Important Chromosomal Aberrations in Hematologic Neoplasms and Key Techniques to Diagnose Them

Introduction to Human Chromosomes

In 1888, Waldeyer was first to introduce the term chromosome (meaning “stainable body,” from the Greek chroma, meaning “color,” and soma, meaning “body”). Waldeyer referred to Walther Flemming, who coined the terms chromatin and mitosis in 1879 at Kiel University. Flemming was also first to describe germinal centers. Since the pioneering studies by Flemming and Waldeyer, a wealth of knowledge on the composition and function of chromosomes has emerged. Each chromosome consists of a DNA double helix bearing a linear sequence of genes, coiled and recoiled around aggregated proteins called histones . Two sister chromatids (each constituting half of a chromosome) are joined together at a junction called a centromere (primary constriction). The full chromosome containing both joined sister chromatids becomes visible only during mitosis, in a phase known as metaphase . Regular human cells have 23 pairs of chromosomes (22 pairs of autosomes, numbered consecutively from 1 to 22, and 1 pair of sex chromosomes, i.e., XX in females and XY in males). Thus, a normal human somatic cell has two complements of 23 chromosomes (2n) for a total of 46 chromosomes, in contrast to a germ cell, which only has one chromosomal complement (1n) of 23 chromosomes. By convention, chromosomes are numbered in descending order according to their size and the position of the centromere (arm ratio), and are arranged into seven groups (from A to G) (Denver classification). On the basis of the centromere location, there are three main types of chromosomes: metacentric, with their arms roughly equal in length, submetacentric, with one arm clearly shorter than the other, and acrocentric, with a centromere located near one end of the chromosome. A band is defined as part of the chromosome that is clearly distinguishable from its adjacent parts by appearing darker or lighter with one or more banding techniques. This banding pattern to some extent reflects the base pair and histone composition of the different chromosome parts. Bands are grouped in regions delimited by specific landmarks, and numbered consecutively from the centromere outward along each chromosome arm, with the first number specifying region and the second band within this region; if sub-bands are discernible, they are numbered with the third number (and fourth in some instances) placed behind a period. Letters p (from French, petite ) and q are used to designate, respectively, the short and long arm of each chromosome. For designation of a particular band, four items are required: (1) chromosome number, (2) arm designation ( p or q ), (3) region number, and (4) band number within that region. For details on banding patterns, naming of chromosomes, and their parts, please refer to the International System of Cytogenetic Nomenclature (ISCN).

Chromosomal aberrations (or abnormalities) are changes in the number of chromosomes (numerical abnormalities; also named aneuploidy when one or a few chromosomes are gained or lost) or in their structure (structural abnormalities). In cancer cytogenetics, somatic (i.e., acquired, tumor-associated) aberrations have to be clearly differentiated from constitutional (i.e., germline) abnormalities. In principle, a chromosomal alteration—particularly if detected in all cells of an investigated individual—could represent a constitutional aberration, as long as its constitutional appearance is compatible with life. Some common examples of constitutional alterations recurrently detected during tumor genetic work-up are numeric changes in the sex chromosomes (e.g., XXY in patients with Klinefelter's syndrome), Robertsonian trans­locations [e.g., t(13;14)(q10;q10)], balanced translocations in phenotypically normal carriers [e.g., t(11;22)(q23;q11)], trisomy 21 in individuals with Down syndrome, or germline uniparental disomy (two different chromosomes from the same parent). The constitutional nature of a suspected abnormality should be confirmed or refuted with cytogenetic analysis of phytohemagglutinin (PHA)-stimulated culture of blood and/or cultured fibroblasts, or another alternative cell system (e.g., buccal swap, sedimented cells from urine). Moreover, occasionally somatically acquired alterations may also occur independently from tumorigenesis. Examples include loss of the Y chromosome in marrow or blood of older male patients or T-cell receptor (TCR) gene loci rearrangements.

