fluorescence in situ hybridization and dna-fish probes
TRANSCRIPT
Fluorescence in situ Hybridization andDNA-FISH ProbesJune 2010www.cancergeneticsitalia.com
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Table of contents
What is the FISH Technique ?
The FISH Procedure
Signal Enumeration
The Cancer Genetics Italia DNA-FISH Probes
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Fluorescence In Situ Hybridization, a widely and globally used clinical test
Molecular cytogenetics test Adjunct to conventional cytogenetics Used for the diagnosis, prognosis and clinical
management of patients in many medical fields. Detection of presence or absence of a specific
chromosomal abnormality (visible or not by conventional cytogenetics) in a single cell
Most cancers show cytogenetic abnormalities
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Conventional Cytogenetics (G-banding)
Chromosomes contain both the heterochromatin (dark bands referred as G and transcriptionaly inactive) and the euchromatin (light bands referred as R and gene rich).
XY
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1. Using Interphase nucleiAlthough G band karyotyping is the clinical gold standard, it requires actively dividing cells (which can be difficult with some disorders)
2. Technical specializationConventional cytogenetics analysis requires a highly trained technician whereas FISH is an easier, and more straightforward technique.
3. TimeThe time it takes to process, hybridize and analyze a FISH sample is shorter than the time it takes to do the same for a G banded karyotype
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Advantages of FISH over Conventional Cytogenetics
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FISH is used in Clinical Management
Tarceva® is a trademark of OSI Pharmaceuticals, Inc.Herceptin® is a trademark from Roche
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Primary exam Diagnosis Prognosis Follow up
Use of DNA-FISH ProbeABL1/BCR (CML)PML/RARA (APL)
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Treatment management
Use of DNA-FISH ProbeMLL Break Apart (ALL/ AML)ERBB2 & Cen17 (Breast carcinoma)
Use of DNA-FISH ProbeEGFR & Cen7 (Gefitinib/Iressa and Erlotinib/Tarceva®)ERBB2 & Cen17 (Response to herceptin®
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Specimen Sources
Peripheral Blood/ Bone Marrow
Paraffin Embedded Tissue (FFPE)
These specimen types are used for the detection of hematological Malignancies (lymphomas, leukemias)
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Used in the cases of solid tumors.Tissue architecture allows for a pathological, cytological and FISH assessment to be made (and adjunct to IHC).
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The FISH procedure: An Overview
Target DNA
Step 2: Hybridization
Fluorescently labeled probe DNA
Step1: Heat denaturation
Specimen
Step 3: Washing and Analysis
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The slides should be prepared according to the laboratory guidelines. Importance should be given to room temperature and hygrometry.
Clean the slide prior to droppng the specimen.
Use gloves at all time when handling specimens. All reagents should be handled as if hazardous.
The FISH Procedure: The Slide Preparation
The specimen can be baked on a temperature controlled hot plate or in an oven.
Ensure the calibration of all apparatus prior to runnning the test.
The specimen can be dropped in many ways, but care should be given to ensure that the nuclei spread properly.
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The FISH Procedure: Dispensing the Probe
Drop the DNA-FISH Probe on the region of interest.
Prior to hybridization, the specimen should be checked under a phase microscope to ensure a proper morphology of the nuclei.
Place a cover slip on the region of interest.
The size of the cover slip can be changed according to the volume of Probe dropped on the slide.
Attention should be given to avoid any air bubbles.
An easy way to get rid of bubbles is to gently roll the tip of a pencil on the cover slip.
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The FISH Procedure: The Hybridization
Prior to hybridization, seal the coverslip on the slide with rubber cement.
Make sure that the rubber cement straddles the edge of the coverslip.
Seal the entire coverslip with the rubber cement.
Place the slide on a controlled hot plate protected from light.
Incubate for 12-18 hours.
Alternatively, the slide can be placed in a dark box at 37oC.
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The FISH Procedure: Post-hybridization
When the hybridization is completed, remove the rubber cement with a forceps by gently pulling one of the corner of the seal.
The coverslip may come along.
Dip the slide in the 2X SSC at room temperature.
If the coverslip did not come out while removing the rubber cement, it may be removed by tapping gently the slide on the Coplin jar.
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The FISH Procedure: Signal Analysis
Perform the washes as recommended in the instructions for use.
Apply the DAPI/Antifade on the region of interest.
Store the slide protected from light at 4oC or -20oC for a long term storage.
Signals should be analyzed under an epi-fluorescence microscope.
Scan the target area with a 10x objective. Scoring should be done with a 63x or 100x objective.
Attention should be given to the lifespan of the mercury lamp. Do not use lamp having over 200 hours of usage.
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Control
Target
13q34
13q
13q34
RB1/D13S1009
Control
Target
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ERBB2
ERBB2/Cen 17
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Signal Enumeration: The Probe Design
Translocation Probes
22der(22)
der(9)9
ABL1/BCR
der(11)
11der?
MLL Break Apart
Fusion ProbeExpected abnormal signal pattern is 1 red, 1 green and 2 fusion signals.
Break Apart ProbeExpected abnormal signal pattern is 1 red, 1 green and 1 fusion signals.
Copy Number Probes
Deletion ProbeExpected abnormal signal pattern is 1 red, and 2 green signals.
Amplification ProbeExpected abnormal signal pattern is 2 greenand over 2 red signals.
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Signal Enumeration: A Fusion Probe
BCRChromosome
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ABLChromosome 9 ABL/BCR
der(9)
BCR/ABLPh or
der(22)
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22
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der(22) der(9)
22der(22)
der(9)9
der(22) der(9)
ABL
BCR
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IGH
centromere telomere14q32
~578 kb ~420 kb
IGH5’ 3’
11 t(11;14)(q13;q2)
t(IGH v)
t(IGH c)
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Normal
t(IGH v)
t(IGH c)14
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t(IGH v)t(IGH c)
Bone Marrow
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Signal Enumeration: A Break Apart Probe
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centromere telomere17
ERBB25’ 3’
100x Zoom view of inset
~160kb
Normal Breast tissue Abnormal Breast tissue
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Signal Enumeration: A Copy Number Probe
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Cancer Genetics Italia DNA-FISH Probes
Filter requirement
Advantages
Ready-to-useWashing time allows for flexibilityShorter processDetection of submicroscopic rearrangements
Fluorophore Excitation max Emission max
Green 496 nm 520 nm
Red 580 nm 603 nm
DAPI 360 nm 460 nm
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Cancer Genetics Italia DNA-FISH Probes Pipeline
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