Breast Cancer Cytogenetics: Clues to Genetic Complexity of the Disease

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<ul><li><p>Breast Cancer Cytogenetics: Clues to Genetic Complexity of the </p><p>Disease </p><p>Marilyn L. Slovak, Ph.D.* and Sandra R. Wolrnant Department of Cytogenetics, City of Hope National Medical Center, </p><p>Duarte, California and tOncor, Inc., Gaithersburg, Maryland </p><p>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- </p><p>~~ </p><p>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. </p><p>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 </p><p>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. </p><p>0 1996 Blackwell Science Inc., 107S-l22Xl96/$10.SOlO The Breast Journal, Volume 2, Number 2, 1996 124-140 </p><p>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. </p><p>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 </p></li><li><p>Breast Cancer Cytogenetics 125 </p><p>for many of the recent data that have increased our un- derstanding of the genetics of breast cancer. </p><p>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. </p><p>At the cellular level, classic cytogenetic studies and DNA content by flow cytometry or image analysis are </p><p>Table 1. Comparisons of Genetic Techniques </p><p>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. </p><p>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- </p><p>Techniaues Strenaths Weaknesses Resolution </p><p>Molecular Defines selected genetic aberrations in cell population </p><p>Sensitive/specific Evaluation of minimal residual disease for </p><p>individual gene mutations, deletions, and amplification </p><p>~l~~~~~~~~~~ in situ Defines complex rearrangements ID abnormalities in interphase Associate genetic alteration with morphology/or </p><p>May use archived tissues </p><p>Detects DNA amplified sequences within tumor </p><p>No need for specific probes No need of prior knowledge of aberrations </p><p>hybridization (FISH) </p><p>tumor area </p><p>Comparative genomic hybridization (CGH) genome </p><p>Cytogenetics Overview of genetic changes in individual cells ID intratumor heterogeneity ID clonality Defines target regions </p><p>Estimate DNA content and proliferative (5- phase) </p><p>Aneuploid detection in paraffin-embedded, fresh, </p><p>Flow Cytometry fraction </p><p>frozen, and formalin-fixed </p><p>Limited to specific probe (gene) alterations 0.2-50 Kb No evaluation of intratumor cell heterogeneity or </p><p>May need polymorphic (informative) markers Tumor clone 325% Need consitutional DNA </p><p>clonality </p><p>Limited probe availability &gt;2.5 Kb </p><p>?&gt;2 Mb Little data regarding intratumor heterogeneity Requires &gt;5 t o 7-fold amplification for detection Compromised by normal cell contamination No data regarding point mutations, transcriptional </p><p>activation, or chromosomal translocation </p><p>Limited genic level data 2-20 Mb Requires mitotic cells Selection due to in vitro culturing </p><p>No specific genetic alterations defined Low sensitivity Loss or gain of small chromosomes not detectable False aneuploidy due t o fixation, stain variations, or </p><p>Chromosome number 2 2 </p></li><li><p>126 S L O V A K AND WOLMAN </p><p>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. </p><p>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. </p><p>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 </p><p>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 &gt;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. </p><p>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. </p><p>DIPLOID TUMORS IN BREAST CANCER </p><p>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 </p></li><li><p>Breast Cancer Cytogenetics 127 </p><p>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. </p><p>CYTOGENETIC CHANGES IN PRIMARY BREAST DISEASE </p><p>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...</p></li></ul>