Genomics Profiles by arrayCGH Support Histomorphologic Classification of

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Genomics Profiles by arrayCGH Support
Histomorphologic Classification of Non-small cell
lung cancer (NSCLC)
Galen Hostetter, M.D. Associate Research
Investigator, TGen Integrative Cancer Genomics
Division Aperio Annual Conference, San Diego,
CA October 26-28, 2008
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Cancer research / histopathological correlates /
clinical application / better treatments

• TGen Biospecimen Core-Science
• innovative technologies to clinical samples
• 244K 60-mer oligo - microarray CGH (aCGH)
• extend protocols, FFPE, downstream genomics
• provide efficient and high quality validation
• TMAs open IHC autostainer
• Aperio high resolution digital scans SHARED
  TRANSPARENCY
• Spectrum Plus digital slide viewing /
  conferencing

• Imaging advances
• radiography
• location
• microscopy
• phenotype
• IHC
• automated stainer
• tumor specific staining

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Improved IHC applications to validate genome wide
modifications HDAC2 staining of NSCLC TMA
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Genomic DNA extracts, FFPE tissues

• Scrape macro-dissection, enriched cellular
• 12 um sections, glass slide
• Sybr-Gold gel electrophoresis
• prolonged Pro-K digest
• phenol / chloroform extraction

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Discovery by aCGH FFPE, NSCLC Cyclin D
amplification - SCC, CDK4 amplification- ACA
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Genomic finding / validation cyclin D /
CDK4 NSCLC cell cycle progression, G0 to S1 phase

• Differential SCC / ACA genomic data
• SCC (4/14), high / broad amplicon cyclin D
  normal copy CDK4
• ACA (3/12) high level / broad amplicon CDK4,
  cyclin D normal copy
• Genotypic correlate to cell cycle control -
  molecular biology
• Validation by in situ analysis IHC / FISH
  molecular pathology
• IHC (below) by non-autostainer and non-Aperio
  scan

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ABI-1
103 - chr. 10
FFPE
Frozen
549 - chr. 10
PTEN
CNV
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Aperio whole section staining, Gene 1, IHC
concordant with aCGH discovery, tumor / normal
interface
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• Gene_1
• classic IHC pattern nuclear
• transcrip. regulator
• min. stain stromal
• min. longit crypt stain
• correlative qRT-PCR
• cell line model with inc. copy
• sustained knock-down by sh-RNAi
• known alteration, hematol. malignancy
• potential drug target

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GH 292
GH1006
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GH1006
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Tumor specific IHC of plasma peptide_1,
uninvolved duct tumor
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D1, intraductal dysplasia ab to peptide_1
Whole section staining of TMA spot from tumor D1,
tumor staining intensity by IHC 3 (range of 0
to 3)
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Acknowledgements
TGen Stephanie Savage Aprill Watanabe Milana
Cypert Sunny Shack Chris Gooden Su Young Kim O-P
Kallioniemi Mike Demure Dan Von Hoff Mike
Bittner Jeff Trent John Carpten
Others David Alberts Elena Martinez Ray
Nagle Doug Lake Bette Wojack Christine
Fleming TGen Foundation Bernice E. Holland
Foundation
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Derivative of Log Ratio (DLR), aCGH on 204 FFPE
samples DLR is particular sensitive QC measure
per individual experiment
DLR gt 0.35, n 11 ( 5.4)
CGH Analytics 3.5 Agilent, Inc. QC Metrics of
good or excellent for DLR lt 0.30 (based on
optimal DNA preps)