Clones and Clonal Evolution

A clone is a cell population derived from a single progenitor cell. At the cytogenetic level, a clone is defined as two metaphase cells with the same structural abnormality or gain of the same chromosome, or three cells with loss of the same chromosome. The presence of a cytogenetically aberrant clone (or clones) at diagnosis usually indicates a neoplastic process. However, a clone does not necessarily prove the presence of a neoplastic disease, as occasionally, a clonal abnormality may be present in non-neoplastic cells, such as in the case of the aforementioned clonal loss of chromosome Y during aging. Moreover, a tumor population is not always homogeneous and in addition to the most basic clone of a tumor cell population, termed stemline, one or more subclones (termed sidelines ), containing new abnormalities in addition to the ones present in the stemline, can appear during tumor development (clonal evolution). Non-clonal aberrations (i.e., those occurring in single cells) are usually not listed in the karyotype description, but if they are indicated, it is done separately from the clonal abnormalities. A single-cell abnormality can sometimes be judged to be of a clonal origin if it represents a typical, cancer-associated aberration and/or its clonality is corroborated by alternative techniques [e.g., fluorescence in situ hybridization (FISH)], or it is found at other time points (e.g., at relapse).

Chromosomal instability is a transient or persistent state that causes a series of mutational events leading to gross genetic alterations. Multiple whole chromosome gains and losses and structural abnormalities present in more than one clone and in non-clonal cells are common manifestations of genomic instability. Determination of chromosomal instability requires approaches capable of monitoring cell-to-cell variability and/or the rate of both numerical and structural chromosomal changes. The most commonly used methods to determine chromosomal instability are conventional cytogenetics, FISH, or copy number (CN) array-based procedures.

Alterations of Cell Ploidy

Cell ploidy alterations are changes in the number of chromosome complements. As outlined earlier, the basic set of human chromosomes is called haploid and contains 23 chromosomes, that is, one copy of each of the 22 autosomes and one sex chromosome. A haploid chromosome set is characteristic for germ cells. A normal somatic human cell has two haploid sets and is called diploid (2n = 46 chromosomes). Cells with an increased number of chromosome sets are called triploid (3n = 69 chromosomes), tetraploid (4n = 92 chromosomes), and so on ( Table 7-1 ).

Systematic cytogenetic analysis of solid tumors and hematologic malignancies has revealed that the chromosome number in cancer cells can be highly variable, ranging from hypodiploidy (<46 chromosomes) to tetraploidy (4n = 92) or even pentaploidy (5n = 115), hexaploidy (6n = 138), or octaploidy (8n = 184). The modal number is the most common chromosome number in a tumor cell population. All changes in chromosome number should be expressed in relation to the appropriate ploidy level. A hyperdiploid karyotype characterizing a subset of acute lymphoblastic leukemia (ALL) patients is thought to arise from a single-step mechanism. Unscheduled tetraploidy can arise by one of three main mechanisms: cell fusion, mitotic slippage, or a failure to undergo cytokinesis. Maintenance of heterozygosity has been demonstrated, suggesting that the hyperdiploidy does not arise from a near-haploid precursor.

Aneuploidy: Monosomy and Trisomy

Monosomy is a term to describe the absence of one member of a chromosome pair, resulting in a clone with 45 chromosomes in the case of a single monosomy. Conversely, the term trisomy describes the presence of an extra chromosome (three copies instead of one pair); a single trisomy results in cells with 47 chromosomes. In the karyotype, a monosomy is usually denoted with a minus sign (e.g., −7 meaning monosomy 7) and a trisomy with a plus sign (e.g., +8 meaning trisomy 8).

Balanced Chromosomal Alterations (Reciprocal Translocations, Insertions, and Inversions)

Balanced chromosomal changes include reciprocal translocations, insertions and inversions. Reciprocal translocations are interchromosomal abnormalities resulting from the exchange of chromosomal material between two chromosomes without apparent gain or loss of chromosome material. Insertions are created when a segment of one chromosome is excised and inserted into one of the arms of another chromosome, whereas inversions constitute intrachromosomal aberrations derived from a 180-degree rotation of a segment within a single chromosome. The majority of recurring reciprocal translocations and inversions in hematologic neoplasms are considered to be primary events. They can lead to generation of gene fusions encoding chimeric transcripts, which contain sequences from both fused genes, or to deregulation of wild-type genes located next to a breakpoint by either promoter substitution or novel regulatory context. Several of these translocations and inversions are highly conserved and can be present in a majority of tumors of a given subtype. This makes various primary genetic alterations valuable diagnostic markers. Few cases carry three-way translocations that similarly involve three chromosomes with one breakpoint in each.

