Upload
asher-nash
View
217
Download
0
Tags:
Embed Size (px)
Citation preview
© 2010 Millennium Pharmaceuticals Inc., The Takeda Oncology Company
Developing quantitative slide-based assays to assess target inhibition in oncology drug
discovery and development
Pathology VisionsOctober 24-27, 2010
Doug BowmanMillennium Pharmaceuticals
Outline
▐ Imaging @ Millennium▐ Technology development & integration▐ Applications in Oncology
▌ Assess in vivo potency▌ Biomarker development▌ Assess clinical activity
Target ID Validation
Assay Dev. HTS Lead
Optimization IIIPhase I II
Preclinical Development
INDDCApproval
Mechanism of Action / Pathway Inhibition / Terminal Outcome
Preclinical PD assays and Clinical Biomarkers
Cell-based imaging assays
Tissue-based imaging enables direct and indirect biomarkers of target inhibition
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
Hit to Lead
•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
* * *
Tissue-Based Imaging @ Millennium
▐ Sample accession to LIMs system▐ Slide preparation
▌ Automated slide processing / staining▐ Slide scanning
▌ Immunofluorescence (IF) and Brightfield▐ Image / data management▐ Image analysis
▌ “Canned” solutions and developed algorithms
Custom developed slide scanning systems
▐ 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
Automated slide-scanning (If & colorimetric)
File Share
MetaMorph
x 4
Slide Loader(200 slide capacity)
XYZ Stage
B/W Camera RGB Camera
BarcodeReader
High resolution and efficient scanning of clinical samples
100um20x 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
aTubulin / pHisH3 / Dapi aTubulin
3 dimensional rotation, +- 30 degrees
Investment in Aperio Technologies platform
Aperio-prd
Automated slide-scanning(Aperio Technologies)
Spectrum
ImageScope
ScanScopeXT
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
▐ MetaMorph Image Analysis▐ Aperio Image Analysis▐ Definiens Developer and Tissue Studio
▐ LIMS database▌ Specimen ID, drug, dose, staining,
patient ID, etc_
Image analysis & visualization stations
Image Analysis
MetaMorph
Workflow integrationAutomated acquistion (If & colorimetric)
File Share
MetaMorph
x 4
Image analysis & visualization stations
Aperio-prd
Automated slide-scanning(Aperio Technologies)
Spectrum
ImageScope
ScanScopeXT
ScanscopeFL
MetaMorphImage Analysis
▐ 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)
LIMS Database
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
SMSSBATLIMS
HistoPathology Corporate Sample Management System
SPECTRUM
DigitalPathology
Sample Accessioning
Cancer Pharmacology Molecular and Cellular Oncology
DSE Clinical
ProjectSpecimenSlide
Barcode scan:Event Trigger
XMLFiles
• Acquire slide/scan barcode• Trigger ‘new record’ event• Retrieve metadata from SMS• Generate XML file• Import metadata to Spectrum
Challenges (multi-month project)
• MPI – Event trigger, read SMS, create XML file
• Aperio – XML import
• Ventana / Aperio: barcode compatibility issues
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
15
Annotation Information
Copy of ROI info and .svs files
Aperio Image Exporter
• Web-based Pipeline Pilot tool• User selectable by project, slide
ID, or image ID• Define destination directory
• Pipeline Pilot protocol finds image location and constructs XML, then copies files to destination directory
• Images and annotation available for image analysis
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
Mitotic Index (dapi / pHH3)
HT29 Xenograft
4hr 8hr 24hr 48hr 72hrControl
~ 9000 slides over 2 year period
Automated analysis
• Count total cells
• Count mitotic cells
0
10
20
30
40
50
Ave
rag
e %
po
siti
ve p
H3
60
2 4 8 16 24 48 72 2 4 8 16 24 48 72 2 4 8 16 24 48 72 hr
6.25 mg/kg 12.5 mg/kg 25 mg/kg
U* P**
