FISH Techniques and Applications PDF

For classroom use only Fluorescence In Situ Hybridization (FISH) Prepared by: Christian Joseph N. Ong, MSc, LPT Department of Biology Genetics Laboratory 1 The Fluorescence Microscope (Part 1) Genetics Laboratory 2 Fluorescen...

For classroom use only Fluorescence In Situ Hybridization (FISH) Prepared by: Christian Joseph N. Ong, MSc, LPT Department of Biology Genetics Laboratory 1 The Fluorescence Microscope (Part 1) Genetics Laboratory 2 Fluorescence Microscope Genetics Laboratory 3 Epifluorescence Microscope Genetics Laboratory 4  Fluorescence Microscopy A type of electromagnetic spectroscopy which analyzes fluorescence from a sample Beam of light (e.g. UV) excites electrons in molecules of certain compounds and causes them to emit light, typically but not necessarily visible light Genetics Laboratory 5  Fluorescence Microscopes A microscope that uses fluorescence to generate an image, whether it is a more simple set up like an epifluorescence microscope, or a more complicated design such as a Confocal microscope, which uses optical sectioning to get better resolution of the fluorescent image Genetics Laboratory 6  Epifluorescence microscopy A mode of fluorescence imaging where light passes through the sample in a straight angle, maximizing the amount of illumination Uses fluorescence and phosphorescence in addition to absorption, scattering, reflection, attenuation. Uses high energy light (xenon, mercury lamp; now LED) Genetics Laboratory 7 Fluorescence Microscopy Excitation filter Genetics Laboratory 8  Applications of Fluorescence Mineralogy, Gemology Chemical sensors (fluorescence spectroscopy) Fluorescent labelling dyes Biological detectors Fluorescent lamps Genetics Laboratory 9  Applications Routine Karyotyping/Cytogenetic analysis Color Karyotyping Spectral Karyotyping (SKY) Multicolor Fluorochorme banding (mBand) Automatic Telomere measurement Genetics Laboratory 10 Imaging Systems Genetics Laboratory 11 Zeiss AxioImager Z2 with ASI Cytogenetics Workstation GENASIS HISKY – HYPERSPECTRAL KARYOTYPING Genetics Laboratory 12 Genetics Laboratory 13  Features of the Imaging Sytem Image Acquisition and Processing Measurement and Analysis Reporting and Data Management Configuration and Updates Genetics Laboratory 14  GenASIs Capture & Analysis (BandView) for Karyotyping Genetics Laboratory 15 Fluorescence in situ hybridization Genetics Laboratory 16  Fluorescence in situ Hybridization (FISH) Powerful technique used in detecting chromosomal abnormalities/mutations Introduced late 1980’s Addressed the limitations of routine chromosome analysis Refined the level of cytogenetic screening Genetics Laboratory 17  Fluorescence in situ Hybridization FISH High specificity and sensitivity Shortened turn around time (TAT) of patients results Genetics Laboratory 18  A B In figures A and B, the Y chromosome (arrows) shows remarkably bright fluorescence and is the most intense in the entire complement. Mayo.edu Genetics Laboratory 19  Basic Steps in FISH *Initially both target and probe are double stranded Denature the chromosomes Denature the probe Hybridization Fluorescence staining Slide Examination (fluorescence microscope); in the dark Genetics Laboratory 20 http://www.abnova.com/images/content/support/Banner_fish.gif Genetics Laboratory 21  Fluorescence In Situ Hybridization (FISH) Hybridization of complimentary DNA with attached fluorophore to target DNA Source: Bishop R. 2010 Genetics Laboratory 22  Types of FISH Metaphase FISH Interphase FISH www.invitrogen.com RBD, SLMC Genetics Laboratory 23  Fiber – FISH High-resolution fluorescence in situ hybridization (FISH) on deproteinized, stretched DNA prepared by in situ extraction of whole cells immobilized on microscope glass slides allows the visualization of individual genes or other small DNA elements on chromosomes with a resolution of approx 1000 bp. Genetics Laboratory 24  Fiber Fish Technique has been successfully used in physical mapping in the 1–300 kb; range as well as for detecting genomic rearrangements. Genetics Laboratory 25 