Identification of HER2 Gene Amplification by SISH: Accessible Molecular Morphology for the Surgical

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Identification of HER2 Gene Amplification by
SISHAccessible Molecular Morphology for the
Surgical Pathologist
Raymond R. Tubbs, DO Professor of Pathology and
Section Head Section of Molecular Genetic
Pathology Department of Molecular
PathologyPathology and Laboratory Medicine
Institute Cleveland Clinic Foundation Cleveland
Clinic Lerner College of Medicineof Case Western
Reserve University School of Medicine
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Unmet Molecular Morphology Need in Diagnostic
Pathology

• High resolution visualization of single gene copy
  in FFPET using FDA approved fully automated
  systemsSilver In Situ Hybridization
• Fully automated, reproducible bright field ISH
  staining
• Second Generation Dual color gene centromere,
  and gene protein
• Bright field ISH systems that are easily
  integrated into conventional microscopy
  workflow-- another slide in the tray
• Reproducible inter-observer interpretation
• HER2 genotyping of breast cancer is an ideal
  system for addressing these unmet needs

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FISH and The Surgical
Pathologist Working in the dark is
counterintuitive Acquisition of new
skills Personnel and instrument
expense Overnight hybridization Workflow
issues

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James Hainfeld, Ph.D.
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SISH HER2 Detection
Enzyme (HRP) catalyzes reduction of silver ions
to metallic silver Metal nanoparticles deposit
at the site of the target hybridization
Dinitrophenol (DNP)
Rabbit anti-DNP
HER2 DNA Probe
HER-2/neu Region of Chromosome 17
horseradish peroxidase (HRPO)
Goat anti-Rabbit HRP
Silver Reagents A, B, C
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AdvantagesHER2 SISH Assay

• Automated
• All steps are automated
• Time and temperature controlled, no in run user
  interaction
• Reagents are stable and pre-formulated - ready
  to run
• Scored by brightfield microscopy
• No fluorescence scope needed
• Interpret along with conventional breast marker
  slides Another slide in the tray with ER/PR/Ki67
  IHC and H Es
• Shorter assay time
• FISH Overnight hybridization
• SISH 6.5 hours (but overnight hybe may be best
  workflow fit)
• Ability to archive and re-examine slides
• SISH reaction products stable over time
• Universal endogenous in tissue control signals
• SISH Image analysis in 2008-- FDA application
  submitted

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HER2 Probe Design

• Three BAC clones (C10, 94L and H17)0.5 million
  base pairs (half are repetitive sequences)
  Repetitive sequences depleted in silico
• 150 PCR products generated from 185 primer pairs
  
• PCR pools (equimolar) for each BAC clone ligated
• Fragments amplified with random priming
• Nick-translation labeling with DNP

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HER2 Assay Design

HER2 Slide Genomic probe DNP Labeled SISH
Detection
CHR17 Slide Oligo probe DNP Labeled SISH
Detection
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SISH HER2
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SISH CHR 17
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HER2 SISH Quantitative vs. AMV PathVysion FISH
97.9 Concordance
Cutoff of 2.0
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Algorithm Development

• Goal Develop interpretation algorithm
  That
• Fits in the laboratorys workflow
• Is highly concordant with FISH
• Is reproducible between laboratories
• Is reproducible between pathologists

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Algorithm Development Study Nine Pathologists

• Six sites
• Cedars-Sinai (Los Angeles)
• Charite (Berlin)
• Cleveland Clinic
• Magee Womens (Pittsburg)
• Roswell Park (Buffalo)
• Sunnybrook and Womens (Toronto)
• USCAP 2007 Lab Invest 8744A, 2007.

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HER2 SISH Quantitative vs. AMV PathVysion FISH
Group 1(n5)
Group 2(n4)

• Review HE
• Review HER2 DNA
• Review CHR17
• Record
• Non-Amplified
• Amplified
• Low Level
• Amplified
• Heterogeneous

• Review HE
• Review, record HER2 DNA
• Review, record CHR17
• Calculate HER2/CHR17 Ratio
• Record
• Non-Amplified
• Amplified
• Low Level
• Amplified
• Heterogeneous

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HER2 SISH Quantitative vs. AMV PathVysion FISH

