Rodak's Hematology: Clinical Principles and Applications, 5th Ed.

CHAPTER 30. Cytogenetics

Gail H. Vance


Reasons for Chromosome Analysis

Chromosome Structure

Cell Cycle

Chromosome Architecture

Metaphase Chromosomes

Chromosome Identification

Chromosome Number

Chromosome Size and Type

Techniques for Chromosome Preparation and Analysis

Chromosome Preparation

Chromosome Banding

Metaphase Analysis

Fluorescence In Situ Hybridization

Cytogenetic Nomenclature

Chromosome Abnormalities

Numeric Abnormalities

Structural Abnormalities

Cancer Cytogenetics


Solid Tumors

Chromosomal Microarray Analysis


After completion of this chapter, the reader will be able to:

1. Describe chromosome structure and the methods used in G-banded chromosome identification.

2. Explain the basic laboratory techniques for preparing chromosomes for analysis.

3. Differentiate between numeric and structural chromosome abnormalities.

4. Discuss the importance of karyotype in the diagnosis of hematologic cancer.

5. Explain the basic technique of fluorescence in situ hybridization (FISH).

6. Discuss the advantage of using FISH analysis in conjunction with G-banded analysis of cells.

7. Describe the types of chromosomal abnormalities that are detectable with cytogenetic methods.

8. Given a diagram of a G-banded chromosome, name the structures identifiable by light microscopy.

9. Given the designation of a chromosome mutation, be able to determine whether the abnormality is numeric or structural, which chromosomes are affected, what type of abnormality it is, and what portion of the chromosome is affected.

10. Define chromosomal microarray analysis.


After studying the material in this chapter, the reader should be able to respond to the following case study:

A 54-year-old man came to his physician with a history of fatigue, weight loss, and increased bruising over a 6-month period. His WBC count was elevated at 200 × 109/L. A bone marrow aspirate was sent for cytogenetic analysis. G-banded chromosome analysis of 20 cells from bone marrow cultures showed all cells to be positive for the Philadelphia chromosome, t(9; 22)(q34; q11.2), as seen in chronic myelogenous leukemia (Figure 30-1). FISH studies using the BCR and ABL1 gene probes (Abbott Molecular, Des Plaines, IL) produced dual fusion signals, one located on the derivative chromosome 9 and one on the derivative chromosome 22, characteristic of the translocation between chromosomes 9 and 22 leading to the rearrangement of BCR and ABL1 oncogenes (Figure 30-2). The patient was treated with imatinib mesylate for the next 2 months. Another cytogenetic study was performed on a second bone marrow aspirate. This analysis showed that 12 of 20 cells analyzed were normal, 46,XY[12]; however, there were still 8 cells positive for the Philadelphia chromosome, 46,XY,t(9; 22)(q34; q11.2)[8].

1. What is G-banded chromosome analysis?

2. Is the described mutation an example of a numeric or a structural abnormality? What type? Which chromosomes are involved? Explain.

3. What is FISH, and how does it complement standard chromosome analysis?


FIGURE 30-1 Karyogram for the patient in the case study showing a translocation between chromosomes 9 and 22, which is characteristic of chronic myelogenous leukemia. Source: (Courtesy the Cytogenetics Laboratory, Indiana University School of Medicine, Indianapolis, IN.)


FIGURE 30-2 Bone marrow metaphase cell from the patient in the case study hybridized with probes for BCR (green) and ABL1 (red) (Abbott Molecular, Des Plaines, IL). The fusion signals (yellow) represent the translocated chromosomes 9 and 22 (der(9) and der(22)). Source: (Courtesy the Cytogenetics Laboratory, Indiana University School of Medicine, Indianapolis, IN.)

Human cytogenetics is the study of chromosomes, their structure, and their inheritance. There are approximately 25,000 genes in the human genome, most of which reside on the 46 chromosomes normally found in each somatic cell.1

Chromosome disorders are classified as structural or numerical and involve the loss, gain, or rearrangement of either a piece of a chromosome or the entire chromosome. Because each chromosome contains thousands of genes, a chromosomal abnormality that is observable by light microscopy involves, on average, 3 to 5 megabases (Mb) of DNA and represents the disruption or loss of hundreds of genes. Such disruptions often have a profound clinical effect. Chromosomal abnormalities are observed in approximately 0.65% of all live births.2 The gain or loss of an entire chromosome, other than a sex chromosome, is usually incompatible with life and accounts for approximately 50% of first-trimester spontaneous abortions.3 In leukemia, cytogenetic abnormalities are observed in more than 50% of bone marrow specimens.4These recurring abnormalities often define the leukemia and frequently indicate clinical prognosis.

Reasons for chromosome analysis

Chromosome analysis is an important diagnostic procedure in clinical medicine. Not only are chromosomal anomalies major causes of reproductive loss and birth defects, but also nonrandom chromosome abnormalities are recognized in many forms of cancer.

Physicians who care for patients of all ages may order chromosome analysis or karyotyping for patients with mental retardation, infertility, ambiguous genitalia, short stature, fetal loss, risk of genetic or chromosomal disease, and cancer (). In the following discussion, basic cytogenetic concepts are presented. Supplementation of this chapter with the material in Table 30-1Chapter 31 is recommended.

