Microsatellite instability (MSI) in stage II and III colon cancer treated with 5FU-LV or 5FU-LV and irinotecan (PETACC 3-EORTC 40993-SAKK 60/00 trial). S. Tejpar, F. Bosman, M. Delorenzi, R. Fiocca, P. Yan, D. Klingbiel, D. Dietrich, E. Van Cutsem, R. Labianca, A. Roth Abst. ID: 4001

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Primary endpoint 5-year DFS rate of stage III (1) Trial: Randomized Phase III Comparing Biweekly Infusional Fluorouracil/Leucovorin Alone or With Irinotecan in the Adjuvant Treatment of Stage II-III Colon Cancer: PETACC 3-EORTC 40993-SAKK 60/00 Primary endpoint 5-year DFS rate of stage III (1) 56.7% with Iri/LV5FU2 54.3% with LV5FU2 log-rank p= 0 .106 3278 patients randomized, stage II and III pathological material of 1564 patients collected Large TR program: alterations in p53, SMAD4, thymidylate synthetase, TERT, KRAS, BRAF, MSI and 18qLOH Outcomes used for the TR study: RFS at 3 and 5 years, OS median 68 mo follow up . Were the stage II high risk stage II, why were they included? (1) J Clin Oncol. 2009 May 18

Microsatellite instability (MSI) is the mutational signature found in colorectal cancers (CRCs) that evolve as a result of inactivation of the DNA mismatch repair (MMR) system. Prognostic: Multiple retrospective studies and a meta-analysis(1) have shown that MSI H tumors have a more favorable stage-adjusted prognosis compared to MSS (2). (1) combined hazard ratio for overall survival: 0.65 (95% CI, 0.59 to 0.71). J Clin Oncol 23:609–618, 2005 Current Opinion in Oncology 2009, 21

Predictive Irinotecan Predictive 5FU + Elsaleh H et al (1) Benefit of 5FU for MSI-H - Ribic CM et al (2) : retrospective, 570 stage II + III, W/0 5FU Benefit of 5FU only in MSS +/- Kim GP et al (3): retrospective, 542 stage II + III, W/O 5FU neutral. -Sargent DA et al (4). retrospective 1000 stage II+III, W/O 5FU Predictive Irinotecan +Bertagnolli et al (5) 702 stage III patients, 5FU/IFL Benefit of Irinotecan in MSI-H Ribic CM et al (2) : retrospective analysis, 570 stage II and III patients, untreated and treated 5FU. 16% MSI H All patients: 5Y DFS according to MSI: yes (p=0.04) All patients : 5 Y OS according to MSI : no (p=0.07) No stage effect In multivariate models for OS adjusted for the stage and grade All patients: 0.61 [95 percent confidence interval, 0.38 to 0.96]; P=0.03) In treated??? In untreated: 0.32 [95 percent confidence interval, 0.14 to 0.75]; P=0.008) 5FU treatment improved OS only in MSS , controlled for stage and grade, test for interaction Kim GP et al (3): retrospective analysis NASPB trials, 542 stage II and III patients, untreated and treated 5FU. 18% MSI H All patients: RFS according to MSI: trend (p=0.1) All patients : OS according to MSI : no (p=0.67) No stage correction done, No association of MSI with stage failed to identify an interaction between MSI status and FULV therapy (but anyhow no effect of Msi on RFS or OS, power?) Bertagnolli et al (5) 702 stage III patients, 5FU, IFL 13% MSI H All cases , OS by MSI: no (HR: 0.86 (95% CI, 0.57 to 1.29; log-rank P = .44) All cases, DFS by MSI: no (HR was 0.61 (95% CI, 0.57 to 0.64; log-rank P= .15) Per treatment arm 5YDFS: in IFL patients: 0.57; 95% CI, 0.42 to 0.71 (p=0.03) In 5FU patients: 0.76; 95% CI, 0.64 to 0.88 (p=0.8) hazard ratio interaction between tumor status and treatment, 0.51; likelihood ratio P = .117 So also significant 5YDFS seen in IFL patients, contrary to sargent, not in 5FU. Specific interaction with iri? Lancet 355:1745-1750, 2000. (2) N Engl J Med 349:247–257, 2003. (3) J Clin Oncol 25:767–772, 2007. (4) abstract #4008 ASCO 2008, J Clin Oncol 2008; 26 (15). (5) J Clin Oncol.27:1814-21, 2009 5

Questions we addressed Incidence of MSI-H in stage II (n= 395) and III (n= 859) Prognostic effect of MSI-H? All our patients treated with at least 5FU (w/o irinotecan) Stage specific prognostic effects of MSI-H? Is MSI-H predictive for irinotecan ? 6

