Fluorescence in situ hybridization (FISH) is a molecular method that allows detection of the number, size, and location of DNA and RNA segments within individual cells in a tissue sample. It is based on the ability of single-stranded DNA to anneal to complementary DNA. In hematologic disorders, the target DNA is the marrow or peripheral blood DNA present in interphase cells or the DNA of metaphase chromosomes that is fixed on a microscope slide.
Fig. 1 shows four types of FISH probes that are used alone or in combination to determine both numeric and structural rearrangements: (a) centromere enumeration probes, (b) whole chromosome painting probes, and (c) subtelomeric probes, while Fig. 2 shows the four FISH probe strategies used in probe design for detection of chromosomal translocations in hematologic malignancies. The first application of FISH technology for detection of chromosomal translocations in hematologic malignancies occurred when the BCR-ABL1 hybrid gene was identified using two-color FISH in interphase cells as well as in metaphase marrow-derived CML cells. In the standard strategy for interphase evaluation of chromosomal translocation, a DNA probe comprising sequences mapped proximal to the breakpoint in one of the chromosomes involved in reciprocal translocations is combined with a differentially labeled DNA probe that includes sequences mapped distal to the breakpoint in the other chromosome. Nuclei positive for the translocation display one dual-color fusion signal, representing one of the derivative chromosomes generated by the translocation, and two single-color signals, one for each of the normal alleles. This standard FISH strategy has been used for detection of translocations in hematologic disorders at diagnosis.
Fig1. TYPES OF CHROMOSOMAL PROBES (SEE TEXT FOR DETAILS). (A) Pair of chromosome 12 (left) and interphase cell (right) after fluorescence in situ hybridization (FISH) study with centromere enumeration probe (CEP) showing two hybridization signals (red) in the centromeric area of chromosome and two tight signals in interphase cell consistent with disomy (normal copy number). CEP probes are most useful for detection of numerical abnormalities. (B) Hybridization with a whole chromosome 8 painting probe showing the hybridization signal (green) along the length of the entire chromosome 8 (left) and hybridization domains in interphase cell (right). Whole chromosome painting probes are useful for identifying unknown chromosomes in metaphase cells. (C) Target of locus-specific indicators are specific gene sequences such as P53 seen after hybridization as two small signals (red) on chromosome 17, band p13. The main applications of locus-specific indica tor (LSI) probes are gene mapping, numerical enumeration in interphase cells, and detection of translocations. Telomeric probe, shown in green for the short arms of chromosome 17, are repetitive probes and are useful for detection of cryptic translocations involving ends of chromosomes. Chromosomes and nuclei are counterstained with DAPI (blue).
Fig2. FOUR DIFFERENT PROBE STRATEGIES FOR DETECTION OF CHROMOSOMAL TRANSLOCATIONS (SEE TEXT FOR DETAILS). (A) Normal cell after in situ hybridization with breakpoint cluster region (green) and ABL (Abelson) (red) showing a normal distribution of two red and two green single signals. (B) Conventional fusion strategy after in situ hybridization shows one fusion (yellow) signal representing derivative chromosome generated by the translocation and one single-color signal, red and green, for normal homologues in positive nuclei. (C) An extra-sensitive fusion approach generates an extra small (red) signal, as well as a fusion signal (yellow) and one signal in single color (green and red) on normal homologues. (D) Dual-fusion strategy generates two fusion signals (yellow) on two derivative chromosomes and one single-color signal on each of two normal chromosomes. (E) Breakapart approach in a normal cell appears as two fusion signals (yellow). In this strategy, the 3′ end and the 5′ part of the gene are labeled in two colors. (F) When the rearrangement occurs, the normal chromosomes show co-localization of red and green (yellow) as a result of the proximity of the sequences on the chromosome, whereas abnormal derivative chromosomes each have one single red and single green signal, indicating that the rearrangement occurred between the two ends of the gene separating the green and red signals on two different chromosomes. The third-color probe (blue) can be used as an internal control (usually centromere enumeration probe) to determine the disomic number of chromosomes.
