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The Cytogenetics of Hematologic Neoplasms 1 5 Aurelia Meloni-Ehrig that errors during cell division were the basis for neoplastic Introduction growth was most likely the determining factor that inspired early researchers to take a better look at the genetics of the The knowledge that cancer is a malignant form of uncon- cell itself. Thus, the need to have cell preparations good trolled growth has existed for over a century. Several biologi- enough to be able to understand the mechanism of cell cal, chemical, and physical agents have been implicated in division became of critical importance. cancer causation. However, the mechanisms responsible for About 50 years after Boveri’s chromosome theory, the this uninhibited proliferation, following the initial insult(s), fi rst manuscripts on the chromosome makeup in normal are still object of intense investigation. human cells and in genetic disorders started to appear, fol- The fi rst documented studies of cancer were performed lowed by those describing chromosome changes in neoplas- over a century ago on domestic animals. At that time, the tic cells. A milestone of this investigation occurred in 1960 lack of both theoretical and technological knowledge with the publication of the fi rst article by Nowell and impaired the formulations of conclusions about cancer, other Hungerford on the association of chronic myelogenous leu- than the visible presence of new growth, thus the term neo- kemia with a small size chromosome, known today as the plasm (from the Greek neo = new and plasma = growth). In Philadelphia (Ph) chromosome, to honor the city where it the early 1900s, the fundamental role of chromosomes in was discovered (see also Chap. 1 ) [ 4 ] . This fi nding stimu- heredity and reproduction was already valued by a number of lated subsequent research on chromosome aberrations in biologists. During that period, the most comprehensive view human neoplasms that still continues to augment our under- of the role played by chromosomes in heredity was held by standing about cancer. This chapter will focus on the visibly Boveri and Sutton, who independently theorized that it was recognizable chromosome abnormalities in human hemato- necessary to have all chromosomes present in the cells for logic neoplasms and their implication in diagnosis, prognosis, proper embryonic development to take place [1, 2 ] . This and therapeutic strategies. innovative concept was later applied to the origin of tumor cells by Boveri himself. Although he never experimented with tumors, Boveri obviously sensed that tumors began Cytogenetic Methods for Diagnosis from a single cell in which defects in the chromosome of Hematologic Neoplasms makeup led cells to divide uncontrollably. He formulated his theories in the book Zur Frage der Entstehung maligner Cytogenetics requires the presence of live cells or at least Tumoren (On the Problem of Origin of Malignant Tumors), intact nuclei (for FISH studies; see Chap. 17 ). Although it is published in 1914 [ 3 ] . This book is probably the most impor- understood that human cancer cells divide spontaneously tant early contribution on the genetics of cancer, as it offered and that culturing might not be a necessary step, it is also true some of the concepts still applicable today, speci fi cally that that neoplastic cells are regulated by different growth cycles, chromosome imbalances, mitotic disturbance, and monoclo- and therefore longer times in culture, as well as mitogen nality are all attributes found in cancer cells. The thought stimulation (in the case of mature lymphoid neoplasms), may be bene fi cial [ 5– 8 ] . Cytogenetics starts with proper sample collection, which in the case of hematologic neoplasms A. Meloni-Ehrig , Ph.D., D.Sc. (*) includes bone marrow aspirate, peripheral blood, as well as Department of Cytogenetics , Ameripath Central Florida, 8150 Chancellor Drive, Suite 110 Orlando, FL, USA various body fl uids and solid tissues in which in fi ltration e-mail: [email protected] by the neoplastic hematologic cells has occurred [ 5, 9 ] . S.L. Gersen and M.B. Keagle (eds.), The Principles of Clinical Cytogenetics, Third Edition, 309 DOI 10.1007/978-1-4419-1688-4_15, © Springer Science+Business Media New York 2013 310 A. Meloni-Ehrig Fig. 15.1 G-banded karyogram of a normal bone marrow cell Collection of samples should be performed aseptically, and in various tissue preparations. Their invention was seen as a the case of solid tissues, samples should be placed in a room potential competitor to conventional cytogenetics, due to their temperature medium, preferably enriched with growth higher resolution. Nevertheless, several years after the intro- factors and antibiotics. Longer transit times (>48 h) might duction of