Cytogenetics — In Color and Digitized
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《新英格兰医药杂志》
Cytogenetics is the genetic analysis of cells, a discipline that has flourished since the chromosome-banding techniques introduced in 1969 by Torbj?rn Caspersson and Lore Zech first provided a simple and inexpensive way to gauge the number and assess the structural integrity of chromosomes. Chromosome banding (see Figure 1A) is probably the most commonly performed genetic test: it is used about 500,000 times each year in North America (most laboratories use G-banding, named after the German chemist Gustav Giemsa). It is used prenatally and postnatally to screen for the presence of constitutional chromosomal aberrations associated with birth defects, as well as for the diagnosis and differential diagnosis of cancer — hematologic cancers in particular.
Figure 1. Examples of Chromosome Banding, Interphase Fluorescence in Situ Hybridization, Spectral Karyotyping, and Array-Based Comparative Genomic Hybridization.
Panel A shows Giemsa-banded chromosomes from a patient with chronic myeloid leukemia. The arrows indicate the sites of a balanced translocation between chromosomes 9 and 22. This chromosomal translocation fuses two genes, BCR and ABL; the robust tyrosine kinase activity of their protein product is the molecular target of imatinib mesylate. Panel B shows interphase fluorescence in situ hybridization with the use of three probes (each a different color) on fine-needle aspirates from a breast carcinoma. The yellow and green signals indicate that chromosomes 7 and 17 are diploid, and the red signal reveals that the HER2/neu oncogene is amplified. Breast carcinomas with HER2/neu amplification respond to adjuvant treatment with herceptin. Panel C shows how spectral karyotyping can reveal massive chromosomal rearrangements in cells of a brain tumor (astrocytoma); distinct chromosome territories appear in the adjacent nucleus in interphase (lower left). Panel D shows an array-based analysis of genomic imbalances in a breast-cancer cell line. Dots that are red indicate increases in the number of copies in the tumor genome; green dots indicate a loss of DNA in the tumor genome.
The application of cytogenetics to the screening of the genomes of patients has led to the linking of specific chromosomal abnormalities (and consequently genetic alterations) with specific diseases; such findings carry enormous implications for medicine. The first such specific correlation was made when Jér?me Lejeune, in Paris, detected extra copies of a small chromosome in patients with Down's syndrome. Other descriptions of constitutional abnormalities, involving both autosomes and the sex chromosomes, followed in rapid sequence.
Such associations were also established in cancer cells. The first report on the so-called Philadelphia chromosome (described by Peter Nowell and David Hungerford in 1960 and characterized by Janet Rowley in 1973), created by an exchange of genetic material between chromosomes 9 and 22, launched the highly prolific field of cancer cytogenetics. (The Philadelphia chromosome reflects the specific target of imatinib mesylate in the treatment of chronic myeloid leukemia.) This work not only confirmed the relevance of cytogenetic abnormalities as disease-initiating events, but also led to the molecular cloning of numerous cancer-associated genes, many of which sit at the junctions of chromosomal translocations.
Cytogeneticists then adapted and combined molecular cloning and hybridization and so arrived at fluorescence in situ hybridization, which has had profound effects on cytogenetic diagnostic testing and research. Specific fluorescently tagged DNA probes could now be used to visualize, with dramatically increased resolution, small deletions and other genetic aberrations, as well as to map the chromosomal locations of genes. Complex clone libraries were developed for the "painting" of entire chromosomes or chromosome arms (see Figure 2), a technique that is useful not only for the confirmation of suspected aberrations in clinical diagnosis, but also for basic research efforts aimed at elucidating the evolution and structure of genomes, relationships between structure and function, mechanisms of DNA repair, and chromosome segregation.
Figure 2. Comparative Genomic Hybridization.
The first step of comparative genomic hybridization involves the differential labeling of tumor and reference genomes with different fluorochromes. After hybridization to normal chromosomes in metaphase, images are acquired with fluorochrome-specific optical filters with the use of a special camera and digital imaging. Appropriate software is used to calculate fluorescence-intensity ratios that reflect genomic imbalances in the tumor genome. A ratio of 1 indicates an equal number of copies of chromosomes or chromosomal regions in the normal reference genome and in the tumor genome. Deviations from this ratio are indicative of either gains or losses in the tumor genome. This procedure permits the mapping of chromosomes or chromosomal regions that are critical for tumorigenesis. In general, genomic amplification indicates the presence of oncogenes, whereas the loss of regions indicates loci that contain tumor-suppressor genes.