TABLE 30-1

Common Translocations in Hematopoietic and Lymphoid Neoplasia and Sarcoma*

Tumor Type



Myeloid Leukemias

CML (and pre-B-ALL)

t(9; 22)(q34; q11.2)


Also see Box 31-1


B Cell Leukemias/Lymphomas

B lymphoblastic leukemia

t(12; 21)(p13; q22)


t(1; 19)(q23.3; p13.3)


t(4; 11)(q21; q23)


Burkitt lymphoma

t(8; 14)(q24; q32.3)


t(2; 8)(p12; q24)


t(8; 22)(q24; q11.2)


Mantle cell lymphoma

t(11; 14)(q13; q32.3)


Follicular lymphoma

t(14; 18)(q32.3; q21.3)


Diffuse large B cell lymphoma

t(3; 14)(q27; q32.3)


Lymphoplasmacytic lymphoma

t(9; 14)(p13.2; q32.3)


MALT lymphoma

t(14; 18)(q32.3; q21)


t(11; 18)(q22; q21)


t(1; 14)(p22; q32.3)


T Cell Leukemias/Lymphomas

T lymphoblastic leukemia



t(7; 11)(q34; p13)



t(2; 5)(p23; q35.1)


Sarcomas and Tumors of Bone and Soft Tissue

Alveolar rhabdomyosarcoma

t(2; 13)(q36.1; q14.1)


t(1; 13)(p36.13; q14.1)


Ewing sarcoma/PNET

t(11; 22)(q24; q12.2)


t(21; 22)(q22.3; q12.2)


t(7; 22)(p22; q12.2)


Clear cell sarcoma

t(12; 22)(q13; q12.2)


Myxoid liposarcoma

t(12; 16)(q13; p11.2)


t(12; 22)(q13; q12.2)


Synovial sarcoma

t(X; 18)(p11.2; q11.2)

SSX1 or SSX2/SS18

Alveolar soft part sarcoma

t(X; 17)(p11.2; q25)


* Modified per the Hugo Nomenclature Database, May 2013

ALCL, Anaplastic large cell leukemia; ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; CML, chronic myelogenous leukemia; CMML, chronic myelomonocytic leukemia; MALT, mucosa-associated lymphoid tissue; PNET, primitive neuroectodermal tumor.

Chromosome structure

Cell cycle

The cell cycle is divided into four stages: G1, the growth period before synthesis of deoxyribonucleic acid (DNA); S phase, the period during which DNA synthesis takes place; G2, the period after DNA synthesis; and M, the period of mitosis or cell division, the shortest phase of the cell cycle (Figure 6-5). During mitosis, chromosomes are maximally condensed. While in mitosis, cells can be chemically treated to arrest cell progression through the cycle so that the chromosomes may be isolated and analyzed.

Chromosome architecture

A chromosome is formed from a double-stranded DNA molecule that contains a series of genes. The complementary double-helix structure of DNA was established in 1953 by Watson and Crick.5 The backbone is a sugar-phosphate-sugar polymer. The sugar is deoxyribose. Attached to the backbone and filling the center of the helix are four nitrogen-containing bases. Two of these, adenine (A) and guanine (G), are purines; the other two, cytosine (C) and thymine (T), are pyrimidines (Figure 31-6).

The chromosomal DNA of the cell resides in the cell’s nucleus. This DNA and its associated proteins are referred to as chromatin. During the cell cycle, at mitosis, the nuclear chromatin condenses approximately 10,000-fold to form chromosomes.6 Each chromosome results from progressive folding, compression, and compaction of the entire nuclear chromatin. This condensation is achieved through multiple levels of helical coiling and supercoiling (Figure 30-3).


FIGURE 30-3 Chromosome structure. The folding and twisting of the DNA double helix. Source: (From Gelehrter TD, Collins FS, Ginsberg D: Principles of medical genetics, ed 2, Philadelphia, 1998, Lippincott Williams & Wilkins.)

Metaphase chromosomes

Metaphase is the stage of mitosis where the chromosomes align on the equatorial plate. Electron micrographs of metaphase chromosomes have provided models of chromosome structure. In the “beads-on-a-string” model of chromatin folding, the DNA helix is looped around a core of histone proteins.7 This packaging unit is known as a nucleosome and measures approximately 11 nm in diameter.8 Nucleosomes are coiled into twisted forms to create an approximately 30-nm chromatin fiber. This fiber, called a solenoid, is condensed further and bent into a loop configuration. These loops extend at an angle from the main chromosome axis.9

Chromosome identification

Chromosome number

In 1956, Tijo and Levan10 identified the correct number of human chromosomes as 46. This is the diploid chromosome number and is determined by counting the chromosomes in dividing somatic cells. The designation for the diploid number is 2n. Gametes (ova and sperm) have half the diploid number (23). This is called the haploid number of chromosomes and is designated as n. Different species have different numbers of chromosomes. The reindeer has a relatively high chromosome number for a mammal (2n = 76), whereas the Indian muntjac, or barking deer, has a very low chromosome number (2n = 7 in the male and 2n = 6 in the female).11

Chromosome size and type

In the 1960s, before the discovery of banding, chromosomes were categorized by overall size and the location of the centromere (primary constriction) and were assigned to one of seven groups: A through G. Group A includes chromosome pairs 1, 2, and 3. These are the largest chromosomes, and their centromeres are located in the middle of the chromosome; that is, they are metacentric. Group B chromosomes, pairs 4 and 5, are the next largest chromosomes; their centromeres are off center, or submetacentric. Group G consists of the smallest chromosomes, pairs 21 and 22, whose centromeres are located at one end of the chromosomes and are designated as acrocentric (). Figure 30-4


FIGURE 30-4 Chromosome morphology. The three shapes of chromosomes are metacentric, submetacentric, and acrocentric. This figure also shows the position of the centromere and telomere as well as the two sister chromatids that comprise a metaphase chromosome.

