2-3), and FES (> lo) were observed (42). Col- lectively, these data indicate that (a) both chromosome rearrangements and gene amplification can be regarded as frequent biologic markers of breast cancers; (b) a pre- cise definition of HSRS/ABRs is needed, perhaps based on molecular cytogenetics (FISH) using whole chromo- some paints; and (c) amplifications observed by classic cytogenetics that are not identified by molecular assays suggest the existence of unknown genes of potential im- portance in breast carcinogenesis. The latter point may be investigated using a combination of CGH, FISH, and chromosome microdissection studies (43).
CYTOG ENETICS IN BENIGN PROLIFERATIVE BREAST DISORDERS
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 (44- 48). A recent study by Dietrich et a1 (49) described clonal cytogenetic abnormalities in 16/30 cases of PBD. The re- current aberrations included del( 1 )( q12), de1(3)( p12-14), r(9)(p24q34), and alterations of chromosomes 1 and 12, especially regions 12pll-13 and 12q13-15. Similar clonal aberrations have been reported by others in smaller subsets of breast fibroadenomas (50,51). Of in- terest, 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. Follow-up studies of women with fibroadenomas char- acterized by clonal cytogenetic aberrations in conjunc- tion with routine mammographic surveillance should reveal whether these women are at increased risk to de- velop breast cancer.
FISH-based assessment has also been applied to pre- neoplastic and proliferative lesions of the breast (52). Using centromeric probes selected for their suggested relevance to breast cancer cytogenetic aberrations, an increase in frequency and extent of chromosomal aber- rations with malignant progression was shown. Similar- ities of specific losses of chromosomes 16, 17, or 18 in hyperplastic and malignant breast lesions fromthe same individual provided evidence that some hyperplasias
contribute to the sequence of progression to malig- nancy. FISH was essential for examination of these dis- crete small lesions that must be defined histologically and therefore are not amenable to conventional metaphase analysis. Proliferative lesions were character- ized mainly by borderline chromosome losses whereas advanced lesions (lobular CIS, ductal CIS, invasive duc- tal cancer) were characterized by unequivocal losses and gains. Sectioning artifact was controlled by establishing expected baseline frequencies for gain and loss in nor- mal tissues from the same breast. Gains of chromosome 1 were noted in both in situ and invasive carcinoma but were absent from proliferative lesions, consistent with the interpretation that this trisomy is probably not an early cytogenetic change in breast cancer tumorigenesis as suggested by others (11,16,20,23).
In the breast cancer cytogenetic studies by Slovak et a1 (15), a subset of 15 cases was further analyzed by a panel of FISH probes (53). Genetic alterations were identified in all 15 cases, including two cases reported as no growth. FISH helped to define additional genetic alterations in nine cases and to resolve questions of clonality in two cases. Normal diploid cells were ad- mixed with karyotypically aberrant cells in four cases that contained numerous infiltrating lymphocytes. In addition, FISH revealed chromosome 1 aneuploidy in diploid tumors, extensive intratumor cell heterogene- ity in 6/15 cases and, surprisingly, confirmed stable clonality in two high-grade tumors. This study under- scores the value of cytogenetics, flow cytometry, and FISH analysis as complementary testing procedures to define genetic alterations in low-mitotic-index tumors.
In summary, chromosomal banding studies of 231 primary breast cancers indicate that the most common numerical 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, 3p, 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. In combination, molecular and cytogenetic studies identify complexity and multi- plicity of genetic alterations and correspond with other evidence of intratumor heterogeneity, variable pheno- types, and perhaps polyclonality in breast cancer. Posi- tional cloning studies are needed to identify the candi- date tumor suppressor and oncogene genes in specific chromosomal regions. Studies to relate the critical genes or regions of interest to clinicopathological correlations
130 S L O V A K AND WOLMAN
are needed and these data will surely lay the foundation for genetically based prognostic subgroups.
