1. Taiwan 2000 PETACC 3 ASCO 2009 Molecular markers in colon cancer have a stage specific prognostic value. Results of the translational study on the PETACC 3 - EORTC 40993 -SAKK 60-00 trial. A. D. Roth, S. Tejpar, P. Yan, R. Fiocca, D. Dietrich, M. Delorenzi, R. Labianca, D. Cunningham, E. Van Cutsem, F. Bosman

2. PETACC 3 ASCO 2009 Rationale Need for new prognostic markers to guide treatment planning in colorectal cancer Lack of large data sets for the validation of potential markers prospectivelyPETACC 3: a large adjuvant trial in colon cancer where the pathological material of 1564 patients out of 3278 was prospectively collected half a ton of material (about 36,000 slides) –20-25 slides/patient or block in bank => ~ half a ton of material (about 36,000 slides) – 669 blocks processed for tissue microarray banking

3. PETACC 3 ASCO 2009 Objectives To assess the frequency of molecular marker alterations: expression of p53, SMAD4, thymidylate synthetase (TS) and hTERT, and mutations of KRAS, BRAF, microsatellite instability (MSI) and 18qLOH in stage II and stage III colon cancer. To assess their prognostic relevance in the whole patient population using RFS as endpoint. To assess possible differences between stage II and stage III disease.

4. PETACC 3 ASCO 2009 Methods FFPE tissue blocks collected and cut in 5-20µ sections. DNA extracted with phenol/chloroform from normal and tumoral tissues after microdissection. Immunohistochemistry (IHC)Immunohistochemistry (IHC) –P53, SMAD4, Thymidylate Synthetase (TS), Telomerase (HTERT) Molecular analysis: –Microsatellite Instability (MSI): assessed on 10 markers (Cf. previous presentation) –18qLOH: multiple SNPs typing by pyrosequencing on Nor/Tu DNA –KRAS exon 2 and BRAF exon 15: Allele specific real time PCR on Tu DNA

5. PETACC 3 ASCO 2009 Statistical Methods Prognostic value of the markers was analyzed per stage by Cox regression for RFS Interactions were tested using a likelihood ratio test p values of the multivariate analysis taken from the full model (unless indicated otherwise)‏ Differences between the stages were assessed by a test of equal proportions

6. PETACC 3 ASCO 2009 Analysis success rate Number of samples with results Total analysed: -1530 patient slides analysed by IHC - DNA successfully extracted from 1404 patient slides (91.2%)

7. PETACC 3 ASCO 2009 Biomarker alteration observed Alterations rate observed Alteration rate reported (literature) p53 overexpression*35%25-76% SMAD4 loss**21%13-63% TS***34%No consistent data hTERT46%No data available MSI15%10-17% 18qLOH68%70% KRAS37%32-40% BRAF8%10% * Intense expression, More than 45% cells positive. ** any loss. *** Positive = more than 25% cell positive.

8. PETACC 3 ASCO 2009 Marker Alteration Rate per stage MarkerStage II (n=420) Stage III (n=984) p value* MSI-H22%12%<0.0001 TS43%29%<0.0001 p5330%37%0.01 SMAD418%23%0.03 18qLOH63%70%0.04 hTERT41%48%0.06 KRAS35%37%0.81 BRAF8% 0.90 * Test of equal proportions

9. PETACC 3 ASCO 2009 Prognostic Value Univariate analysis Marker Stage II (n=420) Stage III (n=984) Interaction* HR § p val**HR § p val** MSI (Hi vs Stable)0.30.0040.70.060.04 18qLOH2.10.0310.910.05 SMAD4 (any loss) 1.40.211.6<0.00010.23 hTERT (High)1.40.321.50.010.92 p53 (High)1.00.981.30.030.37 TS (High)0.50.030.70.020.30 KRAS (Mutated)1.10.841.00.720.32 BRAF(Mutated)0.90.901.20.280.38 * p value from Likelihood ratio test to assess differences between the stages ** p values from the Wald test in a univariate Cox regression § HR = hazard ratio

10. PETACC 3 ASCO 2009 Prognostic Value (RFS) Multivariate Analysis in whole population Markers Stage IIStage III HR § p value*HR § p value* T Stage (T4 vs T3)2.80.00011.60.0006 N Stage (N2 vs N1)N/A 2.2<0.0001 Histologic Grade (3-4 vs 1-2)0.60.551.40.07 Age (>60 vs ≤60)1.80.0261.10.3 MSI (High vs Stable)0.30.0270.70.12 p53 (High)0.70.271.30.015 SMAD4 (any loss)1.00.91.60.0002 Treatment, Sex, Site, KRAS, BRAF,TS, 18qLOH (Stage II: HR 1.4, p=0.33), hTERT: not significant * p values from the Wald test in a multiivariate Cox regression § HR = hazard ratio

11. PETACC 3 ASCO 2009 MSI and 18qLOH in a minimal multivariate model in stage II disease MarkersHR [95% CI]P value T4 v. T32.34 [1.42 - 3.84]0.00085 18qLOH2.02 [1.03 - 3.96]0.041 T4 v. T32.58 [1.56 - 4.28]0.00024 MSI-H v. MSS0.28 [0.10 - 0.72]0.0089 18qLOH1.37 [0.67 - 2.77]0.38

12. PETACC 3 ASCO 2009 Graphical comparison between univariate and multivariate analysis

13. PETACC 3 ASCO 2009 E5202: A Pioneer study!

14. PETACC 3 ASCO 2009 Molecular predictors of survival after adjuvant chemotherapy for colon cancer Watanabe T. et al NEJM 344: 1196-206 (2001) “No marker had predictive value in the analysis of 121 patients with stage II cancer, possibly because of the small sample”

15. PETACC 3 ASCO 2009 Really low risk?

16. PETACC 3 ASCO 2009 PETACC 3: RFS according to MSI or 18qLOH in stage II colon cancer MSI18qLOH

17. PETACC 3 ASCO 2009 PETACC 3: Additional prognostic value of 18qLOH on MSS and MSI-H tumors in stage II disease MSI-H population: –18% 18qLOH (9/51)! – 18qLOH prognostic value not assessable P = 0.527

18. PETACC 3 ASCO 2009 Conclusions Stage II and III colon cancers express different biomarker alteration frequencies with different prognostic effects according to disease stage These data suggest that pooled analysis of stage II and III might be confounded by this heterogeneity The possibility that stage II and III are biologically different rather than sequential evolution steps in a pathological continuum needs to be considered Careful assessment of the relative weight of each prognostic marker in multivariate analysis according to disease stage is needed before translating them into clinic

19. PETACC 3 ASCO 2009 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