Fluorescence in situ hybridization extended the application of cytogenetic diagnosis from chromosomes in metaphase to all stages of the cell cycle. This approach, termed "interphase cytogenetics," has transformed the diagnostic application of cytogenetic techniques because it allows the direct detection of specific genetic rearrangements together with cellular morphology without the need for cell culture, which is difficult and time consuming and is therefore not feasible for most diagnostic applications in solid tumors. Thus, interphase cytogenetics has improved the diagnosis of hematologic cancers and enhanced the interpretation of cytologic samples (e.g., fine-needle aspirates or Papanicolaou smears). The detection of an amplification of the HER2/neu oncogene in breast carcinomas by means of fluorescence in situ hybridization is one example of such an advance (see Figure 1B).
Conceptual and technical developments in molecular cytogenetics now permit the hybridization-based screening of genomes for both numerical aberrations (aneuploidy and genomic imbalances) and structural aberrations. The first such screening technique, called comparative genomic hybridization, changed our perception of genomic instability in cancer genomes. This technique compares the DNA content of cancer cells with that of normal "reference" genomes (see Figure 2) and thus reveals a blueprint of genomic imbalances. It does so through the differential labeling of the cancer genome (e.g., with the use of a green fluorochrome) and a reference genome (with the use of a red fluorochrome). After hybridization to normal chromosomes, gains and losses of DNA in the tumor genome can be mapped according to color ratios — for example, three copies of a specific chromosome in the cancer cell would shift the fluorescence ratio to green. This technique, which can be applied to archived, formalin-fixed, and previously stained tumor samples, provided a refreshed focus on preinvasive dysplastic lesions, which are difficult to study with chromosome-banding techniques, and allowed specific cytogenetic abnormalities in early-stage cancer to be correlated with the prognosis and the cellular phenotype. We now know that the dynamic genomes of carcinomas are defined by the sequential acquisition of chromosomal aneuploidies that are specific to a particular type of tumor and that are continuously selected for under conditions of prevailing genetic instability.
The second such screening technique resulted in the colorization of cytogenetics. With the use of novel imaging formats or improved optical filters, together with the combinatorial fluorescent labeling of chromosome-specific probes, all human chromosomes could be simultaneously discerned in different colors (see Figure 1C). Called spectral karyotyping or multiplex fluorescence in situ hybridization, this method unveiled previously hidden chromosomal aberrations, again with implications for both clinical diagnosis and basic research. The application of comparative genomic hybridization and spectral karyotyping to mouse chromosomes offers a tool for the evaluation of mouse models of human diseases. The presence of related chromosomal aberrations indicates that similar or identical genetic pathways are disrupted in humans and mice, which validates the mouse in question as an appropriate model.
Diagnostic cytogenetic techniques, whether based on banding or hybridization, are not high-throughput techniques and have to compete with those that are. Accordingly, several cytogenetic techniques, such as comparative genomic hybridization, have been converted to array-based formats (see Figure 1D). In conventional comparative genomic hybridization, the chromosomes in metaphase serve as the stationary phase for which the cancer and test genomes compete. In the array-based approach, DNA fragments that represent the chromosome are tethered to a slide or chip, and the results of the hybridization are interpreted as digitized fluorescence-intensity values. Genome screening for genomic imbalances will soon be routinely carried out on arrays of genomic clones or oligonucleotides. And it is conceivable that custom arrays geared toward addressing specific diagnostic challenges (such as the detection of subtle subtelomeric deletions that are frequently associated with mental retardation) will be produced. Such array-based cytogenetic analyses will be faster, more objective, of higher resolution, and more amenable to standardization than chromosome-based approaches. The chromosome itself will become less important as the primary target for diagnostic testing.
I predict, however, that the direct analysis of chromosomes and genes in cytologic preparations will continue to fill two important niches — one in diagnosis and the other in basic research. We will continue to strive to diagnose disease, and in particular cancer, as early as possible. This goal presents a challenge: morphologic alterations are not as well developed in early-stage cancer and premalignant lesions as they are in late-stage cancer, and it is therefore more difficult, and sometimes impossible, to establish a definitive diagnosis. Moreover, the fact that there are smaller numbers of cells at earlier stages makes it more difficult to obtain pure populations of tumor cells, a critical factor for array-based diagnostic formats that are sensitive to dilution with normal cells. These diagnostic challenges can be met by genetic tests that are based on individual cells, and fluorescence in situ hybridization with disease-specific genetic markers offers this very feature. As for basic research, the parallel monitoring of gene expression has greatly contributed to our understanding of mechanisms of disease, but our understanding of the coordination of gene transcription will undoubtedly depend on our understanding of the higher-order architecture and functional compartmentalization of the interphase nucleus. Again, such relationships between structure and function can be elucidated only by direct, high-resolution chromosomal analysis of individual cells.