Fiber-FISH on extended DNA, obtained from subjects C1 and C5. Upper panel: subject C1, utilized as control, shows all 4 DAZ gene copies. Lower panel: the normozoospermic fertile C5 man presents only 3 fluorescent regions of the DAZ cluster (D’amico et al., 2006). Genetics Laboratory 26  Types of FISH Detection DIRECT FISH INDIRECT FISH Reporter + Probe Antibody + Reporter + Probe SLIDE SLIDE Target (DNA) Target (DNA) Genetics Laboratory 27 Schematic representation of in situ hybridization detection using amplification with a dye-labeled anti-dye antibody Genetics Laboratory 28 Schematic representation of mRNA in situ hybridization detection using Tyramide signal amplification (T5A) in the presence of Horseradish peroxidase and hydrogen peroxide; tyyramide radicals are formed (red box) that can covalently react with nearby residues/ Genetics Laboratory 29 Genetics Laboratory 30 Sample DNA Probe DNA Fluorescent In Situ Hybridization Denature Hybridize Direct Label Wash & Detect Indirect Label Genetics Laboratory 31 Different Types of FISH Probes A B C D Chromosome Centromeric Telomeric Gene-specific Painting probe probe probe Repetitive sequences Genetics Laboratory 32 Chromosome arm specific probe. (XCAP, Metasys) Genetics Laboratory 33 Genetics Laboratory 34 Genetics Laboratory 35 Specific FISH probes Genetics Laboratory 36 Examples of FISH patterns. A: Schematic of dual color dual fusion fluorescent in situ hybridization (FISH) probes. B: Schematic of break apart FISH probes. Genetics Laboratory https://oncohemakey.com/molecular-genetic-aspects-of-non-hodgkin-lymphomas/ 37  https://image.slidesharecdn.com Genetics Laboratory 38 Acute Propmyelocytic Leukemia (APL) Transretinoic acid Genetics Laboratory 39 FISH and Cytogenetics : A Tandem for Medical Diagnosis Genetics Laboratory 40 ROUTINE CHROMOSOMAL ANALYSIS also known as karyotyping / cytogenetic analysis preparation of an organized profile of a patient’s chromosomes essential for detecting chromosomal abnormalities associated with congenital disorders/diseases Genetics Laboratory 41  Chromosomal Analysis Differential banding: G-banding Resolution of banding analysis: > 3 Mb of DNA Limited to dividing cells: Metaphase Uses Monochrome Banding pattern: Dark and Light bands Time consuming Technical Skill needed Genetics Laboratory 42 Karyotyping Sample Requirements 10 ml heparinized peripheral blood (for non- malignant cases) 2-3 ml heparinized bone marrow aspirate (for hematological malignancies) Solid parts of affected tissue (aseptically extracted) Genetics Laboratory 43  Sample requirements Bone Marrow aspirate 2 - 3 ml, in GREEN top vacutainer submit to the lab within 24 hours after extraction if sample cannot be submitted immediately, store in refrigerator (4°C) Transport sample in a well insulated container You may use ice but it should not be in direct in contact with the sample so as not to freeze it. Genetics Laboratory 44 NORMAL HUMAN KARYOTYPES 46,XX (female) 46,XY (male) Genetics Laboratory 45 How the results would look like as applied to Leukemia cases Genetics Laboratory 46  BCR ABL e1 b2 a2 BCR BCR-ABL RNA (q11) fusion Derivative 22 Normal 22 PROTEIN ABL ABL-BCR (q34) fusion (p210) Normal 9 Derivative 9 A B Fig. 2. A. Reciprocal translocation in chromosomes 9 and 22; t(9;22)(q34;q11) producing the Philadelphia (Ph) chromosome. The fused gene in the Ph chromosome produces the BCR-ABL protein which cause white blood cells to actively divide causing the malignancy. Genetics Laboratory 47 Genetics Laboratory 48  Chronic myelogenous leukemia (CML) Negative for bcr-abl gene fusion: Positive for bcr-abl gene fusion Green color signals for BCR gene 1F1G2O; (chrom 22); 2 orange signals for ABL gene (chrom. 