Molecular Subtypes: Not Quite Ready for Prime Time

1. Molecular Subtypes:Not Quite Ready for Prime Time Scott Kopetz, MD, PhD. Department of GI Medical Oncology MD Anderson Cancer Center

2. Individual Biomarkers versus Molecular Subtypes YES • Individual biomarkers: • Microsatellite instability in all patients • For adjuvant decisions in Stage II and screening for HNPCC • KRAS, NRAS, BRAF in all metastatic patients • For consideration of EGFR sensitivity and prognosis • Molecular subtypes • 200 gene…400 gene…whole exome sequencing • Gene expression profiles • Proteomic panels NOT YET

3. Why not yet….? • We need studies to evaluate the benefit from extended molecular testing (beyond KRAS, NRAS, BRAF) • We need to define the molecular subtypes by gene expression • We need validated assays to move into the clinic

4. Why not yet….? • We need studies to evaluate the benefit from extended molecular testing (beyond KRAS, NRAS, BRAF) • We need to define the molecular subtypes by gene expression • We need validated assays to move into the clinic

5. “My panel is bigger than yours…”

6. Integrating into Clinical Trials:Increase in Prospective Enrichment 2007-08 2011-12 40% Kopetz, et al JCO ‘08, updated from clinicaltrials.gov

7. Paucity of High-Frequency Targets Means Large Screening Efforts Needed 100’s <3% frequency Currently “actionable” Screening size for a 20 patient proof-of-principle study Not actionable

8. Timeline for Biomarker Testing Median Time: 6 calendar days 28 calendar days 33 calendar days

9. ATTACC Program: Assessment of Targeted Therapies Against Colorectal Cancer S. Kopetz, PI

10. Current Screening Panel • IonTorrent50 gene panel • -IonProton 400 gene • CpG Methylation Panel • Immunohistochemistry • -PTEN, MET, HER2 expression • KRAS, NRAS, EGFR ectodomain mutation in cfDNA/plasma • Microsatellite instability panel EnrichmentTherapeutic Agent(s) Mechanism PTEN Loss or PIK3CAmut Akt inhibitor MK-2206 CpG Island Methylation DemethylatorAzacitadine + XELOX Mitotic inhib Nab-paclitaxel HER2 overexpression HER2 inhibitionTrastuzumab +/- EGFR HER2 mutation ERB family inhib TBA Exon 3,4 KRAS or NRAS mutant RAF inhibition LY3009120 ERK inhibition Biomed Valley MD Anderson ATTACC Program: Biomarker Screening for 5-FU Refractory Metastatic CRC BRAF Mutation BRAF+EGFR+irinoVemurafenib +cetux+ irino PTEN Loss/KRAS WT PI3K-beta inhibitor SAR26031 Aquired RAS mutation MEK + EGFR inhibition Panitum + Trametinib EGFR ectodomain mutation Alternate EGFR Panitumumab KRAS and PIK3CA mutation Dual MEK, PI3K BYL719 and MEK162 MSI High CTLA4 and PD1 Nivolumumab, Ipilumumb N=550 enrolled Triple KRAS/BRAF/NRAS WT EGFR+HER2 Cetuximab + trastuzumab

11. The Reality of Screening Studies1 in 5 Patients Allocation to Enrichment Study 19% enriched companion study Overall, 42% study enrollment, including 23% unenriched study Through 3/1/13, N=250, first new treatment on ATTACC

12. Practical Considerations for Enrichment Studies • Enrichment strategies require… • Consenting patients for screening • Explaining the study • High research staff utilization per “screen failure” • Patient-satisfaction is very dependent on biomarker turn-around time • Obtaining outside paraffin blocks is rate-limiting step • How long should one delay treatment waiting for a 5% frequency biomarker? • Other experimental options need to be available • Enrichment study is hard to justify to patients in isolation

13. 19124802 Example:

14. ASSIGN Study: COLON CANCER TASK FORCE , NCI GI STEERING COMMITTEE FOLFOX/Bev x 8 then Maintenance Pails STUDY DESIGN 5-FU/Bev + drug A Kras Chemo + drug B DNA-based Screening Trial, based on NCI MATCH study Braf 5-FU/Bev + drug C PIK3CA PTEN AKT 5-FU/Bev + drug D BOTH 5-FU/Bev + drug E Endpoint PFS N = TBD but likely 3000 – 5000 Slide from P. O’Dwyer WT/WT

15. SPECTAColor Protocols CLINICAL CENTERS Screening Effort Enrollment  Goal: 600 pts/year Enroll: 10-15% of screened Treating and recruiting patients Answering if patient eligible for study BIOBANKING EORTC Headquarters Sending Tumor tissue Providing clinical data Maintaining Sample Tracking tool, eCRF and results database Centralizing and storing samples Extracting gDNA/RNA Providing results DIAGNOSTICS LABORATORIES Flexibility & Centralization Sending gDNA/cDNA Performing BM analyses Slide from S. Tejpar

16. To date… Limited Prospective Biomarker Success in CRC New or Anticipated Agents/Indications • Bevacizumab (2nd line) • Ziv-aflibercept • Regorafenib • TAS-102 No new biomarker-directed therapy Wrong premise, wrong implementation, or still too early?