SPECIFICITY AND MOLECULAR ASSOCIATIONS
Many of the specific aberrations noted are not unique to breast cancer. Pericentromeric rearrange- ments of chromosome 1 have also been noted in benign proliferative disorders of epithelial and stromal breast tissue (49). The unbalanced 1;16 translocation has been described in multiple myeloma, plasma cell leukemia, myelodysplastic syndrome, Ewings sarcoma, and other solid tumors (54-56). Interestingly, the classical and al- phoid DNA sequences localized to secondary constric- tions (or constitutive heterochromatin regions) of chro- mosomes 1 and 16 could result in uncoiling, elongation, and excess breakage (chromosome instability), leading to exchanges between homologous or near-homologous sequences (57). The recurrent nature of these chromo- somal aberrations resulting in gain of l q and loss of 16q indicates that they may serve as instability markers with functional significance in breast disease evolution. The proposed causal relations between hypomethylation, re- petitive DNA sequences, and resultant pericentromeric exchanges in tumors should be investigated in depth.
Both Hainsworth et a1 (1 7) and Thompson et a1 (1 1) found rearrangements of 16q22-24 in near-diploid cases, suggesting that this region may contain genes im- portant in the early stage of breast cancers. In corrobo- ration, allelic loss of 16q24.2-qter has been reported in up to 60% of sporadic breast tumors irrespective of in- vasion, metastasis, differences in clinical stage, tumor size, histological grade, or estrogen receptor status (58- 60). Alterations of genes in this region such as BBCl (breast basic conserved-1 gene) and DPEPl (dipepti- dase-1 gene) may be relevant to breast cancer develop- ment. Conversely, two of the del( 16q) cases reported by Hainsworth et a1 (17) marked invasive ductal cancers, and 16q abnormalities have been associated with distant metastases (61,62). Interestingly, genes associated with cancer invasion have been mapped to this chromosomal region (i.e., E-cadherin, M-cadherin, C A R ) (63), as well as a gene relevant to metastasis (64).
Karyotypic aberrations involving lp36 (15,16) were consistent with LOH studies in ductal carcinomas (65). Because distal chromosome 1 aberrations are reported frequently in other malignancies, genic alterations of lp36 may imply changes associated with tumor progres- sion. Genes localized to lp36 appear to control cell divi- sion (66-68) and perhaps control amplification of the
M Y C genes (69). Aberrant expression of the cell cycle regulatory genes is a plausible underlying mechanism for intratumor heterogeneity.
GENERAL CORRESPONDENCE OF CYTOGENETIC AND MOLECULAR DATA
Cytogenetic alterations are expected to support loss of heterozygosity (LOH) reported for many chromo- somal arms (IP, 1% 3P, 6% 7% 8P, 9% I l P , 13% 14% 15q, 16q, 17p, 17q, 18q, 22q, and Xp) (for review of al- lelotype studies see 40 and 41). However, although cy- togenetic losses of chromosome 1 are complemented by many LOH studies (70-72), the correspondence of loca- tion of the lesions is imperfect. Although consistent karyotypic deletions are highly suggestive of inactiva- tion of putative tumor suppressor genes, few of the al- tered target genes have been identified. One could argue that some genetic inactivating events are beyond the range of karyotyping procedures (e.g., microdeletions, homologous recombination with a defective allele). The reported frequencies and loci of LOH vary widely and these variations have been attributed to use of different probes (usually a single probe per region; noncompre- hensive analysis), different methods (Southern vs. PCR vs. RFLP) of demonstration, or to breast tumor hetero- geneity (especially important in cases containing 30- 40% stromal tissue). A genetic dissection strategy could combine screening genetic alterations in many tumors to determine the smallest chromosomal region of overlap (aka smallest common deleted region) with molecular assays in precursor or in situ lesions to determine alter- ations that are critical in the early stages of breast tu- morigenesis.
