Molecular Pathology 1.0 - Histoteknikerforeningen

Molecular Pathology – What’s new out there!
Juliana Hughes, Ph.D
Application Specialist
Norway
Dako Nordic Sales
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Agenda
1)
2)
Introduction
Molecular Biology – Genetic errors

Types of genetic errors
3) What technology to use?

In situ hybridization (ISH)
4) Probes
•
Types of probes
5) ISH Method – FISH step-by-step
6) Principle of CISH
7) SureFISH
8) IQ Buffer
9) Dako Solution
10) Summary
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Molecular Pathology
H&E
Special
Stains
IHC
Molecular
Pathology
PharmDx
Workflow & Solution Integration
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Digital
Pathology
Genetic errors
Chromosome
abnormality
Amplification
Deletion
Translocation
Point mutation
A
C
Many types of genetic errors
Different technologies depending on error type
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Types of genetic errors
Chromosome
abnormality
Amplification
Deletion
Translocation
Point mutation
A
C
Atypical number of chromosomes or a structural abnormality in one
or more chromosomes:
•
Down syndrome
•
Turner syndrome
•
Cri du chat syndrome
•
Klinefelter syndrome
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Types of genetic errors
Chromosome
abnormality
Amplification
Deletion
Translocation
Point mutation
A
C
DNA
Increased copy number of
a particular gene
E.g. HER2 amplification
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Types of genetic errors
Chromosome
abnormality
Amplification
Deletion
Translocation
Point mutation
A
C
DNA
Decreased copy number of
a particular gene
E.g. TOP2A deletion
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Types of genetic errors
Chromosome
abnormality
Amplification
Deletion
Translocation
Point mutation
A
C
Large fragments of DNA exchange
locations and thereby end up at
wrong places in the DNA
E.g. ALK-EML4 or BCR/ABL (Philidelphia
translocation, CML)
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Types of genetic errors
Chromosome
abnormality
Deletion
Amplification
Translocation
Point mutation
A
C
A change in the DNA where a single base is added, deleted or
substituted
DNA A T G C C G
A
RNA A U G
Methionine
C
Base A (Adenine) have changed to base C (Cytosine)
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RNA C U G
Leucine
C C G
Proline
C C G
Proline
T C A
U C A
Serine
U C A
Serine
Which technology to choose?
DNA
RNA
Protein
Cell behavior
Altered DNA
Altered RNA
Altered protein
expression
Altered cell behavior
and morphology
ISH
ISH
IHC
Cell
Protein
DNA
Nucleus
RNA
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H&E, Special Stains
In Situ Hybridization (ISH)
Definition: a technique that uses a labeled complementary DNA or RNA
strand (probe) to localize a specific DNA or RNA sequence in a portion or
section of tissue (in situ).
We can use for:
• Selection of patients for targeted therapies (HER2 - PharmDx)
•Detection of genetic anomalies
•Gene copy number >> Amplification, Deletion (Breast cancer)
•Gene Structure >> Translocation (Lymphoma or Leukemia)
•Detection of viral infections
•Human Papillomavirus (Cervical cancer)
• Epstein Barr virus (Burkitts disease)
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What to choose from?
FISH/CISH
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SureFISH
Arrays
PCR
NGS
Molecular Technologies - FISH/CISH
FISH/CISH
SureFISH
Arrays
PCR
ISH: In Situ Hybridization
FISH: Fluorescent ISH > Fluorescence Microscope
CISH: Chromogenic ISH > Bright Field Microscope
SISH: Silver ISH > Bright Field Microscope
A ISH probe is attached to the genomic region of interest
and can be detected in a microscope
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NGS
Molecular Technologies - FISH/CISH
DNA
Denaturation
Add probe
Detect with
flourescence
microscope
Hybridize
Optional
Chromogenic step
(Antibody + Visualization)
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Detect with
bright field
microscope
Probes
Diagnostics of a variety
of cancer types
Leukemia
BCR
ETV6
FIP1L1-PDGFRA
MLL
SIL-TAL1
TCF3
TLX1
TLX3
TCL1
Type
Trans
Trans
Trans
Trans
Trans
Trans
Trans
Trans
Trans
Breast
EGFR/CEN-7 FISH
HER2 IQFISH pharmDx
HER2 CISH pharmDx
MYC/CEN-8 FISH
TOP2A FISH pharmDx
Breast
Leukemia
Lung
Probes
Cervical
Cervical cancer
HPV 16/18
HPV 31/33
HPV 6/11
HPV wide spec
HPV, GenPoint™
Type
Detect
Detect
Detect
Detect
Detect
Lymphoma
Gastric
Gastric
EGFR/CEN-7 FISH
Her2 IQFISH pharmDx
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Type
Amp
Amp
Amp
Amp
Amp
Type
Amp
Amp
Lung
Type
ALK
EGFR/CEN-7 FISH
MYC/CEN-8 FISH
Trans
Amp
Amp
Lymphoma
ALK
BCL2
BCL3
BCL6
BCL10
CCND1
IGH
IGK
IGL
Kappa/Lampda
MALT1
MYC
PAX5
TCRAD
TCRG
TCRB
EBV (EBER)
Type
Trans
Trans
Trans
Trans
Trans
Trans
Trans
Trans
Trans
AMP:
Trans:
Detect:
Trans
Trans
Trans
Trans
Trans
Trans
Detect
Amplification/Deletion
Translocation
Detection
Types of probes
Gene probes attach to the gene so
the gene number can be evaluated
HER2
Tumor cell
with
amplificati
on of
HER2
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Split signal probes attach to either
side of a breakpoint region, so
presence of translocations can be
evaluated
CEN-17
Translocation
t(4;11)(q21;q23)
detected with
Y5401; MLL FISH
DNA Probe, Split
Signal.
FISH Step-by-Step Procedure
 Deparaffinization & Rehydration
 Heat Pretreatment in Microwave Oven or Water Bath
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FISH Step-by-Step Procedure
 Pepsin Digestion
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FISH Step-by-Step Procedure
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FISH Step-by-Step Procedure
 Denaturation & Hybridization
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FISH Step-by-Step Procedure
 Stringent Wash
 Possibility to continue with the CISH procedure
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CISH - an alternative to FISH

