Developing quantitative slide-based assays
to assess target inhibition in oncology drug
discovery and development
Pathology Visions
October 24-27, 2010
Doug Bowman
Millennium Pharmaceuticals
© 2010 Millennium Pharmaceuticals Inc., The Takeda Oncology Company
Outline
▐
▐
▐
Imaging @ Millennium
Technology development & integration
Applications in Oncology
▌
▌
▌
Assess in vivo potency
Biomarker development
Assess clinical activity
Tissue-based imaging enables direct and
indirect biomarkers of target inhibition
Target ID Assay
HTS
Validation Dev.
Hit to
Lead
Lead Optimization
*
DC
*
IND
Preclinical Development
Phase I
II
*
III
Approval
•Drive medicinal chemistry
•Assess pharmacodynamic response in preclinical
in vivo models
Mechanism of Action / Pathway Inhibition / Terminal Outcome
•Assess pharmacodynamic response in variety of
clinical tissues, in use in Phase 1 clinical trials
Cell-based imaging assays
Preclinical PD assays and Clinical Biomarkers
Adopt to variety of tissue and biopsy types
•
•
15 drug candidates in the following areas: protein
homeostasis, angiogenesis, growth signaling inhibition,
hormone regulation, cell cycle inhibition and apoptosis
All stages of development
Tissue-Based Imaging @ Millennium
▐
▐
Sample accession to LIMs system
Slide preparation
▌
▐
Slide scanning
▌
▐
▐
Automated slide processing / staining
Immunofluorescence (IF) and Brightfield
Image / data management
Image analysis
▌
“Canned” solutions and developed
algorithms
Custom developed slide scanning systems
Automated slide-scanning (If & colorimetric)
B/W Camera
RGB Camera
Barcode
Reader
x4
File Share
MetaMorph
▐
▐
▐
▐
▐
4 integrated systems
Multi-mode, multi-channel IF
Developed suite of image
acquisition and analysis tools
3000 slides (IF) per year
7000 slides (IHC) per year
Slide Loader
XYZ Stage
(200 slide capacity)
High resolution and efficient scanning of
clinical samples
100um
20x objective
•Multi-mode
•Multi-channel IF
Capture entire volume of cells for 3D
morphology assays
Z-axis
15 optical sections
@ 0.5 um intervals
aTubulin
Visualization of 3D cellular morphology
3 dimensional rotation, +- 30 degrees
aTubulin / pHisH3 / Dapi
aTubulin
Investment in Aperio Technologies platform
Automated slide-scanning
(Aperio Technologies)
ScanScopeXT
Spectrum
Aperio-prd
ImageScope
ScanscopeFL
▐
▐
▐
▐
ScanScopeXT (~13000 slides, 1.5 yr)
ScanScopeFL (400+ slides)
Spectrum: Image management
ImageScope: Image visualization and
analysis
Image analysis platforms and LIMS
LIMS Database
Image Analysis
MetaMorph
Image analysis &
visualization stations
▐
MetaMorph Image Analysis
Aperio Image Analysis
Definiens Developer and Tissue Studio
▐
LIMS database
▐
▐
▌
Specimen ID, drug, dose, staining,
patient ID, etc_
Workflow integration
Automated acquistion (If & colorimetric)
Automated slide-scanning
(Aperio Technologies)
x4
LIMS Database
ScanScopeXT
MetaMorph
Spectrum
Image Analysis
File Share
MetaMorph
Aperio-prd
ImageScope
ScanscopeFL
▐
MetaMorph
Image analysis &
visualization stations
Challenges
▌
▌
▌
▌
▌
Multiple platforms for acquisition
Integration of image data with Aperio Spectrum
Integration with specimen LIMS system
Barcode issues
Integration with existing analysis tools, (Aperio, Definiens,
Metamorph)
Integration with biorepository database
▐
▐
Problem: Associate specimen metadata with images
Requirements:
▌
▌
▌
▌
Integrate in-house biorepository (i.e. dose, tissue, study) with
Aperio’s Spectrum image database
Utilize Aperio’s Integration Server (support for upgrades)
Barcode must be compatible with both Ventana and Aperio
instruments
Flexibility for future database changes
12
LIMS / Spectrum integration
HistoPathology
Corporate
Sample Management System
SBATLIMS
SMS
Challenges (multi-month project)
•
