Cancer Cytogenetics: Chromosomal and Molecular Genetic Aberrations of Tumor Cells

Preface to the Fourth Edition

Although only six years have passed since the publication of the third edition of Cancer Cytogenetics, the field has undergone marked changes. New information about many tumor types has been gathered using chromosome banding and various molecular cytogenetic techniques, but first and foremost it is the increasing addition of state-of-the-art genomic analyses to the chromosome-level studies of neoplastic cells that has now brought about a more detailed and better understanding of how neoplastic transformation occurs in different disease entities. Inevitably, therefore, this fourth edition contains a wider coverage of the molecular genetic changes that neoplastic cells have acquired than was possible in previous editions. The main focus nevertheless remains unaltered: the genomic aberrations of neoplastic cells as they appear at the chromosomal level of organization. To put the molecular knowledge and studies—especially those involving various ways to search for pathogenetic fusion genes by means of whole genome sequencing—into an integrated molecular genetic–cytogenetic perspective, an entire new chapter was added. Otherwise the overall structure of the book remains the same as it was in the previous edition: the first five chapters, Chapters 1–5, are more generic in nature, Chapters 6–11 deal with hematologic malignancies and lymphomas, and Chapters 12–24 review existing cytogenetic and molecular genetic knowledge on solid tumors.

In all the chapters of this edition, we have strived to emphasize the clinical impact of the various acquired rearrangements, be it diagnostic or prognostic, as much as possible. Cancer cytogenetics has come of age as one of the several means whereby different neoplastic diseases could and should be diagnosed—especially hematologic disorders, malignant lymphomas, and tumors of bone and soft tissue but also increasingly other solid tumors—and it is imperative that cancer cytogeneticists communicate these aspects of their work to the pathologists and clinicians who are in direct charge of the patients. The closer the dialogue with other diagnosticians and clinicians, the more useful the karyotype and other cytogenetic and molecular findings will be in the risk assessment and choice of therapy for individual patients.

At the same time, cancer cytogenetics remains pivotal in the examination of neoplastic cells for research purposes. Chromosome banding analysis is a robust and unbiased method whereby global genetic information can be obtained at the cytogenetic level. All molecular examinations of tumor cells should ideally be viewed against the background of knowledge about the tumor karyotype.

A large number of experts have helped us write the various chapters of Cancer Cytogenetics, Fourth Edition. They have done a better job than we ever could even if we had had unlimited time on our hands, and we are profoundly grateful to all of them. The heterogeneity inevitable resulting from multiple authorship reflects reality within the scientific community and we choose to see it as an advantage rather than a disadvantage. We have nevertheless strived to impart a recognizable common format on the various organ-specific chapters so as to comply with the overall plan of the book. It is our hope that those who read and use this book will agree with us that the final result does the field of cancer cytogenetics the credit that is its due.

Sverre Heim

Felix Mitelman

Oslo and Lund, December 2014

CHAPTER 1

How it all began: cancer cytogenetics before sequencing

Felix Mitelman¹ and Sverre Heim²

¹Department of Clinical Genetics, University of Lund, Lund,Sweden

²Section for Cancer Cytogenetics, Institute for Cancer Genetics and Informatics, Oslo University Hospital, Oslo, Norway

The role of genetic changes in neoplasia has been a matter of debate for more than 100 years. The earliest systematic study of cell division in malignant tumors was made in 1890 by the German pathologist David von Hansemann. He drew attention to the frequent occurrence of aberrant mitoses in carcinoma biopsies and suggested that this phenomenon could be used as a criterion for diagnosing the malignant state. His investigations as well as other studies associating nuclear abnormalities with neoplastic growth were, a quarter of a century later, forged into a systematic somatic mutation theory of cancer, which was presented in 1914 by Theodor Boveri in his famous book Zur Frage der Entstehung maligner Tumoren. According to Boveri’s hypothesis, chromosome abnormalities were the cellular changes causing the transition from normal to malignant proliferation.

For a long time, Boveri’s remarkably prescient idea, the concept that neoplasia is brought about by an acquired genetic change, could not be tested. The study of sectioned material yielded only inconclusive results and was clearly insufficient for the examination of chromosome morphology. Technical difficulties thus prevented reliable visualization of mammalian chromosomes, in both normal and neoplastic cells, throughout the entire first half of the 20th century.

During these dark ages of mammalian cytogenetics (Hsu, 1979), plant cytogeneticists made spectacular progress, very much through their use of squash and smear preparations. These techniques had from 1920 onward greatly facilitated studies of the genetic material in plants and insects, disclosing chromosome structures more reliably and with greater clarity than had been possible in tissue sections. Around 1950, it was discovered that some experimental tumors in mammals, in particular the Ehrlich ascites tumor of the mouse, could also be examined using the same squash and smear approach. These methods were then rapidly tried with other tissues as well, and in general, mammalian chromosomes were found to be just as amenable to detailed analysis as the most suitable plant materials.

