Clinical and technical validation of a genomic classifier (ColoPrint) for predicting outcome of stage II colon cancer patients Josep Tabernero, Vall d’Hebron University Hospital, Barcelona, Spain and Victor Moreno 2, Robert Rosenberg 3, Ulrich Nitsche 3, Thomas Hoffmann-Bachleitner 4, Giovanni Lanza 5, Jeroen van Akker 6, Paul Roepman 6, Iris Simon 6, Ramon Salazar 2 2 IDIBELL, Institut Català d'Oncologia, L'Hospitalet de Llobregat, Spain; 3 Department of Surgery, Klinikum rechts der Isar,T echnische University Munich, Munich, Germany; 4 Department of Surgery, Medical University of Vienna, Vienna, Austria; 5 Istituto di Anatomia e Istologia Patologica, University di Ferrara, Ferrara, Italy; 6 Agendia BV, Amsterdam, Netherlands and Agendia Inc., Irvine, CA, US

Treatment of stage II patients is still debatable Quasar Collaborative Group, Lancet. 2007; 370(9604): ChemoObservation 5 year survival82.5%80.6%

ASCO recommendation “direct evidence from randomized controlled trials does not support the routine use of adjuvant chemotherapy for patients with stage II colon cancer. Features associated with an increased risk of recurrence include inadequate lymph node sampling, T4 disease, perforation and a poorly differentiated histology NCCN guidelines consider 5FU or clinical trial or observation for low risk patients (T3, no high risk features) Consider 5FU/oxaliplatin or clinical trial or observation for high risk patients High risk features: T4, less than 12 LN assessed, perforation, obstructions, positive margins, high grade, lymphatic/vascular invasion Guidelines for Risk Assessment

Whole Genome Array Training Set (stage I-IV) (n=188) Netherlands Cancer Institute, Leiden Medical Center, Slotervaart Selection of Final 18-Gene Set & Algorithm Clinical Validation Study 1 (stage I-III) Institut Catala d’Oncologia Barcelona (J Clin Oncol. 2011;29:17-24) Standardization of Analytical Methods In-silico Validation Study (stage I-III) public datasets (n=322) Development Validation of ColoPrint Clinical Validation Study 2 (stage II) Munich Hospital Rechts der Isar (J Clin Oncol 28:15s (abstract 3513) Clinical Validation Study 3 (stage II) Vall d’Hebron, MedUni Vienna, University of Ferrara PARSC Prospective Study (stage II + III) - ongoing US, Asian, and European Center (N ~600 stage II) Clinical Validation Study 4 (stage II-III) MD Anderson (ongoing) Stage II pooled analysis

STAGE II PATIENTS (N=320) Pooled Analysis

Patient Characteristics *MSI-status based on PCR (n=170) and Genomic Profile (n=150) – see Poster BRD.B37 (R. Salazar et al)

Patient Characteristics (cont’)

ColoPrint identifies patients at risk of distant and local-regional relapse (RFS) Local, Regional and Distant Relapse 5-year RFS Low Risk = 88% (83-93%) High Risk = 71% ( %) 3-year RFS Low Risk = 91% (86-95%) High Risk = 74% (64-83%)

Univariate analysis: 3-yr Relapse-free Survival

Clinical Risk Factors distinguish risk groups but are not sufficient p-value for uncensored time

Subgroup analysis in T3-MSS patients (n=227) VariableHR95% CIP-value ColoPrint Age Localization Grade Gender LN > Univariate Analysis of 3-year RFS 3-year RFS Low Risk = 91% (86-96%) High Risk = 73% (63-83%)

Clinical risk factors are even less sufficient to distinguish low and high risk patients in the T3-MSS subgroup

ColoPrint in combination with clinical factors might give best risk stratification 3-year RFS 93 % Low Risk ColoPrint, low risk NCCN 88 % Low Risk ColoPrint, high risk NCCN 76 % High Risk ColoPrint, low risk NCCN 71 % High Risk ColoPrint, high risk NCCN ColoPrint + NCCN clinical factors All patients T3 MSS 3-year RFS 93% 89% 76% 70%

ColoPrint and MSI-status MSI-High patients have a better prognosis than MSS patients and may suffer worse adverse effects from 5-FU ColoPrint indentifies low risk patients beyond MSI- high status – 67 patients were classified as MSI-H (20.9%) – MSI-H patients are mainly ColoPrint Low Risk (53/67 = 80%)

Technical Validation of ColoPrint as a reproducible and standardized test ColoPrint uses the same technology, methods and QC as FDA-cleared MammaPrint assay Repeated runs of three samples over 20 days performed by different operators = less than 5% variation

Summary The 18-gene genomic signature for patients with colon cancer distinguishes populations with different outcomes The signature was validated in an in-silico study and with independent cohorts In stage II patients: – Over 60% of patients were identified as Low Risk with a 3-year RFS of 91% – It identifies patients at High Risk of developing metastases and who are more likely to benefit from adjuvant CTx – It is better at identifying High Risk patients than any clinical risk factor alone ColoPrint complements clinical-pathological factors for better treatment decisions

PARSC: Prospective Assessment of Risk Stratification by ColoPrint Aim 575 eligible stage 2 patients Status January 2012: – 32 sites open (EU 15, Asia 2, US 15) – 340 eligible stage 2 – 300 eligible stage 3 Expected last patient enrollment: Dec’12 Patient Information & Informed Consent Surgery RNARetain Sample Agendia Treatment at discretion of investigator Quality check sample CRF If eligible: CRF1 Year 1 Year 3 Year 5 ColoPrint analysis ± 4 weeks after surgery See also Poster BRD. G15

Acknowledgements To all patients and participating Institutions