David L' Rimm M'D', Ph'D

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Measuring Protein on Formalin Fixed, Paraffin
Embedded Tissue Slides
David L. Rimm M.D., Ph.D Professor, Dept. of
Pathology Yale University School of Medicine
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Disclosure

• I am a consultant, stockholder and scientific
  founder of HistoRx
• I am an author on the Yale held patent on the
  AQUA technology.

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Context makes the image
Auguste Renoir The Luncheon of the Boating
Party  C.1881
Claude Monet The Stroll, Camille Monet and Her
Son Jean (Woman with a Parasol)C. 1875
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Immunoperoxidase stain

• Uses of IPOX
• Identification (binary)
• Measurement (ordinal)
• Quantification (continuous)

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Case 4
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The human eye is not a great tool for assessment
of intensity
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Use of analyte measurement to determine patient
management
Clinician suspects possible diabetes
Clinician suspects possible breast cancer
Obtain tissue sample (blood)
Obtain tissue sample (core biopsy)
Measure Blood glucose levels in mg/dl (objective
measurement)
Make Histologic Dx then measure estrogen receptor
levels (subjective judgment)
Treat with appropriate therapy
Treat with appropriate therapy
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Fluorescence Microscopy for In situ Measurement
of Protein Expression

• Assessment of features on the basis of molecular
  interactions rather than morphology-based
  (contrast generating) edges (feature extraction).
• Not biased by post hoc human selection of the
  area selected for scoring.
• Broader dynamic range than chromagenic methods
• Emissive signal rather than absorptive to
  facilitate multiplexing

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AQUA method of objective analyte measurement on
a tissue slide based on co-localization
Step 1 Mask (define region of interest, exclude
stroma, blank space, etc) colocalization with
Cytokeratin for carcinoma Step 2 Define the
numerator (target) and denominator (compartment)
S target intensity in compartment pixels
Numerator
Concentration
AQUA score
Denominator
S compartment pixel area
Step 3 Calculate the AQUA score
Step 4 Convert to absolute concentration or
normalize to set of uniform standards
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The numerator is the sum of the intensity of the
of the pixels in the nuclear compartment
The denominator is the sum of the of the pixels
in the nuclear compartment (as defined by DAPI
staining above a threshold)
Marisa Dolled-Filhart
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HT 29 SUM 159 BT 20 ZR 751 BT 474 T47D MCF7 Puro
0 Puro .01 Puro .1 Puro .5 Puro 1 Puro 5 MB 231 H
1666 MB 436 CHO SKBR3 MB 435
ERa levels by western
1 ng 2.5 3.5 5 7.5
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rER
ERa
b-tubulin
Alley Welsh
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Limit of detection is 50 pg/mg
Use these cell lines (present on TMAs) to convert
patient ER AQUA scores from YTMA 49 to an ER
concentration (pg/ug)
Alley Welsh
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Nature Medicine November 2002

• Over 60 peer reviewed papers
• gt10 reviews
• Journals including NEJM, Nature, Science, JNCI,
  Nature Medicine, Cancer Research, JCO and others
• Papers from 12 labs (18 machines worldwide
  outside HistoRx)

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The HistoRx AQUA platformHardware, Software,
Reagents for Quantitative Pathology

• Automated Fluorescence Microscopy
• Expanded dynamic range of measurement
• Multi-parametric
• Commercially available with about 18 current
  placements worldwide
• AQUA analysis software compatible with .tiff
  images
• In use by more than a dozen Pharma companies for
  drug development
• FDA clearance pending on AQUA software using ER
  as the example analyte
• US Patent 7,219,016

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Unsupervised image segmentation
DAPI
Cy3

Gustavson et al. Appl Immuno and Mol Morph. 2009.
(in press)
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AQUA analysis Normalization/Standardization
Example HER2
Operator 1
Operator 2
Mach. 1
Mach. 3
Mach. 2
Mach. 1
Stain 1
Stain 1
Stain 1
Stain 1
CV4.3 Cumulative
HER2 by AQUA analysis by 3 different operators
on 3 different machines with three different
batches.
PM2
PM1
PM1
PM3
PM1
PM1
PM1
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Comparison of traditional IHC to AQUA analysis --
Reproducibility
Estrogen Receptor
Path 1 v. Path 2 Kappa 0.482 (plt0.001) Path 1
v. Path 3 Kappa 0.444 (plt0.001) Path 2 v. Path
3 Kappa 0.400 (plt0.001)
Positive/Negative concordance 92-95
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Other Systems
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Key Issues for Accurate Measurement on Protein on
Slides

• Antibody validation
• Antibody titration
• Fixation
• Tissue and Section preservation
• pre-analytic variables (cold ischemic time)

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Antibody Validation (reproducibility)
HER2 on serial sections
R0.966
c-Met Antibodies
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All Z sections
Spot 251 (60x)
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HT 29 SUM 159 BT 20 ZR 751 BT 474 T47D MCF7 Puro
0 Puro .01 Puro .1 Puro .5 Puro 1 Puro 5 MB 231 H
1666 MB 436 CHO SKBR3 MB 435
ERa levels by western
1 ng 2.5 3.5 5 7.5
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rER
ERa
b-tubulin
Alley Welsh
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Limit of detection is 50 pg/mg
Use these cell lines (present on TMAs) to convert
patient ER AQUA scores from YTMA 49 to an ER
concentration (pg/ug)
Alley Welsh
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Key Issues for Accurate Measurement on Protein on
Slides

• Antibody validation
• Antibody titration
• Fixation
• Tissue and Section preservation
• pre-analytic variables (cold ischemic time)

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Objective Optimization of Antibody Concentration
Mark Gustavson and Marisa Dolled-Filhart
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Key Issues for Accurate Measurement on Protein on
Slides