Reported cytogenetic rearrangements and deletions range from single bands to whole arms (15,16,36,73). Short arm deletions of chromosome 1 appear correlated with poor prognosis (65,72-74) and at least one com- mon region of LOH at lp35-p36 was correlated with the presence of lymph node metastasis, large tumor size, and nondiploid tumors (75). A correlation was also found between LOH of lp32-pter and amplification of the M Y C protooncogene suggesting that inactivation of a putative TSG in this region may result in MYC gene amplification (genetic instability) (69). Similarly, the common l q alterations (16,20) have been refined by molecular analysis. Based on RFLP analysis of 124 hu- man breast tumors, Bieche et a1 (76) defined two l q sub- groups: loss of the entire l q and/or gain of multiple cop- ies of chromosome l q (due to mitotic nondisjunction
Breast Cancer Cytogenetics 131
usually within the constitutive heterochromatin) and structural rearrangements resulting in partial deletions and partial gains of lq. Deletions defined by molecular and cytogenetics approaches narrowed the location of a putative tumor suppressor to within a 16 cM region of lq21-31 (76). Analogously, the smallest region of over- representation localized to 1q41-44 implied activation of an oncogene(s) in this region (76).
Loss of 3p, occasionally noted as the sole abnormal- ity in breast cancer, may thereby be an early or primary event in breast tumorigenesis (21). By cytogenetics, FISH and LOH, three independent deletion clusters were identified: 3pll-14 (1,21,77), 3p14-23 (17), and 3p24-26 (77-79) pinpointing sites for putative tumor suppressor genes (TSG). These data coincide with breakpoints observed in lung and renal adenocarcinoma suggesting that the TSG are not specific for breast cancer.
Allelic losses in three distinct regions of 6q, namely 6q13-21,6q23-24, and 6q27 (80,81) have failed to de- fine relationships between LOH (6q24-27) and the es- trogen receptor (ER) gene (mapped to 6q25.1) or ER content (82,83).
Deletions of 7q have been described infrequently (15) although 2 7 4 1 % of breast tumors exhibit LOH at the MET locus on 7q31, associated with poor prognosis (79,84,85). Association between TP.53 mutations and 7q31 LOH may reflect a partnership effective in either the generation or progression of breast cancer (79). Us- ing a highly polymorphic (C-A)n microsatellite repeat, the putative TSG has been localized to within a 2 cM segment distal to MET near 7q31.1-q31.2 (85) .
With a panel of microsatellite markers for chromosome 11, Carter et a1 (86) identified three regions associated with allelic imbalance in breast cancer (llpl5.5,11q13, and 11q22-q23) and demonstrated loss within 11~1.5 independent of l l q loss. Allelic deletion of the Harvey- RAS gene (11~15) is controversial (87,88) but another putative TSG at 11~15.5 within the TH-HBB region may contain a pleiotropic tumor suppressor gene (89).
Initial lack of concordance for 1 l q loss between cyto- genetic and molecular studies was related to use of mo- lecular probes that mapped proximal to the chromo- somal deletions (16,90). Now, two classes of breast cancer have been recognized with respect to l l q13 al- terations. The first group is characterized by LOH within the INT2 and PYGM region, pinpointing loss of the MEN-1 (multiple endocrine tumor) tumor suppres- sor gene (91). Microdissected in situ and invasive hu- man breast cancer lesions provided evidence for 11q13 loss early in development of sporadic human breast can-
cer and gave molecular support for the hypothesis that invasive breast cancer arises from in situ lesions (91). The second group shows amplification for 11q13 in -4-17% of breast tumors (92,93) which has been asso- ciated with decreased survival (92,94,95), and within the subgroup of node-negative patients, with an in- creased probability of tumor recurrence (96). The l l q 1 3 amplicon extends between 800 kb to 1,500 kb and houses several genes that may contribute to tumor development, CCNDl/PRADl/ BCL-1, E M S l , HSTF1I FGF4, and INT2/FGF3 (92,93,95,97). Of these, EMSl and CCND are the only genes shown to be overex- pressed with or without amplification, indicating several potential mechanisms for oncogene activation (97).
Surprisingly, there appears to be no statistical signifi- cant relationship between the progesterone receptor (PR) gene (at 11q22-23) content or expression and loss of l l q (98). Other candidate genes of interest include the ataxia telangiectasia gene (11q22.3), Fanconi ane- mia group D locus, and NCAM (99). A significant increase of breast cancer in families carrying the constitutional t( 11;22)(q23;qll) translocation is further evidence fa- voring a susceptibility gene on 11q23 (or 22q l l ) for breast cancer (100).