Techniques for chromosome preparation and analysis

Chromosome preparation

Tissues used for chromosome analysis contain cells with an inherently high mitotic rate (bone marrow cells) or cells that can be stimulated to divide in culture (peripheral blood lymphocytes). Special harvesting procedures are established for each tissue type. Mitogens such as phytohemagglutinin or pokeweed mitogen are added to peripheral blood cultures. Phytohemagglutinin primarily stimulates T cells to divide,12whereas pokeweed preferentially stimulates B lymphocytes.13

Chromosomes may be obtained from replicating cells by arresting the cell in metaphase. Cells from the peripheral blood or bone marrow are cultured in media for 24 to 72 hours. In standard peripheral blood cultures, since the cells are terminally differentiated, a mitogen is added to stimulate cellular division. Neoplastic cells are spontaneously dividing and generally do not require stimulation with a mitogen. After the cell cultures have grown for the appropriate period, Colcemid, an analogue of colchicine, is added to disrupt the mitotic spindle fiber attachment to the chromosome. Following culture and treatment with Colcemid, cells are exposed to a hypotonic (potassium chloride) solution that lyses red cells and causes the chromosomes to spread apart from one another. A fixative of 3:1 methanol and acetic acid is added that “hardens” cells and removes proteinaceous material. Cells are dropped onto cold, wet glass slides to achieve optimal dispersal of the chromosomes. The slides are then aged, typically by exposure to heat, before banding.

Chromosome banding

Analysis of each chromosome is made possible by staining with a dye. The name chromosome is derived from the Greek words chroma, meaning “color,” and soma, meaning “body.” Hence chromosome means “colored body.” In 1969, Caspersson and colleagues14 were the first investigators to stain chromosomes successfully with a fluorochrome dye. Using quinacrine mustard, which binds to adenine-thymine–rich areas of the chromosome, they were able to distinguish a banding pattern unique to each chromosome. This banding pattern, called Q-banding, differentiates the chromosome into bands of differing widths and relative brightnesses (Figure 30-5). The most brightly fluorescent bands of the 46 human chromosomes include the distal end of the Y chromosome, the centromeric regions of chromosomes 3 and 4, and the short arms of the acrocentric chromosomes (13, 14, 15, 21, and 22).


FIGURE 30-5 Q-banded preparation. Note the intense brilliance of Yq. Source: (Courtesy the Cytogenetics Laboratory, Indiana University School of Medicine, Indianapolis, IN.)

Other stains are used to identify chromosomes, but in contrast to Q-banding, these methods normally necessitate some pretreatment of the slide to be analyzed. Giemsa (G) bands are obtained by pretreating the chromosomes with the proteolytic enzyme trypsin. GTG banding means “G banding by Giemsa with the use of trypsin.” Giemsa, like quinacrine mustard, stains AT-rich areas of the chromosome. The dark bands are called G-positive (+) bands. Guanine-cytosine–rich areas of the chromosome have little affinity for the dye and are referred to as G-negative (−) bands. G+ bands correspond with the brightly fluorescing bands of Q-banding (Figures 30-6 and 30-7). G-banding is the most common method used for staining chromosomes.


FIGURE 30-6 Normal male metaphase chromosomes.


FIGURE 30-7 Normal male karyogram, GTG-banded preparations. Source: (Courtesy the Cytogenetics Laboratory, Indiana University School of Medicine, Indianapolis, IN.)

C-banding stains the centromere (primary constriction) of the chromosome and the surrounding condensed heterochromatin. Constitutive heterochromatin is a special type of late-replicating repetitive DNA that is located primarily at the centromere of the chromosome. In C-banding, the chromosomes are treated first with an acid and then with an alkali (barium hydroxide) before Giemsa staining. C-banding is most intense in human chromosomes 1, 9, and 16 and the Y chromosome. Polymorphisms from different individuals are also observed in the C-bands. These polymorphisms have no clinical significance (). Figure 30-8


FIGURE 30-8 C-banded male metaphase chromosomes. Note the stain at the centromere and heterochromatic regions of the chromosomes. Source: (Courtesy the Cytogenetics Laboratory, Indiana University School of Medicine, Indianapolis, IN.)

Specific chromosomal regions that are associated with the nucleoli in interphase cells are called nucleolar organizer regions (NORs). NORs contain tandemly repeated ribosomal nucleic acid (RNA) genes. NORs can be differentially stained in chromosomes by a silver stain in a method called AG-NOR–banding.

Chromosome banding is visible after chromosome condensation, which occurs during mitosis. The banding pattern observed depends on the degree of condensation. By examination of human chromosomes early in mitosis, it has been possible to estimate a total haploid genome (23 chromosomes) with approximately 2000 AT-rich (G+) bands.15 The later the stage of mitosis, the more condensed the chromosome and the fewer total G+ bands observed.

Metaphase analysis

After banding, prepared slides with dividing cells are scanned under a light microscope with a low-power objective lens (10×). When a metaphase cell has been selected for analysis, a 63× or 100× oil immersion objective lens is used. Each metaphase cell is analyzed first for a chromosome number. Then each chromosome pair is analyzed for its banding pattern. A normal somatic cell contains 46 chromosomes, which includes two sex chromosomes and 22 pairs of autosomes (chromosomes 1 through 22). The technologist records his or her summary of the analysis using chromosome nomenclature. This summary is called a karyotype. Any variation in number and banding pattern is recorded by the technologist. At least 20 metaphase cells are analyzed from leukocyte cultures. If abnormalities are noted, the technologist may need to analyze additional cells. Computer imaging or photography is used to confirm and record the microscopic analysis. A picture of all the chromosomes aligned from 1 to 22 including the sex chromosomes is called a karyogram.

