1 / 37

Q-CROC -01

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. Potential Conflict of Interest. Dr. Gerald Batist

levana
Download Presentation

Q-CROC -01

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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. 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 Pharma

  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. 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 21 22 -2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 11 chemo cycle 1 2 3 4 5 6 7 8 9 10 12 1 2 3 4 5 6 banked blds pre-chemo 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 liver bx CT CT liver bx? week 21 22 -2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 chemo cycle 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 banked blds pre-chemo

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

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

  15. Technical challenges: e.g. Pt 006 Images of frozen tissue (OCT), fragmented biopsy 1 fragment 100% tumor tissue (left) 1 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 260/280 1.94 1.73 1.98 1.73 2.02 1.81 1,71 1.69 1.67 1.6 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.

  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 A B C D 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. T-199 T-117 3.3 N 2.3 N 2.0 N 2.0 N FLOW SORTING OF TUMOR CLONES IN BIOPSIES

  23. ARRAY CGH ON SORTED TUMOR CLONES P4 P5 P4 P5

  24. RNA quality from core biopsy material

  25. RNAomics Platform (splice variants) UNIVERSITY OF SHERBROOKE

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

  27. 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):pp1981-89

  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 MICRO RNA PROFILING RNA

  30. 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???

  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.

  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 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.

  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?

  34. 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

  35. 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 Ongoing Q-CROC Research Projects

More Related