Karyotypic alterations of chromosome 17 are fre- quent in breast cancer, but complete loss of chromo- some 17 by nondisjunction appears uncommon. Five distinct chromosome 17 LOH regions are estimated to influence breast cancer. Two putative mutually indepen- dent TSG sites on 17p have been localized to 1 7 ~ 1 3 . 3 and within TP.53. Deletion of a 1 7 ~ 1 3 . 3 locus has been reported in -60% of breast tumors as a possible early event (90,101-103), whereas mutations in TP.53 have been found in 17-46% of the investigated tumors (101,104). 27.53 alterations are more common in med- ullary and ductal invasive breast carcinoma (105,106), observed frequently in ER-negative or PR-negative tu- mors (106), and have been strongly associated with poor survival regardless of nodal involvement (106,107). Furthermore, mutations of TP.53 may occur in the earli- est phase of breast cancer and this alteration is con- served during progression from intraductal to infiltrat- ing carcinoma to metastatic disease (108). A cooperative effect of the alteration between LOH at 17p with ampli- fication of the FLG gene ( 8 ~ 1 2 ) or LOH of l p has been hypothesized for tumor progression (109).
Allele loss patterns on the long arm of chromosome 17 defined three distinct regions of interest: proximal 17q21, corresponding to BRCA-1, the gene predispos- ing to hereditary breast and ovarian cancer; 17q21.3-
132 S L O V A K AND W O L M A N
q22; and the distal band 17q25 (101,110-115). Muta- tions of BRCA-1 have been described in a small subset of sporadic primary breast and ovarian cancers (116, 117) but the gene is large and the mutations appear widely distributed. Other genes of interest in breast can- cer reside in the 17q21 region but relevant genes in the more distal regions have not yet been defined.
Amplification of HER-2/neu (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 (93,118-120). ERBB2 alter- ations are present at all clinical stages with equivalent expression in the noninvasive and invasive components of breast tumors. Such data are consistent with role of ERBB2 in the initiation or early progression of a subset of breast tumors (121).
Molecular alterations of chromosome 17 have been reported in 80% of the breast tumors studies (101), a frequency substantially higher than that of cytogenetic aberrations. Primary breast tumors with mutant p53 ex- hibited a significantly higher proportion of complex chromosome aberrations (122), and were more likely to show allelic loss of chromosome 17 and amplification of ERBB2 compared to tumors with wild type p53 (106,122). Cells with abnormal p53 do not show nor- mal GI arrest in response to DNA damage leading to ac- cumulation of multiple unrepaired lesions (123,124). The resultant chromosomal instability and concomitant intratumor heterogeneity are consistent with presumed roles of p53 in cancer predisposition, spontaneous tu- morigenesis, disease progression, and perhaps therapy- related disease.
COMPLEXITY OF GENIC INTERACTIONS
Several studies have focused on interactive cooperat- ing genetic alterations in breast cancer. Tumors carrying a combination of l l p and 17p LOH may have increased metastatic potential (125) and LOH on 1 l p in associa- tion with amplification of either CMYC or ERBB2 has been linked with recurrent disease (109). Both LOH and cytogenetic deletions of the 1 lq22-q23.3 region appear more common in metastatic breast cancers and are sig- nificantly correlated with LOH at 17~13.1 (86,126,127). To determine if chromosomal regions that appear to be differentially involved in breast cancer indeed network with one another, correlation of LOH and specific gene amplification with histopathological and clinical param- eters in a large series of tumors are needed.