Simultaneously, tissue culturing became more widespread and successful, one effect of which was that the cytogeneticists now had at their disposal a stable source of in vitro grown cells. Of crucial importance in this context was also the discovery that colchicine pretreatment resulted in mitotic arrest and dissolution of the spindle apparatus and that treatment of arrested cells with a hypotonic salt solution greatly improved the quality of metaphase spreads. Individual chromosomes could now be counted and analyzed. The many methodological improvements ushered in a period of vivid expansion in mammalian cytogenetics, culminating in the description of the correct chromosome number of man by Tjio and Levan (1956) and, shortly afterward, the discovery of the major constitutional human chromosomal syndromes. Two technical breakthroughs around the turn of the decade were of particular importance: the finding that phytohemagglutinin (PHA) has a mitogenic effect on lymphocytes (Nowell, 1960) and the development of a reliable method for short-term culturing of peripheral blood cells (Moorhead et al., 1960).

Cytogenetic studies of animal ascites tumors during the early 1950s, followed soon by investigations of malignant exudates in humans (Figure 1.1), uncovered many of the general principles of karyotypic patterns in highly advanced, malignant cell populations: the apparently ubiquitous chromosomal variability within the tumor, surmised by pathologists since the 1890s; the stemline concept, first defined by Winge (1930); and the competition between stemlines resulting in labile chromosomal equilibria responsive to environmental alterations. The behavior of malignant cell populations could now be described in Darwinian terms: by selective pressures, a dynamic equilibrium is maintained, but any environmental change may upset the balance, causing shifts of the stemline karyotype. Evolution thus occurs in tumor cell populations in much the same manner as in populations of organisms: chromosomal aberrations generate genetic diversity, and the relative fitness imparted by the various changes decides which subclones will prevail.

Figure 1.1 Camera lucida drawing of tumor cell mitosis from one of the first (early 1950s) human cancerous effusions submitted to detailed chromosome analysis. The modal number was 75. The stemline also contained numerous abnormal chromosome shapes

(Courtesy of Prof. Albert Levan).

The elucidation of these evolutionary principles in numerous studies by a number of investigators, for example, Hauschka (1953), Levan (1956), and Makino (1956), paved the way for the new and growing understanding of the role of karyotypic changes in neoplasia and laid the foundation of modern cancer cytogenetics. In humans as well as in other mammals, the results strongly indicated that the chromosomal abnormalities observed were an integral part of tumor development and evolution (see, e.g., Levan, 1967; Koller, 1972; Hsu, 1979; Sandberg, 1980, for review of the early data). It should be kept in mind, however, that the object of these early investigations was always metastatic tumors, often effusions, that is, highly malignant cell populations. Hence, few, if any, conclusions could be drawn from them as to the role of chromosomal abnormalities in early tumor stages.

Interest in cancer cytogenetics influenced human cytogenetics much more profoundly than is currently appreciated. For example, the main goal behind the study that eventually led to the description of the correct chromosome number in man (Tjio and Levan, 1956) was to identify what distinguished a cancer cell. The motivation was not primarily an interest in the normal chromosome constitution, which at that time had no obvious implications, but the hope that such knowledge would help answer the basic question of whether chromosome changes lay behind the transformation of a normal to a cancer cell.

The first spectacular success in cancer cytogenetics came when Nowell and Hungerford (1960) discovered that a small karyotypic marker (Figure 1.2), the Philadelphia (Ph) chromosome, replaced one of the four smallest autosomes (the G-group chromosomes according to the nomenclature at the time) in the bone marrow cells of seven patients with chronic myeloid leukemia (CML). This was the first consistent chromosome abnormality in a human cancer, and its detection seemed to provide conclusive verification of Boveri’s idea. It was reasonable to assume that the acquired chromosomal abnormality—a perfect example of a somatic mutation in a hematopoietic stem cell—was the direct cause of the neoplastic state.

Figure 1.2 Unbanded metaphase cell from a bone marrow culture established from a patient with chronic myeloid leukemia. The arrow indicates the Ph chromosome (previously called Ph¹); the superscript indicated that this was the first cancer-specific aberration detected in Philadelphia. This naming practice was later abandoned, but the abbreviation Ph has for sentimental reasons been retained, since it was the first consistent chromosome abnormality detected in a human malignancy.

Nowell and Hungerford’s discovery greatly stimulated interest in cancer cytogenetics in the early 1960s, but for several reasons, the Ph chromosome long remained an exceptional finding. The confusing plethora of karyotypic aberrations encountered in other malignancies suggested that the changes were epiphenomena incurred during tumor progression rather than essential early pathogenetic factors. The enthusiasm for tumor cytogenetics as a result gradually faded. With this change of mood, the perceived significance of the Ph chromosome also changed, and the very uniqueness of the marker came to be regarded as a perplexing oddity. Why should there be such a simple association between a chromosomal trait and one particular malignant disease, when more and more data from other neoplasms showed either no chromosome aberrations at all or a confusing mixture of apparently meaningless abnormalities?