Unbalanced Chromosomal Aberrations (Deletions, Duplications, Unbalanced Translocations)

In addition to a whole chromosome gain (trisomy), chromosomal segments can be gained through unbalanced translocations or intrachromosomal duplications. Massive gain of a large number of copies of a small chromosomal region is called amplification, which cytogenetically is manifested as a homogeneous staining region (HSR) if the amplicon sticks together at one chromosomal site or as small acentric structures called double minutes (dmin). Amplifications are known to activate oncogenes and constitute a genetic mechanism leading to the overexpression of the amplification target gene(s). In this sense, several loci of recurrent amplification has been identified in different leukemia and lymphoma types as amplifications of REL/BCL11A at 2p16, BCL2 at 18q21, or MYC at 8q24. The border between chromosome material gain and amplification is sometimes difficult to establish. Complex rearrangements containing amplification of two loci juxtaposed by a chromosomal translocation have been named complicons.

Structural abnormalities resulting in loss of a chromosomal segment are intrachromosomal deletions and unbalanced translocations. The major consequences of deletions in cancer cells are the loss and/or inactivation of tumor suppressor genes, although occasionally deletions can lead to gene fusions and oncogene activation. The most prominent example of deletions in both lymphoid and myeloid neoplasms with a complex karyotype is loss of the short arm of chromosome 17 (17p), which contains the locus of the tumor suppressor gene TP53 . Losses of 6q are present in many types of aggressive lymphoma, such as diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), or mantle cell lymphoma (MCL). In patients with these deletions, the other allele of the target gene is frequently inactivated by a mutation. In some other cases, a homozygous deletion (i.e., deletion of both alleles) can occur. This is recurrently the case in chromosomal region 9p21, in which homozygous deletions involving the CDKN2A gene can be detected in several types of lymphoma and ALL. However, for many recurring deletions, genes presumed to be targets of deletions have not been hitherto identified. It has been suggested that such deletions can play a role in leukemogenesis through haploinsufficiency, that is, decreased expression of genes mapped to the lost segments because of the presence of only one functional allele following a deletion of the second.

In myeloid and lymphoid neoplasms with reciprocal translocations or inversions as primary abnormalities, unbalanced aberrations associated with gain and loss of chromosome material usually represent secondary genetic events and might be present only in a subset of cells of a given tumor. In contrast, in patients diagnosed with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) with a complex karyotype, unbalanced aberrations predominate and have presumed primary significance.

Copy-Neutral Loss of Heterozygosity

Loss of heterozygosity (LOH) means that a constitutionally heterozygous locus loses one allele. The reason of such loss can be a deletion (which is a copy-associated LOH) or LOH without chromosomal loss due to gain of the other allele in the form of a (partial) isodisomy. For this second kind of event, the term copy neutral-LOH (CN-LOH) has been introduced. In this sense, consequences of loss of one allele and duplication of the mutated allele can be functionally similar to a homozygous mutation. CN-LOH is a recurrent oncogenic event in lymphomas and AML. The regions affected by CN-LOH in lymphomas usually include such tumor suppressor genes as TP53 or TNFRSF14 , whereas in AML, CN-LOH often results in homozygous mutations at loci frequently mutated in this disease, such as CEBPA, FLT3, RUNX1, and WT1. Its identification has been useful for characterizing tumor stages and progression in different cancer types. As the gene dosage is not altered, CN-LOH cannot be detected by conventional cytogenetics, FISH, or comparative genomic hybridization (CGH array) analysis.

Chromothripsis

Chromothripsis is a recently described phenomenon identified in cancer cells by whole-genome sequencing that produces catastrophic chromosome reorganization of one or a small number of chromosomes at a single point in time. Some distinctive features of chromothripsis are: (1) alternating regions of copy number aberration with a minimal number of copy number states (one and two copies in its simplest form); (2) loss of heterozygosity of the lower copy number state; (3) derivation of each “new derivative” chromosome from one or a small number of chromosomes. Features 1 and 2 can be assessed by CN array, but this approach does not reveal the full complexity of the interchromosomal rearrangements. Chromothripsis escapes conventional cytogenetic detection, but can be suspected in complex karyotypes with one to three chromosomes participating in complex rearrangements. Multicolor FISH (M-FISH) or spectral karyotyping (SKY) can also identify the involvement of a minimal number of chromosomes (feature 3), but at low resolution. Nevertheless, a combination of both CN array and multicolor karyotyping techniques is currently the appropriate strategy for detection of chromothripsis in routine diagnosis.