MPI – Event trigger, read
SMS, create XML file
•
Aperio – XML import
•
Ventana / Aperio: barcode
Sample Accessioning
compatibility issues
Cancer Pharmacology
Molecular and Cellular Oncology
DSE
Clinical
Digital
Pathology
XML
Files
SPECTRUM
Project
Specimen
Slide
Barcode scan:
Event Trigger
• Acquire slide/scan barcode
• Trigger ‘new record’ event
• Retrieve metadata from SMS
• Generate XML file
• Import metadata to Spectrum
3rd Party Access to Spectrum Images
▐
▐
Problem: Currently there is no easy method to retrieve
images from Spectrum to run analysis with third party
analysis software (MetaMorph, Definiens)
Requirements
▌ Web-based tool
▌ Access to Spectrum information (project, slide ID, or
ImageID)
▌
Select networked destination for images and annotation layers
(XML)
14
Aperio Image Exporter
Annotation Information
Copy of ROI info and .svs files
•
•
Pipeline Pilot protocol finds
image location and constructs
XML, then copies files to
destination directory
Images and annotation available
for image analysis
•
•
•
Web-based Pipeline Pilot tool
User selectable by project, slide
ID, or image ID
Define destination directory
15
Application examples
▐
▐
▐
Direct and indirect pathway
biomarkers
Preclinical biomarkers
Clinical biomarkers
▐
Preclinical biomarker
▌
▌
▌
Lead optimization efforts to measure
potency of compounds
Hit target, affect pathway
Guide clinical: understand temporal
response of biomarker for optimal sampling
point and to help define clinical sampling
Pathway inhibition in pre-clinical models
Control
4hr
8hr
24hr
48hr
72hr
Mitotic Index (dapi / pHH3)
HT29 Xenograft
~ 9000 slides over 2 year period
Automated analysis
• Count total cells
• Count mitotic cells
Preclinical PD: dose and temporal response
60
Average % positive pH3
50
40
30
20
10
0
U* P** 2
4
8
16 24
48 72
6.25 mg/kg
*Untreated control
**Positive control
▐
▐
2
4
8
16
24 48 72
12.5 mg/kg
2
4
8
16
24 48
72 hr
25 mg/kg
Increase in pH3 begins at 8hrs
pH3 continues to rise with increasing dose and peaks at 24 hrs
Average % pH3 area
Average % pH3 area
Evaluation of PD Response in different
models
80
70
60
50
40
30
20
10
0
HT29
U*
80
70
60
50
40
30
20
10
0
P** 24h
HCT116
48h
72h
U*
CWR22RV1
U*
P** 24h
▐
48h
P** 24h
48h
Calu-6
72h
U*
LY19
72h
U*
P** 24h
P** 24h
48h
72h
48h
72h
WSU
48h
72h
U*
P** 24h
PD response in colon, lung, prostate and lymphoma
xenografts after a single 50 mg/kg po dose
▐
MLN8237: Aurora A Kinase inhibitor
▌
▌
Pharmacodynamic evaluation in Phase 1
clinical studies in advanced solid tumors
Includes image-based PD biomarker
strategy to assess activity
Biomarker strategy based on MoA of
Aurora A inhibition
centrosome separation defects
spindle assembly defects
prometaphase delay
Spindle Bipolarity,
Chromosome
Alignment
segregation
errors
Mitotic Index
late and
terminal outcomes
Nuclear
Morphology
multinucleation
monopolar
Direct
Marker
bipolar, misaligned
Aurora A
Target inhibition
apoptosis
multipolar
senescence
Spindle
Morphology
micronucleation
Assess MLN8237 pathway inhibition in clinical
patient biopsies
▐
▐
▐
▐
Mitotic Index in surrogate tissue (skin)
Mitotic Index (tumor)
Spindle bipolarity (tumor)
Chromosome alignment (tumor)
Punch biopsy (skin):
DNA, pH3
Mitotic cells (tumor): DNA, aTubulin
Needle biopsy (tumor): DNA, Ki67, pH3
MLN8237 clinical trials 14001/14002
Biopsy schedules
▐
Two P1 trials in patients with advanced solid tumors
▌
▐
C14001 in US; C14002 in Spain
Secondary Objectives
▌
Evaluate MLN8237 PD effect on Aurora A inhibition in skin / tumor biopsies
Day 1
Day 7
pre-treatment ~6h post-dose ~24h post-dose ~6 post-dose ~24h post-dose
14001
skin
biopsy
14002
skin
biopsy
14002
tumor
biopsy










PT 703, a case study to highlight
pharmacodynamic assays used
▐
▐
▐
▐