FISH STUDIES TARGETING CHROMOSOME 17
Many of the attempts to apply FISH to breast cancers for detection of aberrations and correlations with other types of genetic alteration have been based on study of dissociated cells from fresh or frozen tumors. In these in- stances it is difficult to achieve careful pathologic corre- lation with the genetic results because of inevitable con- tamination by stromal, inflammatory, or nontumor parenchymal cells. Other studies have been based on im- print preparations in which some geographic repre- sentation of the tissue architecture is maintained and cytologic detail may also aid in correlation. Although si- multaneous application of FISH with immunohis- tochemical markers is potentially feasible, many of the antigens available at present are neither highly tumor- specific nor breast-specific and some are not suitable for study of formalin-fixed, paraffin-embedded material.
The same principles of in situ hybridization can be applied to tissue sections after formalin fixation and paraffin embedding. Although histologic assessment is not ideal, basic geographic details are preserved and finer structures can be ascertained by examination of adjacent sections with conventional histologic stains. The chief drawback is sectioning and the resultant par- tial nuclear loss. This disadvantage is more than offset in many instances by maintenance of tissue organization and by access to genetic analysis of lesions otherwise limited by lesion size or complete utilization of the sam- ple for microscopic diagnosis.
Although FISH is preferred by many investigators, in situ hybridization (ISH) with nonfluorescent signals is feasible using the avidin-biotin complex. This approach has the advantage that signals are permanent and non- fading and morphologic detail is improved over fluores- cence. However, the advantages are offset by confounding effects of nonsignal Giemsa-stained nuclear aggregates, particularly in tumor types characterized by nuclear hy- perchromasia and chromatin clumping. An early study (128) demonstrated heterogeneity of centromere 17 sig- nal frequencies corresponding to different morphologic areas in a metastatic breast cancer, illustrating the power of paraffin-section-based applications.
Chromosome 17 is the leading contender as the site for many genes relevant to breast cancer as discussed above. Included are the tumor suppressor TP.53 gene, the ERBB2 oncogene which is amplified in up to 30% of cases and may have prognostic significance, NMEl, the gene that encodes the metastasis inhibitor protein, nm23, two estradiol dehydrogenases, the BRCA-1 gene that denotes high risk of early-onset breast cancer, and
Breast Cancer Cytogenetics 133
possibly others. It is, therefore not surprising that sev- eral FISH studies have focused on this chromosome.
The study by Rosenberg et a1 (129) was intended to examine the temporal and structural relation between enumeration of chromosome 17 by FISH and allelic im- balances of TP53, ERBB2, N M E l , as well as several non-gene-specific loci by Southern blotting and PCR. Tumors were dissociated and DNA retrieved from frag- ments other than those used for histologic evaluation, so that extent of contamination by normal cells could not be estimated. A substantial fraction (from 31% to 89%) of each tumor was disomic for the chromosome 17 cen- tromere. Of 13 samples, 8 were evaluated as showing in- creased copy number and 4 (some overlapping cases) as showing loss by FISH. Only 4 cases were deemed nor- mal by the stated criteria; these cases lacked ERBB2 am- plification and showed allelic imbalances (3/4) limited to l7pter. One of them was the only case that failed to show any molecular aberration. It was also noted that 7/9 aneuploid tumors had lesions beyond the 17pter region and these included all of the cases of gene amplification. Thus, the aneuploid cases appeared more deviant by several measures.
Similarly, Matsumura et a1 (3) used FISH in parallel with Southern blotting to explore and collate different genetic events on the short (p) arm of chromosome 17. They examined imprint preparations from fresh tumors and dissociated cells from paraffin blocks using a peri- centromeric 17 probe for chromosome enumeration and a probe just distal to the TP53 gene for evaluation of gene loss. A probe, YNZ22, that maps to the distal (17~13.3) region of 17p was examined for loss of het- erozygosity (LOH) by Southern blotting to compare chromosome loss or gain with presumed loss of TP.53. All tumors were represented by heterogeneous popula- tions (although some normal cell populations are as- sumed to contribute to the observed heterogeneity). One case with a subpopulation of cells showing loss by FISH was also borderline for LOH, whereas 9/11 cases with LOH of the distal locus displayed loss of the FISH signal near TP53. Monosomy for chromosome 17 appeared to explain 2 cases. Two trisomic cases showed LOH, one with clear demonstration of loss distal to the TP53 lo- cus, and the other unexplained. Similar observations in other systems have been interpreted as nondysfunctional chromosome replication that may be driven by LOH (130). Matsumura et a1 (3) suggest that much of the mo- lecular alteration identified in breast cancer is not in- tragenic but has a physical representation that can be detected at the level of the light microscope, in this in-
stance the distance spanned from 17~13.1 to 17~13.3, with appropriate tools. Evidence emerging from multi- studies indicate that loss from distal 17p is more com- mon than is loss of TP53 (101,102,131,132), as was suggested by the work of both Matsumura et a1 (3) and Rosenberg et a1 (129).