Fluorescence in situ hybridization

The use of molecular methods coupled with standard karyotype analysis has improved chromosomal mutation detection beyond that of the light microscope. DNA or RNA probes labeled with either fluorescent or enzymatic detection systems are hybridized directly to metaphase or interphase cells on a glass microscope slide. These probes usually belong to one of three classes: probes for repetitive DNA sequences, primarily generated from centromeric DNA; whole-chromosome probes that include segments of an entire chromosome; and specific loci or single-copy probes.

Fluorescence in situ hybridization (FISH) is a molecular technique commonly used in cytogenetic laboratories. FISH studies are a valuable adjunct to the diagnostic workup. In FISH, the DNA or RNA probe is labeled with a fluorophore. Target DNA is treated with heat and formamide to denature the double-stranded DNA, which renders it single-stranded. The target DNA anneals to a similarly denatured, single-stranded, fluorescently labeled DNA or RNA probe with a complementary sequence. After hybridization, the unbound probe is removed through a series of stringent washes, and the cells are counterstained for visualization (). Figure 30-9


FIGURE 30-9 General protocol for fluorescence in situ hybridization. PCR, Polymerase chain reaction.

In situ hybridization with centromere or whole-chromosome painting probes can be used to identify individual chromosomes (Figure 30-10). Marker chromosomes represent chromatin material that has been structurally altered and cannot be identified by a G-band pattern. FISH using a centromere or paint probe, or both, is often helpful in identifying the chromosome of origin (Figure 30-11).16 Specific loci probes can be used to detect both structural and numerical abnormalities but are especially helpful in identifying chromosomal translocations or inversions.


FIGURE 30-10 Metaphase preparation is “painted” with multiple probes for chromosome 7, producing a fluorescent signal. Source: (Courtesy the Cytogenetics Laboratory, Indiana University School of Medicine, Indianapolis, IN.)


FIGURE 30-11 Fluorescence in situ hybridization in the clinical laboratory.

The FISH procedure has many advantages and has advanced the detection of chromosomal abnormalities beyond that of G-banded analysis. Both dividing (metaphase) and nondividing (interphase) cells can be analyzed with FISH. Performance of FISH on uncultured cells, such as bone marrow smears, provides a quick test result that can be reported in 24 hours. Also, in cultured bone marrow samples submitted for G-band analysis, the number of dividing cells may be insufficient for cytogenetic diagnosis. In such cases, FISH performed on interphase (nondividing) cells with probes for a specific translocation or structural abnormality may provide the diagnosis. FISH also can be performed on paraffin-embedded tissue sections, specimens obtained by fine needle aspiration, and touch preparations from lymph nodes or solid tumors.

Cytogenetic nomenclature

Banding techniques enabled scientists to identify each chromosome pair by a characteristic banding pattern. In 1971, a Paris conference for nomenclature of human chromosomes was convened to designate a system to describe the regions and specific bands of the chromosomes. The chromosome arms were designated p (petite) for the short arm and q for the long arm. The regions in each arm and the bands contained within each region were numbered consecutively, from the centromere outward to the telomere or end of the chromosome. To designate a specific region of the chromosome, the chromosome number is written first, followed by the designation of either the short or long arm, then the region of the arm, and finally the specific band. Xq21 designates the long arm of the X chromosome, region 2, band 1. To designate a subband, a decimal point is placed after the band designation, followed by the number assigned to the subband, as in Xq21.1 (Figure 30-12).


FIGURE 30-12 Banding pattern of the human X chromosome at the 550 average band level. Arrow indicates the location of Xq21.1.

Cytogenetic (and FISH) nomenclature represents a uniform code used by cytogeneticists around the world to communicate chromosome abnormalities. In this nomenclature each string begins with the modal number of chromosomes, followed by the sex chromosome designation. A normal male karyotype is designated 46,XY, and a normal female karyotype is designated 46,XX. If abnormalities are observed in the cell, the designation is written to include abnormalities of modal chromosome number, sex chromosomes, and then the autosomes. A cell from a bone marrow specimen with trisomy of chromosome 8 (three copies of chromosome 8) in a male is written as 47,XY,+8 (no intervening spaces). The number of cells with this abnormality is indicated in brackets. If 20 cells were examined, trisomy 8 was found in 10 cells, and the remainder were normal, the findings would be written as 47,XY,+8[10]/46,XY[10]. Translocations (exchange of material between two chromosomes) are designated t, with the lowest chromosome number listed first. Thus a translocation between the short arm of chromosome 12 at band p13 and the long arm of chromosome 21 at band q22 is written as t(12; 21)(p13; q22). A semicolon is used to separate the chromosomes and the band designations. A translocated chromosome is called a derivative chromosome. Using the previous example, chromosomes 12 and 21 are referred to as der(12) and der(21). Deletions are written with the abbreviation del preceding the chromosome. A deletion of the long arm of chromosome 5 at band 31 is written as del(5)(q31). No spaces are entered in these designations except between abbreviations.17

Chromosome abnormalities

There are many types of chromosome abnormalities, such as deletions, inversions, ring formations, trisomies, and polyploidy. All these defects can be grouped into two major categories: defects involving an abnormality in the number of chromosomes and defects involving structural changes in one or more chromosomes.

