Colorectal cancer intrinsic subtypes are associated with prognosis, chemotherapy response, deficient mismatch repair and epithelial to mesenchymal transition (EMT) Josep Tabernero, Vall d’Hebron Hospital and Iris Simon, Paul Roepman, Andreas Schlicker, Ian Majewski, Victor Moreno, Christine Chresta, Robert Rosenberg, Ulrich Nitsche, Teresa Macarulla, Gabriel Capella, Ramon Salazar, George Orphanides, Lodewyk Wessels, Rene Bernards

Disclosure Information Relationships relevant to this session Tabernero, Josep VHIO has received research funding from Agendia Please note, all disclosures are reported as submitted to ASCO, and are always available at gicasym.org

Colorectal cancer  different subtypes Colorectal cancer is the second leading cause of cancer death Although several treatments exist we do not have an optimal way to select treatments for individual patients Only KRAS status has been established as a predictor of anti-EGFR treatment activity New technology platforms allow genetic definition of different types of cancer based on gene expression and characterization Unbiased genome-wide analyses of gene expression patterns have been successful for molecular classification of BC & GBM

Molecular classification based on nearest centroid single sample predictor (SSP)  3 gene expression profiles (A, B & C) Development Set (stage I-IV) (n=188) Netherlands Cancer Institute, Leiden Medical Center, Slotervaart Whole Genome Array Development Validation Set (stage II-III) (n=543) Technical University Munich, Institut Catala d’Oncologia & Vall d’Hebron Hospital Barcelona, Medical University Vienna, University of Ferrara Validation Analysis of mutations in BRAF(V600), KRAS (codons 12, 13 & 61) and PIK3CA (exons 9 & 20) – All samples Analysis of 615 (incl. kinome) by NGS – 73 samples All sets Analysis of Epithelial and Mesenchymal genes – All samples OS and Distant Metastasis-free survival (DMS) – All samples Effect of adjuvant treatment – Stage III samples, validation cohort (n=123) MSI analysis by IHC – All samples MSI/dMMR gene expression pattern (64 gene signature 1 ) – All samples 1 Tian S et al. J Pathol. 2012

Development and Validation of the Molecular Subtype Signature Development CohortValidation Cohort N HospitalNKI, LUMC, SlotervaartVall d’Hebron, ICO Barcelona, Munich StageI II III IV 24 (13%) 100 (53%) 56 (30%) 8 (4%) (59%) 223 (41%) - GenderFMFM 104 (55%) 84 (45%) 226 (42%) 317 (58%) SubtypeABCABC 65 (35%) 98 (52%) 25 (13%) 117 (22%) 336 (62%) 90 (16%)

Unsupervised hierarchical clustering of whole genome reveals 3 distinct patient groups Gene profiles were developed to identify these subgroups A-TypeB-TypeC-Type

Unsupervised hierarchical clustering of whole genome reveals 3 distinct patient groups Gene profiles were developed to identify these subgroups A-Type (32 genes) B-Type (53 genes) C-Type (102 genes)

PROGNOSIS, MSI AND BENEFIT FOM ADJUVANT CHEMOTHERAPY Clinical Characterization

Subtypes are significantly associated with prognosis Risk of Distant Metastasis Risk of Death C-Type B-Type A-Type DM Risk Death

Subtypes are significantly correlated with benefit from adjuvant 5-FU-based treatment

Difference in proliferation between subtypes might explain difference in treatment benefit significantly reduced expression of Ki67 and AURKA in C-type compared to A- and B-type –Ki67 p=6.06e-5, AURKA p=4.53e-6 C-Type B-Type A-Type

Subtypes differ significantly in mutation and MSI frequency (Mismatch Repair deficiency) Cancer kinome sequencing (~600 kinases and other cancer related genes) –high mutation frequency in A and C-type (dMMR) –B type represent proficient mismatch repair (pMMR)

Subtypes differ significantly in mutation and MSI frequency (Mismatch Repair deficiency) Cancer kinome sequencing (~600 kinases and other cancer related genes) –high mutation frequency in A and C-type (dMMR) –B type represent proficient mismatch repair (pMMR) TypesMutated genesMSI/dMMR A36%68% B17%1% C34%36% Observed mutations in the cancer kinome

EPITHELIAL VS. MESENCHYMAL Biological characteristics

Epithelial-Mesenchymal Transition

EMT markers are differently expressed in subtypes Mesenchymal markers (higher in C-type) –VIM, CDH2, FN1, FGFR1, FLT1, TWIST1, AXL, TGFB1 Epithelial markers –CDH1, CDH3, CLDN9, EGFR, MET Mesenchymal Character of C-type was confirmed by EMT signature developed at MDACC (Loboda et al. 2011)

CONCLUSION Molecular Subtypes

Biological featuresClinical featuresSubtypeClinical utility Chemotherapy New targeted therapy? (companion Dx) No adjuvant or 5FU Colon molecular subtype model

Acknowledgements All collaborators and patients Vall d'Hebron Hospital Agendia Institut Català d'Oncologia Technische Universität Munich Netherland Cancer Institute Slotervaart Hospital Leiden Medical Center Medical University of Vienna University of Ferrara COLTHERES, EU-FP7