Q-CROC -01A biopsy driven study for

molecular signatures of therapeutic resistance in

metastatic CRC

Gerald BatistMcGill Department of Oncology

McGill UniversitySegal Cancer Centre-JGH

Potential Conflict of InterestPotential Conflict of Interest

Dr. Gerald BatistDr. Gerald Batist

• This study supported by the FRSQ-Pfizer This study supported by the FRSQ-Pfizer Innovation FundInnovation Fund

CreditsCredits

Won ‘best in biomarker category’ Won ‘best in biomarker category’ prize presentation at ESMO, Italy 2010prize presentation at ESMO, Italy 2010

Selected as poster discussion at Selected as poster discussion at ASCO-NCI-EORTC molecular biomarker ASCO-NCI-EORTC molecular biomarker meeting, Oct 2010meeting, Oct 2010

Network and Partners

Q-CROC

GovernmentFRSQ (research)

MDEIE (economic dev.)MSSS (health)

Universities

Hospitals

Pharma

Coalition Priorité Cancer (advocacy group)

Members80 members from Montréal,

Québec City and Sherbrooke

Burning questions:

WHO can be spared unnecessary treatment?WHO can be spared unnecessary treatment?

WHO will predictably not benefit?WHO will predictably not benefit?

HOW can we overcome resistance?HOW can we overcome resistance?

Focus on therapeutic resistance in the metastatic Focus on therapeutic resistance in the metastatic settingsetting

Optimistic view The ‘incremental’ or marginal benefits seen in

many clinical trials is a function doing studies in non-selected population;

This implies that there are sub-groups with major benefits, which are being diluted by the non-responders;

Therefore. Our task, the goal of clinical trials, should include indentifying the sub-groups.

Experience: mCRC biopsy-driven trial

Prospective metastatic tumor biobank• Ethics committee approval for research-driven biopsy• Logistics- image-guided biopsies, standardized

collection and storage• DNA microarrays and proteomics

Genomic signature of resistance (K-ras gene mutation

here) identifies the responder subset of patients with colorectal cancer

The necessary team:The necessary team:

Surgeon-scientist Interventionist radiologist Medical oncologists HTP array technologies Molecular pathology A pharma company…..

Experimental Design: Q-CROC-01

To identify the molecular signature of therapeutic resistance to standard therapy in metastatic CRC.

Model can be applied to other drugs and tumor types.

mFOLFOX6 +Avastin

XELOX + Avastin

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20weekchemo cycle 1 2 3 4 5 6 7 8 9 10

banked bldspre-chemo

1 2 3 4 5

11

6

CT

21 2212

CT

-1-2

liver bx

Oxaliplatin 85 mg/m2, IV over 2 h (Day 1)Leucovorin 400 mg/m2 IV over 2 h (Day 1)5FU 400 mg/m2 bolus (Day 1), 2400 mg/m2 for 46 h, cont. infusionBevacizumab 5mg/kg over 90 min on Day 1

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20week

chemo cycle 1 2 3 4 5 6 7 8

banked bldspre-chemo

1 2 3

CT

21 22

CT

-1-2

liver bx

Oxaliplatin 130 mg/m2, IV over 2 h (Day 1)Capecitabine 1000 mg/m2 daily from days 1-14 Bevacizumab 7.5 mg/kg over 90 min on Day 1

4 5 6 7 8

liver bx?

liver bx?

Integrating Two Approaches

Discovery

High throughput technologiesLarge sets of results

Candidate-BasedApproach

TISSUE SAMPLE PATHWAY

Sample

DNA

RNA

LYSATES

GENE EXPRESSION PROFILING

NEXT GEN SEQUENCING

ARRAY CGH

PHOSPHOPROTEIN PROFILING

TISSUE SECTIONS IMMUNOHISTOCHEMISTRY

SPLICE ISOFORM PROFILING

MICRO RNA PROFILING

Main Technologies Accessible

GenomicsGenomics

• MicroarraysMicroarrays

• Comparative Genomic Comparative Genomic HybridizationHybridization

• RNomics RNomics (RNA variant (RNA variant expression and phenotypic expression and phenotypic analysis)analysis)