9). Reading: 2G2O. (1 fusion of green and orange = yellow) Genetics Laboratory 49 Metaphase spread with BCR-ABL gene fusion Probe: VYSIS LSI BCR/ABL ES Dual Color Translocation Probe The chromosomes and interphase cells are stained with DAPI stain (a nucleic acid stain) Genetics Laboratory 50 Probe: VYSIS LSI BCR/ABL ES Dual Color Translocation Probe A nucleus lacking the t(9;22) translocation will exhibit the two orange, two green (2O2G) signal pattern. In a nucleus possessing the t(9;22), involving the M-bcr, one green (native BCR), one large orange (native ABL), one smaller orange (ES) , and one fused orange/green signal (5’ BCR/3’ ABL), (2O1G1F) will be observed. Genetics Laboratory 51 Probe: VYSIS LSI BCR/ABL DCDF Translocation Probe A nucleus containing simple balanced t(9;22), as shown by one red and one green signal from the normal 9 and 22 chromosome and two red/green (yellow) fusion signals, one each from the derivative 9 and 22 chromosomes, will be observed (1R1G2F). Genetics Laboratory 52  DAPI Stain ▪ When bound to double-stranded DNA DAPI has an absorption maximum at a wavelength of 358 nm (UV) and its emission maximum is at 461 nm (blue). ▪ For fluorescence microscopy DAPI is excited with UV light and is detected through a blue/cyan filter. ▪ Can also bind with RNA (not as strongly fluorescent) DAPI (magenta) bound to the minor groove ▪ Its emission shifts to around of DNA (green and blue 500 nm when bound to RNA. Genetics Laboratory 53 4676 20 yrs old, Male Diagnosis: CML Sample: Bone marrow aspirate Normal BCR-ABL gene fusion Result:19.08% of cells analyzed were found Karyotype: 46,XY positive for BCR-ABL gene fusion. Total # of interphase cell analyzed: 325 # of cells positive for BCR-ABL fusion: 62 Genetics Laboratory 54 4640 31 yrs old, Female Clinical Impression: Myeloproliferative neoplasm;CML Sample: Bone marrow aspirate Normal BCR-ABL gene fusion Result: 60.8% of cells analyzed were found positive for BCR-ABL gene fusion. Karyotype: 46,XX,t(9;22)(q34;q11) Total # of interphase cell analyzed: 250 # of cells positive for BCR-ABL fusion: 152 Genetics Laboratory 55  Imatinib Mesylate (Glivec) Glivec is a molecularly targeted chemo drug for BCR-ABL positive CML patients. Genetics Laboratory 56  Acute Promyelocytic Leukemia (APL) APL = Translocation between Trans Retinoic acid chromosomes 15 and 17. t= (15;17)(q24;q21) Trans Retinoic Acid is a molecularly Targeted drug for patients positive for t(15;17)(q24;q21) or PML/RaRa gene fusion). Genetics Laboratory 57  FLUORESCENCE in situ HYBRIDIZATION (F.I.S.H.) used for determining XX / XY cell ratio in sex-mismatched bone marrow transplants Donor » Male, Recipient » Female Donor » Female, Recipient » Male Genetics Laboratory 58 Probe: CEP X SpectrumOrange/CEP Y SpectrumGreen DNA Probe Kit In a normal male cell, the expected pattern for a nucleus hybridized with the CEP X/Y DNA Probe is the one orange, one green (1O1G) signal pattern. In a normal female cell the two orange (2G) single pattern for female donor cells will be observed. Genetics Laboratory 59 FISH in Solid Tumors Genetics Laboratory 60 Genetics Laboratory 61  Gene Mutations increase susceptibility to Breast Cancer Chromosome 13 Chromosome 17 BRCA2 gene 13q13 BRCA1 gene 17q21 Genetics Laboratory 62  FDA Approved technologies used worldwide for Breast cancer screening 1. Immunohistochemistry (IHC) 2. Fluorescence in situ Hybridization (FISH). 3. Chromogenic in situ Hybridization (CISH) Genetics Laboratory 63 A. Immunohistochemistry (IHC) involves the process of selectively identifying antigens (proteins) in cells of a tissue section based on the principle that antibodies bind specifically to antigens in biological tissues. B. FISH involves the process of hybridizing the specific fluorescence probe to its specific target (fixed on a slide). Genetics Laboratory 64  BRCA1 gene The BRCA1 tumor suppressor gene long arm of chromosome 17 (17q11–21). BRCA1 protein, a multifunctional protein Functions in DNA damage response, and transcriptional regulation, and serves to maintain genomic stability Genetics Laboratory 65  BRCA1 and BRCA2 These women’s risk of ovarian cancer is also increased. Abnormal BRCA1 or BRCA2 genes are found in 5-10% of all breast cancer cases in the United States. Women with an abnormal BRCA1 or BRCA2 gene have about a 60% risk of being diagnosed with breast cancer during their lifetimes (compared to 12-13% for women overall). Genetics Laboratory 