Recently Baca and colleagues also introduced the term chromoplexy to describe another type of coordinated structural genome rearrangement that, different from chromothripsis, can occur in different steps in the evolution of the tumor, not in one single catastrophic event, and the breakpoints are unclustered and include multiple chromosomes. Recent genomic studies have identified these changes in chronic lymphocytic leukemia (CLL).

Fluorescence In Situ Hybridization

In FISH, fluorescently labeled DNA probes are hybridized to interphase nuclei or metaphase spreads prepared for standard cytogenetic analysis. FISH can also be applied to a wide range of cellular preparations such as banded slides, air-dried bone marrow or blood smears, fresh tumor touch prints, frozen or paraffin-embedded tissue sections, or nuclear isolates from fresh or fixed tissues.

A variety of FISH probes, each targeting a specific region or the entire chromosome, are available. Probes routinely used in the analysis of hematologic malignancies include chromosome-specific enumerator (i.e., mostly centromeric) probes, gene- or locus-specific probes, whole chromosome painting probes, arm-specific sequence probes, and telomeric probes.

Chromosome-specific centromeric probes are derived from the highly repetitive mostly alpha-satellite DNA sequences located within the centromeres. Because the target size is several hundred kilobases (kb) in length, the probes exhibit bright, discrete signals and are easy to evaluate in both metaphase and interphase nuclei. Centromeric probes are useful in identifying numerical abnormalities (aneuploidy), dicentric chromosomes, and the origin of marker chromosomes. Clinically important aberrations such as trisomy 12 in CLL (see Fig. 7-2, B ), monosomy 7 in AML, and high hyperploidy in ALL—all of which are detected at a lower incidence by conventional cytogenetics owing to low mitotic index or poor morphology—are routinely evaluated by FISH in many clinical laboratories. Another example is the use of differentially labeled probes specific for chromosomes X and Y in monitoring engraftment in sex-mismatched allogeneic stem cell transplantation.

Whole chromosome painting probes (WCP) or arm-specific sequence probes use mixtures of fluorescently labeled DNA sequences derived from the entire length of the specific chromosome or one of its arms. They are helpful in characterizing complex rearrangements and marker chromosomes. However, cryptic rearrangements affecting terminal regions may remain undetected, because of suppression of the repetitive DNA sequences within these regions. The application of chromosome painting probes is limited to metaphase analysis because the signals are often large and diffuse in interphase. Chromosome-specific telomeric or subtelomeric probes are derived from DNA sequences located at or adjacent to the telomeres and are effective in detecting terminal, interstitial, and cryptic translocations that are below the resolution of conventional cytogenetics and/or are undetectable by WCP probes.

Gene-specific or locus-specific probes are derived from unique DNA sequences or loci within the chromosome. With banding techniques on highly extended chromosomes, the smallest detectable chromosome abnormality is 2000 to 3000 kb, whereas gene- or locus-specific probes can routinely detect regions as small as 0.1 kb. As such, these probes have wide application in both basic and clinical research. Gene-specific or locus-specific probes have been extremely useful in gene mapping and in defining structural rearrangements, amplifications, and origin of marker chromosomes in both metaphase chromosomes and interphase nuclei.

In lymphoid malignancies, locus- or gene-specific probes have also been effective in delineating minimal regions of deletion (e.g., on chromosomes 6q, 11q, and 13q ) and in demonstrating monoallelic losses of such genes as RB1 and TP53.

Although the FISH probes can be easily applied to and analyzed on cytogenetic preparations, paraffin-embedded or frozen tissue sections can be difficult to work with and require additional standardization techniques. Loss of signal due to low hybridization efficiency and high non-specific background autofluorescence can lead to atypical signal patterns, making signal interpretation difficult. Nevertheless, recently adapted FISH protocols have been successfully implemented in the routine diagnosis ( Fig. 7-3 ). The major limitation of detecting losses by FISH in paraffin-embedded tissues is that part of the cell can be lost during the sectioning process, leading to false-positive results. Therefore, for detection of deletions, the cutoff value (i.e., a minimal percentage of cells with deletion detected for calling the case positive) has to be established at a higher level and appropriate negative controls have to be evaluated. Consequently, commercial FISH probes designed for evaluation of losses usually include an internal FISH hybridization control labeled with a different fluorochrome that usually hybridizes to the centromere of the chromosome with the locus of interest, or to a distal region of the same chromosome expected to be preserved if the deletion occurs. In the analysis of deletions by FISH, the evaluation by two different observers is highly recommended, as it is for the evaluation of translocations.

