Fluorescence in Situ Hybridization

Fluorescence in Situ Hybridization

TuDong Nguyen

Arie Perry

Anjum Hassan

Fluorescence in situ hybridization (FISH) utilizes tagged probes that bind to chromosome-specific DNA sequences of interest, thereby allowing for the identification of both structural and numeric aberrations characteristic of certain hematopoietic and nonhematopoietic malignancies. While FISH can be performed on dividing (metaphase) cells, it has several major advantages over conventional cytogenetics in that it can be applied in many clinical settings (Table 59.1), can be performed on nondividing (interphase) cells, can be performed on air-dried or formalin fixed specimens, can facilitate detection of molecular abnormalities in neoplasms with low proliferation rates such as multiple myeloma, and can facilitate detection of numeric abnormalities. In surgical pathology, the technique is used primarily to detect somatic cancer-associated alterations with known diagnostic, prognostic, or therapeutic implications.

FISH provides insight into intranuclear target DNA localization and copy number. Therefore, using locus-specific probes (with the exception of XY sex chromosome determinations in males), two signals per nucleus are expected and so four common alterations are readily detectable: aneusomy (gain or loss of a chromosome), gene deletion, gene amplification, and translocation (e-Figs. 59.1 through 59.4).* Sex chromosome determinations can be useful in patients with sex mismatched bone marrow or organ transplants (e-Fig. 59.5), in order to monitor engraftment success or failure.


    • A. Specimens, retained morphology, and combined FISH/immunohistochemistry. FISH is applicable to a variety of specimen types, including fresh or frozen tissue, cytologic preparations, and formalin-fixed paraffin-embedded (FFPE) tissue (Table 59.2). The latter provides a particularly rich source of archival material.

      In clinical diagnostic testing, morphologic preservation is one of the principal advantages of FISH, particularly for studies on heterogeneous tissue samples in that it eliminates the need for microdissection (e-Fig. 59.6). An extension of this morphologic advantage comes from the possibility of combining FISH with immunohistochemistry, wherein separate assessments can be rendered in immunopositive and immunonegative cellular populations.

    • B. FISH versus other cytogenetic and molecular techniques. When compared with metaphase cytogenetics (see Chap. 58), interphase FISH has several clear advantages. One advantage is the lack of a requirement for mitotically active cells via cell culture, which removes potential artifacts due to in vitro growth selection biases such as overgrowth of nonneoplastic stromal elements. On the other hand, FISH is not a genomic screening tool; it provides a targeted approach for alterations that have been initially identified by more global molecular techniques, such as classic cytogenetics, loss of heterozygosity (LOH) screening, comparative genomic hybridization (CGH), array CGH (aCGH), array single nucleotide polymorphism (SNP) analysis, and gene expression profiling.

      In terms of resolution, FISH is more sensitive than conventional karyotypic analysis and CGH (both of which are limited to alterations of several Mb in size)
      but less sensitive than PCR-based assays for detecting small alterations (which can be designed to detect even single base pair mutations). Since FISH probes are typically at least 20 kb long, and most average 100 to 200 Kb long, alterations need to be fairly large for reliable detection by FISH, and consequently FISH cannot detect small intragenic mutations, deletions, or insertions.

      TABLE 59.1 Examples of Diagnostic Tests by FISH

      Prenatal testing

      Trisomy 13, 18, 21

      XY aneusomies

      Microdeletion syndromes

      Cri-du-Chat (5p)

      Prader-Wili/Angelman (15q)

      Di George syndrome (22q)

      Transplant pathology

      XY FISH on sex mismatched organ transplant

      Disease relapse using known genetic alterations in primary tumor

      Oncology (diagnostic, prognostic, and/or predictive markers)

      Chromosomal aneusomies

      Gene/locus deletions

      Gene amplifications


      Minimal residual disease or early recurrences are better detected by PCR of blood or fresh tissue specimens rather than FISH. Minimal residual disease detection usually involves detection of as few as one abnormal per million normal cells, a level of sensitivity that cannot be attained by FISH techniques to date. In contrast, FISH is very sensitive for identifying gene deletions or amplifications from samples of mixed cellularity, such as neoplasms with clonal heterogeneity or contaminating nonneoplastic elements (J Neuropathol Exp Neurol. 1997;56:999); in this setting, FISH can typically detect gains, translocations, or amplifications in as few as 5% and deletions in 15% to 30% of the cells within a sample.