66 BRCA 1 gene FISH Probes Genetics Laboratory 67  BRCA1 and 2 Gene Deletion Figure 4: Two-color FISH of BRCA1 and chromosome 17 centromere. A sporadic tumor cell showing in (A) a physical deletion of BRCA1. Two-color FISH of BRCA2 and ETB (13q reference probe). A sporadic tumor cell showing in (B) a physical deletion of BRCA2. The probes are visualized in green and red colors (fluorescein and Texas Red, respectively). The probes are marked with the corresponding color in each panel to indicate the color they are visualized in. The nuclei were counterstained with DAPI (blue). The case numbers are marked in each panel in white. Genetics Laboratory 68  Her2/neu HER2/neu (often just shortened to HER2) is a growth-promoting protein on the outside of all breast cells. Also called ERBB2, a known proto-oncogene, located at chromosome 17 (17q12); near BRCA1 gene locus Genetics Laboratory 69 Genetics Laboratory 70  Testing for HER2 Status ▪ An accurate HER-2 assessment can enable the most appropriate therapy decision. ▪ Test should measure either gene copy number or presence of protein receptors. ▪ Use of formalin-fixed, paraffin-embedded breast cancer tissue. ▪ Trastuzumab by Genentech is a targeted monoclonal antibody treatment for women with HER-2 positive metastatic breast cancer. Genetics Laboratory 71 Testing for HER2/neu status Genetics Laboratory 72 http://www.aboutcancer.com/herceptin_0211.htm Genetics Laboratory 73 ❑ Count the HER-2 and Chromosome 17 signals in 20 nuclei, ❑ Calculate the ratio of HER-2 to Chromosome 17. ❑ If the value is 2 or >2, the patient is considered amplified for HER-2 or positive. ❑ If the test shows a normal gene count, she is considered HER-2 negative. ❑ FISH allows the viewer to literally count the genes; help overcome technical and interpretative limitations of other testing methods. ❑ Because this test measures genetic material, which is very stable, tissue Sauter G, et al. Guidelines for preparation has very little effect on test Human Epidermal Growth Factor outcome.1 Receptor 2 Testing: Biologic and Methodologic Considerations. J Clin Oncol 27:1323-1333, 2009. Genetics Laboratory 74  KITS for Bladder Carcinoma Screening designed to detect aneuploidy for chromosomes 3, 7, 17, and loss of the 9p21 locus via fluorescence in situ hybridization (FISH) Urine specimens from persons with hematuria suspected of having bladder cancer. an aid for initial diagnosis of bladder carcinoma monitoring for tumor recurrence in patients previously diagnosed with bladder cancer Genetics Laboratory 75 A schematic diagram of a cross-section of the bladder. The three-layered structure (on the right) show how tumor grade is determined based what later of the tissue is affected. the different tumor grades in bladder cancer. Genetics Laboratory 76 Specific Centromeric Probes used in Bladder Carcinoma Genetics Laboratory 77  Normal cell Abnormal cell with Aneuploidies (extra copies of chromosomes Multiple Probes for Bladder carcinoma: Spectrum red for Chrom 3, Green for Chrom 7; Spectrum Aqua for chrom 17 and Spectrum gold for Chrom. 9. Genetics Laboratory 78  Spectral Karyotyping (SKY) A multi-fluorochrome fluorescence hybridization technique (FISH) in which all the chromosome pairs are simultaneously visualized in different colors in a single hybridization can be used to analyze human, mouse, and rat chromosomes This software is available in the Cytogenetics Workstation iCELL Genetics Laboratory 79  GenASIs HiSKY Hyperspectral Karyotyping a unique technology Multiplexed spectral imaging Each chromosome pair is uniquely colored in a karyotype Use of specialized probe kit developed by ASI Genetics Laboratory 80 Probe: MFISH Genetics Laboratory 81 Computer generated “false color”. Multicolor Banding (M-Band) is an advanced chromosome painting technique that combines multiple chromosome or region- specific paints in one step. The combination of probes that hybridize to a particular chromosome produces a unique pattern for each chromosome. Genetics Laboratory 82 Genetics Laboratory 83 Thank you Genetics Laboratory 84