Taiwan 2000 PETACC 3 ASCO 2009 PETACC 3 ASCO 2010 Molecular and clinical determinants of survival following relapse after curative treatment of stage II- III colon cancer (CC). Results of the translational study on the PETACC 3 - EORTC SAKK trial A. D. Roth, D. Klingbiel, P. Yan, R. Fiocca, M. Delorenzi, R. Labianca, D. Cunningham, E. Van Cutsem, F. Bosman, S. Tejpar

PETACC 3 ASCO 2010 Rationale Our previous results showed that stage II and III colon cancers harbour different biomarker alteration frequencies with different prognostic effects according to disease stage (ASCO proc. 2009, 27 abstr. #4002) There is a lack of data regarding biomarkers prognostic for disease behaviour after relapse Early observations suggest that marker sets prognostic for the risk of relapse might be different from those prognostic for survival after relapse (SAR)

PETACC 3 ASCO 2010 Roth, A. D. et al. J Clin Oncol; 28: Copyright © American society of Clinical Oncology Relapse free survival (RFS)Overal survival (OS) KRAS BRAF BRAF status is prognostic in OS, very little or not in RFS!

PETACC 3 ASCO 2010 Objectives To look for clinical and molecular markers prognostic for survival after relapse (SAR). To assess their respective and relative prognostic relevance in the relapsing patient population using SAR as endpoint. To assess possible differences between markers prognostic for risk of relapse and markers prognostic for SAR.

PETACC 3 ASCO 2010 PETACC3 trial: Patient Distribution (32.5%) (27.9%) 3278 Total Accrual: NO suitable material for biomarkers With suitable material for biomarkers # relapsing Patients treated

PETACC 3 ASCO 2010 Methods (1) FFPE tissue blocks prospectively collected and cut in 5-20µ sections Immunohistochemistry (IHC)Immunohistochemistry (IHC) –P53: mouse mAb clone D07, ABC Basic DAB Detection (Ventana medical systems) –SMAD4: mouse mAb clone B8 (IgG1, Santa Cruz Biotechnology). Novocastra polymer detection kit –Thymidylate Synthetase (TS): Monoclonal antibody TS 106/4H4B1 (IgG1, Zymed). DAKO EnVision detection system –Telomerase (HTERT): Monoclonal antibody NCL-hTERT (IgG2, Novocastra). DAKO EnVision detection system

PETACC 3 ASCO 2010 Methods (2) DNA macrodissectionDNA was extracted with phenol/chloroform from normal and tumoral tissues after macrodissection of FFPE sections. Molecular analysis: –Microsatellite Instability (MSI): assessed on 10 markers (BAT-25, BAT-26, D5S346, D2S123, D17S250, BAT- 40, TGF-ß RII, D18S58, D18S69, D17S787) < 3 positive markers = MSI stable (MSS) ≥ 3 positive markers = MSI high (MSI-H) –18qLOH: multiple SNPs typing by pyrosequencing on Normal and tumor DNA –KRAS exon 2 and BRAF exon 15: Allele specific real time PCR on Tumor DNA

PETACC 3 ASCO 2010 Statistical methods Prognostic value of the markers was analyzed by Cox regression for SAR and adjusted for stage p values of the multivariate analysis taken from the full model (unless indicated otherwise) Time to recurrence (TTR) of ≤18 months defined early relapse (ER), >18 months late relapse (LR) Survival curves were computed according to the Kaplan-Meier method and compared using the log- rank test

PETACC 3 ASCO 2010 PETACC3 trial: Relapser Distribution ER 171 LR ER255 LR 42.6% 43.6% Full set With material (biomarker set) Ø material

PETACC 3 ASCO 2010 Relapsing Patients Characteristics VariablesFull set (n=990) % (.95 CI)Marker set (n=392) % (.95 CI) Early relapse 56457% ( ) % ( ) Age < % ( ) % ( ) Sex Female % ( ) % ( ) Stage III % ( ) 33786% ( ) Left side primary % ( ) % ( ) Treatment arm 5-FU/FA % ( ) % ( )

PETACC 3 ASCO 2010 Univariate Analysis for SAR in the marker set (N=392) HR (95% CI)P-value TTR (ER/LR)1.55 ( ) age1.14 ( )0.045 sex1.13 ( )0.33 Tumor Grade (G-34 / G-12)1.69 ( )0.003 stage (III versus II)1.60 ( )0.015 Tumor site (right/left)1.86 ( )7.85e-07 Treatment group1.09 ( )0.49 MSI (MSI-H / MSS)1.05 ( )0.84 Thymidilate synthetase0.89 ( )0.46 SMAD41.27 ( )0.07 p ( )0.10 hTERT1.33 ( ) qLOH0.75 ( )0.10 BRAF mut/wt3.63 ( )6.15e-10 KRAS mut/wt 1.04 ( )0.76