▐ Increase in pH3 begins at 8hrs▐ pH3 continues to rise with increasing dose and peaks at 24 hrs
Preclinical PD: dose and temporal response
*Untreated control
**Positive control
Av
era
ge
% p
H3
are
a
Av
era
ge
% p
H3
are
a
01020304050607080
U* P** 24h 48h 72h
HT29 HCT116
U* P** 24h 48h 72h
Calu-6
U* P** 24h 48h 72h
01020304050607080
CWR22RV1
U* P** 24h 48h 72h
LY19
U* P** 24h 48h 72h
WSU
U* P** 24h 48h 72h
▐ PD response in colon, lung, prostate and lymphoma xenografts after a single 50 mg/kg po dose
Evaluation of PD Response in different models
▐ 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
centrosome separation defectsspindle assembly defects
prometaphase delaysegregation
errorslate and
terminal outcomes
monopolar
bipolar, misaligned
multipolar
micronucleationmultinucleation
apoptosis
senescenceAurora A
Target inhibition
Biomarker strategy based on MoA of Aurora A inhibition
Direct Marker
Nuclear MorphologyMitotic Index
Spindle Bipolarity, Chromosome
Alignment
Spindle Morphology
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
Needle biopsy (tumor): DNA, Ki67, pH3
Mitotic cells (tumor): DNA, aTubulin
MLN8237 clinical trials 14001/14002 Biopsy schedules
14001skin
biopsy
pre-treatment
14002skin
biopsy
14002tumorbiopsy
Day 1
~6 post-dose~6h post-dose ~24h post-dose ~24h post-dose
Day 7
14001skin
biopsy
pre-treatment
14002skin
biopsy
14002tumorbiopsy
Day 1
~6 post-dose~6h post-dose ~24h post-dose ~24h post-dose
Day 7
▐ 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
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 studySkin mitotic index
Mitotic index (mitotic cells / mm BEL)Day 1; Pre-dose = 0.10Day 1; 6 Hr Post-dose = 0.39Day 7; 6 Hr Post-dose = 3.62Day 7; 24 Hr Post-dose = 8.08
Day 1; Pre-dose = 1
Day 7; 24 Hr Post-dose
MLN8237 skin mitotic index (14002)*Positive values are in a direction consistent with Aurora A inhibition
Day 1 6h minus Pre-dose
-5.00
0.00
5.00
10.00
15.00
20.00
25.00
30.00
0 2 4 6 8
Mito
tic I
ndex
(Day
1 6
h m
inus
Pre
-dos
e)
5 mg QD (n=3)
80 mg QD (n=3)
50 mg BID (n=10)
60 mg BID (n=6)
150 mg QD (n=3)
75 mg BID (n=2)
100 mg BID (n=6)
Day 7 6h minus Pre-dose
-5.00
0.00
5.00
10.00
15.00
20.00
25.00
30.00
0 2 4 6 8 10
Mito
tic I
nd
ex
(Da
y 1
6h
min
us
Pre
-do
se)
5 mg QD (n=3)
50 mg QD (n=4)
80 mg QD (n=3)
50 mg BID (n=9)
60 mg BID (n=6)
150 mg QD (n=3)
75 mg BID (n=3)
100 mg BID (n=6)
Day 7 24h minus Pre-dose
-5.00
0.00
5.00
10.00
15.00
20.00
25.00
30.00
0 2 4 6 8
Mito
tic I
ndex
(Day
1 6
h m
inus
Pre
-dos
e)5 mg QD (n=3)
80 mg QD (n=3)
50 mg BID (n=7)
60 mg BID (n=1)
150 mg QD (n=2)
75 mg BID (n=2)
100 mg BID (n=3)
Semi-automated method to measure mitotic spindle morphology changes in tissue
15 optical sections@ 0.5 um intervals
Z-axis
3D Rotation
Deconvolution Score
ImageAcquisition
ImageAcquisition
Image Processing (Deblur)+ Visualization
Image Processing (Deblur)+ Visualization
ImageRandomization
ImageRandomization
Scoring byblinded scorers
Scoring byblinded scorers
BipolarAligned
BipolarNot Aligned
Not Bipolar
No Call(telophase)
Spindle Morphology• Spindle Bipolarity• Chromosome Alignment
Semi-automated method to measure spindle bipolarity and chromosome alignment
ScoreBipolarAligned
BipolarNot Aligned
Not Bipolar No Call(telophase)
PT 703 tumor biopsiesAligned chromosomes, bipolar spindles
% cells with bipolar spindles
63.64%
23.91%
8.51%
0.00%
10.00%
20.00%
30.00%
40.00%
50.00%
60.00%
70.00%
Pre-dose Day 1 post-dose Day 7 post-dose
% cells with aligned chromosomes
61.76%
2.22%0.00%
0.00%
10.00%
20.00%
30.00%
40.00%
50.00%
60.00%
70.00%
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 biopsiesAligned chromosomes, bipolar spindles, mitotic index
Tumor mitotic index
7.7%
20.6%
32.8%
0.0%
5.0%
10.0%
15.0%
20.0%
25.0%
30.0%
35.0%
Pre-dose Day 1 post-dose Day 7 post-dose
%pH
istH
3 po
sitiv
e ce
lls
.
