Basic Cytogenetics and the Role of Genetics in Cancer Development

Chapter

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Basic Cytogenetics and the Role of Genetics in Cancer Development Alain Verhest and Pierre Heimann

Contents Introduction Historical Background Basic Knowledge of Cytogenetics Cell Cycle The Interphase The Mitosis The Meiosis The Chromosome Structure

Methodology The Karyotype Fluorescent in Situ Hybridization Comparative Genomic Hybridization (CGH)

Introduction This chapter will summarize the knowledge acquired on conventional cancer cytogenetics in the second half of the last ­century and introduces additional applications of fluorescent in situ hybridization available for the study of cancer development and evolution. Other indications of these techniques applied on cytology samples are also described in Chapter 36.

Historical Background As suspected by von Hansemann more than a century ago, cancers are associated with nuclear and mitotic anomalies in their cells. In 1914, Boveri hypothesized his theory on somatic mutations responsible for the origin and development of malignant transformation. He stressed the acquisition of an unbalanced chromosome constitution as a cause of cancer illustrated by mitotic asymmetry and asynchrony, and foresaw the monoclonal origin of the cancer cell. It took at least 40 more years to establish the exact number of human chromosomes. The blood-culturing method became more successful than the squash method when colcemid was discovered to arrest the mitotic cycle in metaphase by poisoning the mitotic spindle and to prevent the centromeres from dividing. The erroneous adjunction of a hypotonic solution to a pellet of harvested cells was an unexpected improvement in the spread of individualized chromosomes rid of their cellular envelope, resulting in a nicer dispersal on the metaphase spread.

Acquired Chromosomal Aberrations in Cancer Introduction Lymphomas Sarcomas Thyroid Carcinomas

Clinical Applications of Conventional Cytogenetics and Fish in Cytology Introduction FISH Strategy Application

Concluding Remarks

In 1956 Tjio and Levan accurately reported that the human somatic cell contains 46 chromosomes, including 22 pairs of autosomes and one pair of sex chromosomes; one X of maternal origin and the other chromosome—X or Y—being from the paternal source.1 Rarely have discoveries had such impact on modern biology and medicine as the description of the 46-chromosome karyotype. The newborn cytogenetic discipline investigated simultaneously the field of inherited diseases and acquired chromosomal anomalies in cancer cells. Trisomies of chromosome 21 in mongolism and of other autosomes or numerical variations of sex chromosomes proved their specificity and consequently their diagnostic value in congenital syndromes. In 1960 Nowell and Hungerford reported the first evidence of a chromosome anomaly specifically associated with a malignant disease, the chronic myelogenous leukemia.2 They showed the recurrent presence in leukemic leukocytes of a deleted small chromosome that they named the Philadelphia (Ph) chromosome in reference to the city where they were working. This proof of a genetic cause in cancer was the starting point to new insights into the pathways of malignant initiation and progression.

Basic Knowledge of Cytogenetics The human somatic cell contains two copies of each chromosome, one from paternal and the other from maternal origin. Therefore the karyotype is diploid with doubled amount of deoxyribonucleic acid (DNA) (2n) compared to the gametes (n) with a single set of 23 chromosomes. 23

PART ONE

General Cytology

The first step is to review the different stages of the cell cycle which are essential to the acquisition of chromosomes suitable for karyotyping.

Cell cycle checkpoint

Cell Cycle The cell cycle is a process of successive cell divisions (mitosis) interrupted by so-called “resting” periods (interphase). Actually, the resting cell is very active metabolically with continuous molecular interactions between DNA, ribonucleic acid (RNA), and proteins.

M G2 G1

G0

S

The Interphase The interphase is the period wherein the cell is in a nondividing state and can be at different stages: the first gap (G1) is between the last mitosis and the S-phase (phase of DNA synthesis) and the second gap (G2) is between the completion of the S-phase and the next mitosis (M). The mitotic division occupies only a short time in the cell cycle. If the cell reaches its ultimate stage of differentiation and will not divide anymore, the cell is said to be in phase G0 of the cycle. G0 applies also for those cells that have temporarily stopped dividing (Fig. 2.1). During the G1 phase, the cell is metabolically active and requires many organelles for protein synthesis while acquiring the potential for the DNA-doubling process. The duration of the entire cycle depends on the time of the G1 phase, which varies­ according to different conditions and tissue types. G1 phase may last from only a few hours to weeks or months, depending on the mitotic rate of the tissue. The phase of DNA synthesis (chromosome replication) has a duration of approximately 8 hours. The replication is not homogeneous throughout the genome, and asynchronism of replication occurs, particularly in the synthesis of the heterochromatin composing the inactivated X chromosome. DNA replication is achieved when all the chromosomes are duplicated in two identical sister chromatids with the consequence that the total amount of DNA is now doubled compared to the normal 2n value of the interphase nucleus. The following phase, G2, takes about 4 hours and accumulates the cytoplasmic organelles necessary to complete the mitosis. This step-by-step progression is controlled by a series of checkpoints which stop the process if the previous phase is not achieved. Different proteins act sequentially on the cell cycle: the cyclin-dependent kinases (CDKs), the cyclins, and the CDK inhibitors (CKIs). Activation of kinases by cyclins positively regulates the cycle by allowing the cell to enter the successive phases. If the quality of DNA synthesis is impaired, CKIs would automatically stop the process and drive the cell to apoptosis.

The Mitosis Although the cell cycle is a continuous process, mitosis has four distinct phases (Fig. 2.2).

Prophase Condensation and fragmentation of the chromatin into chromosomes becomes evident. The nucleolus vanishes and the centrioles, replicated in G2, migrate to opposite poles of the cell. Each chromosome is still attached to the nuclear membrane and composed of a double strand of sister chromatids. A constricted 24

Cell cycle checkpoint Fig. 2.1  Schematic representation of the cell cycle with the four sequential phases (see text). The cell cycle checkpoints are located at the G1/S and G2/M transitions.

area called centromere becomes apparent on the chromosomes and the nuclear membrane disintegrates.

Metaphase The chromosomes are aligned at the equatorial plate of the mitotic spindle and attached by their centromere to the network of microtubules. Metaphase chromosomes are composed of two sister chromatids joined together by the centromere.

Anaphase The centromeres are split into two parts and both strands of the sister chromatids are attracted to opposite pole by shortening of the spindle fibers. The chromosomes, pulled apart, are clustered at each pole of the cell.

Telophase Telophase results in the formation of a nuclear membrane. The constriction of the cellular membrane starts the division of the cytoplasm (cytokinesis). The chromosomes progressively melt back into a chromatin network. At the end, both daughter cells have the same number of chromosomes as the maternal cell.

The Meiosis The meiosis is a more complex process by which the gonad cell undergoes two cellular divisions. The meiosis I follows stages similar to the mitotic division. During prophase I, each chromosome is duplicated. Chromatid exchanges occur between paired homologue chromosomes which are linked together by their sites of junction: the chiasmas. This process, called “crossing over,” results in genetic recombination, with the consequence that genomes between maternal and daughter cells will not be strictly identical. Anaphase I start with the migration of homologue chromosomes to the opposite poles of the cell without splitting of their centromere. Meiosis II arises without previous DNA synthesis and produces the longitudinal separation of the two chromatids, thereby reducing the cell to a haploid n number of 23 single-stranded chromosomes. The fecundation of the ovule by the spermatozoid will ­restitute

2 Basic Cytogenetics and the Role of Genetics in Cancer Development

Interphase

Prophase

approximately 30 nm in diameter observable in electron microscopy. Further condensation makes it optically identifiable as heterochromatin in the interphase and as chromosomes at the late prophase. An animation on cell division and chromosome structure can be found at http://www. johnkyrk.com.3 The extremities of the chromosomes are called telomeres. They preserve the integrity of chromosomal extremities by allowing replication to occur without loss of coding sequences, but undergo repetitive shortenings themselves after each cellular division. The so-called “mitotic clock” counts the number of cell divisions that have occurred and pushes the cell to apoptosis before a critical telomeric shortening is reached. If this should occur, chromosomes would be prone to fuse end to end, giving rise to sticky ends that would favor mitotic aberrations and promote the accumulation of subsequent genetic rearrangements, possibly leading toward the first crucial steps in the development and progression of neoplasia.4

Methodology Metaphase

Anaphase

Telophase

Cytokinesis

Fig. 2.2  The four different phases constituting the mitotic process (cytokinesis being included in the telophase).

the diploid value of somatic cells and provide a complete zygotic genome.

The Chromosome Structure The chromosomes are composed of DNA and associated histone and non-histone proteins. This combination, called chromatin, is individualized into visible chromosomes only during mitosis. The double helix of DNA described by Watson and Crick is supercoiled around protein cores in a complex structure of nucleosomes. Compacted nucleosomes constitute chromatin segments of

The Karyotype In the 1950s and 1960s, human chromosomes were studied with Giemsa or Wright stains, making it possible for these chromosomes to be counted accurately and grouped together according to their length and the position of the centromeric constriction. The 22 pairs of autosomes and the sex chromosomes were thus classified into seven groups, A to G. The largest pairs are numbered 1 to 3 in group A. The centromere is located in the middle of chromosomes 1 and 3 and displaced in a submetacentric position in pair 2. Group B is composed of pairs 4 and 5, both with a subtelomeric centromere. Group C is the largest and is composed of medium-sized chromosomes including pairs 6 to 12 and chromosome X. Most of them are submetacentric and roughly classified by decreasing length. Group D is composed of chromosome pairs 13–15 and characterized by a distal acrocentric centromere. Group E contains the metacentric pair 16 and the submetacentric 17 and 18 sets. Chromosome pairs 19 and 20 are smaller metacentric chromosomes and constitute group F. Group G is composed of small acrocentric chromosomes arbitrarily placed in pairs 21 and 22. The small Y chromosome is included in group G. Accurate individual classification of chromosomes was rendered possible by the banding techniques developed first by applying fluorescent quinacrine mustard on metaphase preparations.5 This fluorescent agent reveals transverse bright bands (Q banding) of different intensities along the chromosome arms. Other procedures using trypsin digestion (which removes proteins from chromatin) and Giemsa staining yield dark G bands superimposed on the bright Q bands. This led to a very precise identification of each individual chromosome (Fig. 2.3). Techniques with heat denaturation in saline solution obtained a reverse staining called R bands with optional enhancing of telomeric ends in T banding. The different banding pattern for each of the 23 different chromosomes allows for a perfect pairing of homologues. The number of bands can be raised up to 800 by the high-resolution staining technique obtained on prometaphase chromosomes. The dark G bands correspond to a compact conformation of the chromatin while the clear bands are composed of rather 25

PART ONE

General Cytology

Fig. 2.3  G banded karyotype from a normal male showing 22 pairs of autosomal chromosomes and two X and Y sexual chromosomes

uncoiled chromatin. The dense Q and G bands are G+C-rich and contain repetitive inactive DNA. Active genes are supposed to be in clear bands; constitutive heterochromatin is located in the pericentromeric regions as revealed by C banding and appears as chromocenters in the nondividing nucleus. Chromosome Y has a unique strong fluorescent appearance visible in the interphase nucleus as a bright dot also visible as a dark C band. With these staining methods, the chromosomes 21, already recognized in the prebanding era because of their known involvement in Down syndrome, remained classified as such, and the minute marker of CML was consequently considered as belonging to pair 22.

