Title: DESC Anatomie Pathologique
1DESC Anatomie Pathologique
- Sarcomes
- Place De La Biologie Moléculaire
- Dr MISHELLANY CJP
2Outlines
- Assessment of Nucleic Acids in Tissue
- In situ hybridization
- Extraction and PCR, Q-RT-PCR or DNA arrays
- Importance of pre-analytic steps
- Issues related to fixation and In Situ analysis
- Issues related to fixation and DNA or RNA
analysis - Dilution of nucleic acids gt micro or
macrodissection - Probe validation
- DNA vs RNA in situ
- Multiplexing probes
3Type of assays
In situ assays
Molecular assays from lysates
4Why ?
- Understanding of tumor biology
- Identification of targets (predictive markers)
example of HER2 - Identification of prognostic or predictive
biomarkers/signatures example of mutation
screening
5Tissue preparation
- Surgical excision of sample
- Slicing of specimen prior to fixation
- Fixation process
- Paraffin embedding
- Tissue sample sectioning and slide mounting
- Selection of tumor areas
6Pre-analytical steps
- For RNA and DNA analysis the quality of the
tissue is the key point - Type of fixative is important for further
interpretation of ISH slides - For RNA, time to fixation or freezing is a major
point too. - The use of RNA preservative solutions (RNA later
or other) is a safe alternative when nitrogen is
not available - The utility of molecular biology friendly
fixatives is under evaluation (RCL2..)
7ISH
- In situ hybridization (ISH) is a type of
hybridization that uses a labeled complementary
DNA or RNA strand (i.e., probe) to localize a
specific DNA or RNA sequence in a portion or
section of tissue (in situ)
8FISH Assays
- First, a probe is constructed. The probe must be
large enough to hybridize specifically with its
target but not so large as to impede the
hybridization process. - The probe is tagged directly with fluorophores,
with targets for antibodies or with biotin.
9Probes
- The differences between the various FISH
techniques are usually due to the construction
and content of the fluorescently-labeled DNA/RNA
probe. - The size, overlap, colour, and mixture of the
probes make possible all FISH techniques.
10Probes
- Probe size is important because longer probes
hybridize more specifically than shorter probes. - The overlap defines the resolution of detectable
features. If the goal of an experiment is to
detect the breakpoint of a translocation, then
the overlap of the probes the degree to which
one DNA sequence is contained in the adjacent
probes defines the minimum window in which the
breakpoint occurs - Centromeric probe can be used in paralel
(polysomic states important for amplifications as
for HER2)
11Example of HER2
- A priori knowledge of the chromosome imbalance
involved.
12Methods for HER2 status assessment
Advantages
Cost
Simple Fast Sensitive Automated
IHC protein
/
Quantitative Standardized Automated
FISH DNA
CISH DNA
? FISH no fluorescence
13HER2 status evaluation
- Morphological approach visualization of the
invasive cells habouring the signal
FISH
CISH
- Immunohistochemistry
- Protein
DNA
14Morphological methods are recommended
- They allow the control of the type of marked
cells -
amplification
CISH
IDC
Only the signal of the invasive component is
significant
normal breast
No amplification
15Morphological methods are recommended
No dilution effect of tumors RNA, DNA or
proteins by stromal cells
FISH
amplification
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17FISH/CISH
Specimen preparation
Denaturation
Hybridization
Detection
Visualization
18Step 1
- 45?m thick sections are cut from the
paraffin-embedded tissue specimen and mounted on
to clean glass microscope slides - The section is deparaffinised in xylene and
dehydrated in a graded series of ethanol - The sections are then treated with protease to
enhance accessibility to target DNA for
hybridisation. This is the most delicate moment
(overdigestion is possible)
19Step 1
protease
Protease digestion 5-10 minutes at 37C Wash
SSC 2X Dry the slides
37C
20Step 2
- Cellular DNA is denatured by placing the slides
in heated formamide solution and dehydrated in a
graded series of ethanol - The probes are added and allowed to hybridise to
the complementary DNA - Slides are then washed stringently to remove
excess probe
21Step 2
denaturation
HCl 0,2N 20 minutes Rinse sterile H2O Wash SSC
2X Pretreat at 80C for 30 minutes Rinse sterile
H2O Wash SSC 2X
22Step 2
Probe addition
LSI HER-2/neu CEP 17 Vysis/AbbottTM
rubber cement
23Step 2
Denaturation hybridation
Denaturation 5 minutes at 85C
minimum hybridization 12 h at 37C
24Step 2
Post hybridization
Remove rubber cement and cover slips
Immerge in SSC/NP40 at 75C for 2 minutes Wash
H2O 2 minutes Dry in the dark 30 minutes Mount
with DAPI (10 to 20 microlitres) Store in the
dark at 20C
25Step 3
- The nuclei are counterstained, i.e. using the
intercalating fluorescent counterstain
4'-6'-diamididino-2'-phenylindole (DAPI) - Probe signals are viewed and enumerated using a
fluorescence microscope with appropriate filters - Positive and negative controls should always be
included with the FISH assay
26Fluorescent microscope
emiss
27Selection of an infiltrative zone at low
magnification Count the spots at X100 in
oil HER2 Count the spots in 30 different nuclei
in 3 different zones CEP17 Count the spots in
30 different nuclei in 3 different zones Give
the ratio HER2/CEP17
Compte rendu N Technique Date Feuille
de comptage
28Storage of the slides
- FISH assayed slides should be stored at 20C or
less. - The fluorescent signal will decay over time so
accurate reporting of score interpretation at the
time of initial analysis is essential.