PT 703, a case study Skin hematoxylin & eosin stain
MitoticMitotic / early apoptoticApoptotic
Day 1; Pre-dose Day 7; 6 Hr Post-dose
Apoptotic index (Apoptotic cells / mm BEL)
Day 1; Pre-dose = 0.00Day 1; 6 Hr Post-dose = 0.13Day 7; 6 Hr Post-dose = 1.96
Day 7; 24 Hr Post-dose = 3.31
MLN8237 skin apoptotic index (14002)*Positive values are in a direction consistent with Aurora A inhibition
Day 1 6h minus Pre-dose
-2.00
0.00
2.00
4.00
6.00
8.00
10.00
12.00
0 2 4 6 8
Mito
tic I
ndex
(Day
1 6
h m
inus
Pre
-dos
e)
5 mg QD (n=3)
80 mg QD (n=3)
50 mg BID (n=10)
60 mg BID (n=6)
150 mg QD (n=3)
75 mg BID (n=2)
100 mg BID (n=6)
Day 7 6h minus Pre-dose
-2.00
0.00
2.00
4.00
6.00
8.00
10.00
12.00
0 2 4 6 8 10
Mito
tic I
ndex
(Day
1 6
h m
inus
Pre
-dos
e)
5 mg QD (n=3)
50 mg QD (n=4)
80 mg QD (n=3)
50 mg BID (n=9)
60 mg BID (n=6)
150 mg QD (n=3)
75 mg BID (n=3)
100 mg BID (n=6)
Day 7 24h minus Pre-dose
-2.00
0.00
2.00
4.00
6.00
8.00
10.00
12.00
0 2 4 6 8
Mito
tic I
ndex
(Day
1 6
h m
inus
Pre
-dos
e)5 mg QD (n=3)
80 mg QD (n=3)
50 mg BID (n=7)
60 mg BID (n=1)
150 mg QD (n=2)
75 mg BID (n=2)
100 mg BID (n=3)
MLN8237 Tumor mitotic index
*Positive values are in a direction consistent with Aurora A inhibition
Day 7 post-dose minus pre-dose
-5.0%
0.0%
5.0%
10.0%
15.0%
20.0%
25.0%
30.0%
5QD
5QD
80QD
50BID
50BID
50BID
60BID
60BID
75BID
150QD
150QD
100BID
100BID
100BID
Mito
tic in
dex
(Day
7 p
ost
-dose
m
inus
pre
-dose
) .
MLN8237 Chromosome alignment / spindle bipolarity
*Positive values are in a direction consistent with Aurora A inhibition
-10
0
10
20
30
40
50
60
70
80
5 5 80 50 BID 150 75 BID 100 BID 100 BID
Dose
Alig
ned
chro
mos
omes
(% p
re-d
ose
- %
day
7)
.Pre-dose minus day 7 post-dose
Sp
ind
le b
ipo
lari
tyC
hro
mo
som
e a
lig
nm
ent
-10
0
10
20
3040
50
60
70
80
5 5 80 50 BID 150 75 BID 100 BID 100 BID
Dose
Alig
ned
chro
mos
omes
(% p
re-d
ose
- %
day
7)
. Pre-dose minus day 7post-dose
Preliminary PK-PD relationshipEmerging results from serial tumor biopsies
Chromosome Alignment
Day 7 AUC(0-24hr) (nM.hr)
0 20000 40000 60000 80000 100000 120000 140000 160000
Per
cent
of
Pre
-dos
e %
Chr
omos
ome
Alig
nmen
t
0
20
40
60
80
100
120
Spindle Bipolarity
Day 7 AUC(0-24hr) (nM.hr)
0 20000 40000 60000 80000 100000 120000 140000 160000P
erce
nt o
f P
re-d
ose
% B
ipol
ar S
pind
les
0
20
40
60
80
100
120
140
▐ 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
Chromosome Alignment Spindle Bipolarity
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
▐ Research Systems / IT▌ David Statham▌ Chris Perkins
▐ Molecular and Cellular Oncology
▌ Jeff Ecsedy▌ Natalie Roy D’Amore
▐ Takeda Development Research
▌ Arijit Chakravarty
▐ MLN8237 Project Team
* POSTER (P25): Details integration work and highlights example using Definiens Tissue Studio and Developer
© 2010 Millennium Pharmaceuticals, Inc.
We Aspire to Cure Cancer™