One of the most significant advances in diagnostic leukemia cytogenetics has been the application of interphase FISH. Interphase cytogenetics is the term used to describe detection of chromosomal abnormalities in non-dividing, interphase nuclei (Fig. 3). Five aspects of interphase FISH are particularly useful: (1) Interphase cytogenetics allows screening of a large number of cells. This permits investigation of hematologic malignancies with a low mitotic yield, such as chronic lymphocytic leukemia (CLL) and multiple myeloma (MM). (2) Interphase FISH permits detection of chromosomal rearrangements in peripheral blood samples, thus obviating the need for marrow aspiration. For instance, in CML, which rarely yields a large number of dividing cells in peripheral blood, conventional cytogenetics usually is uninformative. However, detection of BCR-ABL1, the molecular equivalent of the Philadelphia chromosome (Ph), in peripheral blood using interphase FISH provides reliable, fast, quantitative results. (3) Interphase FISH offers a quantitative assay for monitoring disease progression or detection of minimal residual disease (MRD) following chemotherapy or hematopoietic stem cell transplantation (HSCT). (4) Use of specific probe sets allows detection of specific disease-associated abnormalities such as t(8;21), which denotes the M2 subtype of acute myeloid leukemia (AML), or t(15;17), which is associated with acute promyelocytic leukemia (APL), within 4 hours, allowing for timely and appropriate therapy. (5) FISH nomenclature is described in the International System for Human Cytogenetic Nomenclature.
Multicolor karyotyping permits examination of the entire genome in a single analysis (Figs. 4 and 5). In 1996 it became possible to identify 24 different human chromosomes (22 autosomes and the X and Y sex chromosomes), each with a unique color, with the help of fluorochrome-specific optical filters. This method is called multi color FISH (M-FISH). When interferometer-based spectral imaging is used, the method is called spectral karyotyping. The starting point in both methodologies is the use of whole chromosome painting probes for each chromosome. Thus each chromosome is labeled with a different combination of fluorescent dyes and images are sequentially obtained using five different fluorochrome-specific optical filters. A computer program combines the data and displays each chromosome as if it were stained with a distinct color. Spectral karyotyping is based on the use of an interferometer (used by astronomers to measure the light spectra of distant stars) to determine the full spectrum of light emitted by each stained chromosome. A computer program then displays all the chromosomes simultaneously, each with its own unique color. These methods are applied with increasing frequency to resolve complex karyotypes and to resolve the origin of the marker chromosome.
Fig3. SCHEMATIC REPRESENTATION OF CELL DIVISION. Most clinical FISH studies are performed on non-dividing interphase cells, whereas conventional cytogenetics is performed at the metaphase stage of cell division. FISH, fluorescence in situ hybridization. (Courtesy Dr. Ari Melnik, Cornell Medical Center, New York.)
Fig4. COMPLEMENTARY METHODOLOGIES OF IDENTIFYING VARIOUS GENOMIC ALTERATIONS AND THEIR RESOLUTIONS. See text for detailed descriptions of how these methodologies are applied for detection of genomic abnormalities in hematologic malignancies. Conventional cytogenetic methods detect clonal numerical and structural chromosomal abnormalities on a single cell level, at the resolution of 5 to 7 Mb. Multicolor FISH method with 24 different colors is specifically useful to identify the origin of marker chromosomes, complex 3- or more-way translocations, origin of ring chromosomes and other chromosomal abnormalities present in a complex karyotype such as those in MDS, lymphoma, and multiple myeloma. As mentioned in the text, there are at least five different reasons to perform interphase FISH in non-dividing cells from specimens of patients with hematologic malignancies. Interphase FISH is specifically useful for initial screening of the most recurrent rearrangements associated with CML, AML, ALL, and multiple myeloma as well as for detection of minimal residual disease with a diagnostic abnormality originally determined by conventional cytogenetics. In the lab of the author interphase FISH for PML-RARA fusion for the diagnosis of APL is a stat test with results obtained within 4 hours. Array CGH+SNP is a molecular method for detection of small and cryptic DNA changes at the exon-level resolution to 1 Mb. SNP platform is particularly useful for detection of acquired copy-number neutral chromosomal regions as well as detection of chromothripsis (see text). Next-generation sequencing (NGS) is the most powerful method for detection of acquired somatic mutation at the single nucleotide level. Although not yet used routinely in clinical laboratories, the application of NGS to hematologic malignancies has revolutionized the current knowledge of many leukemic entities. aCGH, array comparative genomic hybridization; ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; APL, acute promyelocytic leukemia; CML, chronic myeloid leukemia; FISH, fluorescence in situ hybridization; MDS, myelodysplastic syndrome; SNP, single-nucleotide polymorphism.
Fig5. Multicolor metaphase FISH of a bone marrow cell from a patient with myelodysplastic syndrome documenting 43,XY, −5, der(8)t(8;8)(p23; q11.2), der(14;16)(p12;p11.1), inv(15)(q21;q24), der(17)t(5;17)(p13;p13), −21 karyotype. The origin of t(8;8) and der(14;16) could not have been determined by conventional cytogenetic study alone. FISH, fluorescence in situ hybridization.