The Standardized Reporting In 1971 at the International Conference in Paris, a nomenclature for banded chromosomes was adopted and extensively explained in a later revised version (ISCN 1985), codifying the way in which all possible numerical and structural chromosome anomalies should be reported.6 On each banded chromosome pair, the upper arms are designated as p arms (petit, meaning “short” in French); the longer arms below are designated as q arms. Regions and bands are numbered starting from the centromere. According to this nomenclature, the Philadelphia (Ph) chromosome was revealed by J. Rowley as being a t(9;22) ­translocation, hence the result of a reciprocal translocation between the q arms of chromosomes 9 and 22, with breakpoints positioned in q34 and q11 chromosomal regions, respectively7 (Fig. 2.4). Rapidly the complexity of ­ neoplasiaassociated ­ chromosome aberrations appeared difficult to adequate with the current nomenclature. Two standing committees proposed in 1991 and 1995 new consensus guidelines to suit the description of tumor karyotypes, including fluorescent in situ hybridization (FISH) methodology. However, the new abnormalities reported with an increasing variety of FISH probes and the new confusing subtleties of ISCN 1995 ­accumulated a greater rate of syntax errors. The heterogeneity of the observations and the variability of the banding resolution made these ISCN nomenclatures not very practical 26

to use for the description of cancer-associated chromosomal abnormalities, and still favors the use of personal simplified nomenclatures.8

Karyotyping Mitoses suitable for karyotyping are obtained more easily from lymphocyte cultures stimulated to grow by phyto­ hemagglutinin. Cells cultures from amniotic fluid or biopsy of chorionic villi allow antenatal diagnoses. Skin fibroblasts or fetal blood samples from the umbilical cord of stillborns are also suitable. Direct examination of bone marrow or short-term cultures are the techniques of choice for hematological disorders. Short-term tissue cultures took advantage of technical improvements such as methotrexate synchronization or collagenase digestion in the analysis of lymphomas and solid tumors. Chromosomes are counted and analyzed on slides. For ­decades, the better metaphase spreads were photographed, and each chromosome was manually cut out before being classified on a sheet of paper. Nowadays, on-screen karyotyping is the commonly used method for routine metaphase analysis. Once acquired by the automated capture device, metaphases can be quickly and accurately presented for chromosome assignment. The CytoVision system (Applied Imaging) used in our laboratory provides classifiers for standard banding methods (Fig. 2.5).

Fluorescent in Situ Hybridization The principle of in situ hybridization (ISH) is an uncoiling of the double DNA strand by heat denaturation followed by subsequent specific hybridization of the targeted DNA molecules with the complementary labeled DNA probe. By this procedure, ISH detects the precise location of unique DNA sequences directly on the chromosomes. The hybridized sequence is then revealed by two layers of fluorescent or chromogenic-labeled antibodies. FISH is preferred to chromogenic ISH (CISH) because of its higher sensitivity and the greater palette of artificial colors available.

2 Basic Cytogenetics and the Role of Genetics in Cancer Development

Fig. 2.4  G banded karyotype showing a t(9;22)(q34;q11) translocation corresponding to the so-called Philadelphia (Ph) chromosome. Arrows indicate the derivative chromosomes 9 and 22 involved in the translocation.

Different types of probes, including the centromeric, the locus specific, and the whole chromosome probes, are described:





• Centromeric probes are the most sensitive. They bind

to highly repetitive juxtacentromeric heterochromatin. Their strong signal remains very easily detectable on tissue sections. They are preferentially used for detecting gains and losses of entire chromosomes, namely aneusomies. • Locus-specific probes are designed to detect unique sequences spanning specific genomic loci. They are used to detect specific gene amplifications, duplications, deletions, or chromosomal translocations. These latter can be revealed by fusion of colors with the use of dual-colour probes flanking chromosomal breakpoints involved in translocations. • Whole chromosome probes, known as chromosome painting, reveal the whole chromosome except the centromeric region. They are used to identify the origin of chromosomal markers such as ring ­chromosomes and to refine complex chromosomal translocations.

In congenital diseases, the use of probes has advantageously circumvented the metaphase search for frequent trisomies or microdeletion syndromes. Although banding analysis remains the standard for identifying acquired chromosome abnormalities in cancer, FISH is now used as an easy and reliable technical substitute to search for well-documented specific chromosomal abnormalities in metaphase or interphase cells. Nowadays, FISH is used on a regular basis as a complementary tool to conventional cytogenetics, justifying the term “molecular cytogenetics.”

The FICTION Technique Fluorescent immunophenotyping and interphase cytogenetics as a tool for the investigation of neoplasms (FICTION) is a combination of fluorescent immunophenotyping and in situ hybridization, making it possible to study genetic ­abnormalities

Fig. 2.5  G-banding karyotype compared to the ideogram according to ISCN 1985.

in phenotypically selected cells.9 The technical principle and main practical applications of this method will be discussed later in this chapter. Suffice to say it is currently used to detect recurrent chromosomal abnormalities in multiple myeloma at diagnosis. This method may be applied to any type of tumor cell 27

PART ONE

General Cytology

Fig. 2.6  Multicolor FISH (SKY) allowing the identification of every human chromosomal pair with an individual color using 24 different painting probes (22 pairs of autosomes plus the two sexual chromosomes). Normal chromosomes are uniform in color, whereas rearranged chromosomes will display two or more colors. This method makes it possible to detect cryptic rearrangements and marker chromosomes in complex karyotypes as demonstrated here.

displaying a specific immunophenotype but is of little value for minimal residual disease.

Multicolor Metaphase FISH Multicolor-FISH includes mainly two different methods called spectral karyotyping (SKY) and multiplex-FISH (M-FISH). In SKY, the chromosomes are first stained with a mixture of 24 chromosome-specific painting probes; each one being labeled with a different combination of five fluorochromes. The spectral pattern of chromosomes is then classified using computer software to identify individual chromosomes. M-FISH uses a combinatorial labeling scheme with only five fluorochromes having different emission spectra. Those fluorochromes are similar to that used for SKY but the method for detecting and discriminating the different combinations of fluorescence signals is different. Both methods are useful in characterizing complex chromosomal rearrangements and in documenting ambiguous marker or ring chromosomes10 (Fig. 2.6).

Comparative Genomic Hybridization (CGH) This method has the advantage of circumventing the need for tumor cell metaphases. Total genomic tumoral DNA is labeled in green and the normal reference DNA in red. Both differentially labeled tumor and normal DNA will be hybridized together to normal human metaphases and will compete with one another. The ratio of the fluorescent green and red intensities is measured along every chromosome, making it possible to give an overview of DNA sequence copy number changes—gains and losses—in the neoplastic cells mapped on normal chromosomes. CGH is thus able to detect amplified and deleted genomic regions harboring oncogenes or tumor suppressor genes, respectively. The limitation of this method is that it can identify DNA imbalances but not balanced chromosomal translocations (hence, without loss or gain of chromosomal material subsequent to translocation). 28

Acquired Chromosomal Aberrations in Cancer Introduction It has long been agreed that tumor cells carry chromosomal aberrations, but their causes have only recently been more deeply explored.11,12 Until the seventies, cytogeneticists were dealing with malignant effusions or long-term cell cultures yielding roughly recognizable multiple chromosome changes, with very large amounts of rearranged DNA in complex aneuploidies. Consequently, this situation led to disillusion in the literature seeded by a plethora of reports with confusing malignant karyotypes, suggesting to most scientists that the chromosomal rearrangements observed were just epiphenomena accompanying the process of malignancy. At that time, no method was able to show molecular changes at the gene level. Karyotype analysis based on banding techniques renewed interest in the characterization of cytogenetic abnormalities in malignant tumors. It appeared evident that nonrandom primary changes involved specific chromosome regions, and were subsequently overwhelmed by secondary more massive variations affecting randomly all chromosomes. This state of overall genomic instability developed during the malignant clonal progression. Cytogenetic investigations focused initially on leukemias. They identified a constantly increasing number of characteristic chromosomal patterns after the Ph/CML association was detected. The FAB classification of leukemias was consequently enriched by the addition of prototypic karyotypic profiles. Beside leukemias, other cytogenetic and molecular information emerged with studies of lymphomas and sarcomas. In those tumors, relatively simple balanced rearrangements often appeared as fingerprints for a unique tumor type. These specific chromosomal abnormalities were rapidly considered as reliable diagnostic, prognostic, and predictive parameters on daily routine. The importance of chromosomal identification in the diagnosis of human leukemias, lymphomas, and mesenchymal

2 Basic Cytogenetics and the Role of Genetics in Cancer Development

A

B

Promotor A Promotor B

Gene A Gene B

Transcription

Fig. 2.7  Main molecular mechanisms subsequent to chromosomal translocations encountered in cancer. (A) In the first mechanism, breakpoints on both chromosomes will spare the coding sequence of the targeted genes. The translocation will lead to the juxtaposition of strong promoter/enhancers elements (blue lozenge) from one gene (A) with the entire intact coding sequence of another gene (B), leading to overexpression of this latter. In the classical example, promoter/enhancers are brought by Ig or TCR coding genes and the targeted coding sequence is oncogenes such as BCL2 or BCL1. (B) The second mechanism is characterized by chromosomal breakpoints occurring within the coding sequence of both genes involved in the translocation, leading to a chimeric gene translated into a hybrid protein with altered function. White vertical bars denote chromosomal breakpoints.