That an orderly pattern existed in what had hitherto been seen as chaos was suggested independently in the mid-1960s by Levan (1966) and van Steenis (1966). Surveying chromosomal data available in the literature, mainly on ascitic forms of gastric, mammary, uterine, and ovarian carcinomas, they found clear evidence that certain chromosome types tended to increase and others to decrease in number in the tumors. Soon afterward, the nonrandomness of karyotypic changes was also demonstrated beyond doubt in specific types of human hematologic disorders and solid tumors; for example, trisomy of a C chromosome in acute myeloid leukemia (Hungerford and Nowell, 1962), deletion of an F-group chromosome in polycythemia vera (Kay et al., 1966), loss of a G chromosome in meningioma (Zang and Singer, 1967), and a C–G translocation in acute myeloid leukemia (Kamada et al., 1968). The results of comprehensive cytogenetic studies of experimental tumors, including more than 200 primary sarcomas induced by the Rous sarcoma virus in mice, rats, and the Chinese hamster, supported the same conclusion (Mitelman, 1974). In both humans and animals, the karyotypic abnormalities seemed to be of two essentially different kinds: nonrandom changes preferentially involving particular chromosomes and a frequently more massive random or background variation affecting all chromosomes. To differentiate between the two could be exceedingly difficult, however. As a consequence, in spite of painstaking efforts, little progress was made in cancer cytogenetics during this period.

The situation changed dramatically in 1970 with the introduction by Caspersson and Zech of chromosome banding techniques (Caspersson et al., 1970a). The new methodology completely revolutionized cytogenetic analyses. Each chromosome could now be precisely identified on the basis of its unique banding pattern; whereas formerly identification was restricted to chromosome groups, all descriptions of chromosome deviations immediately became more precise and the conclusions based on them more stringent. As a consequence, a steadily increasing number of cancer cases, initially predominantly malignant hematologic disorders, were investigated with the new techniques, and a number of characteristic, specific, sometimes even pathognomonic changes were soon discovered (Table 1.1). Caspersson et al. (1970b) first used banding in this context and identified the Ph chromosome as a deleted chromosome 22, and in 1972, three of the nonrandom aberrations described in the 1960s were clarified: the additional C-group chromosome in acute myeloid leukemia was identified as trisomy 8 (de la Chapelle et al., 1972), the lost G-group chromosome in meningioma corresponded to monosomy 22 (Mark et al., 1972; Zankl and Zang, 1972), and the deleted F-group chromosome in polycythemia vera was a del(20q) (Reeves et al., 1972). A previously unrecognized recurrent abnormality, a 14q + marker chromosome in Burkitt lymphoma (BL), was also described the very same year (Manolov and Manolova, 1972). The first recurrent balanced rearrangements were reported shortly afterward: a reciprocal translocation between chromosomes 8 and 21, that is, t(8;21)(q22;q22), was found in the bone marrow cells of some patients with acute myeloid leukemia (Rowley, 1973a), and the Ph chromosome of CML was demonstrated to stem from a t(9;22)(q34;q11), not a deletion of chromosome 22 as was previously thought (Rowley, 1973b). Among other important translocations also soon identified were t(8;14)(q24;q32), t(2;8)(p12;q34), and t(8;22)(q24;q11) in BL (Zech et al., 1976; Berger et al., 1979b; Miyoshi et al., 1979; van den Berghe et al., 1979), t(15;17)(q22;q21) in acute promyelocytic leukemia (Rowley et al., 1977), t(4;11)(q21;q23) in acute lymphoblastic leukemia (Oshimura et al., 1977), and t(14;18)(q32;q21) in follicular lymphoma (Fukuhara et al., 1979). Ohno et al. (1979) identified two characteristic translocations—t(6;15) and t(12;15)—in mouse plasmacytomas (MPC), the first specific rearrangements in experimental neoplasms and, as it turned out (see below), the perfect equivalents of the characteristic translocations in human BL. In total, more than 1200 neoplasms with clonal abnormalities were reported during this first decade of banding cytogenetics, and more than 60 recurrent chromosomal aberrations were identified.

