Molecular Pathology:

FISH I

Luigi Tornillo

PathoBasic 12.08.2014

Pathology

• Introduction

• Indications

• Techniques/Procedures

• Examples

*ISH

„The use of DNA (or RNA) probes to

detect complementary genetic material in

cells or tissue. ISH involves hybridizing a

labeled nucleic acid to suitably prepared

cells or tissues on microscope slides to

allow visualization in situ (the normal

location)“

Pathology

*ISH

• FISH

• SISH

• CISH

Pathology

PNAS, 1969: some kind of SISH?

FISH

• Numeric alterations

–Gains (Amplifications)

–Losses (Deletions)

• Translocations (balanced

aberrations)

• Interphase cytogenetics

Comparison of cytogenetic techniques for identifying chromosomal abnormalities.

Bishop R Bioscience Horizons 2010;3:85-95

© The Author 2010. Published by Oxford University Press.

FISH and pathology: indications

• Diagnostics

– Soft tissues • MDM2, CDK4

• USP6

– Lymphomas • CMYC

• CCND1

– Urothelium • Aneusomy

– Etc...

• Prediction

– Breast, stomach • ERBB2 (HER2)

– Lung • ALK, ROS1, RET

– Etc...

Pathology

Pathology

Centromeric probes (CEP)

• High repetitive sequences

• 250-5000 kb

• Aneuploidy

• In combination with locus-specific

for gain/losses (dual-color)

• Sometimes tricolor

Pathology

Locus-specific probes

• 500bp-102 kb

• Very specific

• Amplifications, deletions

• Translocations

• LSI®

Other probes

• Telomeric

–100 kb, unique subtelomeric

• Painting

–Chromosome or chromosomal arm

–Gross aberrations on metaphase

spread (FISH-banding)

Pathology

Choosing the slides

• Pathologist

• Tumor architecture + cytology

• No necrosis

• No overlapping nuclei

Pathology

Prehybridization step

• Manual/automated

Pathology

Protease digestion

• Tissue permeabilization, protein

digestion

• Overdigestion: less intensity, poor

morphology

• Underdigestion: autofluorescence, not

distinguishable signals

• Poorly differentiated > less protease

• Mucinous/fibrotic > more protease

Pathology

Pathology

Un-FISH-able fixatives

• Unbuffered formalin in archives

• B5, Bouin, heavy metal

• Sousa

• Schäfer

• All acid-containing fixatives

• “Stark” decalcifying media

–Prefer EDTA

Pathology

Denaturation/Hybridization

• Manual/automated

• Kit/“In-house“

• ≈ 73°C-75°C in ≈50% formamide

• 37°C in humid chamber 12h overnight

• Cot-DNA

• Repetitive sequences

Pathology

Denaturation/Hybridization

Tm = 81.5 + 16.6 * log

[Na+] + 0.41 * (%GC) -

0.63 (% formamide v/v)

– (300 + 2000* [Na+])/N

??

Pathology

• Temperature of annealing (usually 72-

75°C)

Lower T°C lower sensitivity & specificity

• Na+ concentration

Lower higher specificity

• Formamide concentration

Higher higher specificity

Denaturation/Hybridization

Pathology

Posthybridization (washing)

• Remove only non-specifically bound probe

• Cytology/fresh more stringent (lower [Na+])

• FFPE less stringent (higher [Na+])

– Smaller DNA fragments, proteins

• DAPI

• Antifade (photobleaching)

Pathology

Fluorescence microscopy

Pathology

Fluorescence microscopy

Stokes shift

Pathology

Pathology

• FISH

– Dark room

– Morphology difficult to recognize

– „Fading“

– Pictures

– Several probes with different stains

• SISH

– Light microscopy

– Automatized

– Morphology preserved

– Perpetual

– 2 stains

– ERBB2 (HER2), CMET, MDM2

Pathology

CEP probes

• Aneuvysion

– 13q14, 18, 21q22.13-q22.2 (green, aqua,

red)

– X, Y (green, red)

• Urovysion

– 3, 7, 17, 9p21 (red, green, aqua, gold)

• All chromosomes

13 18

21 X, Y

3, 7, 17, 9p21

Gene gain/loss tests

• Dual color tests (LSI + CEP, usually

green + red/orange)

• CEP reference

• At least 20 cells (at IfP 50)

• Ratio LSI/CEP signals

• Cut-off?

Pathology

HER-2 FISH: Amplification

HER-2 SISH: Amplification

HER-2 FISH: No amplification

HER-2 SISH: No amplification

Translocation: bap

• 600-1500 kb

• Relatively sensitive

• Binds the normal chormosone flanking

the break site

• The two probes separate

Translocation: bap

• Translocation partner unknown

• Detects unusual translocations

• Relatively reliable and easy to

interprete

Translocation: fusion

• 600-1500 kb

• Spezifisch

• Breakpoint of both

translocation

partners

• The two probes fuse

Pathology

Translocation: fusion

• Identifies (only) the translocation

partner

• Create non-standard patterns

• May find non-standard breakpoints

• Very demanding technically

• Useful as accessory

Pathology

NORMAL TUMOR

Truncation of nuclei in histological sections Thickness of section: Thickness of nuclei: variable, often larger than thickness of section

Literatur

• Pfeifer D, Molecular Genetic Testing in

Surgical Pathology. LWW, 2006

• Cagle PT, Craig Allen T, Basic Concepts

in Molecular Pathology. Springer, 2009

• Wiktor A et al., Preclinical validation of

fluorescence in situ hybridization

assays for clinical practice. Gen Med,

2006; 8 (1):16-23