tumors is now recognized as a component of the current subclassifications in the World Health Organization (WHO) fascicules dealing with classification of tumors of hematopoietic and lymphoid tissues and soft-tissue tumors.12,13 Nowadays, the well-accepted opinion is that cancer is a genetic disease with two main genetic events triggering cancer initiation: the activation or deregulation of oncogenes as a consequence of point mutation, amplification, or chromosomal translocation; and the inactivation of tumor suppressor genes due to chromosomal deletion, mutation, or epigenetic mechanisms. In malignant epithelial tumors, the prevailing view is that they do not exhibit tumor-specific genetic alteration but rather complex karyotypes with multiple abnormalities shared by carcinoma of different histological subtypes and origins. However, single and specific chromosomal translocations are encountered in some epithelial malignancies such as thyroid carcinoma, kidney carcinoma of childhood and young adult, aggressive midline carcinoma, and a surprisingly great number of prostate cancer.14 Recurrent and specific chromosome abnormalities can be easily investigated by FISH at diagnosis. The method originally used on metaphase plates is also applicable on nondividing cells (interphase cells) provided by smears, cytospins, or paraffin-embedded tissues. It has proved to be suitable for the detection of numerical deviations on previously stained slides or fresh smears and to be feasible for improving the sensitivity of conventional cytology yielding “atypical cells” in cell suspensions.15 As it will be illustrated below, FISH may be more sensitive than conventional cytology. FISH combined with cytology can improve the diagnostic sensitivity of detecting malignancy in bronchial brushing and washing specimens.16 FISH has many

more applications in all fields of diagnostic cancer cytology, with significant improvement in tumor classification and a critical value in selection of patients who will benefit from targeted therapies (see Chap. 36). In the following sections, we will review the chromosomal abnormalities observed in lymphoma and sarcoma with their relationship to tumor development. We will mainly focus on specific aberrations that can be used as diagnostic tools in complement to cytology. In the same way, the examples of chromosomal markers in carcinoma will be limited to thyroid carcinoma. In a second part, we will discuss the applications of FISH in the field of cytology, again limiting our comments to lymphoma and sarcoma. The contribution of FISH in multiple myeloma will also be mentioned because of the novel and promising FICTION technique used to detect chromosomal abnormalities in selected nondividing plasma cells.

Lymphomas Recurrent chromosomal abnormalities in lymphoma are mainly represented by balanced chromosomal translocations that exert their tumorigenic action by two alternative molecular mechanisms (Fig. 2.7). In the first mechanism, the breakpoints on both chromosomes will occur adjacent to two genes and bring them close together but will not alter the protein produced by one of the targeted genes, mainly an oncogene. This latter is translocated close to strong promoter/enhancer elements of the other gene involved, hence the immunoglobulin (Ig) or T-cell receptor (TCR) genes. The functional consequences are constitutive activation of the oncogene through its overexpression 29

PART ONE

General Cytology

driven by Ig or TCR enhancers. In the second mechanism, the chromosomal breakpoints occur within the coding sequence of each gene, such that the two broken genes are fused, leading to a chimeric gene translated into a new chimeric protein with dysregulated function. The first mechanism accounts for the majority of lymphoma diseases while the second molecular event predominates in sarcoma. There is an abundant literature demonstrating good correlations between chromosomal abnormalities and different lymphoma subtypes.12 Identification of specific genetic aberrations has several meaningful implications in non-Hodgkin lymphoma’s (NHL). First, it may help in accurately diagnosing NHL. For example, identification of the t(11;14) translocation makes it possible to distinguish mantle cell lymphoma (MCL) from small lymphocytic lymphoma/chronic lymphoid leukemia (SLL/CLL). The presence of the t(2;5) translocation is the characteristic genetic feature of a subgroup of anaplastic large cell lymphoma (ALCL). Second, demonstration of chromosomal translocations may help in prognostic assessment of NHL; ­marginal zone lymphoma of MALT type with a t(11;18) is unlikely to respond to antibiotic therapy. By contrast, the MALTNHL negative for the t(11;18) is most often associated with Helicobacter pylori gastritis and more often responds to antibiotic therapy. The presence of the t(2;5) translocation and its consecutive anaplastic lymphoma kinase (ALK) overexpression in ALCL is associated with good prognosis. Third, identification of genetic abnormalities in NHL may serve as markers for staging assessment and for studies of minimal residual disease. As Hodgkin’s lymphoma does not exhibit any consistent or specific genetic abnormality detectable by cytogenetics or FISH analysis, the following topic will focus on non-Hodgkin’s lymphoma. Within this last group, we will restrict our talk to NHL subtypes exhibiting characteristic chromosomal aberrations that can be used as diagnostic tools on a regular basis, and will organize this section according to the REAL/WHO morphological classification, hence from small cell lymphomas to large cell lymphomas.

Follicular Lymphoma Follicular lymphoma (FL) is characterized by the t(14;18) (q32;q21) translocation (Fig. 2.8), which juxtaposes the B-cell lymphoma/leukemia 2 (BCL2) oncogene at 18q21 into the heavy chain immunoglobulin (IgH) gene locus at 14q32, leading to upregulated expression of the BCL2 protein.17 BCL2 is an antiapoptotic gene, and its overexpression leads to prolonged cell survival that may make the cell more vulnerable to additional genetics events, leading to cell overgrowth and cancer. In a minority of cases, variant translocations such as t(2;18)(p11,q21) and t(18;22)(q21;q11), which relocate the BCL2 oncogene to the kappa light chain immunoglobulin (IgL kappa) gene locus and lambda light chain immunoglobulin (IgL lambda) gene locus, respectively, have also been observed. This translocation t(14;18) and its variants are observed in up to 85% of FL which are mainly represented by histological grades 1, 2, and 3A. The remaining 15% cases do not exhibit a t(14;18)(q32;q21) translocation and are essentially constituted by FL grade 3B.18,19 Among them, a minority (~30%) exhibit BCL2 overexpression on immunohistochemistry, resulting from a non-Ig-related mechanism. The origin of this BCL2 gene overexpression is still unknown but could be due to duplication of chromosome 18 as observed in some karyotypes, or could involve other unknown mechanisms favoring BCL2 overexpression.19 30

Fig. 2.8  Karyotype of follicular lymphoma showing the balanced t(14;18)(q32;q21) translocation (arrows). The gains for chromosomes 3, 12, 15, and X as well as deletion 13q are additional abnormalities associated with clonal evolution.

The clinical outcome of this subgroup seems to be similar to that of follicular lymphoma with t(14;18). The major subgroup (~70%) does not show any BCL2 overexpression but presents a recurrent translocation of the 3q27 chromosomal region, resulting in a disruption of the B-cell lymphoma/leukemia 6 (BCL6) oncogene located at this breakpoint. This abnormality is also observed in diffuse large B-cell lymphomas (DLBCL), a feature that will be discussed later. Of interest, these 3q27+ FL grade 3B show peculiar clinicopathologic features distinct from their t(14;18)+ counterparts20: a stage III/IV disease as well as a bulky mass are less frequently observed, and they usually disclose a CD10− phenotype. Finally, this genetic subgroup seems to have a better survival rate and have clinically more in common with de novo 3q27+ DLBCL.19,20 These findings indicate that the search for BCL2 and BCL6 rearrangement status by genetic analysis may be clinically warranted for all cases of follicular lymphoma. Although the t(14;18) translocation is an early event and is critical for lymphomagenesis, it is by itself insufficient to produce FL. As said before, the prolonged cell survival provided by BCL2 overexpression allows the acquisition of further genetic events that contribute to the development of FL.21 These genetic events occur as a series of chromosomal gains and losses that can be detected at diagnosis as complex and heterogeneous karyotypes. It is not the karyotypic complexity but rather the type of abnormalities exhibited that underlies the varied clinical outcome observed in FL. Recurrent cytogenetic aberrations that have been noted to correlate with a more aggressive disease include chromosomal gains such as +7, +12 or gain of 12q1314, +18 and chromosomal losses including del 6q, del(9)(p21), and del(17)(p13), the two last aberrations corresponding to loss of tumor suppressor genes p16 and p53. Beside a complex karyotype, 3q27/BCL6 translocations can subsequently occur in t(14;18)+ FL, less frequently in low than in high grades, and have been shown to correlate with a risk of transformation to diffuse large B-cell lymphoma.22

Mantle Cell Lymphoma According to the REAL/WHO classification, the diagnosis of mantle cell lymphoma should be based on clinicomorphological­ but also cytogenetic or molecular features.12 The genetic hallmark of MCL is the t(11;14)(q13;q11) translocation (Fig. 2.9) that ­ juxtaposes

2 Basic Cytogenetics and the Role of Genetics in Cancer Development

Fig. 2.9  Karyotype of mantle cell lymphoma displaying the t(11;14)(q13;q11) chromosomal translocation (arrows), associated with multiple additional abnormalities such as interstitial deletion involving one chromosome 13, loss of the normal chromosome 14, and marker chromosome (A). This profile is observed in aggressive cases.

part of the IgH locus on chromosome 14q32 to the entire coding sequence of BCL1 oncogene, also named Cyclin D1 or PRAD1, located on chromosome 11q13.23 BCL1 gene is thus brought under the control of an IgH enhancer, leading to overproduction of cyclin D1 protein, a mechanism similar to that observed for the BCL2 oncogene in FL. Cyclin D1 is one of the key regulators of the cell cycle, and complexes with CDK-4 and -6 in order to promote the G1/S-phase transition of the cell cycle. Increased Cyclin D1 production in MCL will dramatically induce cells to enter the S-phase and, therefore, tumor cell proliferation, by inhibiting the cell cycle inhibitory effects of the retinoblastoma (Rb) and CDK inhibitors p27kip1 proteins.24 Concurrent disruptions of other cell cycle-associated genes contribute also to the pathogenesis of MCL. In particular, homozygous deletions of the CDK inhibitor p16INK4 were observed in aggressive variants of MCL. p16INK4 is an inhibitor of CDK-4 and -6 and thus maintains the Rb protein activity by preventing its phosphorylation. p16INK4 deletion and an increased level of Cyclin D1 may therefore work together in promoting the G1/S-phase transition in MCL cells. The t(11;14) translocation is very specific to MCL among other B-NHL and is detected by conventional cytogenetics in 60–75% of MCL cases, but this number rises to nearly 100% with the use of FISH. Beside the presence of t(11;14) translocation, the study of the overall cytogenetic profile brings prognostic meanings. Normal karyotype or karyotype with a single t(11;14) is associated with the typical form of MCL and is a good prognostic factor. In the majority of aggressive cases, t(11;14) is associated with a complex karyotype including numerous structural and numerical alterations of chromosomes 1, 2, 3, 9, 11, 13, 17 as well as unidentified chromosomal aberrations (markers).25 Also, near-tetraploid karyotypes (hence ±92 chromosomes) seem to be characteristic for the blastoid variant MCL. These karyotypic features occurring in aggressive MCL cases reflect the existence of alterations in both the DNA damage response pathways and mitotic checkpoints that may constitute another important pathogenetic mechanism in this lymphoma subtype. Indeed, one of the most frequently additional cytogenetic aberrations observed in MCL is deletion in the 11q22-23 chromosomal region where the ATM (ataxia-telangiectasia mutated) gene is located. ATM gene plays

a key role in genomic stability by activating gatekeeper and caretaker genes such as p53 and BCRA1 in response to DNA damage.26 ATM inactivation in MCL is associated with a high number of chromosomal alterations, suggesting that it may, at least in part, be responsible for the chromosomal instability in these lymphomas.26

