DESC Anatomie Pathologique

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DESC Anatomie Pathologique

• Sarcomes
• Place De La Biologie Moléculaire
• Dr MISHELLANY CJP

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Outlines

• 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

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Type of assays
In situ assays
Molecular assays from lysates
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Why ?

• Understanding of tumor biology
• Identification of targets (predictive markers)
  example of HER2
• Identification of prognostic or predictive
  biomarkers/signatures example of mutation
  screening

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Tissue 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

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Pre-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..)

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ISH

• 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)

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FISH 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.

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Probes

• 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.

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Probes

• 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)

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Example of HER2

• A priori knowledge of the chromosome imbalance
  involved.

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Methods for HER2 status assessment

Advantages
Cost
Simple Fast Sensitive Automated
IHC protein
/
Quantitative Standardized Automated

FISH DNA
CISH DNA
? FISH no fluorescence

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HER2 status evaluation

• Morphological approach visualization of the
  invasive cells habouring the signal

FISH
CISH

• Immunohistochemistry
• Protein

DNA
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Morphological 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
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Morphological methods are recommended
No dilution effect of tumors RNA, DNA or
proteins by stromal cells
FISH
amplification
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FISH/CISH
Specimen preparation
Denaturation
Hybridization
Detection
Visualization
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Step 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)

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Step 1
protease
Protease digestion 5-10 minutes at 37C Wash
SSC 2X Dry the slides
37C
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Step 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

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Step 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
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Step 2
Probe addition
LSI HER-2/neu CEP 17 Vysis/AbbottTM
rubber cement
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Step 2
Denaturation hybridation
Denaturation 5 minutes at 85C
minimum hybridization 12 h at 37C
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Step 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
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Step 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

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Fluorescent microscope
emiss
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Selection 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 
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Storage 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.

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High amplification
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Low amplification
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Polysomy
Polysomy Chr 17
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vysis
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vysis
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dako
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ventana
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Ventana
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FISH 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

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Time 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.

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Type 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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Results
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Thickness 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 ).

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LIMITATION OF FISH/CISH deletion
The only nuclei to be counted have to have at
least 2 spots
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Alternative methods to FISHnon fluorescent ISH
technologies CISH/SISH or ..BrISH
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Methodology of the CISH assay
HER2 gene
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Methodology of the CISH assay (contd)
HER2 gene
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Methodology of the CISH assay (contd)
HER2 gene
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Chromogenic 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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Weak amplification
6 spots
Use probe with centromere 17
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SISH 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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SISH
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SISH
Chromosome 17 polysomy
Chr 17
HER2
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SISH
Amplification
Chr 17
HER2
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Dual color BrISH
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DAKO
Image Magali Lacroix
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ZYTOVISION
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Silver 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)
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Non-Amplified
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Amplified with normal stroma
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HSR Amplification
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Heterogeneity
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Chromosome 17 monosomy
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IHC-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)
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Mutation screening from paraffin embedded tissue

• Example of KRAS

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Identification of mutation

• Sequencing
• classical
• microsequencing
• Pyrosequencing
• Snap-shop
• Allele specific amplification
• primers specific of mutation
• Allelic discrimination

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KRAS mutation screening
Screening of the 7 most frequent mutations (96)

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MUTATION 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
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KRAS 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
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À sélectionner
Tumor fixed in formalin or AFA
Selection of tumor area
macrodissection
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Contrôle histologique
Detection of the tumors areas by a pathologist
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À ne pas sélectionner
Avoid low cellularity
Avoid bouins fixation
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Dilution effect
tumor tissue
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• 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).
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• 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.

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Methods Validation
direct sequencing versus TaqMan?
48 tested DNA normal and with a selection of the
different possible mutation
100 of concordance
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Sequencing
TaqMan
G35T G12V
G38A G13D
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DNA 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
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Frozen
Paraffine embedded tissus
121 GC
121 GT
121 GA
122 GC
122 GT
122 GA
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Advantages 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

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How to look at several markersAssessment of
Nucleic Acids in Tissue
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CO DETECTION OF HER2 AND TOPO2
                                               
                                                  
                                                  
                                               
                                                  
    
ORANGE TOPO2 GREEN HER2 BLUE CEP17
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Multiplex-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

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• 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.

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RNA 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

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PNA (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.

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Examples 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
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Example of Comparative Genomic Hybridization or
CGH

• No a priori knowledge of the probe

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(CGH)
Laser microdissection
Area of interest
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Comparative Genomic Hybridization
DOP-PCR control of the quality of DNA
(Degenerate Oligonucleotide Primed PCR)
Only five tumors are correct
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CGH
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CGH

• Grade I ER
• Gain 1q
• Deletion 6q 16q 17q 22q

grade I and Basal-like Deletion 2q 6q 9q
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CGH

• 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.

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Profiling
Puces à ADN
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Gene-based biomarker

• Mammaprint
• Oncotype DX (FFPE tissues)
• MapQuant
• several publications on predictive and/or
  prognostic signatures,
• no prospective clinical validation

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Conclusion

• 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