29High amplification
30Low amplification
31Polysomy
Polysomy Chr 17
32vysis
33vysis
34dako
35ventana
36Ventana
37FISH assay
- It is recommended that an HE section is assessed
alongside the FISH assay. - three main steps to the FISH protocol, which
takes approximately 2 days to complete. - Whichever commercially available FISH kit is
used, the manufacturers protocol should be
strictly followed
38Time to fixation
- The time from excision to fixation can affect
HER2 assay results, as can the thickness of the
tissue slices prior to fixation, by delaying the
penetration of the fixative. - Therefore, care should be taken to ensure that
specimens are fixed optimally by slicing at
0.51.0cm and fixing as soon as possible after
excision, but preferably within 1 hour.
39Type of fixative
- Alcohol-based fixatives, can generate
false-positive results when using IHC. HER2
assessment by FISH can also be hampered by use of
alcohol-based fixatives such as AFA, Z-5 and
Pen-Fix or formalin free fixatives such as
Finefix (overdigestion). - Moreover, the use of Bouins solution, which was
commonly used in France, has the disadvantage of
making retrospective FISH testing impossible.
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41Results
42Thickness of tissue section
- Can affect the visualisation and interpretation
of assay results. - Cut to a standard width of 35 microns for IHC
analysis and 45 microns for FISH analysis. - It is also recommended that an HE section is
evaluated in parallel to the HER2 assay to ensure
the presence of adequate tumour versus normal
tissue and to confirm the presence of the
invasive component of the tumour (FISH ).
43LIMITATION OF FISH/CISH deletion
The only nuclei to be counted have to have at
least 2 spots
44Alternative methods to FISHnon fluorescent ISH
technologies CISH/SISH or ..BrISH
45Methodology of the CISH assay
HER2 gene
46Methodology of the CISH assay (contd)
HER2 gene
47Methodology of the CISH assay (contd)
HER2 gene
48Chromogenic in-situ hybridization (CISH)
- CISH detects HER2 gene amplification and follows
the same principles as FISH - However, signal detection similar to IHC
- no fluorescence microscope required
- allows histological evaluation
- allows regular slide storage
- less expensive than FISH
- CISH can be used as analternative to FISH
- Testing kit available(approved by EU Commission)
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50Weak amplification
6 spots
Use probe with centromere 17
51SISH detection
- Enzyme (HRP) catalyzes reduction of silver ions
to metallic silver - Metal nanoparticles deposit at the site of the
target hybridized to the probe
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53SISH
54SISH
Chromosome 17 polysomy
Chr 17
HER2
55SISH
Amplification
Chr 17
HER2
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57Dual color BrISH
58DAKO
Image Magali Lacroix
59ZYTOVISION
60Silver Enhanced In Situ Hybridization
Double in situ Hybridisation
Gene Normal Protein Level 0
Gene Normal Protein Level 1
Gene Amplified Protein Level 3
MCF7
ZR-75-1
BT-474
? HER2 Gene ? Chromosome 17 Centromere
provided by Hiro Nitta (Tucson)
61Non-Amplified
62Amplified with normal stroma
63HSR Amplification
64Heterogeneity
65Chromosome 17 monosomy
66IHC-ISH combined Triple Staining
Gene Normal Protein Level 0
Gene Normal Protein Level 1
Gene Amplified Protein Level 3
MCF7
ZR-75-1
BT-474
HER2 Protein ? HER2 Gene ? Chromosome 17
Centromere
provided by Hiro Nitta (Ventana, Tucson)
67Mutation screening from paraffin embedded tissue
68Identification of mutation
- Sequencing
- classical
- microsequencing
- Pyrosequencing
- Snap-shop
- Allele specific amplification
- primers specific of mutation
- Allelic discrimination
69KRAS mutation screening
Screening of the 7 most frequent mutations (96)
70MUTATION SCREENING
IMPORTANCE OF PATHOLOGISTS SELECTION
KRAS mutated in 50 of allels
Tumor and 50 of normal cells and stroma
KRAS mutated in 25 allels
Importance of sample preparation and use of
sensitive technique
71KRAS methods
- Tumor DNA
- 1/ From paraffin embedded tissues
- 2/ Enriched for tumor cells
- gt50 of tumor cells macrodissection of one slide
of 10µM - 3/ DNA extracted
- Standardized methods
Tumor area
Stroma inflammatory cells
72À sélectionner
Tumor fixed in formalin or AFA
Selection of tumor area
macrodissection
73Contrôle histologique
Detection of the tumors areas by a pathologist
74À ne pas sélectionner
Avoid low cellularity
Avoid bouins fixation
75Dilution effect
tumor tissue
76- Zone tumorale repérée sur la lame HES
- Évaluation approximative du de cellules
tumorales - 3. "Macro dissection" sur le bloc paraffine de la
zone tumorale à couper au microtome.
les blocs avec moins de 50 de tissu tumoral
seront transmis mais les résultats ne seront
validés que si la tumeur est mutée (pour éviter
de faux négatifs).