• PharmacogenomicsPharmacogenomics

• MicroRNA ScreeningMicroRNA Screening

• Methylation ProfilingMethylation Profiling

Proteomics i-TRAQ

TMA

Others Radiobiology/medical

physics core lab

Various Cores (including the LDI/SCC ones)

1

2

3

formalin

1.5 x 0.1 x 0.1 cm

RNAlater

RNAlater

Liver Biopsy from a Metastatic Lesion

JGH

HDQ

HDQ

OCT embedding

Verification of % tumor

>70% tumor

RNA / DNA

<70% tumor

Discovery platforms

rejected

Immunohistochemistry

Images of frozen tissue (OCT), fragmented biopsy

1 fragment 100% tumor tissue (left) 1 fragment 100% necrosis (right)

100X 100X

Technical challenges: e.g. Pt 006

Preliminary results: biopsiesPreliminary results: biopsies

Profiling of biopsiesProfiling of biopsies

• 14-16 gauge needle14-16 gauge needle• 11.4 ug total RNA (median)11.4 ug total RNA (median)• 22 ug DNA (median)22 ug DNA (median)• Required: 600 ng DNA for aCGH and 1 ug for Required: 600 ng DNA for aCGH and 1 ug for

expression arrays, 300 ng for microRNA expression arrays, 300 ng for microRNA profiling, 300 ng for RNA splicing profilingprofiling, 300 ng for RNA splicing profiling

DNA quality control obtained with Bioanalyer (Agillent) for A) DNA extracted from needle biopsies form breast and colon cancers. Samples 1-3 are from primary breast cancer specimens and samples 4-5 are from colon cancer metastasis. Note that samples 2 and 5 have less DNA, and sample 5 DNA quality may be poorer. B) same DNA amplified using Illustra Genomiphi V2 DNA amplification Kit (GE Healthcare #25-6600-31). Samples 7-9 are amplified DNAs from breast cancer specimens and samples 10-11 are amplified DNAs from colon metastases. The values below the image are the ratio of absorbance at 260 nm/280 nm further confirming the overall good quality of both non-amplified and amplified DNA.

A B

260/280 1.94 1.73 1.98 1.73 2.02 1.81 1,71 1.69 1.67 1.6

Array CGH from core biopsy material

Focal amplifications on q arm of chromosome 11 from specimen 1

Global changes detected in colon metastasis using array CGH analysis.

DNA obtained from a liver metastasis from colon cancer shows DNA copy number gains on chromosomes 8 (MYC), 13 and 20. This results further validate our methodology and suggest that DNA obtained from biopsies can be amplified if necessary for array CGH analysis.

Feasability: DNA quality excellent for amplification, aGCH

Small amplified region found in 3 breast cancer biopsies (A-C), but not in a liver metastasis from colon cancer (D)

The presence of a relatively small amplification (about 240 Kb) in three different breast cancer specimens validate the precision of the array CGH methodology for the analysis of DNA copy number changes in amplified DNA from needle biopsy samples of cancers. Note that this narrow area of increased DNA copy number is the site of a common copy number polymorphism.

A

C

B

D

Specificity of aCGH findings in core needle biopsies of breast vs colon mets in liver

The analysis of tumor subclones

MOLECULAR PROFILING

FLOW SORTING OF TUMOR CLONES IN BIOPSIES

T-117

2.0 N

3.3 N

2.0 N

2.3 N

T-199

ARRAY CGH ON SORTED TUMOR CLONES

P4

P5

P4

P5

RNA quality from core biopsy material

RNAomicsPlatform (splice variants)

UNIVERSITY OF SHERBROOKE

MicroRNA expression in core biopsy

material

(M. Simard, Univ. Laval)