The HER-2heu (ERBB2) oncogene itself is detectable by FISH and both the evidence and nature of amplifica- tion of this gene were shown by Kallioniemi et a1 (133) in breast cancers and cell lines. FISH results were consis- tent with measures of amplification by Southern and slot blotting, and intrachromosomal localization of the amplified genes was demonstrated in metaphase prepa- rations of cell lines. In two cases FISH revealed amplifi- cation that was undetected by slot blot, probably be- cause of confounding effects of nontumor cells. The authors emphasized the power of FISH in distinguishing between increased signal due to extra chromosome cop- ies and that related to true gene amplification. FISH in imprint preparations from fresh or frozen breast tissues may also aid in diagnosis when morphology is ambigu- ous (5) . An added advantage when cosmid or regional probes such as that for the HER-2/neu oncogene are em- ployed is that the frequency of 3- and 4-signal cells re- flects the fraction of G2M cells present and, thus, af- fords an approximation of the DNA-synthetic fraction in the tumor cell population (134).
COMPARATIVE (OR COMPETITIVE) GENOMIC HYBRIDIZATION (CGH)
Evidence indicates that other yet unknown genes play a role in breast cancer. Two groups of investigators have applied this technique to the study of breast cancer. Kal- lioniemi et a1 (135), report common regional increases in 17q and 20q. They used cell lines as reference for studies of 33 fresh primary surgical breast cancers. The extent of the abnormalities found in the primary tumors is remarkable, especially in light of the comment that amplification in the range of two- to fivefold was insuf- ficient for detection, and that evidence of losses was not presented. Sixty-four percent of the cases in this study showed greater than fivefold gains of whole chromo- somes or whole chromosome arms with the most common increases of 1q and Sq, 36% and 27%, respectively. Re- gional increases were most frequent in sequences origi- nating from 17q22-24 and 20q13 in cell lines, and to a lesser extent in the primary cancers. The amplified re- gion on 17q was distal to the HER-2heu (ERBB2) lo- cus and was amplified independently of it in cell lines with known amplifications by Southern blot. The rela-
134 SLOVAK AND WOLMAN
tively high (5-1OX) copy numbers suggest that un- known genes important in breast cancer are present in these regions. The investigators extended this study by examination of the amplified region on 20q13, using FISH for mapping and several candidate genes localized to the region (136). They used an ordered series of paired 2-color probes as references to narrow the region of greatest amplification to a 1.5 Mb length in 20q13.2 that included one of the reference probes. All of the can- didate genes examined could be excluded on the basis of distance from the maximally amplified probe and by ab- sence of high copy numbers for the genes in question. The importance of amplification at this site was empha- sized because of association to aggressive tumors (137).
The other group that has utilized CGH to study breast cancer focused on tumors already assumed to have gene amplification based on prior recognition of HSRs by conventional karyotyping (34,37,3 8). Their findings are very similar to those cited above with whole chromosome arm overrepresentations more frequent than regional amplifications and with minor distinctions as to which sites were most common (especially with re- spect to 20q13). They also were limited to recognition of fivefold or greater replications. Finally, they also ob- served HER-2/neu (ERBB2) gene amplification in asso- ciation with CGH of a more distal locus on chromo- some 17. The discrepancies between their CGH and HSR results were explained as possible heterogeneity or karyotypic misinterpretation. In a substantial number of cases, the CGH results indicated nonnative origin of the observed HSRs.