Marginal Zone B-cell Lymphoma Several chromosomal abnormalities are encountered in marginal zone B-cell lymphomas (MZL) and are distributed according to the three different subtypes: extranodal MZL of MALT type, nodal MZL, and splenic MZL. In MALT lymphoma, four main recurrent chromosomal translocations have been observed and demonstrate a site-specificity in terms of their incidence: t(11;18)(q21;q21), t(1;14) (p22;q32), t(14;18)(q32;q21), and the recently described t(3;14) (p14.1;q32) (Table 2.1). The latter, limited to MALT lymphoma of the thyroid, skin and ocular adnexa regions, leads to the juxtaposition of the transcription factor Forkhead box-P1 (FOXP1) next to the enhancer region of the IgH gene.27 This molecular event results in FOXP1 gene overexpression but the patho­ genetic relevance of this translocation is still not known. The three other translocations affect a common signaling pathway, resulting in the constitutive activation of the nuclear factor-κB (NF-κB), a transcription factor which plays a major role in cellular activation, proliferation and survival.28 The t(1;14)(p22;q32) is detected in approximately 5% of MALT lymphoma, arising in localizations such as stomach, intestine, and lung. This translocation results in overexpression of the BCL10 gene (chromosome 1p22) due to its juxtaposition with the IgH gene enhancer. The t(14;18)(q32;q21) translocation, cytogenetically identical to the t(14;18)(q32;q21) involving BCL2 gene in follicular lymphoma, is observed in more or less 20% of MALT lymphoma, especially in non-gastrointestinal localizations such as liver, lung, salivary glands, skin, and ocular adnexa. This translocation brings the mucosae-associated lymphoid tissue (MALT1) gene, also involved in antigen-receptor-mediated NF-κB activation, under the control of the IgH enhancer region, with subsequent MALT1 overexpression. The t(11;18)(q21;q21) represents the most common translocation, accounting for 31

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General Cytology

Table 2.1  Four main recurrent chromosomal translocations observed in MALT lymphomas

t(11;18)(q21;q21)

t(1;14)(p22;q32)

t(14;18)(q32;q21)

t(3;14)(p14.1;q32)

Product

cIAP2-MALT1 fusion protein

Overexpression of BCL10

Overexpression of MALT1

Overexpression of FOXP1

% of cases

15–40

1–2

20

Unknown

Main lymphoma localizations

Stomach, lung, intestine

Stomach, lung

Salivary glands, skin, ocular adnexa, liver, lung

Thyroid, ocular adnexa, skin

Fig. 2.10  Karyotype showing an interstitial deletion of chromosome 7q as observed in splenic marginal zone lymphoma. In the present case, the deletion involves the 7q22q32 chromosomal segment.

15–40% of cases, and is observed in stomach, intestine, and lung MALT lymphoma cases. It results in the reciprocal fusion of the API2 and MALT1 genes. API2 (cellular inhibitor of apoptosis protein 2) gene is believed to be an apoptosis inhibitor by inhibiting the biological activity of caspases 3, 7, and 9. The pathogenesis of those three translocations sharing the same molecular pathway is beginning to be understood.28,29 NF-κB activation is driven by stimulation of cell-surface receptors, such as Bor T-cell receptors. In unstimulated lymphocytes, NF-κB proteins are bound with inhibitory κB (IκB) proteins and sequestered in the cytoplasm. Phosphorylation of the IκB proteins by the IκB kinase (IKK) heterodimer leads to ubiquitylation and degradation of IκB, allowing NF-κB to migrate to the nucleus and transactivate genes involved in cellular activation, proliferation and survival, and induction of effector function of lymphocytes. In MALT lymphoma with t(1;14) translocation and BCL10 overexpression, BCL10 is able to complex with MALT1 and trigger aberrant NF-κB activation without the need for upstream signaling. With the t(14;18) translocation causing MALT1 overexpression, MALT1 interacts and stabilizes BCL10, leading to its cytoplasmic accumulation. Both proteins in high cellular concentration will then synergistically favor a constitutive NF-κB activity. In t(11;18) positive MALT lymphoma, the API2-MALT1 chimeric protein activates NF-κB through self-­oligomerization, and bears a gain of function when compared to wild type MALT1. This higher activation is also due to the API2 protein partner. Indeed, wild-type API2 downregulates BCL10 expression by ubiquitylation and degradation, a mechanism used to regulate BCL10 activity after antigen receptor stimulation. 32

The API2–MALT1 protein is no longer able to ubiquitylate it and high BCL10 expression will synergistically increase API2– MALT1’s intrinsic capacity for NF-κB activation, independently of any antigen-receptor activation. Because of their specificity, the identification of these chromosomal translocations can be of interest for diagnostic purposes. They have also an immediate impact on treatment decisions, at least for two of them. Indeed, a causal relationship between H. pylori infection in the stomach and development of gastric MALT lymphoma has been clearly demonstrated, and 75% of these lymphomas can be successfully treated with appropriate antibiotics targeting H. pylori.28 However, the presence of either the t(11;18) or t(1;14) translocation defines patients who will not respond to H. pylori eradication. At the opposite, gastric MALT lymphoma without these chromosomal translocations, sometimes carrying trisomies of chromosomes 3, 12, and 18, can be effectively treated by antibiotic treatment, at least at their early stages. However, they can progress, become H. pylori-independent and transform into high-grade tumors following the acquisition of additional genomic alterations (such as TP53 and CKN2A inactivation). Intriguingly, t(11;18) positive MALT lymphomas will rarely develop into highgrade tumors, unlike their t(1,14) counterparts. These clinical features indicate that chromosomal abnormalities in some MALT lymphoma can also serve as prognostic parameters. In splenic marginal zone lymphoma (SMZL), cytogenetic alterations include mainly partial or complete trisomy 3, and interstitial deletion of chromosome 7q involving segments of variable size, usually centered around the 7q31q32 region (Fig. 2.10). Recent gene expression profiling revealed that genes

2 Basic Cytogenetics and the Role of Genetics in Cancer Development

A

B

Fig. 2.11  (A) Karyotype of a diffuse large B-cell lymphoma exhibiting a characteristic t(3;14)(q27;q32) chromosomal translocation (arrows). Other abnormalities such as additional material of unknown origin attached to the 1p36 chromosomal region of one chromosome 1, to the 6p24p25 chromosomal regions of both chromosomes 6, and deletion of the 7p21 segment of one chromosome 7 are additional aberrations reflecting clonal evolution. (B) Metaphase FISH with the use of a dual-color (green and red) break-apart probe specific to the BCL6 gene. The yellow signal (juxtaposition of green and red colors) identifies the normal BCL6 gene, whereas splitting of the green and red signals indicates a disruption of the other BCL6 gene subsequent to the t(3;14) chromosomal translocation.

mapping to the 7q31 chromosomal region were consistently downregulated, among which three were found to be very SMZL-specific: ILF1, Senataxin, and CD40.30 Nodal marginal zone lymphoma (NMZL) is a very rare disease. However, local regional lymph node of MALT lymphoma is virtually indistinguishable from NMZL, requiring clinical information and, in some respect, cytogenetic data to diagnose it. NMZL is characterized by frequent trisomies of ­chromosomes 3, 7, and 18, but the characteristic translocations of MALT ­lymphoma are never seen.12

Small Lymphocytic Lymphoma The histology, immunophenotypic and cytogenetic features of small lymphocytic lymphoma are indistinguishable from the more common CLL.12 Chromosomal aberrations observed in SLL include thus trisomy 12, 11q, and 17p deletions—all of them being poor-risk cytogenetic parameters—and a 13q14 deletion which is considered as a marker of good prognosis. A t(14;19)(q32;q13) translocation occurs infrequently in SLL and juxtaposes the BCL3 gene located on chromosome 19 next to the enhancer region of the Ig-heavy-chain gene, leading to BCL3 overexpression. When present, it confers a more aggressive behavior.31

Lymphoplasmacytic Lymphoma Lymphoplasmacytic lymphoma (LPL) is a rather uncommon entity but its diagnosis remains challenging for most pathologists. Cytogenetic investigations had previously considered the t(9;14)(p13;q32)—juxtaposing the PAX5 transcription factor with the Ig-heavy-chain gene enhancer—as characteristic of LPL, but more recent studies question the accuracy of this association. Firstly, no PAX5 rearrangement was detected in a series of 13 LPL.32 Secondly, PAX5/IgH rearrangement was observed in

other types of lymphoma including T-cell-rich B-cell lymphoma, post-transplantation diffuse large B-cell lymphoma, and some cases of SMZL.33

Diffuse Large B-cell Lymphoma Diffuse large B-cell lymphoma is a very heterogeneous clinicopathologic entity, displaying numerous and disparate chromosomal aberrations. In this section, we will only focus on the most frequent cytogenetic aberrations observed in DLBCL, hence chromosomal translocations involving BCL6, BCL2 and C-MYC oncogenes. The translocations involving the 3q27 chromosomal region are the most characteristic and frequent cytogenetic aberrations, detected in 30 to 40% of DLBCL12 (Figs 2.11A and 2.11B). The 3q27 breakpoint involves the BCL6 gene, which is required for germinal center (GC) formation and the B-cell immune response. The gene partners of the BCL6 chromosomal translocations are multiple. They most often involve the Ig-heavy- or -light-chain (κ and λ) genes on chromosome bands 14q32, 2p11 and 22q11, but more than 20 non-Ig partners have also been described, a phenomenon termed “promiscuous translocation”.34 Whatever the partner is, the chromosomal translocation brings the entire coding sequence of BCL6 under the control of a replaced promoter that will cause its deregulated expression during B-cell differentiation. BCL6 plays a key role in the generation of a germinal center by B cells. It encodes a transcriptional repressor protein that downregulates the expression of the B-lymphocyte-induced maturation protein 1 (BLIMP1) gene necessary for plasma cell differentiation, and also the expression of p27kip1, cyclin D2, and P53 which control the cell cycle, apoptosis, DNA repair, and maintenance of genomic stability.35 In a normal situation, BCL6 expression is tightly regulated during B-cell ontogenesis, being 33