Numeric abnormalities

Numeric abnormalities often are subclassified as aneuploidy or polyploidy. Aneuploidy refers to any abnormal number of chromosomes that is not a multiple of the haploid number (23 chromosomes). The common forms of aneuploidy in humans are trisomy (the presence of an extra chromosome) and monosomy (the absence of a single chromosome). Aneuploidy is the result of nondisjunction, the failure of chromosomes to separate normally during cell division. Nondisjunction can occur during either of the two types of cell division: mitosis or meiosis. During normal mitosis, a cell divides once to produce two cells that are identical to the parent cell. In mitosis, each daughter cell contains 46 chromosomes. Meiosis is a special type of cell division that generates male and female gametes (sperm and ova). In contrast to mitosis, meiosis entails two cell divisions: meiosis I and meiosis II. The end result is a cell with 23 chromosomes, which is the haploid number (n).

In polyploidy, the chromosome number is higher than 46 but is always an exact multiple of the haploid chromosome number of 23. A karyotype with 69 chromosomes is called triploidy (3n) (Figure 30-13). A karyotype with 92 chromosomes is called tetraploidy (4n).


FIGURE 30-13 Triploid karyotype, 69,XXY. Source: (Courtesy the Cytogenetics Laboratory, Indiana University School of Medicine, Indianapolis, IN.)

In cancer, numerical abnormalities in the karyotype may be classified further based on the modal number of chromosomes in a neoplastic clone. Hypodiploid refers to a cell with fewer than 46 chromosomes;near-haploid cells have from 23 up to approximately 34 chromosomes (Figure 30-14); hyperdiploid cells have more than 46 chromosomes. High hyperdiploidy refers to a chromosome number of more than 50.18Finally the term pseudodiploid is used to describe a cell with 46 chromosomes and structural abnormalities.


FIGURE 30-14 Hypodiploid karyotype with 36 chromosomes (arrows indicate missing chromosomes).

Structural abnormalities

Structural rearrangements result from breakage of a chromosome region with loss or subsequent rejoining in an abnormal combination. Structural rearrangements are defined as balanced (no loss or gain of genetic chromatin) or unbalanced (gain or loss of genetic material). Structural rearrangements of single chromosomes include inversions, deletions, isochromosomes, ring formations, insertions, translocations, and duplications. Inversions (inv) involve one or two breaks in a single chromosome, followed by a 180-degree rotation of the segment between the breaks with no loss or gain of material. If the chromosomal material involves the centromere, the inversion is called pericentric. If the material that is inverted does not include the centromere, the inversion is called paracentric (Figure 30-15).


FIGURE 30-15 A, Pericentric inversion involves the centromere. B, Paracentric inversion occurs in either the short or long arm of the chromosome.

Interstitial deletions arise after two breaks in the same chromosome arm and loss of the segment between the breaks. Terminal deletions (loss of chromosomal material from the end of a chromosome) and interstitial deletions involve the loss of genetic material. The clinical consequence to the individual with a deletion depends on the extent and location of the deleted chromosomal material (Figure 30-16).


FIGURE 30-16 A, Interstitial deletion. B, Isochromosome. C, Ring chromosome. D, Insertion.

Isochromosomes arise from either abnormal division of the centromeres in which division is perpendicular to the long axis of the chromosome rather than parallel to it or from breakage and reunion in chromatin adjacent to the centromere. Each resulting daughter cell has a chromosome in which the short arm or the long arm is duplicated.

Ring chromosomes can result from breakage and reunion of a single chromosome with loss of chromosomal material outside the break points. Alternatively, one or both telomeres (chromosome ends) may join to form a ring chromosome without significant loss of chromosomal material.

Insertions involve movement of a segment of a chromosome from one location of the chromosome to another location of the same chromosome or to another chromosome. The segment is released as a result of two breaks, and the insertion occurs at the site of another break.

Duplication means partial trisomy for part of a chromosome. This can result from an unbalanced insertion or unequal crossing over in meiosis or mitosis.

Translocations occur when there is breakage in two chromosomes and each of the broken pieces reunites with another chromosome. If chromatin is neither lost nor gained, the exchange is called a balanced reciprocal translocation. A reciprocal translocation is balanced if all chromatin material is present. The loss or gain of chromatin material results in partial monosomy or trisomy for a segment of the chromosome, which is designated an unbalanced rearrangement.

Another type of translocation involving breakage and reunion near the centromeric regions of two acrocentric chromosomes is known as a Robertsonian translocation. Effectively this is a fusion between two whole chromosomes rather than exchange of material, as in a reciprocal translocation. These translocations are among the most common balanced structural rearrangements seen in the general population with a frequency of 0.09% to 0.1%.19 All five human acrocentric autosomes (13, 14, 15, 21, and 22) are capable of forming a Robertsonian translocation. In this case, the resulting balanced karyotype has only 45 chromosomes, which include the translocated chromosomes (Figure 30-17).


FIGURE 30-17 Balanced Robertsonian translocation between chromosomes 14 and 21. The nomenclature for this karyogram is written 45,XY,der(14; 21)(q10; q10). Source: (Courtesy the Cytogenetics Laboratory, Indiana University School of Medicine, Indianapolis, IN.)

Cancer cytogenetics

Cancer cytogenetics is a field that has been built upon discovery of nonrandom chromosome abnormalities in many types of cancer. In hematologic neoplasias, specific structural rearrangements are associated with distinct subtypes of leukemia that have characteristic morphologic and clinical features. Cytogenetic analysis of malignant cells can help determine the diagnosis and often the prognosis of a hematologic malignancy, assist the oncologist in the selection of appropriate therapy, and aid in monitoring the effects of therapy. Bone marrow is the tissue most frequently used to study the cytogenetics of a hematologic malignancy. Unstimulated peripheral blood and bone marrow trephine biopsy samples also may be analyzed. Cytogenetic analysis of cancers involving other organ systems can be performed using solid tissue obtained during surgery or by needle biopsy. Chromosomal defects in cancer include a wide range of numeric abnormalities and structural rearrangements, as discussed earlier (Table 30-1).