Table 1.1 Characteristic neoplasia-associated cytogenetic aberrations detected by banding analyses 1970–1979

The following decade saw a rush of data coming from studies of solid tumors, initially in particular mesenchymal neoplasms. The chromosome abnormalities of more than 2000 solid tumors were reported between 1980 and 1989, and almost 200 recurrent structural changes were identified. Several of them were no less specific than those previously found among hematologic disorders (Table 1.2), for example, t(2;13)(q36;q14) in alveolar rhabdomyosarcoma (Seidal et al., 1982), t(11;22)(q24;q12) in Ewing sarcoma (Aurias et al., 1983; Turc-Carel et al., 1983), and t(12;16)(q13;p11) in myxoid liposarcoma (Limon et al., 1986a). At this time, it also became clear that many benign tumors carried characteristic aberrations, including reciprocal translocations, for example, t(3;8)(p21;q12) in salivary gland adenoma (Mark et al., 1980), t(3;12)(q27;q13) in lipoma (Heim et al., 1986; Turc-Carel et al., 1986), and t(12;14)(q14;q24) in uterine leiomyoma (Heim et al., 1988; Mark et al., 1988; Turc-Carel et al., 1988).

Table 1.2 Characteristic cytogenetic aberrations detected by banding analyses of solid tumors 1980–1989

The identification of specific cytogenetic aberrations enabled meaningful clinical–cytogenetic association studies, the most important of which were the International Workshops on Chromosomes in Leukemia established in the late 1970s. The workshops provided an arena for a fruitful and at the time unique collaboration among cytogeneticists, clinicians, and pathologists who shared their data and insights in order to find diagnostically and prognostically interesting associations between cytogenetic aberrations and clinical characteristics in various hematologic disorders. The results obtained by this collaborative study group over a 10-year period showed that cytogenetics could subdivide phenotypically identical leukemias and lymphomas into distinct subgroups on the basis of specific abnormalities and that this classification had important clinical implications. For example, the workshop collaborators demonstrated that the diagnostic karyotype in childhood acute lymphoblastic leukemia was of greater prognostic importance than any hitherto known risk factor, such as patient age, white blood cell count, or immunophenotype (Bloomfield et al., 1986). The studies performed by the Workshops on well-characterized patient materials from different parts of the world were thus instrumental in consolidating cytogenetics as clinically well-nigh indispensable in hematology. A similar collaborative study group dedicated to the genetic analysis of mesenchymal tumors—the Chromosomes and Morphology (CHAMP) study group—was formed a decade later and has identified several important clinical–cytogenetic associations among different bone and soft tissue tumors (e.g., Mertens et al., 1998).

Technological advances at the same time made it possible to supplement cytogenetic investigations by molecular genetic studies of the same tumor types. Analyses in the early 1980s of the specific translocations in MPC, BL, and CML proved particularly pivotal for our understanding of how chromosome aberrations contribute to neoplastic transformation not only in these specific disorders but also generally (Mitelman et al., 2007). The picture to emerge was that reciprocal translocations exert their effects by one of two main alternative mechanisms: deregulation, usually resulting in overexpression, of a seemingly normal gene in one of the breakpoints (the BL scenario) or the creation of a hybrid, chimeric gene through fusion of parts of two genes, one in each breakpoint (the CML scenario). Deregulation of an oncogene by juxtaposition to a constitutively active gene region was predicted by Klein already in 1981, and the principle was soon demonstrated in MPC (Adams et al., 1982; Harris et al., 1982; Kirsch et al., 1982) and human BL (Dalla Favera et al, 1982; Taub et al., 1982; Croce et al., 1983; Erikson et al., 1983). The breakpoints of the characteristic translocations in mice and humans were found to be located within or close to the MYC oncogene and one of the immunoglobulin heavy- or light-chain genes (IGH, IGK, or IGL). As a consequence of the translocations, the entire coding part of MYC is juxtaposed to one of the immunoglobulin genes, resulting in deregulation of MYC because the gene is now driven by regulatory elements of the immunoglobulin genes. The alternative mechanism—the creation of a fusion gene—was documented at the same time in CML with the demonstration that the Ph chromosome, that is, the der(22)t(9;22)(q34;q11), contains a fusion in which the 3′ part of the ABL oncogene from 9q34 has become juxtaposed with the 5′ part of a gene from 22q11 called the BCR gene, resulting in the creation of an in-frame BCR–ABL fusion transcript (de Klein et al., 1982; Heisterkamp et al., 1983; Groffen et al., 1984; Shtivelman et al., 1985).

These and similar molecular insights into how cancer-specific chromosomal abnormalities act pathogenetically sparked an enormous interest in cytogenetics as a powerful means to pinpoint the locations of genes important in tumorigenesis (Heim and Mitelman, 1987). An impressive amount of information has been accumulated through these efforts. More than 65 000 neoplasms with at least one clonal cytogenetic change have been identified, and more than 700 gene fusions have been found by genomic characterization of breakpoints in cytogenetically identified aberrations in various leukemias, lymphomas, and solid tumors (Mitelman et al., 2015). We now know that practically all acquired balanced rearrangements lead to in principle the same consequences as the ones originally elucidated in BL and CML, that is, deregulation of a seemingly normal gene or the creation of a hybrid gene. In addition to oncogene activation via translocations and other balanced rearrangements (inversions, insertions), gene fusions may also be produced by unbalanced changes such as deletions leading to fusion of genes in the deletion edges.