• This lead us to two conclusions
• Pathologists SISH interpretation concordance
  with FISH when the HER2/CHR17 ratio is between
  1.4 and 4.0 is not so great.
• Accountant approach improves concordance

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Algorithm Overview
Method 1
Method 2
Semi-Quantitatively determine HER2/CHR17 Ratio
Quantitatively determine HER2/CHR17 Ratio
Ratio?
Ratio?
lt1.8
gt2.2
1.4 Ratio 4.0
lt1.4
gt4.0
1.8 Ratio 2.2
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Fits in the Pathologists Workflow

• USA
• Germany

• UK
• Norway

Semi-quantitative
Fully quantitative
1.4
4.0
1.8
2.2
5.0
6.0
1.0
2.0
3.0
5.0
6.0
1.0
2.0
3.0
HER2/Chr17 Ratio
HER2/Chr17 Ratio
Negative
Positive
Negative
Positive
Gray Area
Go to Method 2 (5 15 of the time)
2 Methods. Same Result
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Charite SISH StudyProfessor Manfred Dietel
Virchows Archives 45119-25, 2007

• 99 invasive breast carcinomas
• AMV PathVysion FISH
• HER2 SISH
• 2 FISH scorers
• 5 Pathologists trained to SISH scoring algorithm
• Each pathologist independently scored all
  cases
• Consensus FISH compared with consensus SISH
• 96 overall concordance between FISH SISH

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SISH at USCAP 2008

• 172 Comparison of Automated Silver Enhanced In
  Situ Hybridization (SISH) and Fluorescent In Situ
  Hybridization (FSIH) for Assessment of Her2/neu
  Gene Status in Invasive Breast Carcinomas.
  Authors HJ Kahn et al. Session Poster 11,
  Wednesday AM
• 211 Silver In Situ Hybridization (SISH) for
  Detection of HER2 Amplification in Breast
  Carcinoma International Inter-Observer
  Interpretive Reproducibility Study of 305 Cases.
  Authors B Papouchado, et al Session Poster
  20, Wednesday Morning
• 210 HER2 Gene Amplification in Breast
  Carcinoma A Methods Comparison Study between
  Silver In Situ Hybridization (SISH) and FISH.
  Authors B Papouchado, et al. Session Poster
  24, Wednesday Morning
• 107 High Inter-Laboratory Reproducibility of
  the Silver In Situ Hybridization Assay (SISH) for
  the Detection of HER2 Gene Status in Breast
  Carcinoma A Study Reporting Results from Five
  Italian Cancer Institutes. Authors A Carbone,
  et al. Session Poster 19, Wednesday Morning
• 334 Molecular Cytopathology Use of ThinPrep
  Preparation for HER2 Tests in Breast Cancer
  IHC, FISH, CISH, and SISH. Authors H Itoh, et
  al. Session Poster 37, Monday Afternoon
• 117 A Comparison of a New Technology Against
  the Established Protocols for HER2 Gene
  Assessment Using Tissue Microarrays. Authors S
  Di Palma, et al Session Poster 13, Wednesday
  Morning
• Heavy Metal and Your Pathology Practice
  Visualizing Genes with Brightfield Microscopy.
  Presenter R Tubbs. Session Companion Society
  Meeting Association for Molecular Pathology
  Sunday evening

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SUMMARY OF EIGHT METHOD COMPARISION STUDIES SISH
versus PathVysion FISH (ASCO/CAP
Scoring) (EQUIVOCAL CASES EXCLUDED)
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107 High Inter-Laboratory Reproducibility of
the Silver In Situ Hybridization Assay (SISH) for
the Detection of HER2 Gene Status in Breast
Carcinoma A Study Reporting Results from Five
Italian Cancer Institutes(Milan, Naples,
Aviano, Bari, and Genoa) Authors A Carbone,
et al Session Poster 19, Wednesday MorningNow
published JMD 10527-536, 2008

• 89 cases, each of five laboratories
  performedFISH, CISH, IHC (4B5) and automated
  SISHAll 5 sites stained and interpreted the
  casesStain failures in 3.6 of the cases
• Excellent inter-observer interpretative
  reproducibilitySite SISH vs Consensus SISH kappa
  0.86 - 1.00Excellent concordance among FISH,
  CISH, SISH (p 0.001 Fisher exact test)
• Discordance Monosomic/Polysomic states
  Genomic heterogeneity Low level/equivocal
  amplification