Cancer results from multiple and sequential genetic mutations occurring in a somatic cell. At some juncture, a critical mutation occurs, and the cell becomes self-perpetuating or clonal. A clone is a cell population derived from a single progenitor.17 A cytogenetica clone exists if two or more cells contain the same structural abnormality or supernumerary marker chromosome or if three or more cells are missing the same chromosome. The primary aberration or stemline of a clone is a cytogenetic abnormality that is frequently observed as the sole abnormality associated with the cancer. The secondary aberration or sideline includes abnormalities additional to the primary aberration.17 In chronic myelogenous leukemia, the primary aberration is the Philadelphia chromosome resulting from a translocation between the long arms of chromosomes 9 and 22, t(9; 22)(q34; q11.2). A sideline of this clone would include secondary abnormalities, such as trisomy for chromosome 8, written as +8,t(9; 22)(q34; q11.2).


Leukemias are clonal proliferations of malignant leukocytes that arise initially in the bone marrow before disseminating to the peripheral blood, lymph nodes, and other organs. They are broadly classified by the type of blood cell giving rise to the clonal proliferation (lymphoid or myeloid) and by the clinical course of the disease (acute or chronic). The four main leukemia categories are acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myelogenous leukemia (CML). The World Health Organization (WHO) classification for myeloid malignancies has categorized AML into seven subtypes: AML with recurrent genetic abnormalities; AML with myelodysplasia-related changes; therapy-related myeloid neoplasms; AML not otherwise specified; myeloid sarcoma; myeloid proliferations related to Down syndrome; and blastic plasmacytoid dendritic cell neoplasm (Chapter 35).20 “AML with recurrent genetic abnormalities” is a classification based on the cytogenetic abnormalities observed (Box 30-1). Some of the divisions of the French-American-British (FAB) classification21 are included in the “not otherwise classified” category. The WHO has classified lymphoid leukemias by precursor cell type, B or T (Chapter 35).

BOX 30-1

Acute Myeloid Leukemia (AML) with Recurrent Genetic Abnormalities*

AML with t(8; 21)(q22; q22); RUNX1T1/RUNX1

AML with inv(16)(p13.1q22) or t(16; 16)(p13.1; q22); /MYH11/CBFB

Acute promyelocytic leukemia with t(15; 17)(q24.1; q21.1); PML/RARA

AML with t(9; 11)(p22; q23); MLLT3/MLL(KMT2A)

AML with t(6; 9)(p23; q34); DEK/NUP214

AML with inv(3)(q21q26.2) or t(3; 3)(q21.3; q26.2); RPN1/MECOM

AML (megakaryoblastic) with t(1; 22)(p13; q13); RBM15/MKL1

AML with normal chromosomes and mutated NPM1(5q35.1)

AML with normal chromosomes and mutated CEBPA(19q13.1)

*Updated per Hugo Nomenclature Database, May 2013

Modified from Swerdlow SH, Campo E, Harris NL, et al, editors: WHO classification of tumours of haematopoietic and lymphoid tissues, ed 4, Lyon, France, 2008, IARC Press.

Chronic myelogenous leukemia

The first malignancy to be associated with a specific chromosome defect was CML, in which approximately 95% of patients were found to have the Philadelphia chromosome translocation, t(9; 22)(q34; q11.2) by G-banded analysis.2223 The Philadelphia chromosome (derivative chromosome 22) is characterized by a balanced translocation between the long arms of chromosomes 9 and 22. At the molecular level, the gene for ABL1, an oncogene on chromosome 9, joins a gene on chromosome 22 named BCR. The result of the fusion of these two genes is a new fusion protein of about 210 kD with growth-promoting capabilities that override normal cell regulatory mechanisms (Figures 30-18 and 30-19) (Chapter 33).24 The fusion protein activates tyrosine kinase signaling to drive proliferation of the cell. This signaling can be blocked by imatinib mesylate (Gleevec; Novartis Pharmaceuticals, East Hanover, NJ) or another tyrosine kinase inhibitor.25 Patient response to imatinib is monitored by cytogenetic analysis and FISH. At diagnosis, the characteristic karyotype is the presence of the Philadelphia chromosome in all cells analyzed. After treatment with imatinib for several months, the karyotype typically has a mixture of abnormal and normal cells indicating patient response to therapy. Complete response is defined as a bone marrow karyotype with only normal cells. Therapeutic response is often monitored using peripheral blood instead of a bone marrow aspirate. In contrast to the bone marrow, the peripheral blood does not contain spontaneously dividing cells. As a result, chromosomal analysis of a specimen of unstimulated peripheral blood may be unsuccessful because of the absence of dividing cells. In these cases, FISH with probes for the specific abnormality is performed on 200 or more interphase (nondividing) cells of the peripheral blood specimen to search for chromosomally abnormal cells. The detection of cytogenetic abnormalities in interphase (nondividing) cells is an important advantage of FISH technology.


FIGURE 30-18 Normal bone marrow interphase cell hybridized with the BCR (green) and ABL1 (red) genes (Abbott Molecular, Des Plaines, IL). The two red and two green signals represent the genes on the normal chromosomes 9 and 22. Source: (Courtesy the Cytogenetics Laboratory, Indiana University School of Medicine, Indianapolis, IN.)


