© 2010 Millennium Pharmaceuticals Inc., The Takeda Oncology Company Developing quantitative slide-based assays to assess target inhibition in oncology

© 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)


-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)


-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%


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%


%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


-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)


-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)


-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™

Documents

© 2010 Millennium Pharmaceuticals Inc., The Takeda Oncology Company Developing quantitative slide-based assays to assess target inhibition in oncology