PART ONE

General Cytology

Fig. 2.12  Karyotype showing a t(8;14)(q24;q32) chromosomal translocation (arrows) characteristic of Burkitt’s lymphoma (or ALL L3). Segmental duplication of chromosome 1q and loss of chromosome 17p are recurrent additional chromosome aberrations in this type of lymphoma.

restricted to B cells in the GC. In contrast, the heterologous Ig and non-Ig promoters exhibit a broader spectrum of activity in B-cell ontogenetic stages and will prevent BCL6 downregulation in post-GC cells. A block in the normal downregulation of BCL6 might thus favor differentiation arrest, continuous cell proliferation, survival, and genetic instability, all of which allowing neoplastic transformation. Indeed, the 3q27/BCL6 rearrangement is sufficient in itself to produce lymphoma as demonstrated by transgenic mice studies. In addition and independently of BCL6 translocations, point mutations and small deletions of BCL6 have been reported in approximately 70% of DLBCL, leading also to its deregulated expression.35 The clinical relevance of BCL6 gene translocations has been initially a subject of controversy with studies reporting improved survival in patients with BCL6 translocation, and other failing to show any statistically significant impact of such rearrangements on the clinical outcome of DLBCL.36 More recently, a cDNA microarray analysis demonstrated that DLBCL patients with the germinal center B-cell-like (GCB) gene expression profile had a better overall survival than those with the activated B-cell-like (ABC) expression pattern.37 As BCL6 is a marker of the GCB-type signature, its mRNA and protein levels were correlated to clinical outcome of DLBCL patients: high-level expression of BCL6 was associated with significantly longer overall survival and shown to be a predictor of a favorable treatment outcome in cases of DLBCL.36 In some cases, 3q27/BCL6 translocation coexists with other translocations in a single clone, including t(14;18)(q32;21) and t(8;14)(q24;q32), involving BCL2 and c-MYC oncogenes, respectively. This coexistence of two to three chromosomal translocations seems not necessarily to have a significant impact on the clinical features.38 Finally, it must be added that around 20% of DLBCL exhibit a t(14,18)(q32;q21) similar to that associated with follicular lymphoma and mutually exclusive of BCL6 rearrangements.

Burkitt’s Lymphoma Burkitt’s lymphoma (BL) and its leukemic equivalent, the L3 variant of acute lymphoblastic leukemia, are characterized in 34

nearly 90% of cases by a reciprocal chromosomal translocation that juxtaposes the c-Myc oncogene (chromosome 8q24) to one of the immunoglobulin genes located on chromosome 14q32 (IgH), chromosome 22q11 (Igλ), or chromosome 2p12 (Igκ) (Fig. 2.12). All three chromosomal translocations lead to overexpression of the c-Myc gene product. C-Myc gene is a transcription factor that regulates a very large number of genes through heterodimerization with the partner protein Max.39 The genes targeted by the c-Myc/Max heterodimer complexes are involved in cell proliferation, differentiation, and apoptosis. Such global transcriptional regulatory function may explain why c-Myc overexpression is sufficient in itself to promote lymphoma diseases as demonstrated in transgenic mice studies. The so-called “Burkitt-like” form is characterized by three cytogenetic categories: one with an 8q24/c-MYC ­translocation, a second with associated 8q24/c-MYC and 18q21/BCL2 translocations, and a third with miscellaneous rearrangements, frequently including an 18q21/BCL2 chromosomal translocation. Recurrent chromosome aberrations associated with the 8q24 translocations include duplications of the 1q21q25 chromosomal region, 6q11q14 and 17p chromosomal deletions, and trisomies for chromosomes 7, 8, 12, and 18. A recent cytogenetic and CGH study on BL has demonstrated that the presence of abnormalities on chromosome 1q (demonstrated either by cytogenetics or by CGH) and gains of 7q (ascertained only by CGH) were associated with adverse prognosis.40

Anaplastic Large Cell Lymphoma Anaplastic large cell lymphoma is a CD30+ T-cell NHL that can be divided in two majors groups according to the WHO classification: (1) systemic nodal ALCL and (2) primary cutaneous ALCL. As this latter group does not exhibit specific chromosomal alteration, it will not be pursued further in this review. In this section, we will only focus on systemic nodal ALCL, more particularly on anaplastic lymphoma kinase-positive ALCL where a characteristic t(2;5)(p23;q35) translocation is observed in approximately 60% of cases. This translocation fuses the nucleophosmin (NPM) gene on chromosome 5q35 to the ALK gene on chromosome 2p23, leading to the NPM-ALK chimeric

2 Basic Cytogenetics and the Role of Genetics in Cancer Development

gene.41 It is present in approximately 75% of ALCL with ALK gene rearrangement. In the remaining approximately 25% of cases, 2p23/ALK locus translocates with various partner genes.41 The common molecular features of all ALK rearrangements is the fusion of the ALK tyrosine kinase domain to the 5’ region of partners which provide a strong promoter and most likely an oligomerization motif allowing constitutive activation and aberrant expression of the ALK kinase. ALK gene encodes for a receptor tyrosine kinase normally expressed in fetal and mature nervous systems but not in lymphoid cells. As any receptor tyrosine kinase and in normal situation, ALK protein will activate signaling pathway and cell cycle after oligomerization induced by binding with its ligand. In ALK rearrangements, the partner gene brings to ALK the ability to self-associate in a ligand-independent fashion, leading to its constitutive activation. In addition, the gene partner brings a strong promoter, driving illegitimate and high levels of ALK receptor tyrosine kinase fusion gene expression in lymphoid cells. The functional consequence is to exaggerate and dysregulate otherwise normal downstream signals which will promote cell growth and inhibit apoptosis.41 Clearly, ALK activation is a critical step in the development of ALCL of T cell origin. As ALK gene is not expressed in normal lymphoid cells, the immunodetection of ALK protein in a lymphoid tumor represents a highly sensitive test for identification of lymphoma with ALK rearrangement, correlating in nearly 100% of cases with the presence of such abnormality. Regardless of other clinical and biological prognostic parameters, the outcome for patients with ALK-positive ALCL is significantly better than that for patients with ALK-negative ALCL with the 5-year survival rates ranging between 79 and 88% and 28 and 40%, respectively.42 Additional information on lymphomas is found in Chapter 24 Lymph Nodes and Flow Cytometry.

Table 2.2  Translocations associated with sarcomas

Translocation

Genes

Type of fusion gene

t(11;22)(q24;q12)

EWSR1-FLI1

Transcription factor

t(21;22)(q22;q12)

EWSR1-ERG

Transcription factor

t(7;22)(p22;q12)

EWSR1-ETV1

Transcription factor

t(17;22)(q21;q12)

EWSR1-ETV4

Transcription factor

t(2;22)(q33;q12)

EWSR1-FEV

Transcription factor

EWSR-ATF1

Transcription factor

Ewing’s sarcoma

Clear-cell sarcoma t(12;22)(q13;q12)

Desmoplastic small round-cell tumor t(11;22)(p13;q12)

EWSR-WT1

Transcription factor

Myxoid chondrosarcoma t(9;22)(q22-31;q11-12)

EWSR-NR4A3

Transcription factor

Myxoid liposarcoma t(2;16)(q13;p11)

FUS-DDIT3

Transcription factor

t(12;22)(q13;q12)

EWSR1-DDIT3

Transcription factor

Alveolar rhabdomyosarcoma t(2;13)(q35;q14)

PAX3-FKHR

Transcription factor

t(1;13)(p36;q14)

PAX7-FKHR

Transcription factor

SYT-SSX

Transcription factor

Synovial sarcoma t(X;18)(p11;q11)

Dermatofibrosarcoma protuberans t(17;22)(q22;q13)

COL1A1-PDGFB

Growth factor

Congenital fibrosarcoma

Sarcomas Although sarcomas are relatively rare neoplasms in adulthood, they represent the most frequent malignant tumors in childhood and young adults. Abundant genetic studies have revealed that a significant number of sarcoma are associated with specific chromosomal abnormalities (mainly chromosomal translocations) that can be used as practical diagnostic markers in histological equivocal cases.13,14,43,44 A typical example is the so-called “small round blue cell” undifferentiated pattern shared by disparate tumor entities such as embryonal or alveolar rhabdomyosarcoma, Ewing’s sarcoma, neuroblast­ oma, and lymphoma. Two major genetic groups distinguishable at the cytogenetic level are observed in sarcomas. One group is characterized by a near-diploid karyotype with a single or few chromosomal abnormalities, whereas the second exhibits complex karyotype with numerous aberrations that reflect severe disturbance in genomic stability. Sarcoma with genetic abnormalities not detectable by conventional cytogenetics and/or FISH means—such as GIST and its specific c-KIT mutation—will not be discussed in this section.

Sarcomas with Single Karyotypic Abnormalities This group is characterized by karyotype harboring single and tumor-specific chromosomal translocations (Table 2.2). Most of these translocations lead to fusion genes encoding aberrant transcription factors but a small subset creates aberrant chimeric genes related to growth-factor signaling pathway.

t(12;15)(p13;q25)

ETV6-NTRK3

Transcription factorreceptor

Inflammatory myofibroblastic tumor 2p23 rearrangements

TMP3-ALK; TMP4-ALK

Growth factor-receptor

Alveolar soft-part sarcoma t(X;17)(p11.2;q25)

ASPL-TFE3

Transcription factor

The Ewing’s family of tumors, which includes Ewing’s sarcoma and primitive neuroectodermal tumor (ES/PNET), are characterized by a t(11;22)(q24;q12) translocation leading to the EWSR1-FLI1 fusion gene and observed in nearly 90% of cases of ES/PNET (Fig. 2.13). The remaining cases show alternative chromosomal translocations fusing the EWSR1 gene (chromosome 22q12) with partner genes other than FLI1 and that belong to the same ETS family of transcription factors. EWSR1 gene is also involved in chromosomal translocations arising in several other tumoral entities such as the intraabdominal desmoplastic small round-cell tumor (DSRCT), myxoid chondrosarcoma, and clear cell sarcoma. However, EWSR1 gene fused in each case with gene partners not encountered in the Ewing’s family of tumors, giving rise to specific fusion genes suitable for diagnostic purposes.13,43,44 Some new data indicate that soft-tissue tumors can no longer be classified only on basis of their site of origin but also 35