The advent of molecular genetics in the 1980s and the development of a range of powerful molecular cytogenetic technologies during the last three decades, such as fluorescence in situ hybridization (FISH), multicolor FISH, comparative genomic hybridization (CGH), various array-based genotyping technologies, and DNA and RNA sequencing (Lander, 2011; Ozsolak and Milos, 2011; Le Scouarnac and Gribble, 2012; Mwenifumbo and Marra, 2013; Mertens and Tayebwa, 2014), have dramatically widened our knowledge and understanding of the molecular mechanisms that are operative in neoplastic initiation and progression. The new techniques have enabled researchers to investigate tumor cells at the level of individual genes, even at the level of single base pairs, and the molecular consequences of an ever increasing number of cancer-associated genomic aberrations have thus been laid bare (Vogelstein et al., 2013).

It is obvious that the cross-fertilization between cytogenetics and molecular genetics has led to conceptually new advances and insights into the fundamental cell biology mechanisms that are disrupted when neoplastic transformation occurs. At the same time, the clinical usefulness of cytogenetic abnormalities as diagnostic and prognostic aids in cancer medicine has been increasingly appreciated. The ultimate goal is to arrive at specific therapies individualized to counter those molecular mechanisms that have gone awry in each patient’s cancerous disease. The development of imatinib (Druker, 2008) as a therapeutic agent for CML—the first example of a targeted therapy against a specific fusion gene in cancer—is a wonderful example of how progress in cytogenetics and molecular biology has led to a qualitatively new treatment approach: the discovery of the Ph chromosome, the finding that the Ph chromosome results from a reciprocal translocation, the identification of the two genes in the breakpoints of the translocation, and the subsequent characterization of the fusion gene and its protein product. Similar targeted therapies are presently being developed against a number of fusion genes, and some have already turned out to be successful, for example, crizotinib targeting the EML4–ALK fusion gene generated by an inversion on the short arm of chromosome 2 in a subset of patients with non-small cell lung cancer (Shaw and Engelman, 2013). While it took 40 years from the discovery of the Ph chromosome to the development of imatinib, it only took a few years from the description of the EML4–ALK fusion in lung cancer to the development of crizotinib. We are convinced that many similar success stories are unfolding as we write; cancer genetic research helps obtain more effective and less toxic treatments for malignant diseases. Thus, in the 100 years since Boveri first postulated that chromosome change may initiate the carcinogenic process, cancer cytogenetics has come of age. It is no longer a purely descriptive discipline but one that attempts to synthesize information from several investigative approaches. Cancer cytogenetics has become both a central methodology in basic cancer research and an important clinical tool in oncology.

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CHAPTER 2

Cytogenetic methods

David Gisselsson

Department of Clinical Genetics, University of Lund, Lund, Sweden

The human chromosome complement consists of 22 pairs of autosomes and one pair of sex chromosomes, XX in females and XY in males. The autosomes are numbered after their relative lengths, with the exception of chromosomes 21 and 22. For stable function of a chromosome, a centromere somewhere along its length and a telomere at each terminus are required. The centromere is associated with the kinetochore protein complex necessary for anchoring of the spindle fibers and for separation of sister chromatids at the metaphase–anaphase transition. Centromeric regions contain large areas of repetitive DNA sequences, some of which contribute to the segments of constitutive heterochromatin found around the centromeres of all chromosomes, though most prominently in 1, 9, 16, and Y. Another type of repetitive DNA element is located at the telomeres. These tandemly repeated TTAGGG hexamer units maintain the structural integrity of chromosome termini and ensure complete replication of the most terminal nonrepetitive sequences.

Since the correct chromosome number of man was reported more than half a century ago (Tjio and Levan, 1956), our possibilities to analyze the human chromosome complement have improved steadily. This chapter is an attempt to outline the methods currently employed in cancer cytogenetics, spanning from chromosome banding to array- and sequencing-based techniques. Cytogenetic methods have traditionally been based on microscopic examination of individual cells, and it can be argued that next-generation sequencing (NGS) and genomic arrays are not cytogenetics. However, also these techniques can be used to obtain significant data on overall genome architecture in cells and could therefore with all rights be considered high-resolution cytogenetics. The practical details and protocols of the specific methods will only be touched upon, and the reader is referred to the individual articles cited in this and later chapters for more detailed information.