FIGURE 30-19 Abnormal bone marrow interphase cell with one BCR (green) and one ABL1 (red) signal (Abbott Molecular, Des Plaines, IL) representing the normal chromosomes and two fusion signals from the derivative chromosomes 9 and 22. Source: (Courtesy the Cytogenetics Laboratory, Indiana University School of Medicine, Indianapolis, IN.)

Acute leukemia

The Philadelphia chromosome is also observed in acute leukemia. It is seen in about 20% of adults with ALL, 2% to 5% of children with ALL, and 1% of patients with AML. In childhood ALL, chromosome number is critical for predicting the severity of the leukemia. Children whose leukemic cells contain more than 50 chromosomes (hyperdiploid karyotype) have the best prognosis for complete recovery with therapy. Recurring translocations observed in ALL include t(4; 11)(q21; q23), t(12; 21)(p13; q22), and t(1; 19)(q23; p13.3). Each translocation is associated with a prognostic outcome and assists oncologists in determining patient therapy. The t(4; 11) translocation is the one most commonly found in infants with acute lymphoblastic leukemia. Rearrangements of the AFF1 gene on chromosome 4 and the MLL gene on chromosome 11 occur in this translocation.2930 Disruption of the MLL gene is seen in both ALL and AML (Figures 30-20 and 30-21).


FIGURE 30-20 Bone marrow metaphase cell with fusion MLL(KMT2A) signal on the normal chromosome 11 and split red and green signals on the translocated chromosomes, representing a disruption of the MLL(KMT2A) gene. Source: (Courtesy the Cytogenetics Laboratory, Indiana University School of Medicine, Indianapolis, IN.)


FIGURE 30-21 Bone marrow interphase cell with a fusion signal (normal chromosome 11) and split red and green signals from the MLL(KMT2A) gene representing a rearrangement. Source: (Courtesy the Cytogenetics Laboratory, Indiana University School of Medicine, Indianapolis, IN.)

The AMLs are subdivided into several morphologic classifications ranging from M0 to M7 according to the FAB classification (Chapter 35).3132 Characteristic chromosome translocations are associated with some subgroups and were incorporated into the WHO classification. Among them is a translocation between the long arms of chromosomes 8 and 21, t(8; 21)(q22; q22), which is representative of AML with maturation. Acute promyelocytic leukemia is associated with a translocation between the long arms of chromosomes 15 and 17, t(15; 17)(q24; q21) (Figure 30-22). A pericentric inversion of chromosome 16, inv(16)(p13.2q22), is seen in AML with increased eosinophils. The inversion juxtaposes the core the binding factor beta (CBFB) gene on 16q with the myosin heavy chain gene (MYH11) on 16p to form a new fusion protein (Figure 30-23).33 These recurring translocations have enabled researchers to localize genes important for cell growth and regulation. As with acute lymphoblastic leukemia, the specific translocationin AML often predicts patient prognosis and response to therapy. Understanding the molecular consequences of the cytogenetic mutations, such as the BCR/ABL1 translocation, provides the fundamental information for the development of targeted therapies.


FIGURE 30-22 Bone marrow metaphase chromosomes 15 and 17 homologues showing a translocation between the long arms of chromosomes 15 and 17, t(15; 17)(q24.1; q21.1), diagnostic of acute promyelocytic leukemia. The abnormal chromosomes are on the right. Source: (Courtesy the Cytogenetics Laboratory, Indiana University School of Medicine, Indianapolis, IN.)


FIGURE 30-23 Bone marrow karyogram for a patient with acute myeloid leukemia (AML) showing a translocation, t(9; 22)(q34; q11.2), and an inverted chromosome 16, inv(16)(p13.1q22).Source: (Courtesy the Cytogenetics Laboratory, Indiana University School of Medicine, Indianapolis, IN.)

Solid tumors

Just as recurring structural and numeric chromosome defects have been observed in the hematologic malignancies, a wide range of nonrandom abnormalities have also been found in solid tumors. Most of these abnormalities confer a proliferative advantage on the malignant cell and serve as useful prognostic indicators. Amplification (increased copy number) of the gene HER2 (also called ERBB2) on chromosome 17, a transmembrane growth factor receptor, is associated with an aggressive form of invasive breast cancer.3435 FISH with probes for the HER2 gene and an internal control (17 centromere) can determine if there is gene amplification in the tumor (Figure 30-24).36 If FISH testing shows amplification to be present, the patient is eligible for targeted therapy with a monoclonal antibody, trastuzumab (Figure 30-25).37 FISH forHER2 typically is performed on tissue sections from the paraffin-embedded tumor block.


FIGURE 30-24 Normal interphase nuclei from a paraffin-embedded tissue section hybridized with probes for HER2 (red) and the chromosome 17 centromere (green) (Abbott Molecular, Des Plaines, IL). Two green and two red signals are seen per cell.


FIGURE 30-25 Tissue section from a breast cancer demonstrating amplification of HER2. The tissue was hybridized with fluorescence in situ hybridization probes for HER2 (red) and the chromosome 17 centromere probe (green) (Abbott Molecular, Des Plaines, IL). The number of HER2 signals exceeds the number of centromere signals, which indicates selective amplification of HER2.