During the past 40 years, cytogenetics has played a pivotal role in the identification, characterization, and diagnosis of genetic abnormalities in human diseases. It will be exciting to follow its colorful, digitized future.
Source Information
From the National Cancer Institute, Bethesda, Md.(Thomas Ried, M.D.)
Figure 1. Examples of Chromosome Banding, Interphase Fluorescence in Situ Hybridization, Spectral Karyotyping, and Array-Based Comparative Genomic Hybridization.
Panel A shows Giemsa-banded chromosomes from a patient with chronic myeloid leukemia. The arrows indicate the sites of a balanced translocation between chromosomes 9 and 22. This chromosomal translocation fuses two genes, BCR and ABL; the robust tyrosine kinase activity of their protein product is the molecular target of imatinib mesylate. Panel B shows interphase fluorescence in situ hybridization with the use of three probes (each a different color) on fine-needle aspirates from a breast carcinoma. The yellow and green signals indicate that chromosomes 7 and 17 are diploid, and the red signal reveals that the HER2/neu oncogene is amplified. Breast carcinomas with HER2/neu amplification respond to adjuvant treatment with herceptin. Panel C shows how spectral karyotyping can reveal massive chromosomal rearrangements in cells of a brain tumor (astrocytoma); distinct chromosome territories appear in the adjacent nucleus in interphase (lower left). Panel D shows an array-based analysis of genomic imbalances in a breast-cancer cell line. Dots that are red indicate increases in the number of copies in the tumor genome; green dots indicate a loss of DNA in the tumor genome.
The application of cytogenetics to the screening of the genomes of patients has led to the linking of specific chromosomal abnormalities (and consequently genetic alterations) with specific diseases; such findings carry enormous implications for medicine. The first such specific correlation was made when Jér?me Lejeune, in Paris, detected extra copies of a small chromosome in patients with Down's syndrome. Other descriptions of constitutional abnormalities, involving both autosomes and the sex chromosomes, followed in rapid sequence.
Such associations were also established in cancer cells. The first report on the so-called Philadelphia chromosome (described by Peter Nowell and David Hungerford in 1960 and characterized by Janet Rowley in 1973), created by an exchange of genetic material between chromosomes 9 and 22, launched the highly prolific field of cancer cytogenetics. (The Philadelphia chromosome reflects the specific target of imatinib mesylate in the treatment of chronic myeloid leukemia.) This work not only confirmed the relevance of cytogenetic abnormalities as disease-initiating events, but also led to the molecular cloning of numerous cancer-associated genes, many of which sit at the junctions of chromosomal translocations.
Cytogeneticists then adapted and combined molecular cloning and hybridization and so arrived at fluorescence in situ hybridization, which has had profound effects on cytogenetic diagnostic testing and research. Specific fluorescently tagged DNA probes could now be used to visualize, with dramatically increased resolution, small deletions and other genetic aberrations, as well as to map the chromosomal locations of genes. Complex clone libraries were developed for the "painting" of entire chromosomes or chromosome arms (see Figure 2), a technique that is useful not only for the confirmation of suspected aberrations in clinical diagnosis, but also for basic research efforts aimed at elucidating the evolution and structure of genomes, relationships between structure and function, mechanisms of DNA repair, and chromosome segregation.
Figure 2. Comparative Genomic Hybridization.
The first step of comparative genomic hybridization involves the differential labeling of tumor and reference genomes with different fluorochromes. After hybridization to normal chromosomes in metaphase, images are acquired with fluorochrome-specific optical filters with the use of a special camera and digital imaging. Appropriate software is used to calculate fluorescence-intensity ratios that reflect genomic imbalances in the tumor genome. A ratio of 1 indicates an equal number of copies of chromosomes or chromosomal regions in the normal reference genome and in the tumor genome. Deviations from this ratio are indicative of either gains or losses in the tumor genome. This procedure permits the mapping of chromosomes or chromosomal regions that are critical for tumorigenesis. In general, genomic amplification indicates the presence of oncogenes, whereas the loss of regions indicates loci that contain tumor-suppressor genes.
Fluorescence in situ hybridization extended the application of cytogenetic diagnosis from chromosomes in metaphase to all stages of the cell cycle. This approach, termed "interphase cytogenetics," has transformed the diagnostic application of cytogenetic techniques because it allows the direct detection of specific genetic rearrangements together with cellular morphology without the need for cell culture, which is difficult and time consuming and is therefore not feasible for most diagnostic applications in solid tumors. Thus, interphase cytogenetics has improved the diagnosis of hematologic cancers and enhanced the interpretation of cytologic samples (e.g., fine-needle aspirates or Papanicolaou smears). The detection of an amplification of the HER2/neu oncogene in breast carcinomas by means of fluorescence in situ hybridization is one example of such an advance (see Figure 1B).