77- 4. Réalisation de 5 "copeaux" de tumeur en
paraffine de 10 ?m mis en tubes Nalgène - 5. Classement des documents dans une pochette
dédiée. - 6. Retour du bloc par Poste au Pathologiste
dorigine après aval du laboratoire
d'Oncopharmacologie.
78Methods Validation
direct sequencing versus TaqMan?
48 tested DNA normal and with a selection of the
different possible mutation
100 of concordance
79Sequencing
TaqMan
G35T G12V
G38A G13D
80DNA extracted from embedded fixed tissues vs DNA
extracted from frozen tissues
15 patients
100 of concordance
8 non mutated 4 mutated G12D 2 mutated G12V 1
mutated G12S
81Frozen
Paraffine embedded tissus
121 GC
121 GT
121 GA
122 GC
122 GT
122 GA
82Advantages and drawbacks
- Sequencing
- Classical
- fast,
- not very sensitive at least 30 of tumors cells,
- possible risk of non amplification due to the
size of the amplicon in paraffin embedded tissues - Microsequencing
- fast,
- sensitive
- need of specific apparatus
- Allele Specific Amplification
- Very sensitive less than 1
- complex
- risk of contamination
- Allelic Discrimination
- the fastest (2 hours)
- Amplification and allelic discrimination in one
step - sensitive 1 of tumor cells
- need of specific apparatus
- 2 different methods are useful in the same lab
83How to look at several markersAssessment of
Nucleic Acids in Tissue
84CO DETECTION OF HER2 AND TOPO2
ORANGE TOPO2 GREEN HER2 BLUE CEP17
85Multiplex-FISH (M-FISH)
- Multiplex-FISH (M-FISH) on metaphase chromosome
spreads and with a resolution of 5 Megabases
(Mb) is widely used for analyses of genome
instability and chromosomal rearrangement. - Using this technique, each chromosome pair can be
stained with specific probes, spanning across the
genome. - This allows a wide range of uses including the
characterization of structural interchromosomal
aberrations and complex chromosomal
rearrangements, which are often observed in tumor
cells
86- BUT obtaining anaphase chromosomes from patient
samples is extremely difficult for solid tumors
and thus its use remains elusive in clinical
cancer research. - Thus, M-FISH is mostly used for cell line
analyses.
87RNA FISH
- The study of intracellular RNA localization using
RNA FISH provides insights into the in situ
physical characteristics of transcription and
intracellular RNA transport in individual cells - Works better on fresh tissues
- Combination with DNA FISH is possible (especially
on cell lines) using a two steps technique
88PNA (peptide nucleic acid) FISH
- PNA (peptide nucleic acid) probe-based FISH
techniques can be used to assess of lengths of
specific chromosomal region. - For example, telomere length can be analyzed
using PNA probes coupled to a fluorochrome,
targeting of TTAGGG repeats. Theoretically, the
amount of PNA probe hybridized is directly
proportional to telomere length. Thus, the level
of fluorescence can be linked to the telomere
length itself. - This technique gives a relative rather than
absolute quantification of the telomere length.
89Examples of FISH. Double hybridization of an
hTERT probe (green) and 5q31 probe (red) used as
a reference on lung interphasic cells. The number
of green spots per nucleus reflects the
amplification of hTERT in a lung cancer sample.
Raynaud et al 2008
90Example of Comparative Genomic Hybridization or
CGH
- No a priori knowledge of the probe
91(CGH)
Laser microdissection
Area of interest
92Comparative Genomic Hybridization
DOP-PCR control of the quality of DNA
(Degenerate Oligonucleotide Primed PCR)
Only five tumors are correct
93CGH
94CGH
- Grade I ER
- Gain 1q
- Deletion 6q 16q 17q 22q
grade I and Basal-like Deletion 2q 6q 9q
95CGH
- CGH has been adapted for use with limited amounts
of genetic material including small subregions of
microdissected tumor samples. - CGH has also been developed for use with paraffin
embedded specimens and even for single cell
analysis - The development of array CGH marked a major
development of this technique. - In array CGH, target metaphase chromosomes are
replaced by large numbers of mapped clones
spotted onto a standard glass slide. - Higher resolution than previous CGH techniques.
- Array CGH facilitates automated, high-throughput
analyses.
96Profiling
Puces à ADN
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98Gene-based biomarker
- Mammaprint
- Oncotype DX (FFPE tissues)
- MapQuant
- several publications on predictive and/or
prognostic signatures, - no prospective clinical validation
99Conclusion
- Tissue is the key
- Freeze when possible
- Buffered formalin fixation
- Combined efforts by big companies (image
analyses, device producer, biochemistry etc.) in
cooperation with selected research institutes are
necessary to move forward and to climb up to
another level of importance in clinical
diagnostic and pharmaceutical development
overcoming today's limitations - Molecular pathology