17. Why not yet….? • We need studies to evaluate the benefit from extended molecular testing (beyond KRAS, NRAS, BRAF) • We need to define the molecular subtypes by gene expression • We need validated assays to move into the clinic

18. Two Approaches to Biomarker Integration • Individual Biomarker Perspective • Biomarkers are paired with individual drugs • Taxonomy Perspective • Move to a “Taxonomy” Perspective Biomarker A Drug X Drug X

19. Two Approaches to Biomarker Integration • Individual Biomarker Perspective • Biomarkers are paired with individual drugs • Taxonomy Perspective • Move to a “Taxonomy” Perspective Biomarker A Drug X Drug X

20. Definitions Taxonomy = Grouping based on common patterns Taxidermy = Stuffing

21. CRC Taxonomy Hasn’t Been Defined To Date Breast Cancer Lymphoma Colorectal Cancer ? Basal Her2-pos Luminal A Luminal B Sotiriou et al NEJM, 2009; Alizadeh et al, Nature 2000

22. Gene Expression Tests are “Fit for Purpose” ≠ Prognostic Assays Taxonomy / Molecular Classification Assays

23. KRAS Poorly Recapitulates Taxonomy Budinska et al ASCO ‘12

24. Published Molecular Subtypes of Colorectal Cancer CIN (30%) TCGA T:220 MSI/CIMP (30%): BRAFm, hypermutated Invasive (40%) TA cetux res (14%): MSS, stem cell, MET-inh sensitive, worse survival Goblet (14%): MSI, crypt top, Wnt low, no benefit adj CT, good prognosis TA cetux sensitive (18%): MSS, high EGFR ligands, good prognosis Swiss T:445 V:774 30 genes Enterocyte (18%): crypt top ,Wnt low Inflammatory (18%): MSI, benefit FOLFIRI Mesenchymal (19%): EMT/ CSC high Wnt low, poor prognosis, BRAFm, desmoplastic CIMP+ (11%): MSI, BRAFm, immune up, mucinous Mixed (14%): Wnt high, CSC high, tubular PETACC3 T:1113 V:720 54 genes Surface crypt (26%): KRASm, EMT low, Wnt low, papillary or serrated phenotype Lower crypt (30%): EMT low, Wnt high, tubular phenotype AMC-AJCCII-90 T:90 V:1074 146 genes CCS3 (25%): poorly dif, EMT, invasion, migration and TGF-β signaling, no benefit cetuximab CCS1 (50%): CIN+, KRASmand TP53m, left colon, Wnt high CCS2 (25%):MSI, CIMP+, BRAFm, right colon CIN normal (10%): serrated, poor survival KRASm (10%): serrated, CIMP+ CSC (10%): serrated, poor survival French T:443 V:1058 57 genes dMMR (20%): sessile serrated precursor, BRAFm, immune up CIN immune down (20%): conventional precursor CIN Wnt up (30%): conventional precursor Agendia T:188 V:543 32/53/102 genes A-type (22%): BRAFm, MSI/dMMR, epithelial proliferative C-type (16%): mesenchymal, no benefit CT A-type (62%): low mutation, MSS, epithelial proliferative, benefit adjuvant CT Melbourne T:209 V:443 128 genes Poor prognosis (60%): immune down/ cell signaling, ECM and focal adhesion pathways up Good prognosis (40%) Slide from Rodrigo Dienstmann

25. PIs: Justin Guinney Rodrigo Dienstmann

26. Consensus clusters CLUSTER 2 CLUSTER 3 CLUSTER 4 CLUSTER 1 ASCO 2014 Clinical Symposium: Colorectal Cancer: Not Just One Disease

27. Why not yet….? • We need studies to evaluate the benefit from extended molecular testing (beyond KRAS, NRAS, BRAF) • We need to define the molecular subtypes by gene expression • We need validated assays to move into the clinic

28. Development of Validated Assay is Nontrivial “The same rigor that we use for development of the drug has to go into the biomarker development” R. Pazdur (FDA)

29. Conclusion • Everyone should be testing for MSI and KRAS, NRAS, BRAF • We need to do the studies to demonstrate benefit of more extended molecular profiling • Low yields for actionable mutations • Need more and better novel therapies • A consensus is building for defining the subsets • The assays need to be built, and moved into clinical labs.