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Molecular Subtypes: Ready for Prime Time Wells Messersmith, MD, FACP Professor Director, Gastrointestinal Medical Oncology Program Co-Head, Division of Medical Oncology Program co-Leader, Developmental Therapeutics March 2014

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Conflict of Interest: 1.No employment, speakers bureaus, stock ownership, royalties, patents, etc 2.Data Safety Monitoring Board for OncoMed 3. PI or Local PI of clinical trials by Genentech/Roche, GSK, Pfizer, Millenium, Bayer, Onconova, and NIH/CTEP.

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Outline/Objectives: 1.Introduction 2.Molecular subtypes already in use 1.KRAS/NRAS (added wrinkle: L v R?) 2.BRAF/MSI (L v R) 3.Risk Stratification in Adjuvant Setting 4.PI3K Molecular Subtypes

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Colorectal Diagnostics: What Was State of Art in 2008? CEA recommended for staging, post-operative, monitoring response DNA ploidy or proliferation assays not recommended p53, TS, DPD, TP insufficient data Ras insufficient data MSI not recommended 18q- or DCC - not recommended FDA approved tests for colorectal cancer (not widely used): - EGFR Immunohistochemistry (Dakocytomation PharmDx) - UGT1A1 Invader Assay (irinotecan glucuronidation) - toxicity - Circulating tumor cells (CellSearch test) - prognosis Copyright 2009 ASCO

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Where have we gone wrong with predictive molecular tests in past? -EGFR staining and EGFR-targeting mAbs -Used to be REQUIRED for insurance approval -Predictive tests for 5-FU efficacy -Thymidylate Synthase (TS) expression and polymorphisms -Methylenetetrahydrofolate reductase (MTHFR) polymorphisms -Dihydropyrimidine Dehydrogenase (DPD) Deficiency (*2A variants) -Topoisomerase, ercc1 (studies pending), UGT1A1.

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Tests for Cytotoxics: Irinotecan UDP-glucuronosyltransferase (UGT) 1A1 - Irinotecans active metabolite, SN-38, is eliminated via glucuronidation by UDP-glucuronosyltransferase 1A1 (UGT1A1). - Patients homozygous for *28 allele (7 vs 6 TA repeats) have 70% reduction in UGT1A1 activity - According to U.S. FDA, patients homozygous for *28 should be considered for dose reduction based on small retrospective studies (increased diarrhea in some, neutropenia in others) - large studies (FOCUS, N9741, PETACC3) have failed to confirm clinical usefulness http://www.fda.gov/cdrh/pdf5/k051824.pdf

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Outline/Objectives: 1.Introduction 2.Molecular subtypes already in use 1.KRAS/NRAS (added wrinkle: L v R?) 2.BRAF/MSI (L v R) 3.Risk Stratification in Adjuvant Setting 4.PI3K Molecular Subtypes

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Distinct Biology of R v. L CRC BRAF mut MSI KRAS PIK3CA Mucinous differentiation Right Left 18q loss 20q Gain EREG expression EGFR gain HER2 gain Poor Prognosis Sensitive to Cetuximab Good Prognosis High mutation Frequency Analysis of PETACC-3 samples (n=2849) Missiaglia, ASCO 2013

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N0147 Trial: Testing Adjuvant Cetux Stage 3 ColonCancer (N = 3768) PREREGISTER RANDOMIZE Centralized K-ras analysis K-ras WT K-ras Mut REGISTER Arm G Arm G Adjuvant therapy per primary oncologistAdjuvant therapy per primary oncologist Report therapy givenReport therapy given Annual status through year 8Annual status through year 8 Arm A mFOLFOX6 Arm D mFOLFOX6 + Cetuximab Note: in analysis, segregated proximal versus distal with splenic flexure as cut-off Alberts, JAMA 2012

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Negative Adjuvant Trial: N0147 Alberts, JAMA 2012 - No difference in DFS with the addition of cetuximab; detrimental effect for patients >70, even in KRAS WT group. - However, trial should yield other useful information