33 year old woman with neural sheath sarcoma
150 mg QD dose group (Spain)
Completed 4 cycles of treatment
Usable tissue and high dose make this a good case
study
PT 703, a case study
Skin mitotic index
Day 1; Pre-dose = 1
Day 7; 24 Hr Post-dose
Mitotic index (mitotic cells / mm BEL)
Day 1; Pre-dose
Day 1; 6 Hr Post-dose
Day 7; 6 Hr Post-dose
Day 7; 24 Hr Post-dose
= 0.10
= 0.39
= 3.62
= 8.08
MLN8237 skin mitotic index (14002)
*Positive values are in a direction consistent with Aurora A inhibition
Day 1 6h minus Pre-dose
30.00
25.00
Mitotic Index
(Day 1 6h minus Pre-dose)
5 mg QD (n=3)
80 mg QD (n=3)
20.00
50 mg BID (n=10)
60 mg BID (n=6)
15.00
150 mg QD (n=3)
75 mg BID (n=2)
10.00
100 mg BID (n=6)
5.00
0.00
0
2
4
6
8
-5.00
Day 7 6h minus Pre-dose
30.00
25.00
5 mg QD (n=3)
50 mg QD (n=4)
20.00
80 mg QD (n=3)
50 mg BID (n=9)
15.00
60 mg BID (n=6)
150 mg QD (n=3)
10.00
75 mg BID (n=3)
100 mg BID (n=6)
5.00
5 mg QD (n=3)
Mitotic Index
(Day 1 6h minus Pre-dose)
Mitotic Index
(Day 1 6h minus Pre-dose)
25.00
80 mg QD (n=3)
20.00
50 mg BID (n=7)
60 mg BID (n=1)
15.00
150 mg QD (n=2)
75 mg BID (n=2)
10.00
100 mg BID (n=3)
5.00
0.00
0.00
0
-5.00
Day 7 24h minus Pre-dose
30.00
2
4
6
8
0
10
-5.00
2
4
6
8
Semi-automated method to measure mitotic
spindle morphology changes in tissue
Image
Acquisition
Image Processing (Deblur)
+ Visualization
Z-axis
Image
Randomization
Scoring by
blinded scorers
Score
Deconvolution
3D Rotation
Bipolar
Aligned
15 optical sections
@ 0.5 um intervals
Bipolar
Not Aligned
Spindle Morphology
•
Spindle Bipolarity
•
Chromosome Alignment
Not Bipolar
No Call
(telophase)
Semi-automated method to measure spindle
bipolarity and chromosome alignment
Score
Bipolar
Aligned
Bipolar
Not Aligned
Not Bipolar
No Call
(telophase)
PT 703 tumor biopsies
Aligned chromosomes, bipolar spindles
% cells with aligned chromosomes
70.00%
% cells with bipolar spindles
70.00%
61.76%
60.00%
60.00%
50.00%
50.00%
40.00%
40.00%
30.00%
30.00%
20.00%
20.00%
10.00%
2.22%
63.64%
23.91%
8.51%
10.00%
0.00%
0.00%
0.00%
Pre-dose
Day 1 post-dose Day 7 post-dose
Pre-dose
Day 1 post-dose
Day 7 post-dose
Measure Aurora A pathway modulation in
clinical tumor needle biopsies
Needle biopsy
PanKeratin / pHisH3 / Dapi
•
•
•
Automated analysis
Find tumor portion of
sample
Count total cells
Count mitotic cells (tumor
only)
PT 703 tumor biopsies
Aligned chromosomes, bipolar spindles, mitotic index
%pHistH3 positive cells .
35.0%
Tumor mitotic index
32.8%
30.0%
25.0%
20.6%
20.0%
15.0%
10.0%
7.7%
5.0%
0.0%
Pre-dose
Day 1 post-dose Day 7 post-dose
PT 703, a case study
Skin hematoxylin & eosin stain
Day 1; Pre-dose
Day 7; 6 Hr Post-dose
Apoptotic index
(Apoptotic cells / mm BEL)
Day 1; Pre-dose = 0.00
Day 1; 6 Hr Post-dose = 0.13
Day 7; 6 Hr Post-dose = 1.96
Day 7; 24 Hr Post-dose = 3.31
Mitotic
Mitotic / early apoptotic
Apoptotic
MLN8237 skin apoptotic index (14002)
*Positive values are in a direction consistent with Aurora A inhibition
Day 1 6h minus Pre-dose
12.00
10.00
Mitotic Index
(Day 1 6h minus Pre-dose)
5 mg QD (n=3)
80 mg QD (n=3)
8.00
50 mg BID (n=10)
60 mg BID (n=6)
6.00
150 mg QD (n=3)
75 mg BID (n=2)
4.00
100 mg BID (n=6)
2.00
0.00
0
2
4
6
8
-2.00
Day 7 6h minus Pre-dose
12.00
5 mg QD (n=3)
50 mg QD (n=4)
8.00
80 mg QD (n=3)
50 mg BID (n=9)
6.00
60 mg BID (n=6)
150 mg QD (n=3)
4.00
75 mg BID (n=3)
100 mg BID (n=6)
2.00
0.00
10.00
5 mg QD (n=3)
Mitotic Index
(Day 1 6h minus Pre-dose)
Mitotic Index
(Day 1 6h minus Pre-dose)
10.00
80 mg QD (n=3)
8.00
50 mg BID (n=7)
60 mg BID (n=1)
6.00
150 mg QD (n=2)
75 mg BID (n=2)
4.00
100 mg BID (n=3)
2.00
0.00
0
-2.00
Day 7 24h minus Pre-dose
12.00
2
4
6
8
10
0
-2.00
2
4
6
8
MLN8237
Tumor mitotic index
Day 7 post-dose minus pre-dose
Mitotic index
(Day 7 post-dose
minus pre-dose) .