• Antibody validation
• Antibody titration
• Fixation
• Tissue and Section preservation
• pre-analytic variables (cold ischemic time)

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Over-fixation
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DeMarzo et al, Human Path, 2002
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Materials and Methods
Am J Clin Pathol. 200312086

• Timed fixation experiment
• 24 carcinomas with strong ER expression on Bx
• Multiple sections from the excisional specimens
  were placed immediately in NBF
• Fixed for 3, 6, 8, 10, 12, 24, 48 hrs and 7 days
  followed by 90 min in processor
• ER staining performed using 25 and 40 minutes of
  HIAR assessed using the Q score
• Intensity (0-3) proportion of stained cells
  (0, 0 1, 1 to 25 2, 26 to 50 3, 51
  to 75 or 4, gt75)
• Range, 0-7

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Results
Am J Clin Pathol. 200312086
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Results
Antigen retrieval time
Am J Clin Pathol. 200312086
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Underfixation Issue
3 hrs
6 hrs
Am J Clin Pathol. 200312086
8 hrs
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Key Issues for Accurate Measurement on Protein on
Slides

• Antibody validation
• Antibody titration
• Fixation
• Tissue and Section preservation
• pre-analytic variables (cold ischemic time)

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Yale Pathology ArchivesOld Blocks
Circa 1982
Circa 1952
Circa 1962
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Pathology Archives 1932 - 1982
Tissue gt60 years old retains antigenicity
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Cytokeratin Green, DAPI Blue, E.R. Red
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Storage Condition StudyFresh vs aged for 3
months
Score of fresh cut cases
Estrogen receptor nuclear stain
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Preservation of ER antigenicity using paraffin
and nitrogen storage
Percentage of cases
N 176
conventional
AQUA
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Preservation of cytokeratin antigenicity using
paraffin and nitrogen storage
Percentage of cases
N 176
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Paraffin coating preserves individual sections
Lori Charette
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Nitrogen Storage to prevent tissue oxidation
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Key Issues for Accurate Measurement on Protein on
Slides

• Antibody validation
• Antibody titration
• Fixation
• Tissue and Section preservation
• pre-analytic variables (cold ischemic time)

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Summar Siddiqui and Robert Camp
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Whole section analysis of core biopsy vs.
resection to assess affect of ischemic time on
stability
Number of fields examined on each tumor
Yalai Bai and Eirini Pectacides
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Lower Expression of pAKT in the resections than
in CNBs
Wilcoxon Signed Ranks test p0.004
Yalai Bai
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Lower Expression of pERK in the resections than
in CNBs
Wilcoxon Signed Ranks test p0.38
Yalai Bai
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Incidental finding for phospho-p70S6K
mTOR protein expression by AQUA correlates with
phospho-p70S6K in both lung cancer and melanoma
(routine specimen collection, assessed by TMA)
suggesting that the phosphorylation of p70S6K may
be insensitive to cold ischemic times
Lung Ca
Melanoma
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Decreased Level of ER Expression In Surgical
Resections
Wilcoxon Signed Ranks test p0.014
Yalai Bai
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Cytokeratin exhibit no significantly change from
CNBs to resection.
Wilcoxon Signed Ranks test p0.23
Yalai Bai
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Normalization by CK expression results in loss of
statistically significant decrease in ER
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Thanks to
Rimm Group Robert Camp Elsa Anagnostou Bonnie
Gould Rothberg Veronique Neumeister Seema
Agarwal Huan Cheng Maria Baquero Alley
Welsh Jason Hanna Jennifer Bordeaux Matt
McRae Bill Bradley Camil Liceaga Summar
Siddiqui Liz Killiam Yalai Bai Arun Gopinath
Tissue Microarray Facility Lori Charette Joe
Salame Sudha Kumar Peter Gershkovich
Former Rimm Group Marisa Dolled-Filhart Maciej
Zerkowski Kyle DeVito Melissa Cregger Tony
McCabe Satish Avvaru Greg Tedeschi Mark
Gustavson Gretchen Graff Carola Zalles Aaron
Berger Sharon Moulis Malini Harigopal Chris
Moeder Jena Giltnane Eirini Pectasides
Yale Collaborators Annette Molinaro Gina
Chung Harriet Kluger Ruth Halaban Lyndsay
Harris John McClaskey
Outside Yale Lisa Ryden (Lund) Karin Jirstrom
(Malmo) David Huntsman (BCCA) Karen Gelmon
(BCCA) Elaine Alarid (UW)
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www.tissuearray.org
Rimm Lab Summer 08
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Tissue Microarray (TMA) Construction
Dolled-Filhart and Rimm (2002) Principles and
Practice of Oncology Technology Update
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Strategy to Evaluate the Optimal Spot Size for
TMAs
Elsa Anagnostou
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Elsa Anagnostou
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Regression Analysis of AQUA scores as a Function
of Spot Size on Six Different Proteins
A
D
C
B
H
G
F
E
Elsa Anagnostou
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Multiplexing

• True Multiplexing (X markers using X
  fluorophores)
• Allows assessment of multiple markers on a single
  slide
• Allows assessment of co-localization or
  co-compartmentalization

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Validation of Multiplexing
Her2 (Cy5) and ER (Cy7) both tyramide amplified
with NaAzideperoxide quenching
Malini Harigopal
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Validation of Multiplexing
Her2 (Cy5) and ER (Cy7) both tyramide amplified
with NaAzideperoxide quenching
Malini Harigopal
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Validation of True Multiplexing
P53 (Cy5) and PR (Cy7) both tyramide amplified
with NaAzideperoxide quenching
Malini Harigopal
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Validation of Multiplexing
P53 (Cy5) and PR (Cy7) both tyramide amplified
with Benzoic hydrazide quenching
Malini Harigopal