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N0147: KRAS and BRAF worse For stage IIII CRC, magnitude of detrimental effect of KRAS and BRAF mutation is lower than other analyses. KRAS MT (HR=1.45, p

Popovic, ASCO 2013 PETACC-3 (5-FU vs. FOLFIRI): Worse OS (and RFS) for BRAF Mutants: L>R - independent of MSI status, left-sided tumors do worse. leftright

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BRAF/MSI Biology: TCGA The Cancer Genome Atlas Network Nature 487, 330-337 (2012) doi:10.1038/nature11252 BRAF mutation associated with hypermutated (MSI-H) tumors.

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Ogino, Clin Can Res 2012 - BRAF mutation analyzed in 506 colon cancer patients from CALGB 89803 (n=75 BRAF mutated patients) - Worse overall survival in BRAF mutated patients (HR=1.66); mainly in microsatellite stable (MSS) patients - Trend to improvement with irinotecan added to 5-FU/LV (HR=0.52) in BRAF MT patients, albeit low numbers.

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Ogino, Clin Can Res 2012 - Due to confounding effect of MSI status in BRAF MT patients, Ogino proposed this strategy for classification. Must split, rather than lump, BRAF MT patients

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What about dMMR and FOLFOX? What about dMMR and FOLFOX? Although n

PI3K Mutations (Samuels, Science 2004) - Colorectal and gastric cancers frequently harbor mutations. - Not found in 76 polyps (except two >5cm tubulovillous adenomas) - Co-existent with KRAS and BRAF mutations (distinct pathway) Functionally important: - Nontruncating - Nonsynonymous - Conserved residues - Higher PI3K activity 2004 www.sciencemag.org

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How strong is PI3K data? Report Drugs Findings Lievre C 7% with PI3K mutn Can Res 2006, n=30 (97% chemo) no effect Sartore-Bianchi C and P 13.6% with PI3K mutn Can Res 2009, n=110 (67% chemo) lower RR, survival Perrone C 13% with PI3K mutn Ann Oncol 2009, n=32 (100% chemo) lower RR, survival De Roock (#1896) C and P 12% with PI3K mutn AACR 2009, n=200 no effect Di Nicolantonio C and P 13% with PI3K mutn AACR 2009, n=132 lower RR, survival Retrospective clinical cases; conflicting.

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PI3K as a predictive marker Prenen, Clinic Can Res 2009, n=200, 12% PIK3CA MT - Used sequenome MALDI-TOF MassArray system - No relationship between PIK3CA and KRAS - No relationship between PIK3CA and response, survival Exon 9 - 20 Note: PIK3CA mutations have been associated with resistance to trastuzumab in breast cancer (possible increased dependence on her2/her3 dimerization). Burns, Cancer Cell 2007 2009 American Association for Cancer Research

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Additive Prediction DeRoock, Lancet Oncol 2010 Response rate increase as markers are added (retrospective database)

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PIK3CA mutations and aspirin Liao X et al. N Engl J Med 2012;367:1596-1606 Mutant = aspirin benefit wildtype

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Possible Mechanism? Di Popolo et al, Oncogene 2000 PI3K signal transduction pathways regulate COX-2 expression and PGE2 synthesis. PD = PD098059 (MAPK inhibitor) LY = LY294002 (PI3K inhibitor) COX2 expression PGE2

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Conclusions -The revolution of genetic information on tumors over the last 5 years has greatly increased understanding of tumor biology. -But this has not yet translated into the clinic, since most anti-cancer drugs are used empirically. -Ignorance (lack of research) is costly and deadly; without knowledge of KRAS, for instance, we would be spending $700,000,000 per year harming patients in the U.S. -Knowledge of relevant biomarkers is critical in caring for colorectal cancer patients; however, important to use markers that have been validated/qualified across multiple studies.

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Thank You! wells.messersmith@ucdenver.edu