Sampling for cytogenetic analysis

A correct sampling procedure is the basis for correct scientific and diagnostic conclusions. A first issue to consider is whether the sample is sufficient for the planned analyses. Chromosome preparation requires live cells, whereas in situ hybridization at least requires intact nuclei, and genome arrays as well as sequencing rely on DNA that has not been extensively degraded. Another issue to consider is whether the sample is representative of the lesion to be investigated. Cytogeneticists rarely know precisely which cells they study. Exceptions to this are when in situ hybridization is combined with immunohistochemical staining of intact cells or when DNA is extracted for analysis from fixed microdissected solid tumor components, from reviewed cryosections, or from flow-sorted cells. When analysis is performed on cultured cells, it is further important to consider whether the results are representative of the in vivo situation. Two main types of heterogeneity can be expected at cytogenetic analysis of a tumor sample: that between neoplastic and nonneoplastic cells and that among various neoplastic cells (Pandis et al., 1994 ; Lindgren et al., 2011). In vitro overgrowth of normal cells or of neoplastic subclones can bias the cytogenetic results. This is a major reason why the use of established cell lines can have serious disadvantages. Pronounced selection may occur among clones that were present already in vivo, and chromosomal aberrations that emerge in vitro may be mistaken for in vivo changes (Gisselsson et al., 2010). Finally, many human tumor cell lines are contaminated by other human or animal cells (Lacroix, 2008). Direct preparations or short-term cultures are therefore usually preferred for chromosome banding analysis.

Chromosome banding

Chromosomes are typically studied at the metaphase stage of the cell cycle when the chromatin is highly condensed and the chromosome morphology is well defined. In most banding methods, individual chromosomes are identified by their relative size, the position of the centromere, and the patterns of transverse striations. Based on this, the short (p) and long (q) chromosome arms are divided into different morphological regions, which in turn can be subdivided into bands and subbands, their number depending on the resolution of the preparation technique. The first of these methods to be invented was Q-banding (Caspersson et al., 1970 ), for which metaphase chromosomes are stained with quinacrine mustard and examined through a fluorescence microscope. A partial explanation of the Q-banding pattern is that quinacrine stains AT-rich sequences brighter than GC-rich sequences (Weisblum and De Haseth, 1972). Most striking are the very bright Q-bands containing highly AT-rich satellite DNA, particularly in the distal part of the Y chromosome. G-banding (Figure 2.1A) is obtained when the chromosomes are pretreated with a salt solution or a proteolytic enzyme before staining with Giemsa or equivalent stains. G-banding yields approximately the same information as Q-banding; bands that fluoresce intensely by Q-banding stain darkly by G-banding. R-banding is obtained by pretreatment with hot alkali and subsequent staining with Giemsa or acridine orange (Dutrillaux and Lejeune, 1971). As the name indicates, R-banding yields a pattern that is the reverse of that obtained by G-and Q-banding. However, since R-banding stains the chromosome ends intensely, this technique may be preferable to G- or Q-banding when it comes to detecting terminal chromosome rearrangements.

Figure 2.1 Examples of how different cytogenetic techniques can be used to delineate chromosome aberrations at different levels of resolution. A supernumerary ring chromosome (arrow) is identified by G-banding (A) in a soft tissue tumor and shown by multicolor FISH paint (B) to contain sequences from chromosomes 9 (arrowhead) and 12 (arrow). Whole-chromosome painting (C) of chromosomes 9 (red) and 12 (green) corroborates these findings, and multicolor chromosome 12 banding with single-copy probes (D) shows that sequences from the MDM2 (yellow) and CDK4 (violet) genes in 12q13–15 are amplified in the rings. Further analysis with SNP array (E) defines the boundaries of the 12q-amplified segments, which include CDK4 and MDM2. The y-axis of the upper panel corresponds to relative gene copy number (log2 ratio). The y-axis of the lower panel shows the mirrored B-allele frequency (mBAF), which is shifted toward homozygosity (mBAF = 1) in the amplified regions. Array images are courtesy of Dr. K. Hansén Nord.

G-banding and other whole-genome banding methods are still used in routine cytogenetic diagnostic investigations and in research. In fact, chromosome banding remains the only truly low-cost genome screening technique allowing the identification of balanced as well as unbalanced genomic rearrangements in single cells (Table 2.1). Besides these whole-genome banding methods, there are several sequence-specific techniques, of which C-banding is most commonly used. This is produced by denaturing the chromosomes prior to Giemsa staining (Sumner, 1972). The method labels the constitutive heterochromatin, thus especially demarcating the variable heterochromatic blocks on chromosomes 1, 9, 16, and Y.

Table 2.1 Comparison of cytogenetic methodologies

*Applies to whole-genome sequencing with optimal coverage.

†Order of magnitude of maximal functional resolution according to Coe et al. (2007).

‡Library-based single-copy probes are typically defined on physical genome maps, but painting probes and satellite probes are not.

§Requires single cell preparation or specific bioinformatic interpretation.

¶Requires bioinformatic/statistical analysis of read numbers.

††Possible if SNPs are included in the array.