    • C. Tissue microarray-FISH. This technology takes advantage of multi-specimen paraffin blocks (tissue microarrays, or TMAs) constructed from hundreds of 0.6 to 2.0 mm neoplastic, nonneoplastic, and control tissue cores of interest. TMA-FISH markedly increases efficiency by reducing data acquisition time, as well as probe, reagent, and storage space requirements. TMA studies have shown excellent morphologic, antigenic, and genomic concordance compared to the traditional whole slide approaches (Adv Anat Pathol. 2001;8:14), and although complications due to tumor heterogeneity can be problematic, adequate sampling can be optimized by incorporating multiple cores from each specimen. TMA-FISH is an excellent method for new probe validation, proficiency testing, interlaboratory comparisons, and quality assurance/quality control (J Histochem Cytochem. 2004;52:501).

      TABLE 59.2 Examples of Specimen Types Applicable to FISH

      Fresh/Frozen tissue

      Cytology specimens

      Body Fluids (e.g., urine)

      Intraoperative smears

      Cell culture preparations

      Formalin fixed paraffin embedded tissue

      Thin sections (4-6 µm)

      Disaggregated nuclei

      Archived unstained sections

      Previously stained sections (e.g., negative immunohistochemistry controls)

    • D. Disadvantages and pitfalls of FISH. Although recent technical advances have greatly enhanced the clinical applicability of FISH, a number of limitations remain. Signal fading is one of the main disadvantages. Clinical labs typically circumvent this pitfall by capturing digital images as a permanent record of each case; a permanent record is not otherwise possible unless chromogenic detection (CISH) is used. Unfortunately, multicolor CISH is not as simple as multicolor FISH; currently available chromogens lack the spectral versatility, sensitivity, and spatial resolution attainable with fluorochromes. Some commercial CISH applications bypass this problem by providing the test and reference probes separately, so that in place of one dual-color FISH assay, two single-color CISH experiments are performed. Recently developed photostable quantum dots offer a potential alternative for permanent fluorescent signals (J Histochem Cytochem. 2003;51:981). Other limitations include a variety of artifacts, particularly common in paraffin sections, that make correct interpretation of FISH results dependent on significant experience.

      • 1. Truncation artifact. This artifact is due to the underestimation of copy number because of an incomplete DNA complement within transected nuclei, and it is therefore important to assess controls cut at the same thickness.

      • 2. Aneuploidy and polyploidy. Artifacts due to aneuploidy and polyploidy can result in confusing signal counts and are a particularly common finding in malignant and even some benign neoplasms. Although the simplest approach is to interpret absolute losses (<2 copies) and gains (>2 copies), “relative” losses and gains can also be delineated on the basis of a reference ploidy, obtained either by flow cytometry or the assessment of multiple chromosomes by FISH. For example, cells with four chromosomes, nine centromeres, and two copies of the p16 gene region on 9p21 would be interpreted as having polysomy 9 and a hemizygous p16 deletion (e-Fig. 59.7A); a similar tumor with no p16 signals would be interpreted as polysomy 9 with homozygous p16 deletion (e-Fig. 59.7B).

      • 3. Autofluorescence. This is a particularly common problem in FFPE tissue sections. Although autofluorescent tissue fragments are usually larger and more irregular than true signals, some fragments have just the right size to mimic true nuclear signals. The use of multiple filters is helpful, since autofluorescence will often appear at several wavelengths of light, whereas true signals only fluoresce at one wavelength.

      • 4. Partial hybridization failure. This issue is most problematic when combining a highly robust probe (e.g., centromere) with a comparatively weak probe (e.g., small locus specific probe). This artifact can be minimized by counting only in regions where the majority of cells have discernible signals. Signals from both probes should be seen in normal cells (e.g., endothelial cells) within the region for the counts to be considered reliable.