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211 Silver In Situ Hybridization (SISH) for
Detection of HER2 Amplification in Breast
Carcinoma International Inter-Observer
Interpretative Reproducibility Study of 305
Cases Authors B Papouchado, et al Cleveland
Clinic Session Poster 20, Wednesday Morning

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HER2 SISHInter-Observer Interpretative
Reproducibility
Consecutively FISHed Series 305 consecutive
primary breast carcinomas International
interpretative reproducibility study 10
pathologists scored using the algorithm Conse
nsus SISH vs FISH by conventional scale
Overall Agreement 97.0 Kappa
0.87 Observers vs consensus SISH Overall
agreement 94.0 - 98.0 Kappa 0.69 - 0.87

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Inter-Observer Interpretive Reproducibility Study

• Jon Myles Cleveland
• Ric Lloyd Rochester
• Andre Oliveira Rochester
• Bettina Papauchado Cleveland
• Manfred Dietel Berlin
• Mark Stoler Charlottesville
• Ray Nagle Tucson
• Adrienne Morey Sydney
• Michael Bilous Sydney
• Ray Tubbs Cleveland

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Certification Programhttp//www.HER2SISH.com/trai
ning.php

• To ensure reproducibility among laboratories and
  between pathologists, each site should be
  pre-certified prior to using the HER2 SISH assay
• Training and Certification available online for
• Pathologists
• Technicians
• Oncologists
• Continuing Medical Education credits
  (ACCME-Accredited) will be awarded

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HER2 SISHInter-Observer Interpretative
Reproducibility
Consecutively FISHed Series 305 primary breast
carcinomas International interpretative
reproducibility study 10 pathologists completed
scoring Consensus SISH vs FISH Overall
Agreement 97.0 Kappa
0.87 Observers vs consensus SISH Overall
agreement 94.0 - 98.0 Kappa 0.69 -
0.87 Average Kappa 0.83 Observers vs
FISH Overall agreement 91.0 - 96.0

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HER2 SISH Assay

• What is the role of SISH in HER2 clinical
  Testing?

SISH role will be determined by local laboratory
operations, pathologists preferences, and
oncologists expectations HER2 IHC equivocal
(2) cases can be immediately reflexed to
automated SISH instead of sending out to an off
site reference laboratory May be an ideal system
for initial HER2 testing
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HER2 SISH Assay

• May provide the best approach for INITIAL HER2
  clinical Testing

High level concordance with FISH Objective
interpretation (vs IHC subjectivity) Interpretati
ve reproducibility Ubiquitous internal positive
controls Best fit with workflow
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DUAL COLOR HER2 CHR17 Diag Pathol 20083(1)41

• Historical Scoring Method
• Consensus concordance vs FISH
  98.9
• Kappa 0.9736
• Sensitivity 96.3, Specificity 100
• Individual scorer concordance vs FISH
  Ranged from 97.8 to 100
• Kappa ranged from 0.9466 to 1.0000
• ASCO/CAP scoring method and equivocal cases
  included
• Consensus concordance vs FISH 95.7
• Consensus Kappa 0.8993
• Individual scorer concordance vs FISH
• Ranged from 92.5 to 95.7
• Kappa ranged from 0.8275 to 0.9069
• ASCO/CAP scoring method and equivocal cases
  excluded
• Consensus concordance vs FISH 100
• Consensus Kappa 1.0000
• Sensitivity 100, Specificity 100
• Individual scorer concordance vs FISH
• Ranged from 97.7 to 100

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Immunophenotypic Genotypic Heterogeneity
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Brightfield Double In Situ Hybridization for EGFR
Chromosome 7 Centromere (CEN7)
EGFR
CEN7
A431 EGFR Gene Amplified
MCF7 EGFR Gene Non-amplified
? EGFR ? CEN7
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Other Roles for SISH

• Moderately Complex Signatures in Breast Cancer?
• FGFR1 STARD3 GRB7 IKBK3 PROCCADAM9
  FNTA ACACA PNMT NR1D1
• Cancer Cell 2006 10529-541Genes Chromosomes
  Cancer 2006 45761-769

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Co-Amplification FrequenciesCCND1, HER2, MYC,
MDM2 and EGFR Amplified Breast Carcinomas

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HER2 MYC CoAMP
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Thanks For Your Attention