fluorescence in situ hybridization and dna-fish probes

Fluorescence in situ Hybridization andDNA-FISH ProbesJune 2010www.cancergeneticsitalia.com

Cancer Genetics Italia 2010 2

Table of contents

What is the FISH Technique ?

The FISH Procedure

Signal Enumeration

The Cancer Genetics Italia DNA-FISH Probes

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2Cancer Genetics Italia, 2010

Cancer Genetics Italia 2010 3

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


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

Cancer Genetics Italia, 2010

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)


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



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.


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.


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.


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.


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


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.

Presenter

Presentation Notes

Break apart schematic = IGH C/V, Dual fusion = ABL/BCR, Locus-specific control = D13 = RB1 (CLL probe)

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Signal Enumeration: A Fusion Probe

BCRChromosome

22

ABLChromosome 9 ABL/BCR

der(9)

BCR/ABLPh or

der(22)

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22

9

9

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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14

14

14

14

Normal

t(IGH v)

t(IGH c)14

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t(IGH v)t(IGH c)

Bone Marrow


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


Signal Enumeration: A Copy Number Probe


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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fluorescence in situ hybridization and dna-fish probes

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