For the detection of translocations, two types of FISH probes are widely used, break-apart probes (BAPs) and double-color double fusion (DCDFs) probes. The BAPs detect gene rearrangements with differently labeled DNA probes that are complementary to sequences distal and proximal to the breakpoint within the target gene. The DCDF are designed for proving the juxtaposition of two loci and are used for the identification of reciprocal translocations. For this purpose, two DNA probes labeled with different colors located at the respective breakpoints of both translocation partners are used. The juxtaposition of both genes is translated into a third color under the microscope (fusion signal) (see Fig. 7-1, B ; Fig. 7-4, B ). BAP probes are less informative than DCDF probes because although they can reveal breakage within a specific locus, they do not define the other gene involved. In addition, because they flank the locus of interest, small insertions could remain unidentified. Nevertheless, their advantage lies in their ability to detect translocations involving different partners of such promiscuous genes as MYC, KMT2A (formerly known as MLL ), or BCL6, and they are easier to evaluate because the separation of two signals is easily recognizable. However, because some normal signals can be vaguely separated when BAP probes are used, the normal signal pattern has to be carefully defined according to probe design, locus interrogated, the material investigated, and so on. On the other hand, a positive result with DCDF probes consists of two fusion signals, an event that is very unlikely to occur by chance. For both kinds of probes, variant signal constellations caused by complex or unbalanced changes need to be considered.

Among the critical factors affecting an accurate interpretation of FISH is the establishment of proper cutoff values for the different probes used. For DCDF, the cutoff is usually clear below 5%, but it might be significantly higher for some variant signal patterns, for example, when it is caused by the loss of one derivative chromosome involved in the translocation. On the other hand, because BAPs are variable based on the location of the breakpoints and the probe design, the FISH evaluator needs to visually estimate the relative distance between the different color probes in normal controls. A break is usually recognized if the distance between the two signals is at least twice the estimated signal diameter. Ideally, the BAP cutoff should be between 1% and 5%, although it might be higher, again depending on the probe design and locus investigated.

ISCN standard nomenclature is also established for description of chromosomal changes detected by FISH. In interphase FISH, the abbreviation nuc ish is followed by the locus designation in parentheses, a multiplication sign (×), and the number of signals seen, for example, nuc ish(D13S319×2). The presence of an extra signal is reported as nuc ish(D13S319×3), whereas loss of one copy is reported as nuc ish(D13S319×1). If loci of two separate chromosomes are tested and they are juxtaposed (translocation), the results are expressed as follows: nuc ish(ABL1×2),(BCR×2),(ABL1 con BCR×1) or alternatively nuc ish(ABL1,BCR)×2(ABL1 con BCR×1).

Probe sets for the detection of most rearrangements associated with specific subtypes of leukemia or lymphoma are now available commercially and routinely used in cytogenetic laboratories to establish a diagnosis, select therapy, and monitor the effects of therapy ( Table 7-3 ). However, it is important to underline that in contrast to conventional cytogenetic analysis, which allows the simultaneous recognition of all microscopically detectable abnormalities in tumor cells regardless of whether they are numerical, structural, balanced, or unbalanced, FISH can be currently used only to detect the presence or confirm the absence of specific abnormalities that the probes used are designed to identify.

Similar to FISH, the chromogenic in situ hybridization (CISH) technique relies on the ability of DNA probes to hybridize specifically to complementary target DNA, but for signal identification CISH uses chromogens instead of fluorochromes used by FISH. An advantage of CISH is that evaluation can be performed with a conventional bright-field light microscope instead of fluorescence microscopy with multiband pass filters. This allows comparison of CISH results with the tumor area routinely stained. The limitations of CISH include a relatively low number of commercially available probes and, in contrast to FISH, difficulty in evaluating more than two different probes simultaneously.

FISH can also be combined with immunophenotyping, which is particularly useful in identifying the cell lineage of a cytogenetically aberrant neoplastic clone. Simultaneous fluorescence immunophenotyping (FICTION technique) allows visualization of antigen expression of cells with chromosomal aberrations directly correlating phenotypic and genotypic cell features. Different studies have demonstrated the application of combining FISH and cell-sorting techniques, as magnetic-activated cell sorting (MACS), in the diagnosis of plasma cell myeloma.