    • E. Additional technical considerations. Many different FISH protocols are available; they vary depending on individual preferences and specimen type. Simple protocols are generally better, requiring less “hands on” time, fewer opportunities for error, and fewer troubleshooting requirements. Automated instruments are now available to minimize hands on time, though they are expensive. In general, the basic steps of the protocols are similar to those of immunohistochemistry and include deparaffinization, pretreatment/target retrieval, probe and target
      DNA denaturation, hybridization (a few hours to overnight), posthybridization washes, detection, and microscopic interpretation/imaging. FISH is therefore typically a 2-day assay, although same day assays are possible if the probes are particularly robust (e.g., centromere probes).

      Similar to immunohistochemistry, microwave or heat-induced target retrieval often enhances hybridization more effectively than chemical forms of pretreatment (Anal Cell Pathol. 1994;6:319). Nonetheless, optimal pretreatment and digestion varies from specimen to specimen and depends on a number of variables, including method of fixation and processing. Some hybridization buffers are also significantly more efficient and may lower probe concentration requirements considerably, which can be particularly beneficial with expensive commercial probes. Lastly, a variety of amplification steps are available for enhancing weak signals, although such steps are rarely necessary with robust commercial probes. One exciting advancement made possible by high-level signal amplification techniques is the potential use of smaller probes, down to the level of 1 kb or less (Biotechniques. 1999;27:608).


    • A. Centromere enumerating probes (CEPs) were among the first types of probes developed and remain ideal for detecting whole chromosome gains and losses, such as monosomy, trisomy, and other polysomies. CEPs target highly repetitive 171 bp sequences of α-satellite DNA, and so are associated with excellent hybridization efficiencies and typically produce large, bright signals. Unfortunately, sequence similarities in some pericentromeric regions result in cross-hybridization artifacts with the potential for overestimating signal counts. Another artifact is caused by the interesting phenomenon observed in nonneoplastic brain specimens in which certain chromosomes in interphase nuclei are packaged such that paired centromeres are in close proximity, a process known as somatic pairing (Hum Genet. 1989;83:231; Cytogenet Cell Genet. 1991;56:214); because of the close proximity, FISH yields an unexpected fraction of cells harboring a single large signal rather than two smaller ones, potentially leading to overinterpretation of monosomy. Despite these technical limitations, CEPs remain extremely useful for detecting aneusomies and are still among the best FISH probes available. The presence of repetitive DNA sequences in subtelomeric regions has led to the development of commercially available probes for each chromosomal arm as well.

    • B. A whole chromosome paint (WCP) probe consists of a cocktail of DNA fragments that targets all the nonrepetitive DNA sequences of an entire chromosome. Because a WCP covers such a large region, it produces a diffuse signal in interphase nuclei (although some of the smaller acrocentric chromosomes yield sufficiently discrete signals for enumeration, even in interphase nuclei). For this reason, WCPs are not often used in interphase FISH, but instead are primarily utilized in advanced cytogenetic applications (see Chap. 58). C. Currently, the most versatile FISH probes are locus-specific identifier probes (also known as LSI, or gene-specific, probes). These probes target distinct chromosomal regions of interest and utilize single copy rather than repetitive DNA sequences. In order to yield signals of sufficient size in interphase FFPE nuclei, the probe typically needs to be at least 20 kb long; the largest LSI probes are >1 Mb long, although most fall into the 100 to 300 kb range. The assortment of LSI probes available commercially has expanded greatly over the last few years. Additionally, cloning vectors, such as cosmids, bacterial artificial chromosomes (BACs), P1 artificial chromosomes (PACs), and yeast artificial chromosomes (YACs) are excellent sources for developing analyte specific (i.e., homemade) FISH probes. In the past, development of LSIs required a rather lengthy and tedious process of screening vector libraries, but the BAC libraries generated
      as part of the human genome project have made it possible to rapidly identify vectors that contain sequences of interest, gene names, or physical maps of individual chromosomes (http://www.genome.ucsc.edu). Similarly, mapped BAC clones spread throughout the human genome at 1-Mb intervals have also become available (http://mp.invitrogen.com). However, regardless of how a probe is obtained, it is important to verify its identity, either by screening for the DNA sequence of interest by PCR or by performing metaphase FISH to determine that the probe localizes to the appropriate cytogenetic band (e-Fig. 59.8).

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Oct 20, 2016 | Posted by in PATHOLOGY & LABORATORY MEDICINE | Comments Off on Fluorescence in Situ Hybridization

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