Methods MSI MSI was analyzed at 10 different microsatellite loci containing mono- or di-nucleotide repeated sequences. The panel consisted of the five markers from the Bethesda reference panel, with the addition of five markers which were also suggested during the International Workshop on HNPCC in 1997. (BAT-25, BAT-26, D2S123, D5S346, TGFBR2, BAT-40, D17S787, D18S69, D17S250 and D18S58) The amplified PCR products were analyzed using the automated ABI Prism Sequencer Model 3100 Genetic Analyzer (Applied Biosystems, Foster City, USA). A locus was called unstable if unequivocal instabilities were seen in the tumour sample in comparison to the paired normal DNA of the same patient. The analysis was successful in 1327/1405 assessable FFPE blocks MSI was graded as high (MSI-H) when 30% or more of markers were unstable, low when 10-20% of markers were unstable and stable when all markers were negative. For the statistical analysis MSI-L and MSS populations were pooled and identified as MSS 7

Statistical Methods Survival curves (and rates) were determined using the Kaplan-Meier estimator and compared using the log-rank test. Frequencies were compared using chi-squared contingency table tests and chi-squared goodness-of-fit tests. Hazard Ratios (with 95% Confidence Intervals plus p-values from the Wald-Test) were computed with (multivariate) proportional hazard models (Cox regression), and adjusted for stage where indicated.

Results: Frequency 22% (86/395) 12% (104/859) <0.0001 Frequency Analysis Stage II Stage III P value MSI-H 22% (86/395) 12% (104/859) <0.0001 in accordance with recent literature: J Clin Oncol.27:1814-21, 2009 : 13.2% in 700 stage III #4008 ASCO 2008, J Clin Oncol 2008; 26 (15):12-19% in 1020 pooled stage II+III

MSI as a suppressor of lymph node and distant metastasis? Frequency Analysis Stage II Stage III Stage IV MSI-H 22% (86/395) 12% (104/859) 3.5% * *Br J Cancer. 2009 100(2):266-73.

Results: Univariate Associations- Clinical Marker Frequency Analysis MSI-H MSS P value Stage (II) 85/188 (45.2%) 306/1045 (29.3%) <0.0001 N stage (N0 vs N1-2) Higher proportion of MSI in N0 tumors T stage (T1/2 vs T3 vs T4) The higher the T stage the higher proportion of MSI 0.037 Grade ( 1-2) 137/188 (72.9%) 942/1045 (94%) Site (Right vs Left) 143/188 (76%) 344/1045 (33%) Age (≤ 60 ) 125/188 (66.5%) 486/1045 (46.5%) Sex (female) 80/188 (42.6%) 437/1045 (41.8%) 0.91 For comparison:Bertagnolli: Relationships Between Tumor MMR Status and Clinicopathologic Factors Overall median follow-up was 6.65 years.. Patients with MMR-D/MSI-H tumors had significantly more proximal tumors ( 2 P < .0001), poorly/undifferentiated tumors ( 2 P < .0001), and tumors containing extracellular mucin (>50%; 2 P < .0001). The nodal ratio (number of positive nodes divided by number of nodes sampled) was significantly lower among patients with MMR-D/MSI-H tumors (Satterthwaite t test P = .005), and women had more MMR-D/MSI-H tumors ( 2 P = .03). There were no significant differences between MMR-D and MMR-I tumors by age, treatment, or number of positive lymph nodes Also compare to Kim et al (Allegra) 11

Results: Univariate Associations- Molecular Marker Frequency Analysis MSI H MSS P value Braf mutation 45/186 (24%) 53/1037 (5%) <0.0001 P53 intense (>45% of cells) 15/188 (.8%) 409/1045 (39%) 18q LOH 20/110 (18%) 515/675 (76%) TS intense (75% of cells staining intensely) 121/181 (67%) 253/958 (26%) Smad4 complete loss 11/188 (0.6%) 249/1045 (24%) kras 55/186 (30%) 389/1028 (38%) 0.038 Telomerase >15% 50/105 (48%) 270/603 (45%) 0.664 refer to: -MSI-high tumours were more likely to originate in the proximal colon and possess BRAF mutations and CIMP-high status, and less likely to present with stage III or IV disease. (Ogino S et al:Gut 2009;58:90–96.) Kim et an and Westra et al for P53 12