Multicolor Fluorescence In Situ Hybridization Techniques

SKY and M-FISH enable the simultaneous visualization of each of 22 pairs of autosomal chromosomes and both sex chromosomes in different colors. To prepare probes used for multicolor hybridizations, flow-sorted chromosomes are labeled with one to five fluorochromes to create a unique color for each chromosome pair. In SKY, image acquisition is based on a combination of epifluorescence microscopy, charge-coupled device imaging, and Fourier spectroscopy. In M-FISH, separate images are captured for each of five fluorochromes with narrow band-pass microscope filters; these images are then combined by dedicated software. Both methods have the ability to characterize complex rearrangements, define marker chromosomes, and identify cryptic translocations (see Fig. 7-4 ).

Multicolor images of metaphase cells hybridized with the SKY/M-FISH probe mixture are analyzed together with electronically inverted and contrast-enhanced DAPI images producing G-banding–like patterns that enable specific breakpoint assignments both in interchromosomal and intrachromosomal rearrangements. The final identification of chromosome aberrations and assignment of breakpoints in structural rearrangements is based on a combination of spectral classification, and G-banding ( Fig. 7-5 ). Additional FISH experiments are often required to clarify ambiguous results, and to confirm or refute the suspected involvement of specific genes located near breakpoints in structural abnormalities. The resolution of SKY/M-FISH for the detection of interchromosomal rearrangements is between 500 and 2000 kb and depends significantly on the quality of the metaphases and the resolution of the chromosomes involved in the rearrangement. As with banding techniques, subtle, subtelomeric translocations cannot be detected by SKY or M-FISH.

Comparative Genomic Hybridization

CGH is designed to scan the entire genome for gains, losses, and amplifications. In this method, test (tumor) and reference (normal) DNAs are differentially labeled and cohybridized to normal metaphase spreads (chromosomal CGH) or to microarrays (array CGH).

CGH has the advantage of requiring only tumor DNA extracted from either fresh or archived material. The reference DNA does not need to be from the same patient. The tumor DNA is usually labeled with a green fluorochrome (FITC/spectrum-green), and the reference DNA is labeled with a red fluorochrome (TRITC/spectrum-RED). The differences in CN between the tumor and normal DNA are reflected by differences in green and red fluorescence along the length of the chromosome. A number of hematologic malignancies have been analyzed by chromosomal CGH to identify genomic imbalances. One valuable finding has been the identification of high-level amplification of genes such as REL, MYC, and BCL2 in B-cell lymphomas. The importance of gene amplification as a genetic mechanism in the biology of lymphomas remained unrecognized by studies with G-banding alone. A caveat related to this assay is its inability to detect balanced genomic aberrations. Moreover, to be reliably detected, a gain or loss must usually be present in at least 35% of the tumor cells, and the altered regions must be at least 10 Mb. For detection of high-level amplifications, the size of a given amplicon must amount to at least 2 Mb.

Array-Based Copy Number Determination

Genetic complexity of cancer cells requires use of sensitive techniques that facilitate detection of small genomic changes in a mixed cell population and segmental regions of homozygosity. CGH arrays rely as conventional CGH on the difference in the CN between differentially labeled test and reference DNAs. The spots on the array are either DNA isolated from clones such as bacterial artificial chromosomes (BACs) containing human genomic DNA or oligonucleotides synthesized directly on the glass slide. For the CGH array, the DNAs are directly labeled with Cy3 and Cy5 fluorescent dyes, for example, with display tumor DNA pseudocolored red and reference DNA green. Again, through competition between test and control, a scanner detects the ratios of the fluorescence intensities of both dyes at each spot.

High-density oligonucleotide arrays have improved the ability to detect gains and losses of fewer than 5 kb, thus permitting the identification of smaller amplicons and microdeletions that were previously undetectable ( Fig. 7-6, A ). Moreover, application of paired germline DNA from the same individual can exclude germline variants, and differences will reflect only somatic lesions acquired by the tumor cells. Nevertheless, one of the limitations of the CGH arrays is that they do not allow detection of regions of homozygosity. The genome-wide SNP arrays rely on oligonucleotide probes corresponding to the allelic variants of selected SNPs covering the whole genome. Hybridization of test DNA to both probe variants indicates heterozygosity, whereas the signal for only one allele is consistent with homozygosity. The fluorescence emitted from individual probes allows the analysis of gene CN ( Fig. 7-6, B , upper panel ). The major advantage of SNP arrays over other CN platforms is the ability to detect diploid stretches of homozygosity ( Fig. 7-6, B , lower panel ). The detection of LOH and other chromosomal changes with large numbers of SNP markers has enabled identification of patterns of allelic imbalances with potential prognostic and diagnostic utility.