Chromosomal microarray analysis

Chromosomal microarray (CMA) is a fluorescence-based molecular technique for submicroscopic analysis of genomic DNA. CMA testing increases the detection of clinically significant imbalances over a karyotype.38 CMA is performed utilizing a glass slide or chip platform. Chromosomal microarrays, like standard cytogenetic analysis, look at the entire genome but with higher levels of resolution (base pair or kilobase level) determined by the number and composition of targets on the array. Using a SNP-based array, patient DNA is hybridized to a chip composed of greater than 2 million markers that detect copy number variation (gains and losses) and SNP polymorphisms. Single nucleotide polymorphism (SNP) probes detect position-specific markers that have different forms (polymorphic). Analysis of the SNP data from a specimen allows for detection of copy neutral loss of heterozygosity or uniparental disomy, as well as gains and losses of genomic DNA. Regions of imbalance (copy gain or copy loss) in the patient specimen are assessed relative to a reference control. The yield of detection of abnormalities is increased from an average of 3% to 11% due to the high resolution of the array (Figures 30-26 and 30-27).38-40 This technique is presently used primarily for diagnosis of constitutional (inherited) disorders, but emerging applications for cancer are in development.


FIGURE 30-26 Chromosomal microarray diagram demonstrating approximately a 32.4-Mb gain of genetic material between bands 7q21 and 7q31. Source: (Courtesy of the Cytogenetics Laboratory, Indiana University School of Medicine, Indianapolis, IN.)


FIGURE 30-27 Chromosomal microarray diagram demonstrating approximately a 4.6 Mb loss at band 7q31.1. (Courtesy of the Cytogenetics Laboratory, Indiana University School of Medicine, Indianapolis, IN.)


• Cytogenetics is the study of chromosome structure and inheritance.

• Chromosome disorders are secondary to structural or numeric chromosomal abnormalities involving the rearrangement or the loss or gain of a piece of a chromosome or the entire chromosome.

• Nonrandom chromosome abnormalities are associated with cancer.

• A chromosome is composed of a double helix strand of DNA. Attached to the backbone of deoxyribose are adenine (A), guanine (G), cytosine (C), and thymine (T).

• During mitosis, cells can be chemically treated to arrest cell progression in metaphase so that chromosomes can be analyzed.

• Q-banding differentiates chromosomes into bands of different widths and relative brightness, revealing a banding pattern unique to each individual chromosome.

• Other stains used to identify chromosomes may require pretreatment of the slide for analysis. These include G-banding, C-banding, and AG-NOR–banding.

• FISH, a molecular cytogenetic technique, uses DNA or RNA probes and fluorescence microscopy to identify individual chromosomes and targeted chromosomal loci. Metaphase and interphase cells can be analyzed by FISH.

• Tissues used for chromosome analysis typically include bone marrow cells and peripheral blood lymphocytes, amniotic fluid, nonneoplastic tissue, and tumors.

• A normal cell contains 46 chromosomes, which includes 2 sex chromosomes (XX or XY).

• Defects in chromosomes can be categorized as numeric or structural. Numeric abnormalities can be subclassified as aneuploidy and polyploidy.

• Structural rearrangements include inversions, deletions, isochromosomes, ring formations, insertions, translocations, and duplications.

• Specific structural rearrangements are associated with distinct subtypes of leukemias and may assist in diagnosis, prognosis, and monitoring of therapy. Solid tumors also may be analyzed using cytogenetics.

• Chromosomal microarray testing utilizes a microarray platform to detect abnormalities at a submicroscopic level of resolution. The higher resolution increases the detection of chromosomal abnormalities.

Now that you have completed this chapter, go back and read again the case study at the beginning and respond to the questions presented.

Review questions

Answers can be found in the Appendix.

1. G-banding refers to the technique of staining chromosomes:

a. To isolate those in the G group (i.e., chromosomes 21 and 22)

b. In the G0 or resting stage

c. Using Giemsa stain

d. To emphasize areas high in guanine residues

2. Which of the following compounds is used to halt mitosis in metaphase for chromosome analyses?

a. Imatinib

b. Fluorescein

c. Trypsin

d. Colchicine

3. One arm of a chromosome has 30 bands. Which band would be nearest the centromere?

a. Band 1

b. Band 15

c. Band 30

4. Which of the following is not an advantage of the use of FISH?

a. It can be used on nondividing cells.

b. It can be used on paraffin-embedded tissue.

c. It can detect mutations that do not result in abnormal banding patterns.

d. It must be performed on dividing cells.

5. Which of the following types of mutations would likely not be detectable with cytogenetic banding techniques?

a. Point mutation resulting in a single amino acid substitution

b. Transfer of genetic material from one chromosome to another

c. Loss of genetic material from a chromosome that does not appear on any other chromosome

d. Duplication of a chromosome resulting in 3n of that genetic material

6. Which of the following describes a chromosomal deletion?

a. Point mutation resulting in a single amino acid substitution

b. Transfer of genetic material from one chromosome to another

c. Loss of genetic material from a chromosome that does not appear on any other chromosome

d. Duplication of a chromosome resulting in 3n of that genetic material The chromosome analysis performed on a patient’s leukemic cells is reported as 47, XY,+4,del(5)(q31)[20]. Answer questions 7 to 9 based on this description.

7. This patient’s cells have which of the following mutations?

a. Loss of the entire number 31 chromosome

b. Loss of the entire number 5 chromosome

c. Loss of a portion of the short arm of chromosome 4

d. Loss of a portion of the long arm of chromosome 5

8. What other mutation is present in this patient’s cells?

a. Polyploidy

b. Tetraploidy

c. An extra chromosome 4

d. Four copies of chromosome 5

9. This patient’s leukemic cells demonstrate:

a. Structural chromosomal defects only

b. Numeric chromosomal defects only

c. Both structural and numeric chromosomal defects

10. Aneuploidy describes the total chromosome number:

a. That is a multiple of the haploid number

b. That reflects a loss or gain of a single chromosome

c. That is diploid but has a balanced deletion and duplication of whole chromosomes

d. In gametes; diploid is the number in somatic cells


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