CGH is clearly a powerful approach to identify the overall gains and amplifications, with the caveat that heterogeneity within the sampled tissue (both within the tumor and from adjacent normal cells) may dilute such identification. The large size of affected regions may re- flect simultaneous activatiodalteration of many, possibly related functions. The extent and number of sites in- volved may also provide a rough measure of comparative genetic instability among tumors of similar histotype, grade, and stage. Its most important value at present may lie in the ability to recognize sites of involvement not identified by other means.
INHERITED BREAST DISEASE
Lynch et a1 (138) were the first to recognize a domi- nant pattern of inheritance in breast cancer in a small subgroup (-4-5%) 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 (139,140). Mutations of these genes confer a high risk of early on- set breast cancer. BRCA-I confers an increased risk of ovarian cancer that is not associated with BRCA-2 (116). BRCA-2 mutations have been associated with male breast cancer, whereas male breast cancers are rare in BRCA-2 families (140). These genes confer risk, not 100 % expression in a carrier, and loss of function of the BRCA-1 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 tumors, and breast cancers (141).
The etiology of breast cancer is complicated by dis- ease heterogeneity accompanied by numerous genetic changes. A productive route to detect genes that are causal of or contributory to cancer is the recognition of frequent and specific chromosome aberrations associ- ated with particular tumors or tumor-prone individuals. Ten years ago, we understood little about the role of ge- netics in breast cancer. Collectively, the data gleaned from cytogenetics, flow cytometry, FISH, and molecular testing approaches have identified specific gene alter- ations in breast tumors. However, no single common ge- netic alteration is unique or common to all breast can- cers. Although ordering and/or determining 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 can- cers. Thus, genetic events in breast cancer are likely to involve a diverse range of genes in different subsets of tumors.
Early genetic alterations appear to involve losses on 3p, 7931, 11q13, 16q22-24, 17~13 .1 or gain of lq. Clonal evolution of breast cancer shows preferential as- sociation with lp36, 6q24-27, or 11q22-23 aberra- tions. A poor prognosis has been reported for mutations of TP.53, loss within bands 7q31 and 11q13 or amplifi- cation of 1 lq13. Breast cancer susceptibility loci have been mapped to 13q12-13, 17q21, 17~13.1 , and per- haps 11~15.5. Of course, the potential interactions of environmental influences of this multifactorial (poly- genic) disease remain to be determined. Rearrangements of lp36,17p13.1, and perhaps other alterations of DNA repair genes appear to have an effect on intra- and inter- tumor heterogeneity resulting from genetic instability.
Genetic information is being incorporated into thera-
Breast Cancer Cytogenetics 135
peutic strategies. Harada et a1 (61) has proposed to treat patients with multiple genetic ( 2 3 ) alterations as high risk for purposes of postoperative management and long-term outcome (regardless whether lymph node sta- tus is negative or tumor is T1 stage). Their study is de- signed to measure recurrence and survival rates over 5-1 0 years and may prove the value of genetic markers as routine prognostic indicators in clinical breast cancer management. Hormonal therapy appears advantageous in p.53 negativelbct-2 positive breast tumors (142). ERBB2 antisense olignucleotides have been shown to inhibit the proliferation of ERBB2 amplified breast tu- mor cell lines, indicating potential use for new class of potential pharmacological agents in those breast tumors (-25%) overexpressing the ERBB2 gene product (143). Other gene therapy approaches may exploit TSGs, since it is conceivable that introduction of a functional tumor suppressor gene into a tumor cell may retard its growth (assuming the gene can be successfully introduced and achieve stable expression in somatic cells) (144).
We still lack information on genetic alterations in precursor lesions such atypical hyperplasia or PBD and the later stages of ductal carcinoma in situ and lobular carcinoma in situ. Associations between inherited pre- disposition and the increasing risk of breast cancer are still poorly understood but surely will provide critical 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 the most effec- tive strategy of overcoming breast cancer.
Acknowledgments We thank the City of Hope Cancer Research Center, which
is supported by Public Health Service Grant CA-33572 (MLS), and Trudy Trimmer for manuscript preparation.
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