Molecular inversion probe (MIP) technology offers a potential solution to the challenges of CN and genotype assessment in formalin-fixed paraffin embedded (FFPE)-derived DNA samples. The small intact target DNA sequence footprint required by MIP probes (~40 bp) makes the MIP platform well suited to work with degraded FFPE DNA. The OncoScan assay uses molecular inversion probe (MIP) technology, which has been optimized for highly degraded FFPE samples (probe interrogation site of just 40 bp). Assay performance has been extensively validated with archived FFPE samples (10 years or older) and has been shown to be compatible with all major solid-tumor tissue types.

Application of these CN technologies, which use only single indirectly labeled tumor DNA for hybridization, has revealed that many normal copy number variations occur throughout the genome within the general population. Information on these regional variations must be taken into account when normal DNA from the patient whose tumor sample is tested is not available.

Although many molecular cytogenetic techniques are available, conventional cytogenetics and FISH are the most widely used techniques in the clinic (see Table 7-2 ). Nevertheless, CN arrays initially introduced in prenatal and postnatal diagnosis are increasingly used in the diagnosis of hematologic and oncologic disorders, especially in hematologic malignancies with a low mitotic index that does not allow conventional cytogenetics analysis. Moreover, CN arrays allow the detection of segmental regions of homozygosity and small genomic changes in a mixed cell population, and their use has identified novel genomic abnormalities that escaped detection with other methods. Moreover, CN arrays are a comprehensive tool for identification of chromothripsis, which requires the detection of at least seven switches between two or more CN states detected on an individual chromosome.

Thus, these array-based technologies have become a complementary tool in cases with existing cytogenetic information, and are used as the diagnostic tool (together with FISH) in cases without diving cells. CN arrays specifically designed for analyzing CN and LOH alterations on DNA from FFPE (MIP-assay) have been introduced recently.

Myeloproliferative Neoplasms

Among several entities included in the myeloproliferative neoplasms category in the WHO classification, only CML is strongly associated with a specific chromosome abnormality, t(9;22)(q34;q11.2), which creates the BCR - ABL1 fusion gene whose chimeric protein product is a target of therapy with tyrosine kinase inhibitors. The derivative chromosome 22 generated by the t(9;22) translocation is for historical reasons named the Philadelphia chromosome and designated as Ph. A vast majority, approximately 90% to 95%, of CML patients carry a standard t(9;22) translocation at diagnosis, whereas in the remaining patients, BCR - ABL1 arises as a result of either three-way or even four-way variant translocations involving, respectively, one additional chromosome, for example, t(1;9;22)(p36;q34;q11.2), or two extra chromosomes, e.g., t(3;17;9;22)(q26;q21;q34;q11.2); or through cryptic insertions such as ins(9;22)(q34;q11.2q11.2) or ins(22;9)(q11.2;q34q34). These cryptic insertions can be detected with FISH or PCR. At diagnosis, secondary abnormalities accompanying the t(9;22) translocation or variants, such as −Y, +8, i(17)(q10), +19, and +der(22)t(9;22)(q34;q11.2), are rare and are only detected in approximately 5% to 10% of patients. However, their presence has been reported to represent a poor prognostic factor in patients treated with imatinib, since patients who harbored any secondary abnormality had lower overall cytogenetic and molecular response rates and longer time to response to therapy. Patients who had the so-called major route abnormalities, that is, +8, i(17)(q10), +19 and +der(22)t(9;22), also had significantly shorter progression-free and overall survival (OS). Acquisition of chromosomal abnormalities, especially the major route ones, in a clone with a t(9;22) translocation during therapy with tyrosine kinase inhibitors (i.e., clonal cytogenetic evolution) indicates disease acceleration and has been associated with shorter OS in patients receiving imatinib. On the other hand, clonal chromosome aberrations, most often −Y and +8, occurring in cells without the t(9;22) translocation in 5% to 10% of CML patients during treatment with a tyrosine kinase inhibitor, appear not to affect patient outcomes. However, acquisition of −7 has been linked to the increased risk for developing MDS or AML, thus indicating the need for more frequent cytogenetic monitoring of such patients.

Myelodysplastic Syndromes