30.0%
25.0%
20.0%
15.0%
10.0%
5.0%
0.0%
-5.0%
5
5
80 50 50 50 60 60 75 150 150 100 100 100
QD QD QD BID BID BID BID BID BID QD QD BID BID BID
*Positive values are in a direction consistent with Aurora A inhibition
MLN8237
Aligned chromosomes
(% pre-dose - % day 7) .
Chromosome
alignment
Chromosome alignment / spindle bipolarity
Pre-dose minus day 7 post-dose
80
70
60
50
40
30
20
10
0
-10
Aligned chromosomes
(% pre-dose - % day 7) .
Spindle
bipolarity
5
5
80
70
60
50
40
30
20
10
0
-10
80
50 BID
150
75 BID
100 BID
100 BID
Dose day 7post-dose
Pre-dose minus
5
5
80
50 BID
150
75 BID
100 BID
100 BID
Dose
*Positive values are in a direction consistent with Aurora A inhibition
Preliminary PK-PD relationship
Emerging results from serial tumor biopsies
Chromosome Alignment
Spindle Spindle
Bipolarity
Bipolarity
140
120
Percent of Pre-dose % Bipolar Spindles
Percent of Pre-dose % Chromosome Alignment
Chromosome Alignment
100
80
60
40
20
120
100
80
60
40
20
0
0
0
20000
40000
60000
80000
100000
120000
Day 7 AUC(0-24hr) (nM.hr)
▐
▐
140000
160000
0
20000
40000
60000
80000
100000
120000
Day 7 AUC(0-24hr) (nM.hr)
Eight patients with steady-state PK and tumor biopsy measurements
Proof of mechanism - evidence for an exposure-related decrease in
chromosome alignment and spindle bipolarity in mitotic cells
140000
160000
How has the PK/PD data guided future
decisions?
▐
Demonstrated proof of mechanism – MLN8237 inhibits Aurora
A in patients
▌ Clinical responses likely related to Aurora A inhibition
▌ Use of pHistH3 as marker of mitotic accumulation confirmed
selectivity for Aurora A relative to Aurora B in patients
▌ Allows for rational drug development based on Aurora A
mechanism
• Combination selection, response marker identification
▐
▐
Demonstrated that RP2D (50 mg BIDx7d) results in biologically
active exposures
▌ Same assays applied to MLN8054 demonstrated that
biologically active exposures achieved at doses greater than
the MTD (defined by somnolence)
PD data informing future decisions
▌ Guide dose and schedule decisions for combination studies
Summary
▐
▐
Developed and integrated imaging technologies for
use in multiple drug discovery and development
programs
Leveraged tissue-based assays and technologies
▌
▌
▌
Drive medicinal chemistry
Assess pharmacodynamic response in preclinical in
vivo models
Assess pharmacodynamic response in variety of
clinical tissues, in use in Phase 1 clinical trials
Acknowledgements
▐
Slide-based Assay Team
▌
▌
▌
▌
▌
Krissy Burke
Alice McDonald
Vaishali Shinde
Yu Yang
Brad Stringer
▐
Molecular and Cellular
Oncology
▌
▌
▐
Takeda Development
Research
▌
▐
Research Systems / IT
▌
▌
David Statham
Chris Perkins
▐
Jeff Ecsedy
Natalie Roy D’Amore
Arijit Chakravarty
MLN8237 Project Team
* POSTER (P25): Details integration work and
highlights example using Definiens Tissue Studio
and Developer
We Aspire to Cure Cancer
™
© 2010 Millennium Pharmaceuticals, Inc.