1. Q-CROC -01
A biopsy driven study for molecular signatures of therapeutic resistance in metastatic CRC
Gerald Batist
McGill Department of Oncology
McGill University
Segal Cancer Centre-JGH

2. Potential Conflict of Interest
Dr. Gerald Batist
This study supported by the FRSQ-Pfizer Innovation Fund

3. Credits
Won ‘best in biomarker category’ prize presentation at ESMO, Italy 2010
Selected as poster discussion at ASCO-NCI-EORTC molecular biomarker meeting, Oct 2010

4. 80 members from Montréal, Québec City and Sherbrooke
Network and Partners
Members
80 members from Montréal, Québec City and Sherbrooke
Coalition Priorité Cancer
(advocacy group)
Government
FRSQ (research)
MDEIE (economic dev.)
MSSS (health)
Q-CROC
Hospitals
Universities
Same comment as before.
Pharma 4

5. Burning questions: WHO can be spared unnecessary treatment?
WHO will predictably not benefit?
HOW can we overcome resistance?
Focus on therapeutic resistance in the metastatic setting

6. 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.

7. 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

8. Genomic signature of resistance (K-ras gene mutation here) identifies the responder subset of patients with colorectal cancer
The necessary team:
Surgeon-scientist
Interventionist radiologist
Medical oncologists
HTP array technologies
Molecular pathology
A pharma company…..

9. 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.

10. mFOLFOX6 + Avastin XELOX + Avastin
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. infusion
Bevacizumab 5mg/kg over 90 min on Day 1
mFOLFOX6 +
Avastin
liver bx CT CT
liver bx?
week -2 -1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
chemo cycle 1 2 3 4 5 6 7 8 9 10 11 12
banked blds
pre-chemo 1 2 3 4 5 6
XELOX +
Avastin
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
CR: 36 à 38, can be reduced to only mention the significance entry criteria (if you want to event keep those slides in. I don’t think I would go into so much details)
liver bx CT CT
liver bx?
week -2 -1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
chemo cycle 1 2 3 4 5 6 7 8
banked blds
pre-chemo 1 2 3 4 5 6 7 8 10

11. Integrating Two Approaches
Discovery
High throughput technologies
Large sets of results
Candidate-Based
Approach

12. TISSUE SAMPLE PATHWAY ARRAY CGH DNA NEXT GEN SEQUENCING
GENE EXPRESSION PROFILING
Sample
RNA
MICRO RNA PROFILING
SPLICE ISOFORM PROFILING
PHOSPHOPROTEIN PROFILING
LYSATES
TISSUE SECTIONS
IMMUNOHISTOCHEMISTRY

13. Main Technologies Accessible
Genomics
Microarrays
Comparative Genomic Hybridization
RNomics (RNA variant expression and phenotypic analysis)
Pharmacogenomics
MicroRNA Screening
Methylation Profiling
Proteomics
i-TRAQ
TMA
Others
Radiobiology/medical physics core lab
Various Cores (including the LDI/SCC ones)

14. Liver Biopsy from a Metastatic Lesion
OCT embedding
HDQ
Verification of % tumor 1
RNAlater
>70% tumor
<70% tumor 2
RNA / DNA
rejected
HDQ
RNAlater
Discovery platforms
1.5 x 0.1 x 0.1 cm 3
JGH
Immunohistochemistry
formalin

15. Technical challenges: e.g. Pt 006
Images of frozen tissue (OCT), fragmented biopsy
1 fragment 100% tumor tissue (left) fragment 100% necrosis (right)
100X
100X

16. Preliminary results: biopsies
Profiling of biopsies
14-16 gauge needle
11.4 ug total RNA (median)
22 ug DNA (median)
Required: 600 ng DNA for aCGH and 1 ug for expression arrays, 300 ng for microRNA profiling, 300 ng for RNA splicing profiling

17. A B
260/ ,
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 # ). Samples 7-9 are amplified DNAs from breast cancer specimens and samples 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.

18. Array CGH from core biopsy material
Focal amplifications on q arm of chromosome 11 from specimen 1

19. Feasability: DNA quality excellent for amplification, aGCH
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.

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

21. The analysis of tumor subclones
MOLECULAR PROFILING

22. FLOW SORTING OF TUMOR CLONES IN BIOPSIES

23. ARRAY CGH ON SORTED TUMOR CLONESP4 P5 P4 P5

24. RNA quality from core biopsy material

25. Platform (splice variants)
RNAomics
Platform (splice variants)
UNIVERSITY OF SHERBROOKE

26. MicroRNA expression in core biopsy material (M. Simard, Univ. Laval)

27. REVERSE PHASE LYSATE PROTEIN MICROARRAYS
PROTEOMIC ANALYSIS
REVERSE PHASE LYSATE PROTEIN MICROARRAYS
High throughput phosphoprotein profiling
Using reliable phospho-antibodies
Wulfkuhle JD et al. J Proteome Res 2008
Paweletz CP et al , Oncogene 2001 (20):pp

28. 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

29. BLOOD SAMPLE PATHWAY DNA LYMPHOCYTES POLYMORPHISMS (CANDIDATE GENES)
CYTOKINE PANEL
Blood
sample
PLASMA
SPECIFIC ELISA
MRM-MS
CELLS
CTCs, EPCs
RNA
MICRO RNA PROFILING

30. K-EDTA plus cocktail of protease inhibitors
Pre-analytical studies on blood collection: clarifying and ? simplifying
K-EDTA plus cocktail of protease inhibitors
Stabilize the blood proteome
$22 per tube: IS IT NECESSARY??? 30

31. New BD protocol validation study shows no difference in levels of several cytokines between P100 and k-EDTA collection tubes
Higher protein levels with old protocol are likely to be an
artefact of the processing protocol resulting in high
platelet levels and their activation. 31

32. The effect of time delays in processing on blood biomarker levels
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
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. 32

33. SERUM/PLASMA RPPMs If tissues can be spotted, why not plasma/serum?
Surface and buffers?
Volumes = 0.7 nanoliters
Limit of detection?
Measureable change?
Reproducibility?
Shelf-life?

35. 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

36. Ongoing Q-CROC Research Projects
Prospective Study to Identify Molecular Mechanisms of Clinical Resistance to Standard First-Line Therapy in Patients with Metastatic Colorectal Cancer (Q-CROC-01)
Needle core biopsies of liver metastasis at D0 and at acquired resistance
Blood samples collected and banked during treatment
Biopsy-driven study in Non-Hodkins Lymphoma (NHL) using rituximab in combination with a drug involved in epigenetic modifications (Q-CROC-02)
Lymph node biopsies D0, D15 and optional at 24 hrs
PBMC isolation at D0 and D15
The genomics and proteomics of Triple Negative Breast Cancer drug resistant breast cancer (Q-CROC-03)
Neoadjuvant: Biopsy: breast before treatment and at surgery
Metastatic: Biopsy: liver, skin, lung, pleura; before and after treatment 36