PART ONE

General Cytology

Fig. 2.13  Karyotype of an Ewing’s tumor with the characteristic t(11;22)(q24;q12) chromosomal translocation (arrows) leading to the EWS-FLI1 fusion gene. Secondary recurrent chromosomal abnormalities such as monosomies 6 and 15 and trisomies 2 and 14 are also observed.

according to their genetic aberrations.43,45 Congenital fibrosarcoma and mesoblastic nephroma were thought to be unrelated tumors until cytogenetic analysis revealed a common aberration, hence the t(12;15)(p13;q25) translocation with subjacent ETV6-NTRK3 fusion gene, indicating that they are simply the same tumoral entity that develops in different locations. Another similar example is illustrated by the t(X;17)(p11.2;q25) translocation shared by the alveolar soft-part sarcoma (ASPS) and a cytogenetic subset of childhood papillary renal cell carcinoma (PRCC).46 Although this translocation is cytogenetically unbalanced in ASPS and balanced in PRCC, it gives rise at the molecular level to the same ASPL-TFE3 fusion transcript in both tumoral types. Therefore, some fusion genes can exert their oncogenic properties in more than one target cell type and seems not to play any role in cell differentiation. On the other hand, in vitro experiments showed that fusion proteins such as EWS-FLI1 contribute to the phenotypic features of ES/PNET by subverting the differentiation program of its neural crest precursor cell to a less differentiated and more proliferative state. In synovial sarcoma, the SYT gene on chromosome 18q11 can fuse with various members of the SSX cluster located on chromosome Xp11 (Fig. 2.14). The SSX2 translocation partner is more likely observed in monophasic synovial sarcoma, whereas SSX1 is much often associated with the biphasic forms, indicating that this latter gene partner may drive epithelial differentiation in synovial sarcoma. Finally, some data support the hypothesis that the gene fusion occurs in an already established lineage that imposes constraints such that the target cell selects the fusion gene. In contrast, other observations suggest that this fusion will modulate the phenotypic features of the undifferentiated precursor harboring this fusion gene.45 Sarcoma-associated chromosomal translocations and/or their respective fusion genes may have some prognostic impacts.14,43 In Ewing’s sarcoma, several molecular variants are observed in the EWS-FLI1 fusion gene due to various breakpoint junctions. The most common, designated type 1 (linking exon 7 of EWS with exon 6 of FLI1) is associated with a better prognosis than other variants. The SYT-SSX fusion type in synovial sarcoma appears to 36

be a significant prognostic factor since patients with the SYT-SSX2 variant have an improved overall survival when compared with SYT-SSX1 positive patients, independent of the histological type. Patients with metastatic alveolar rhabdomyosarcoma having the PAX7-FKHR fusion gene show a substantially better prognosis than those with the PAX3-FKHR translocation. These variations in behavior could be due to subtle differences in the biochemical activities of the variant fusion proteins, with a better prognosis associated with variants having a less transcriptional activity. The close association between specific translocations and distinct sarcoma types indicates that they are early events in tumorigenesis but their exact role in tumor development remains often difficult to assess. In the small subset of translocations with aberrant chimeric genes related to growth-factor signaling pathways (see Table 2.2), the pathogenesis arises through cell cycle activation although this is probably not sufficient per se to induce full transformation. The great majority of chromosomal translocations in sarcoma involve transcription factors without obvious putative oncogenic properties at first sight. Transcription factors are proteins that directly interact with the DNA strand of their target genes, and regulate the expression of these genes by binding their promotor regions upstream of RNA transcription sites. A translocation will lead to aberrant gene fusion composed of the DNA (or RNA)-binding domain of a transcription factor fused with the transactivation domain of another transcription factor. The functional consequence is that the transcriptional activity of the latter will be deviated toward downstream genes targeted by the DNA-binding domain provided by the transcription factor partner. Moreover, most of these chimeric proteins show enhanced transcriptional activity compared with their constitutive normal protein, providing eventually a gain of function mechanism. It is thus believed that these phenomena lead to dysregulation of gene expression, accounting for the tumoral properties of fusion genes in sarcoma. This general opinion can be illustrated by the t(2;13)(q35;q14) and t(1;13)(p36;q14) translocations arising in alveolar rhabdomyosarcoma and corresponding to the PAX3/FKHR and PAX7/FKHR fusion genes, respectively.43,47 Both translocations

2 Basic Cytogenetics and the Role of Genetics in Cancer Development

Fig. 2.14  Karyotype of a synovial sarcoma with the specific t(X;18)(p11;q11) chromosomal translocation (arrows). Losses and gains of other chromosomes represent additional secondary changes.

fuse the DNA-binding domain of PAX3 or PAX7 to the transactivation domain of FKHR. PAX genes activate myogenesis, and fork head in rhabdomyosarcoma (FKHR) is though to have pro-apoptotic activities. The resulting PAX-FKHR fusion gene is a highly potent activator with a transcriptional activity 10 to 100 times as high as that brought by wild-type PAX gene. This enhanced transcriptional activity is further amplified by mechanisms of PAX-FKHR fusion genes amplification. As the DNA-binding of FKHR is lost in the PAX-FKHR fusion, any DNA-binding specificity of the fusion gene is directed by the PAX sequence, leading to dysregulated expression of downstream target genes of PAX genes. Consequently, PAX3/FKHR will be able, firstly, to inhibit cellular apoptosis through a PAX3 target gene, the anti-apoptotic protein BCL-XL and, secondly, to activate c-MET, PDGFαR, or c-RET oncogene, downstream targets of PAX3 involved in migration and proliferation of myogenic precursors. Other sarcoma-associated fusion genes have been shown to get tumoral properties by activating growth factor receptors. MET oncogene has been recently shown to be a direct transcriptional target of the ASPL-TFE3 fusion gene. Induction of MET by ASPL-TFE3 results in strong MET autophosphorylation and activation of downstream signaling in the presence of hepatocyte growth factor.48 Another question that remains a matter of debate is whether these chromosomal translocations are sufficient for neoplastic transformation.14,43 Although expression of certain gene fusions can induce sarcoma in primary mesenchymal progenitor cells, secondary mutations are likely to be required for full malignancy as observed in the context of hemato­ logical disorders. Loss of tumor suppression genes expression (such as P16 and RB) is observed in more than 50% of various sarcoma. Activation of common growth-factor pathways not directly due to chromosomal translocation is described in sarcomas including the insulin-like growth factor 1 (IGF1) pathway in alveolar RMS, the platelet-derived growth factor receptor (PDGFR) in DSRCT, and the c-KIT receptor pathway in Ewing’s tumors. Parallel to the situation observed in childhood leukemia,49 it is possible that some chromosomal translocations associated with childhood tumors arise during

Table 2.3  Sarcomas with complex karyotypes

Type of sarcoma Fibrosarcoma (other than congenital) Leiomyosarcoma Malignant fibrous histiocytoma Osteosarcoma Chondrosarcoma (types other than extraskeletal myxoid) Liposarcoma (types other than myxoid) Embryonal rhabdomyosarcoma Malignant peripheral nerve-sheath tumour Angiosarcoma Neuroblastomaa a Neuroblastoma is quoted in this table as it belongs to the “small round blue cell tumours” group.

fetal development, leading to a “pre-malignant state” preceding the sarcomatous transformation induced by additional genetic aberrations.

Sarcomas With Complex Karyotypes This group of sarcoma does not exhibit any specific and recurrent chromosomal translocation but rather complex karyotypes with multiple numerical and structural aberrations characteristic of severe genetic and chromosomal instability43 (Table 2.3) (Fig. 2.15). The underlying genetic mechanisms frequently include alterations in cell-cycle genes such as P53, INK4A, and RB1 as well as genes directly involved in DNArepair pathways. Oncogene amplifications occur in cytogenetically complex sarcoma. MDM2 and MYCN gene amplifications are well-known examples. MDM2 amplification is observed in liposarcoma (other than myxoid) and malignant fibrous histiocytomas. MDM2 is a p53 inhibitor and its amplification 37

PART ONE

General Cytology

Fig. 2.15  Typical example of a complex karyotype as observed in embryonal rhabdomyosarcoma showing multiple numerical and structural abnormalities. The latter (isochromosome 17q and two chromosome markers) are marked with arrows.

will lead to inability of p53 to induce apoptosis in cells with DNA damage, which, in turn, will induce genomic instability. MYCN amplification (Fig. 2.16) is used as a genetic parameter for better therapeutic stratification of patients suffering from neuroblastoma, one of the most frequent malignant tumors in childhood.50 MYCN is a member of the MYC family of protooncogenes which are transcription factors promoting cell proliferation and inhibiting terminal differentiation. In view of its function, MYCN is involved in the genesis of a wide range of cancers including neuroblastoma, small cell lung carcinoma, some cases of medullary thyroid carcinoma, retinoblastoma, and breast cancers. A forced expression of MYCN in central nervous system cells in mouse leads to the development of a subgroup of neuroblastomas, indicating that it is sufficient for malignant transformation. Additional information on ­sarcomas is found in Chapter 18.

Thyroid Carcinomas Among epithelial malignancies, two histological types of thyroid carcinoma, namely the papillary and follicular thyroid carcinoma, deserve to be mentioned as they exhibit specific genetic aberrations that represent reliable diagnostic parameters.

Fig. 2.16  Interphase FISH demonstrating amplification of the MYCN oncogene (red signals) in a case of neuroblastoma. The two green spots correspond to centromeric probes specific for the chromosome 2 and used as control for diploid status assesment of the analyzed cell.

Papillary Thyroid Carcinoma Papillary thyroid carcinoma (PTC) is characterized by rearrangements of the RET oncogene, a receptor tyrosine kinase (RTK) gene located on chromosomal region 10q11.2. These activating rearrangements, called RET/PTC, are caused by either paracentric inversion of chromosome 10 or balanced translocations involving chromosome 10 and various chromosome partners51 (Table 2.4). The molecular consequences are fusion of the tyrosine kinase domain of RET with the 5’ part of the various gene partners with subsequent release of the extracellular ligand­binding and juxtamembrane domains of RET receptor. As the juxta­membrane domain negatively regulates RET mitogenic signaling, its deletion contributes to RET/PTC activation, which is 38

further enhanced by dimerization potential brought by the gene partner.52 This leads to ligand-independent activation of the RET kinase, signaling pathway stimulation and cell-cycle activation; a well-known oncogenic process in tumoral cells harboring RTK rearrangements. As part of its oncogenic effect, RET/PTC directly modulates genes involved in inflammation/invasion of the cell such as various cytokines (GM-CSF, M-CS, IL6, etc.), chemo­­ kines (CCL2, CXCL12, etc.), and chemokine receptors (CXCR4). The induction of an inflammatory-type reaction may explain the chronic inflammatory reaction observed in this type of cancer.52 The prevalence of RET/PTC in papillary thyroid carcinoma is highly variable (0–87%), depending on age of patient,

2 Basic Cytogenetics and the Role of Genetics in Cancer Development

Table 2.4  Characteristics of different types of RET/PTC rearrangement in papillary thyroid carcinoma

Type of RET/PTC

Gene fused with RET

Mechanism of rearrangement

Prevalence among all RET/PTC

RET/PTC1

H4 (D10S170)

inv(10)(q11.2;q21)

60–70%

RET/PTC2

Ria

t(10;17)(q11.2;q23)

<10%

RET/PTC3 (and RET/PTC4)

ELE1 (RFG, ARA70)

inv(10)(q11.2)

20–30%

RET/PTC5

GOLGA5

t(10;14)(q11.2;q?)