PETACC 3 ASCO 2010 Multivariate Analysis for SAR in the marker set (N=392) HR (95% CI)P-value TTR (ER/LR)1.60 ( ) age1.00 ( )0.98 sex1.24 ( )0.11 Tumor Grade (G-34 / G-12)1.52 ( )0.04 stage (III versus II)1.53 ( )0.051 Tumor site (right/left)1.69 ( ) Treatment group1.09 ( )0.52 MSI (MSI-H / MSS)0.51 ( )0.034 Thymidilate synthetase0.99 ( )0.95 SMAD41.21 ( )0.18 p ( )0.76 hTERT1.37 ( ) qLOH0.86 ( )0.43 BRAF mut/wt3.61 ( )1.24e-07 KRAS mut/wt 1.13 ( )0.40

PETACC 3 ASCO 2010 SAR according to BRAF mutation status Median survivals (95% CI): - BRAF mut: 7.49 m ( ) - BRAF wt: 25.2 m ( ) (p = 1.9e-11)

PETACC 3 ASCO 2010 SAR according to primary tumor site Full set (n=990)Marker set (n=392) LEFT side: 28.4 m (26.5 – 32.3) 27.6 m (22.9 – 33.6) RIGHT side: 16.2 m (14.4 – 18.5) p=4.52e m ( 12.6 – 19.0) p=2.71e-06 Median survivals (95% CI)

PETACC 3 ASCO 2010 SAR after relapse according to TTR Full set (n=990)Marker set (n=392) LR: 30.5 m (27.1 – 34.7) 30.0 m (24.7 – 38.4) ER: 18.4 m (16.4 – 20.5) p=2.18e m ( 15.5 – 20.3) p= Median survivals (95% CI)

PETACC 3 ASCO 2010 Multivariate Analysis for SAR omitting BRAF in the marker set (N=392) HR (95% CI)P-value TTR (ER/LR)1.69 ( )9.54e-05 age1.09 ( )0.21 sex1.20 ( )0.17 Tumor Grade (G-34 / G-12)1.45 ( )0.07 stage (III versus II)1.51 ( )0.06 Tumor site (right/left)1.76 ( )4.39e-05 Treatment group1.08 ( )0.55 MSI (MSI-H / MSS)0.69 ( )0.24 Thymidilate synthetase0.98 ( )0.90 SMAD41.29 ( )0.08 p ( )0.94 hTERT1.28 ( ) qLOH0.81 ( )0.27 KRAS mut/wt 0.97 ( )0.82

PETACC 3 ASCO 2010 SAR: MSI interactions with BRAF, Tumor Grade and Tumor Site Cox regression model type Variables HR (95% CI)P-valueInteraction HR (95% CI), p-value Univariate MSI (MSI-H / MSS)1.02 ( )0.94NA Bivariate BRAF mut/wt3.95 ( )5.53e (0.15 – 1.37) p=0.16 MSI (MSI-H / MSS)0.71 ( )0.19 Bivariate Grade (G-34 / G-12)1.91 ( ) (0.17 – 1.37) p=0.17 MSI (MSI-H / MSS)0.75 ( )0.28 Bivariate Tumor site (right/left)1.87 ( )1.14e (0.17 – 1.47) p=0.21 MSI (MSI-H / MSS)0.75 ( )0.25 Multivariate 4 variables Grade (G-34 / G-12)1.73 ( )0.002 Tumor site (right/left)1.70 ( )4.68e-05 MSI (MSI-H / MSS)0.48 ( )0.005 BRAFmut/wt3.75 ( )1.38e-09

PETACC 3 ASCO 2010 Conclusions Whereas BRAF status and tumor site were not prognostic for RFS of stage II-III colon cancer ( Roth, A. D. et al. J Clin Oncol; 28: ), BRAF status, tumor site and TTR are highly prognostic for colon cancer survival after relapse Tumor stage at diagnosis and MSI status have a weaker impact on survival after relapse BRAF status, tumor site and TTR should imperatively be used to stratify studies in metastatic colon cancer The independent effects of tumor site and TTR might be indicative of additional as yet non-identified prognostically significant molecular markers

PETACC 3 ASCO 2010 Thank You For All Your Efforts!! Austria, Belgium, Bulgaria, Croatia, Czech Republic, Denmark, Egypt, Finland, France, Germany, Greece & Cyprus, Hungary, Ireland, Iceland, Israel, Italy, Netherlands, Norway, Poland, Portugal, Russia, South Africa, Slovakia, Slovenia, Spain, Sweden, Switzerland, Taiwan, Turkey & UK We would also like to thank Pfizer for facilitating the execution and analysis of the PETACC3 study