these sophisticated technologies, conventional affect the viability of the neoplastic cells and should be cytogenetic analysis is still the best method for the diagnosis avoided when possible [ 5 ] . Analyzable chromosome prepa- of most hematologic neoplasms since it has the advantage rations are obtained by fi rst exposing the cells to mitotic of an overall examination of all chromosomes, compared to inhibitors and subsequently treating them with hypotonic the more focused detection of abnormalities with the other solution and fi xation [ 10, 11 ] . Chromosome preparations are molecular genetic methods. Undisputed, in fact, is the ability then subjected to banding techniques, the most widespread of conventional cytogenetics to identify related and distinct of which is the trypsin-Giemsa banding method [ 12, 13 ] . See clonal populations, which is challenging for FISH and practi- Fig. 15.1 ; see also Chap. 4 . The terms used in cancer cytoge- cally impossible for array CGH [24, 25 ] . Furthermore, the netics are listed in Table 15.1 , and the karyotypes are presence of abnormalities acquired during clonal evolution, described according to An International System for Human an important indicator of disease progression, might be Cytogenetic Nomenclature (the most recent version appeared missed during a targeted FISH analysis [26– 29 ] . in 2009; see also Chap. 3 ) [ 14 ] . Chromosome Abnormalities in Hematologic Importance of Conventional Cytogenetics in Neoplasms the Diagnosis and Prognosis of Hematologic Neoplasms Cytogenetics began in 1956, when Tijo and Levan, and soon after them Ford and Hamerton declared that normal human There is no question that the development of sophisticated cells contained 46 chromosomes and not 48, as previously techniques such as fl uorescence in situ hybridization (FISH), believed (see Chap. 1 ) [ 30, 31 ] . From that point on, experi- multicolor karyotyping (M-FISH, SKY), and, to some extent, mental work on cell cultures and banding was geared to the array comparative genomic hybridization (array CGH) has improvement of chromosome spreading and morphology and enhanced the knowledge of chromosome abnormalities in was presented in subsequent publications [ 4, 32 ] . It was the hematologic neoplasms [15– 23 ] (see also Chaps. 17 and 18 ). detection of the Philadelphia chromosome by Nowell and These techniques have immensely contributed to the discov- Hungerford, however, that de fi nitively established that chro- ery of signi fi cant cryptic rearrangements as well as to the mosome abnormalities in leukemia are acquired and as such detection of such rearrangements in nondividing cells of they are present exclusively in the neoplastic cells [4 ] . But it 15 The Cytogenetics of Hematologic Neoplasms 311 Table 15.1 Glossary of cytogenetics terminology used in this chapter Acentric fragment A chromosome fragment lacking a centromere and therefore incapable of attaching to the spindle. Acentric chromosomes are distributed randomly among daughter cells Aneuploidy Deviation of the chromosome number that is characteristic for a particular species caused by either gain or loss of one or more chromosomes Autosome Any chromosome other than the sex chromosomes Banding Alternating intrachromosomal light and dark segments along the length of chromosomes Breakpoint Speci fi c band on a chromosome containing a break in the DNA as the result of a chromosome rearrangement Centromere An area of chromosomal constriction that holds the two chromatids together and is needed for spindle site attachment. Based on the position of the centromere, chromosomes are classi fi ed as metacentric (middle position), submetacentric (above the middle), and acrocentric (extremely small short arm consisting of satellites and stalks) Chromosome Arrangement of nuclear genetic material into formations containing a centromere and two chromosome arms. The normal chromosome number in human somatic cells is 46, whereas in germ cells it is 23 Chromosome rearrangement Structural aberration in which chromosomes are broken and rejoined. These rearrangements can occur on a single chromosome or involve multiple chromosomes Clonal evolution A stepwise evolution characterized by the acquisition of new cytogenetic abnormalities Cytogenetics The examination of chromosomes Deletion Loss of a chromosome segment. Deletions can either be terminal or interstitial Dicentric A chromosome containing two centromeres Diploid Normal chromosome complement (two copies of each autosome and two sex chromosomes) in somatic cells Double minute Cytogenetic visualization of gene ampli fi cation. So called because of their appearance as two adjacent dots. Each double minute is thought to contain hundreds of copies of a particular oncogene Duplication Two copies of the same segment present