Interpretation by bright field microscope

Preserves morphology
 Enables easy and fast identification of invasive tissue and internal control

Stained sections stored at room temperature without loss of signals
 Provides the opportunity to archive and re-evaluate cases
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Principle of CISH
HER2 FISH
Texas Red labeled DNA probe
Target DNA (gene)
HER2
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FITC labeled PNA probe
Target DNA (reference)
CEN-17
Principle of CISH
Incubation with Antibody Mix
Anti-Texas Red
conjugated to alkaline
phosphatase (AP)
Texas Red labeled DNA probe
Target DNA (gene)
HER2
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AP
Anti-FITC conjugated to
horse radish peroxidase
(HRP)
FITC labeled PNA probe
Target DNA (reference)
CEN-17
Principle of CISH
Incubation with chromogens
Blue chromogen
Red chromogen
Anti-Texas Red
conjugated to alkaline
phosphatase (AP)
Texas Red labeled DNA probe
Target DNA (gene)
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AP
Anti-FITC conjugated to
horse radish peroxidase
(HRP)
FITC labeled PNA probe
Target DNA (reference)
Dot-til-dot conversion
The green FISH signals are converted to blue CISH signals (CEN-17)
The red FISH signals are converted to red CISH signals (HER2)
FISH
CISH
Excellent concordance between FISH and CISH
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Molecular Technologies - SureFISH
FISH/CISH
SureFISH
Arrays
PCR
NGS
Oligonucleotide probes
(Oligo – Greek: Few/little)
A short sequence of nucleotides synthesized to match a region of DNA
In general, same use as for the Dako FISH/CISH
Difference is the length of the probes, selection and manufacturing
method
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SureFISH probe production – Oligo based
Target genes
Repetitive
elements
Target sequences
SureFISH probes (print and label)
BAC probes (obtain BAC clone, grow, purify DNA, and label)
SureFISH Oligo’s are repeat free probes
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SureFISH – differences to ”normal” FISH
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Unique micro-gap design gives tight yellow signals
Sample1
Sample2
Sample3
60X
SureFISH ALK BA probe with negative samples
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Formamide vs. IQFISH Fast Hybridization Buffer
Formamide (traditional buffer)
• Lowers denaturation temperature
- by interfering with DNA base paring
• Hybridization of FISH probe slowed down
 => Slow hybridization (overnight)
• Teratogenic (toxic)
IQFISH Fast Hybridization Buffer
• Lowers denaturation temperature
- by diminishing DNA base stacking
• Hybridization NOT slowed down
 => Fast hybridization (75-90 min)
• Brighter signals
• Non-toxic (Ethylene Carbonate)
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Instant Quality – IQFISH Fast Hybridization Buffer
 IQFISH results in less than 4 hours
• IQFISH Buffer reduces hybridization time to 75 – 90 min
Enables you to report FISH results the same day
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Dako Solution
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Histology FISH Accessory Kit (K5799)
Dako DuoCISH (SK108)
Probes
IQ Buffer
Hybridizer
SureFISH (Agilent)
Molecular Pathology - Summary
• Studying cancer on DNA level (where IHC is on protein level)
• Molecular Pathology is supplementary to IHC, Special Stains and H&E
• Method based on DNA probes attaching to complementary DNA
• Dako have solutions based on FISH and CISH
• Probes for applications in breast, lung, lymphoma, gastric, cervical, leukemia
• Instant Quality buffer reduces hybridization ’s time from 14-20h to 1,5h.
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