In situ hybridization

In situ hybridization techniques are based on the inherent organization of DNA into two antiparallel complementary strands. After denaturation of target DNA in metaphase spreads or interphase nuclei, single-stranded DNA probes are allowed to form hybrid double-stranded complexes with their complementary genomic sequences. Before hybridization, probes can be labeled by fluorophores to allow direct detection by fluorescence microscopy (Pinkel et al., 1986 ; Cremer et al., 1988). This fluorescence in situ hybridization (FISH) strategy allows simultaneous detection of several genomic sequence targets as fluorophores of different wavelengths can be combined in the same hybridization experiment and concurrently detected (Figure 2.1B–D). However, probes can also be labeled with nonfluorescent haptens, allowing secondary detection by enzymatic methods analogous to those used in immunohistochemistry. This chromogenic in situ hybridization (CISH) technique avoids the problem of tissue autofluorescence and can therefore be advantageous for direct analysis of fixed tissue sections (Tanner et al., 2000; Hsi et al., 2002). However, the number of colors available for chromogenic detection is still quite limited.

One great advantage of FISH is its versatility with respect to target sequences. Whole chromosomes or large parts of the chromosomes can be targeted by painting probes, whereas centromeres, telomeres, and polymorphic satellites can be detected by specific repeat sequence probes. Furthermore, single-copy probes can be manufactured by amplification of genomic DNA cloned into libraries of various vectors such as cosmids, fosmids, and bacterial artificial chromosomes. These probes can target unique sequences down to the gene level. Single-copy probes are highly useful to map chromosome breakpoints in metaphase preparations or to search for copy number changes in metaphase or interphase cells. All these FISH applications require preexperimental selection of which sequences to examine; one only gets information about those sequences one probes for. Differential labeling of several probes may circumvent this problem and thus make whole-genome screening by FISH possible. In multicolor karyotyping, simultaneous hybridization of differently labeled painting probes allows chromosome identification by assigning each pair of homologous chromosomes a certain spectral signature (Schröck et al., 1996; Speicher et al., 1996; Tanke et al., 1999). This application is particularly useful for resolving the composition of complex structural aberrations but may have limited resolution when very small chromosome fragments are involved (Lee et al., 2001). Similar combinatorial labeling of single-copy probes can also be used to create color banding along the length of one or several chromosomes. Some of these methods are based on cross-species hybridization or microdissection of chromosome segments (Müller et al., 1998; Chudoba et al., 2004), whereas others are based on smaller sequences that can be traced back directly to the human genome sequence map (Lengauer et al., 1993; Gisselsson et al., 2000). For routine cancer genetic purposes, FISH is typically used either to assess amplicon status of importance for risk stratification and/or treatment such as ERBB2 amplification in breast cancer and MYCN amplification in neuroblastoma.

Genomic arrays

Genomic arrays are highly efficient tools for obtaining data on genomic imbalances present in a tumor sample. In its most original form, DNA was first extracted from a tumor specimen and a normal reference sample, respectively, differentially labeled with fluorophores, mixed, and allowed to hybridize competitively to complementary target sequences (Kallioniemi et al., 1992 ). Originally, the target sequences were normal chromosome spreads where the ratio of test-to-reference fluorescence along the chromosomes was quantified using digital image analysis. Defined DNA fragments fixed in a matrix system provide much higher resolution and have now largely replaced this approach. Currently available genomic array platforms are based on single-stranded oligonucleotides (oligonucleotide arrays), which may or may not include polymorphic probes to detect single nucleotide polymorphisms (SNP arrays). Apart from efficiently detecting gains and losses of genome sequences at a very high resolution, SNP arrays can be employed to assess genomic imbalances not reflected as copy number abnormalities, such as loss of heterozygosity by uniparental disomy (Bignell et al., 2004; Zhao et al., 2004).

The resolution of arrays primarily depends on their underlying design concept, for example, the total number of DNA target sequences, their individual lengths, and the distance between them (Aradhya and Cherry, 2007). Even within a specific array platform, the resolution is often different for different parts of the genome. Furthermore, the resolution for an individual tumor sample depends on unique experimental conditions such as the degree of contamination by DNA from nonneoplastic tissues, which often makes the practical resolution of genomic arrays lower than expected from theoretical calculations (Coe et al., 2007). Another disadvantage of the array methodology is that it reflects a theoretical average of a tumor sample so that intercellular variability is difficult to assess. Techniques have long been established for unbiased PCR amplification of small amounts of DNA, for instance, from microdissected tumor samples (Speicher et al., 1993, 1995; Wiltshire et al., 1995) or from single cells (Klein et al., 1999; Wells et al., 1999). This has made it possible to compare genomic profiles from different parts of a single tumor. These techniques are sensitive to differences in sample quality and are rarely used in a clinical setting. Through the use of relatively simple bioinformatic tools, it is now possible to determine the relative prevalence of different genomic aberrations in a single sample (Lindgren et al., 2011). This, in turn, enables indirect characterization of clonal hierarchies in a tumor (Lundberg et al., 2013). However, a remaining disadvantage of genomic array techniques is their failure to detect completely balanced aberrations, such as inversions, insertions, and translocations. Nevertheless, the typically high resolution of genomic arrays, their potential for robotic streamlining, and the fact that they do not require living cells for analysis (Table 2.1) have quickly made them ubiquitous tools in cancer cytogenetics.