Rare

RET/PTC6

HTIF1

t(7;10)(q32;q11.2)

Rare

RET/PTC7

RFG7

t(1;10)(p13;q11.2)

Rare

RET/ELKS

ELKS

t(10;12)(q11.2;p13)

Rare

RET/KTN1

KTN1

t(10;14)(q11.2;q22.1)

Rare

RET/RFG8

RFG8

t(10;18)(q11.2;q21-22)

Rare

RET/PCM-1

PCM-1

t(8;10)(p21-22;q11.2)

Rare

g­ eographic regions, and sensitivities of the detection methods used (polymerase chain reaction versus FISH), particularly if the rearrangement is present only in a small proportion of tumor cells or if the RET/PTC transcripts is expressed at low levels. The average prevalence is 20–30% in sporadic adult cases and rises to 45–60% among tumors from children and young adults. It is higher (50–80%) in papillary carcinoma associated with radiation exposure, and it is thought that the close association between RET/PTC translocations and irradiation is due to spatial proximity of the participating chromosomal loci in the nuclei of thyroid cells, providing a structural basis for radiation-induced illegitimate recombination of the genes.14 Most studies concur that RET/PTC rearrangements are rare or absent in benign adenomas, and not observed in other types of thyroid carcinomas. They are more frequent in PTC exhibiting a classic architecture and in microcarcinomas.52 Among the different variants of RET/PTC translocations, RET/PTC1 and 3 are the most frequent, accounting for more than 90% of all rearrangements.53 A small subset of PTC (around 10%) is characterized by ­rearrangement of the NTRK gene, another receptor tyrosine kinase, located on chromosome 1q22 and encoding one of the receptors for the nerve growth factor. NTRK gene activation is due to chromosome 1 inversions or balanced translocations between chromosome 1 and 10, resulting in fusion of the NTRK tyrosine kinase domain to 5’-end sequences from at least three different genes: tropomyosin (TPM3) or TPR gene, both on chromosome 1, and TFG gene located on chromosome 3.51,53

Follicular Thyroid Carcinoma Follicular thyroid carcinomas are characterized by PPARγ (peroxisome proliferator-activated receptor γ) gene rearrangements in 25–50% of cases, mainly under the form of a distinctive t(2;3)(q13;p25) chromosomal translocation.53 This translocation leads to the fusion of the PAX8 gene (paired box gene 8) with PPARγ gene, resulting in a fusion protein designed PPFP. PAX8 is a transcription factor expressed at high levels in thyrocytes and necessary for normal thyroid development. PPARγ encodes a nuclear hormone receptor transcription factor whose activity is related to adipocyte differentiation, lipid and carbohydrate metabolism, and cellular proliferation and differentiation. PPFP is thought to exert its oncogenic properties through a mechanism in which it acts as a dominant-negative inhibitor of wild-type PPARγ. This results in inhibition of apoptosis and promotion of proliferation as well as anchorage-independent growth of

t­ hyroid follicular cells. PPARγ has mainly been observed in lowstage follicular carcinomas with vascular invasion and has been identified at apparent lower frequency in adenomas.

Clinical Applications of Conventional Cytogenetics and fish in Cytology Introduction Cytological assessment of a fine-needle aspiration (FNA) specimen remains the first-line morphological investigation of any suspected mass but cytomorphology alone—hence, without tissue architecture—is not always sufficient for a definitive diagnosis.54,55 For example, small-to-intermediate cell lymphomas such as MCL, FL, or MZL can show overlapping cytomorphologic features with one another as well as with reactive lymph node hyperplasia. Limitations of FNA are also encountered in soft-tissue neoplasms, especially in the diagnostic management of small round-cell tumors. Most of the diagnostic problems can be solved with the help of immunocytochemistry but limitations can be encountered mainly due to immunophenotypic heterogeneity among small B-NHL subtypes.56 For examples, the intensity of CD10 expression in FL has been shown to be variable, and even negative in some cases.57 MCL and SLL can be distinguished by differences in CD23 expression but CD23 can be weakly expressed in both subtypes.58 CD5 expression may not systematically be used as a diagnostic criterion between MCL and SLL, and some FL can also exhibit a CD5 positivity.59 It is thus necessary that FNA examination be supplemented with ancillary methods such as karyotype, FISH, or polymerase chain reaction (PCR). Conventional cytogenetics allows complete karyotype analysis and, as such, remains the historic gold standard by which everything is started. However, it is a cumbersome and time-consuming procedure requiring adequate fresh tissue and special cell culture techniques. PCR and interphase FISH (I-FISH) methods are more practical in that they can bypass the need of cell culture. They have both their own advantages and disadvantages, and must be considered as complementary rather than competing with one another. It is therefore not surprising that both have been included in a combined diagnostic algorithm proposed in the literature.60–62 However, I-FISH remains a less sophisticated laboratory technique than PCR and offers a greater qualitative sensitivity in studies of tumor-­associated chromosomal abnormalities as will be 39

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i­ llustrated later. This technique is advantageous for FNA specimens because it requires only a few cells. It allows also retention of cellular morphology, which permits simultaneous evaluation of morphology and chromosomal alterations. Moreover, recent studies have demonstrated the feasibility of FISH on Papanicolaou-stained archival cytology slides, highlighting the good flexibility of such method.63–66 These advantages probably explain why FISH is becoming more and more popular in cytopathology laboratories.

FISH Strategy Interphase FISH requires simple material such as cytospins from FNA specimens. Cytospin is an optimal preparation for I-FISH because the monolayer allows excellent hybridization results. Cytospin preparation can be made by Ficoll-Hypaque gradientseparation technique and then fixed in methanol-glacial acetic acid (3:1) for 20 minutes at −20°C. The slides will be then airdried and stored at −20°C prior current FISH procedure. Specimen handling is thus very simple, but it is critical to avoid delays in specimen processing in order to prevent possible degradation of the target DNA and subsequent poor hybridization results.67 Subsequent FISH steps can be then easily performed without further manipulation of the samples, with the use of commercially available kit sets including the premixed probes, and according to the protocol recommended by the manufacturer. At least, 200 nonoverlapping and intact nuclei per case and at least two different areas on the same slide should be scored before giving a result. The great advantage of working on an interphase cell can nevertheless be a source of interpretative pitfalls in that random chromosome colocalizations occur not infrequently in normal nuclei and can mimic chromosomal translocations. Although most of the commercial probes have been designed to limit the risk of false-positive profiles, it remains critical to determine the frequency of such false-positive cells in order to define a cutoff level. Normal lymphocyte nuclei can be used as negative control to assess hybridization efficiency, and the cutoff level for positivity should be set at the mean (%) ± 3 standard deviations. Beside this pitfall, other good practice recommendations are needed and must be known by the user. Such guidelines are detailed in an excellent overview recently published that we highly recommend to the reader.68 The commercial probes are usually several hundred kilobases in length and yield large, bright and easily detectable signals. They are currently available to detect many of the relevant chromosomal abnormalities described in the previous section and are known to be highly sensitive.69 For detection of chromosomal translocations, three different kinds of probes are available, including the dual-fusion probes, the single-fusion extra-signal probes, and the break-apart probes, all being dual-color probes (Figs 2.17A and 2.17B). Dual-fusion and extra-signal probe sets are made of two differentially labeled (green and red) DNA segments, each of these segments identifying one of the chromosomal loci involved in the translocation. For the dual-fusion probes, an abnormal pattern will be represented by one red and one green signal (representing the normal homolog) and by two fusion or colocalization signals corresponding to the chromosomal translocation and its reciprocal (“2F,1R,1G” pattern). Typical examples are probes designed to detect lymphoma­associated chimeric genes subjacent to translocation such as the BCL2-IgH or BCL1-IgH in follicular or mantle cell lymphoma, respectively (Fig. 2.18A). Such probes make it ­ possible to 40

Normal cell Gene A

Gene B

Cell with translocation

A Dual fusion probe

Normal cell

Gene Cell with translocation

B Break apart probe Fig. 2.17  Schematic representation of two different types of dual-color probes, the dual fusion and break-apart probes. (A) Left: Dual-fusion probes are composed of two differentially labeled (green and red) DNA segments, each of these segments identifying one of the genes/loci involved in the chromosomal translocation. The probes are usually several kilobases in length and extend largely on both sides of the gene of interest. Right: a normal pattern will show two red and two green spots, whereas a cell harboring a chromosomal translocation will demonstrate two fusion or colocalization signals corresponding to the chromosomal translocation and its reciprocal; the red and green spots indicate the two remaining normal chromosomes (“2F,1R,1G” pattern). (B) Left: break-apart probes are made of two differentially labeled (green and red) DNA segments flanking the breakpoint cluster region of a gene involved in chromosomal translocations. Right: a normal cell will show two yellow fusion signals corresponding to two copies of a normal gene. The disruption of one of these two copies subsequently to a chromosomal translocation will lead to split of one yellow signal into two red and green signals (“1R,1G,1F” pattern).

s­ ignificantly reduce the risk of false positives as the possibility that two overlapping signals are due to random spatial proximity of the participating chromosomal loci remains very low. The abnormal pattern for extra-signal translocation probes will be represented by a single fusion (corresponding to one derivative chromosome) plus a small extra signal representing the residual portion of one of the loci involved in the translocation. Again, the probability that such pattern is observed in a normal nucleus is very low. A well-known example is the probe used to detect the BCR-ABL chimeric gene in chronic myeloid leukemia. Such a probe has not been designed for detection of recurrent chromosomal translocations in lymphoma or sarcoma and will not be illustrated here. Dual-color break-apart probes are made of differentially labeled (green and red) DNA segments located on either side of a breakpoint cluster region within a target gene. The separation of green and red signals indicates break between the 5’ and 3’ regions of the rearranged gene. In normal cells, the two probes colocalize to produce two yellow fusion

2 Basic Cytogenetics and the Role of Genetics in Cancer Development

A Fig. 2.19  Interphase FISH using centromeric probes for chromosomes 7 (green) and 18 (red). Both cells show three signals for each probe indicative of trisomies 7 and 18 as observed in nodal marginal zone lymphoma.