Paweletz CP et al , Oncogene 2001 (20):pp1981-89

PROTEOMIC ANALYSISREVERSE PHASE LYSATE PROTEIN MICROARRAYS

Wulfkuhle JD et al. J Proteome Res 2008

High throughput phosphoprotein profiling

Using reliable phospho-antibodies

BLOOD BIOMARKERS

Accessible

Patients very willing to undergo blood test: least invasive procedure

Can be followed in time

Proven biomarkers (CEA, PSA) that anticipate disease recurrence in cancers

Host-factors as well as tumor-derived factors

BLOOD SAMPLE PATHWAY

Bloodsample

DNALYMPHOCYTES

PLASMA

CELLS

CYTOKINE PANEL

POLYMORPHISMS (CANDIDATE GENES)

CTCs, EPCs

RNA MICRO RNA PROFILING

MRM-MS

SPECIFIC ELISA

K-EDTA plus cocktail of protease K-EDTA plus cocktail of protease inhibitorsinhibitors

Stabilize the blood proteomeStabilize the blood proteome $22 per tube: IS IT NECESSARY???$22 per tube: IS IT NECESSARY???

Pre-analytical studies on blood collection: clarifying and ? simplifying

Higher protein levels with old protocol are likely to be anartefact of the processing protocol resulting in highplatelet levels and their activation.

New BD protocol validation study shows no difference in levels of several

cytokines between P100 and k-EDTA collection tubes

*

*

*

*

• There was a consistent trend towards an increase in cytokine levels with time in all tube types.

• Significant increases were seen for 22% of cytokines in P100 tubes and 44% in k-EDTA tubes; fold changes ranged between 1.5-2.85.

• Differences in P100 tubes were all between time 0 and 6h while in k-EDTA they were between time 2h and 6h except for one cytokine.

The effect of time delays in processing on blood biomarker levels

SERUM/PLASMA RPPMs

If tissues can be spotted, why not If tissues can be spotted, why not plasma/serum?plasma/serum?

• Surface and buffers?Surface and buffers?• Volumes = 0.7 nanolitersVolumes = 0.7 nanoliters• Limit of detection?Limit of detection?• Measureable change?Measureable change?• Reproducibility?Reproducibility?• Shelf-life?Shelf-life?

Q-CROC-01 Project Update• Beginning of the project: September 2009

• Funded by competitive grant and pharma partnerships

• Multiple sites opening, accrual proceeding (17)

• Study of patient and physician attitudes is linked

• Biopsy material is being processed for the platforms

Prospective Study to Identify Molecular Mechanisms of Clinical Prospective Study to Identify Molecular Mechanisms of Clinical Resistance to Standard First-Line Therapy in Patients with Resistance to Standard First-Line Therapy in Patients with Metastatic Colorectal Cancer (Q-CROC-01)Metastatic Colorectal Cancer (Q-CROC-01)

• Needle core biopsies of liver metastasis at D0 and at acquired resistanceNeedle core biopsies of liver metastasis at D0 and at acquired resistance• Blood samples collected and banked during treatmentBlood samples collected and banked during treatment

Biopsy-driven study in Non-Hodkins Lymphoma (NHL) using Biopsy-driven study in Non-Hodkins Lymphoma (NHL) using rituximab in combination with a drug involved in epigenetic rituximab in combination with a drug involved in epigenetic modifications (Q-CROC-02)modifications (Q-CROC-02)

• Lymph node biopsies D0, D15 and optional at 24 hrsLymph node biopsies D0, D15 and optional at 24 hrs• PBMC isolation at D0 and D15PBMC isolation at D0 and D15

The genomics and proteomics of Triple Negative Breast Cancer drug The genomics and proteomics of Triple Negative Breast Cancer drug resistant breast cancerresistant breast cancer (Q-CROC-03)(Q-CROC-03)

•Neoadjuvant: Biopsy: breast before treatment and at surgeryNeoadjuvant: Biopsy: breast before treatment and at surgery•Metastatic: Biopsy: liver, skin, lung, pleura; before and after treatmentMetastatic: Biopsy: liver, skin, lung, pleura; before and after treatment

Ongoing Q-CROC Research Projects