Large-scale sequencing

The advent of massive parallel/second-generation/NGS technology has radically transformed the field of cancer cytogenetics. More than 800 genomes of more than 25 cancer types have now been sequenced (Mwenifumbo and Marra, 2013 ). This has provided a flood of novel cancer genetic data, including but not limited to the identification of novel driver mutations, somatic mutational signatures correlating with carcinogenic exposures, and further details on clonal evolution through ultra-deep sequencing or multisample/multiregion sequencing. NGS has also increased our possibilities to perform refined mapping of chromosomal rearrangements down to the single nucleotide level (Table 2.1). This has enabled the delineation of complex structural rearrangements such as combined deletion–inversion events, in particular through the methodology of whole-genome sequencing using paired-end reads (Inaki and Liu, 2012). Also, novel architectural features of the cancer genome have been described through NGS, such as the tandem duplicator phenotype (Stephens et al., 2009; Hillmer et al., 2011; McBride et al., 2012), chromothripsis (massive numbers of genomic rearrangements involving several chromosomes with minimal corresponding change in copy numbers; Stephens et al., 2011), and kataegis (clusters of somatic single nucleotide variants that often colocalize with structural rearrangements; Nik-Zainal et al., 2012).

From a methodological standpoint, NGS at its current state has higher requirements with respect to sample tissue than does genome arrays. This mainly stems from the need to separate constitutional variants from somatic events. Not only is a high (60–80%) content of neoplastic cells recommended but also nonneoplastic control tissue from each patient (Mwenifumbo and Marra, 2013). Further requirements for robust ascertainment of somatic single nucleotide variations include a high number of redundant reads (high coverage), critical filtering against databases of constitutional variants, and some extent of confirmative resequencing. Taken together, this demands not only a robust infrastructure for material collection but also extensive bioinformatic skills and resources. NGS technology has thereby transformed cancer cytogenetics to a multidisciplinary field. Considering the continuously decreasing costs for NGS analysis together with its unprecedented level of resolution, it can be expected to largely replace older cytogenetic methods, not only as a research tool but also as a clinical instrument. However, NGS should not uncritically be regarded as a method that surpasses other whole-genome analysis technologies in all respects as it still faces significant limitations. For example, in contrast to banding and in situ hybridization, it does not on its own allow single cell resolution; even though the presence of multiple neoplastic clones can be ascertained by high-coverage sequencing, it provides no direct information about which genetic aberrations inhabit the same cells. Furthermore, compared to in situ methods, NGS provides limited topographical information, making it difficult to precisely locate significant aberrations, such as amplicons, in the genome. This in turn could make it hard to dissect precisely the mechanisms behind complex genomic changes by virtue of NGS alone.

Interpretation of cytogenetic data

Even though the technical possibilities to detect genomic rearrangements in neoplasms have increased tremendously in recent years, several principal issues regarding the interpretation of cytogenetic data remain unresolved. One example is the finding of a normal karyotype/flat genomic array profile in a neoplastic sample (Pandis et al., 1994 ). In many cases, particularly when the cells were cultured prior to analysis, this could be explained by the presence of nonneoplastic cells in the sample. Another explanation is that the studied cells were truly neoplastic but that their pathogenic mutations were below the resolution level of the method used for analysis. Finally, the possibility remains that no mutation was present in the neoplastic cells, leaving the alternatives that pathogenic abnormalities exist at the epigenetic level or were caused by nonhuman DNA, for example, oncogenic viruses, not picked up by, for example, genomic arrays.

Another question is whether all abnormalities found upon analysis of a seemingly representative sample are actually significant. Considering the high resolution of array- and NGS-based approaches and the high degree of polymorphic variation in the human genome, both at the single nucleotide level and at the copy number level, it is of great importance to determine which changes are of pathogenic importance and which are part of the normal spectrum of interindividual genome variation (Rodriguez-Revenga et al., 2007). This is stressed by the fact that clonal chromosome changes have also been found in several reactive/inflammatory lesions that are classified as nonneoplastic by morphological and clinical criteria (Ray et al., 1991; Johansson et al., 1993; Broberg et al., 1999). Recent data have also indicated that genomic imbalances commonly accumulate with age in somatic nonneoplastic tissues (Forsberg et al., 2013), suggesting that such human somatic mosaicism may constitute a confounding factor in the cancer context. It is evident that delineation of the extent of somatic genetic variation in humans of different ages and with different nonneoplastic conditions is becoming increasingly important for the interpretation of cancer genetic data.

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