B Fig. 2.18  (A) Interphase FISH of follicular lymphoma cells with the use of a dual-fusion BCL2-IgH probe. The two fusion/colocalization signals indicate the existence of a BCL2-IgH oncogene and its reciprocal while the green and red signals correspond to the remaining normal IgH and BCL2 genes respectively. (B) Interphase FISH with the use of a dual-fusion breakapart probe specific to the EWSR1 locus. The lower nucleus shows a normal pattern, whereas the upper one displays split of one EWSR1 gene copy as it can be observed in Ewing’s tumors.

signals (corresponding to two copies of nonrearranged genes), whereas in the case of translocation involving one of the two genes, one of the fusion signal splits, resulting in a characteristic 1 red–1 green–1 yellow fusion (“1R1G1F”) signal pattern. The break-apart strategy offers the advantage of detecting in a single experiment all recurrent rearrangement of a gene involved in translocations with different gene partners. A typical example is the EWSR1 gene which can fuse with no less than nine different gene partners (Fig. 2.18B). Interphase FISH is also able to identify submicroscopic chromosomal deletions as well as numerical chromosomal abnormalities such as trisomy or monosomy. The probes used to detect entire chromosomal gains or losses are juxtacentromeric alphoid DNA sequences while submicroscopic deletions will be identified with locus-specific probes. To ensure the quality of hybridization (mainly the hybridization properties of the tumor cells being analyzed), a control probe, labeled with a different fluorophore and identifying any other chromosome, will be cohybridized with the probe of interest. For detection of microdeletion, the control probe will also serve to identify the chromosome harboring the deleted region. Examples are trisomy 3 (Fig. 2.19) and deletion of chromosome 7q in marginal zone lymphoma.

Application Lymphomas Studies demonstrating the feasibility and diagnostic utility of FISH in FNA specimens have focused on the most frequent lymphoma such as FL and, to a certain extent, diffuse large B-cell

lymphoma. Although less common, MCL has also been a subject of interest because of the clinical relevance and difficulties to differentiate it cytologically from other small cell NHLs. Among the latter, small lymphocytic lymphoma may be difficult to diagnose when it presents as isolated lymphadenopathy. As mentioned earlier, FISH and PCR remain complementary methods for detecting predictable chromosomal abnormalities in lymphoma, but comparative studies on specimens such as tissue imprints, cytospins, or smears have demonstrated a higher qualitative sensitivity of I-FISH. In follicular lymphoma, the detection rate of the t(14;18) translocation with PCR was 70% at best, whereas a positive result could be achieved in around 90% of cases with FISH.65,66,70–73 The low detection rate encountered with the PCR technology is due to mutation involving primer binding sequences and to the fact that the current PCR method applicable in routine use is not able to detect breakpoints outside the known major breakpoint region (MBR) and minor cluster region (mcr). A similar situation is encountered in MCL where the sensitivity of FISH analysis for the direct detection of the t(11;14) translocation largely exceeds DNA-PCR methods; the detection rate reaching nearly 100% according to FISH studies,64,69,72,74,75 while it falls in the range of 40% with the second method.74,60 The lower qualitative sensitivity offered by DNA-PCR is mainly due to the wider variation of BCL1 gene breakpoints that are difficult to span with primers. FISH analysis circumvents these limitations by using IgH/BCL2 and IgH/BCL1 dual-fusion probes covering the entire BCL2 and BCL1 gene, respectively. Moreover, these results highlight the greater applicability of FISH since all known BCL2 and BCL1 breakpoints can be covered and detected with a single-probe set. Among other small-cell lymphoproliferative disorders, SLL/CLLs are characterized by recurrent chromosomal abnormalities such as trisomy 12 or interstitial deletion involving 13q14 chromosomal region. Interphase FISH can easily detect these aberrations63,76 and, together with negative results for BCL2 and BCL1 rearrange­ ments, can help in assessing an accurate diagnosis of SLL/CLL. In FNA specimens displaying monomorphous lymphoid population composed of medium-sized or large cells, a proper diagnosis of Burkitt, diffuse large B-cell, or anaplastic lymphoma can be easily reached with the use of specific break-apart probes targeting C-MYC, BCL6, or ALK gene, respectively.72,77 Beside the detection of lymphoma-associated specific translocations, atypical patterns revealed by interphase FISH can help in better classifying lymphoma. For example, a current IgH/BCL1 dual-fusion probe can identify hypotetraploid profiles with extra copies of BCL1 signals as observed in blastoid variants of MCL.74,78 41

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General Cytology

A

B

Fig. 2.20  Illustration of the FICTION method on multiple myeloma cells. The plasma cells are detected with the use of antibodies directed against the cytoplasmic immunoglobulins λ or κ. These antibodies are colored with fluorochrome AMCA (blue color). (A) Two plasma cells with deletion of the p53 locus (chromosome 17p13) demonstrated by the absence of one red signal, whereas the existence of both chromosomes 17 is confirmed by a specific chromosome 17 centromeric probe (green signals). (B) Plasma cell harboring the FGFR3/MMSET-IgH fusion genes corresponding to the t(4;14)(p16;q32) translocation. The two yellow fusion signals are due to red and green signals relocating next to each other, and indicate the FGFR3/MMSET-IgH fusion gene and its reciprocal. The green and red signals correspond to remaining normal IgH and FGFR3/MMSET genes respectively.

A recent study aimed at comparing the utility of I-FISH and flow cytometry immunophenotyping (FCM) in a series of FL and DLBCL.71 They found that detection of t(14;18) by FISH was a slightly more sensitive (85%) diagnostic marker than identification of the typical CD19+/CD10+ immunophenotype profile by FCM (75%). FISH appeared to be more sensitive because it could also detect FLs with an atypical CD19+/CD10- pattern. In the same study, a BCL2 gene rearrangement was detected by FISH in 29% of DLBCL cases, whereas FCM was able to identify a CD10+ monoclonal population in only 23% of such lymphoma cases. I-FISH appeared thus to be slightly more sensitive than FCM in identifying germinal center B-like DLBCL.

Sarcomas Most (if not all) chromosomal translocations described in softtissue sarcomas (STS) are detectable by I-FISH. This method is thus particularly useful in diagnostically difficult cases such as small blue cells tumors. Several studies aimed at comparing the efficiency of both reverse transcriptase (RT)-PCR and FISH techniques for a molecular diagnosis in sarcoma.79,80 Both methods were complementary and had their own advantages and disadvantages in terms of specificity and qualitative and quantitative sensitivity. However, the FISH break-apart approach appears to be very practical in that the use of a single­ break-apart probe can recognize each specific translocation such as the t(X;18) in synovial sarcoma, the t(2;13) or t(1;13) in alveolar rhabdomyosarcoma, and the t(12;16) in myxoid liposarcoma. The potential disadvantage of such an approach would be its inability to distinguish Ewing/PNET from other sarcomatous types harboring EWS gene rearrangements (see Table 2.2) since the partner gene is not detected. In most cases, these neoplasms are nevertheless distinguishable from each 42

other on the basis of clinical data or immunocytochemical differences. Several studies have demonstrated the usefulness of cyto­ genetics81,82 or I-FISH83–85 as an adjunct in making a definitive diagnosis of sarcoma by FNA. As our knowledge about the specific chromosomal abnormalities associated with sarcoma is constantly increasing, there is good hope that I-FISH will allow accurate diagnosis on more cases investigated by FNA, obviating open surgical biopsy preceding therapy.

Multiple Myeloma Multiple myeloma (MM) is characterized by numerous chromosomal abnormalities which have been shown to significantly impact survival in patients with such disease.86 The most relevant alterations include hyperdiploidy, monosomy 13/deletion 13q14, deletion 17p, t(11;14)(q13;q32), and t(4;14)(p16;q32) translocations, giving rise to the BCL1-IgH and FGFR3/MMSETIgH chimeric genes, respectively. Hyperdiploidy is associated with a favorable prognosis but all other abnormalities represent unfavorable parameters, among which the t(4;14) and deletion17p appear to be the most important. Their identifications have implications for the design of risk-adapted treatment strategies. Historically, testing for abnormalities in MM was based on conventional chromosomal analysis performed on bone marrow, but results were often falsely normal since the actively normal myeloid cells were analyzed rather than the monoclonal plasma cells, which infrequently enter mitosis. Standard FISH studies were thus employed to detect the classical abnormalities associated with MM. Again, erroneously normal results were most often obtained since this method is not able to ­distinguish between normal cells and small clones of monoclonal plasma cells. A novel FICTION method, which is a combination of

2 Basic Cytogenetics and the Role of Genetics in Cancer Development

­ uorescent immunophenotyping and in situ hybridization, has fl thus been developed.9 First, antibodies against the cytoplasmic immunoglobulins λ or κ are applied in order to specifically ­identify the plasma cells thanks to their cytoplasmic fluorescence. Second, FISH probes will be hybridized to all cell types, but only specifically target plasma cells will be analyzed. This FICTION method is thus capable of detecting chromosomal abnormalities in bone marrow specimens even when few plasma cells are present (Fig. 2.20A and 2.20B).

Concluding Remarks Over the past two decades, conventional cytogenetics has made it possible to identify nearly all chromosomal abnormalities associated with specific histological subtypes of ­lymphoproliferative

disorders and soft-tissue tumors. These chromosomal aberrations made it possible, in a second step, to pinpoint the underlying oncogenes and to study the pathogenesis of tumors bearing such abnormalities. In addition to their role in fundamental research, these alterations rapidly appeared to be powerful diagnostic and prognostic parameters relevant to use on a regular basis. The constant emergence of commercial probes yielding large, bright, and easily detectable signals made the FISH method a reliable tool for detecting specific chromosomal abnormalities on nondividing cells provided by cytology specimens such as smears, cytospin, or liquid-based samples. At present time, there is enough evidence in the specialized literature demonstrating that I-FISH, in conjunction with other ancillary tools such as immunocytochemistry and molecular biology, constitutes a suitable complementary approach in the cytological diagnosis of cancers detailed in this chapter.

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