Conceptual and technical developments in molecular cytogenetics now permit the hybridization-based screening of genomes for both numerical aberrations (aneuploidy and genomic imbalances) and structural aberrations. The first such screening technique, called comparative genomic hybridization, changed our perception of genomic instability in cancer genomes. This technique compares the DNA content of cancer cells with that of normal "reference" genomes (see Figure 2) and thus reveals a blueprint of genomic imbalances. It does so through the differential labeling of the cancer genome (e.g., with the use of a green fluorochrome) and a reference genome (with the use of a red fluorochrome). After hybridization to normal chromosomes, gains and losses of DNA in the tumor genome can be mapped according to color ratios — for example, three copies of a specific chromosome in the cancer cell would shift the fluorescence ratio to green. This technique, which can be applied to archived, formalin-fixed, and previously stained tumor samples, provided a refreshed focus on preinvasive dysplastic lesions, which are difficult to study with chromosome-banding techniques, and allowed specific cytogenetic abnormalities in early-stage cancer to be correlated with the prognosis and the cellular phenotype. We now know that the dynamic genomes of carcinomas are defined by the sequential acquisition of chromosomal aneuploidies that are specific to a particular type of tumor and that are continuously selected for under conditions of prevailing genetic instability.
The second such screening technique resulted in the colorization of cytogenetics. With the use of novel imaging formats or improved optical filters, together with the combinatorial fluorescent labeling of chromosome-specific probes, all human chromosomes could be simultaneously discerned in different colors (see Figure 1C). Called spectral karyotyping or multiplex fluorescence in situ hybridization, this method unveiled previously hidden chromosomal aberrations, again with implications for both clinical diagnosis and basic research. The application of comparative genomic hybridization and spectral karyotyping to mouse chromosomes offers a tool for the evaluation of mouse models of human diseases. The presence of related chromosomal aberrations indicates that similar or identical genetic pathways are disrupted in humans and mice, which validates the mouse in question as an appropriate model.
Diagnostic cytogenetic techniques, whether based on banding or hybridization, are not high-throughput techniques and have to compete with those that are. Accordingly, several cytogenetic techniques, such as comparative genomic hybridization, have been converted to array-based formats (see Figure 1D). In conventional comparative genomic hybridization, the chromosomes in metaphase serve as the stationary phase for which the cancer and test genomes compete. In the array-based approach, DNA fragments that represent the chromosome are tethered to a slide or chip, and the results of the hybridization are interpreted as digitized fluorescence-intensity values. Genome screening for genomic imbalances will soon be routinely carried out on arrays of genomic clones or oligonucleotides. And it is conceivable that custom arrays geared toward addressing specific diagnostic challenges (such as the detection of subtle subtelomeric deletions that are frequently associated with mental retardation) will be produced. Such array-based cytogenetic analyses will be faster, more objective, of higher resolution, and more amenable to standardization than chromosome-based approaches. The chromosome itself will become less important as the primary target for diagnostic testing.
I predict, however, that the direct analysis of chromosomes and genes in cytologic preparations will continue to fill two important niches — one in diagnosis and the other in basic research. We will continue to strive to diagnose disease, and in particular cancer, as early as possible. This goal presents a challenge: morphologic alterations are not as well developed in early-stage cancer and premalignant lesions as they are in late-stage cancer, and it is therefore more difficult, and sometimes impossible, to establish a definitive diagnosis. Moreover, the fact that there are smaller numbers of cells at earlier stages makes it more difficult to obtain pure populations of tumor cells, a critical factor for array-based diagnostic formats that are sensitive to dilution with normal cells. These diagnostic challenges can be met by genetic tests that are based on individual cells, and fluorescence in situ hybridization with disease-specific genetic markers offers this very feature. As for basic research, the parallel monitoring of gene expression has greatly contributed to our understanding of mechanisms of disease, but our understanding of the coordination of gene transcription will undoubtedly depend on our understanding of the higher-order architecture and functional compartmentalization of the interphase nucleus. Again, such relationships between structure and function can be elucidated only by direct, high-resolution chromosomal analysis of individual cells.
During the past 40 years, cytogenetics has played a pivotal role in the identification, characterization, and diagnosis of genetic abnormalities in human diseases. It will be exciting to follow its colorful, digitized future.
Source Information
From the National Cancer Institute, Bethesda, Md.(Thomas Ried, M.D.)