Results: Prognostic impact MSI - Univariate HR MSI-H (95% CI) 5FU (n= 625) 5FU/iri (n= 608) Both arms Stage II (n= 391) RFS 0.228 (0.05-0.955) P= 0.043 0.296 (0.091-0.968) P=0.044 0.265(0.107- 0.661) P= 0.0044 OS 0.18 (0.02-1.34) P= 0.095 0.143 (0.02-1.06) P=0.057 0.159( 0.039- 0.659) P=0.011 Stage III (n= 842) 0.596 (0.344-1.03) P=0.064 0.815 (0.478-1.39) P=0.45 0.693 (0.473-1.02) P= 0.06 0.515 (0.261-1.02) P=0.055 0.939 (0.515-1.71) P=0.84 0.699 (0.446- 1.09 ) P= 0.12 Both stages * P are stage corrected 0.501 (0.300-0.837) P=0.0083 0.642 (0.394-1.05) P=0.076 0.569 ( 0.400 -0.811) P=0.0018 0.437(0.229-0.833) P= 0.012 0.676 (0.380-1.20) P= 0.18 0.548 (0.357-0.842) P=0.006 RFS | SII | SIII ------+---------+--------- MSI-H | 5(85) | 29(103) MSS | 61(306) | 285(739) OS | SII | SIII MSI-H | 2(85) | 21(103) MSS | 42(306) | 211(739)

Results: Prognostic impact MSI - Univariate HR MSI H (95% CI) 5FU (n= 625) 5FU/iri (n= 608) Both arms Stage II (n= 391) RFS 0.228 (0.05-0.955) P= 0.043 0.296 (0.091-0.968) P=0.044 0.265(0.107- 0.661) P= 0.0044 OS 0.18 (0.02-1.34) P= 0.095 0.143 (0.02-1.06) P=0.057 0.159( 0.039- 0.659) P=0.011 Stage III (n= 842) 0.596 (0.344-1.03) P=0.064 0.815 (0.478-1.39) P=0.45 0.693 (0.473-1.02) P= 0.06 0.515 (0.261-1.02) P=0.055 0.939 (0.515-1.71) P=0.84 0.699 (0.446- 1.09 ) P= 0.12 Both stages * P are stage corrected 0.501 (0.300-0.837) P=0.0083 0.642 (0.394-1.05) P=0.076 0.569 ( 0.400 -0.811) P=0.0018 0.437(0.229-0.833) P= 0.012 0.676 (0.380-1.20) P= 0.18 0.548 (0.357-0.842) P=0.006 Conclusion: Effect lost in Stage III, only in stage II. Number of evenst stage II versus III In stage III the effect is lost , maybe due to both bad outcome of the MSIH irinotecan treated arm (1.15 HR)and improvement of the MSS (O.85 HR) . In multivariate no effect at all in stage III for RFS, some for OS Note also RFS and OS, both stages for arm B(5FU), quite the contrary of D Sargent 14

Take home message prognostic effects When considering stage II and III combined there is a strong prognostic effect of MSI-H both on RFS and OS. When analyzed per stage the effect is very strong in stage II RFS (P= 0.0044) OS (P=0.011) but there is only a trend in stage III RFS (P= 0.06) OS (P= 0.12) A regression containing Stage, MSI H and their interaction shows a bordeline significant interaction both for RFS (p=0.058) and OS (p=0.051)

Questions we addressed Incidence of MSI in stage II (n= 395) and III (n= 859) Prognostic effect of MSI-H? All our patients treated with at least 5FU (w/o irinotecan) Stage specific prognostic effects of MSI-H? Is MSI-H predictive for irinotecan ? 16

abstract #4008 ASCO 2008, JCO 2008; 26 (Suppl 15) Pooled Data stage II + III patients : 5y DFS By MSI Status Treated 5FU (N=512) Untreated (N=515) Sargent Asco 2008: We believe that the strength of the available evidence is sufficient at the present time to recommend that MSI-H stage II colon cancer patients not receive adjuvant chemotherapy given that such tumors have a favorable prognosis and exhibit resistance to 5-FU Msi status validated as a prognostic marker in untreated patients, No suggestion of benefit from 5-FU based treatment in dMMR patients MSI-H MSS HR (95% CI) P value 5Y DFS rate 70% 67% 0.79 (0.49-1.25) p=0.30 MSI-H MSS HR (95% CI) P value 5Y DFS rate 80% 56% 0.51 (0.29-0.89) p=0.009 17

compare ASCO 2008 with our data Data stage II + III patients : 5y DFS By MSI Status Treated 5FU (N=512) Sargent Treated 5FU (N=625) Petacc3 Sargent Asco 2008: We believe that the strength of the available evidence is sufficient at the present time to recommend that MSI-H stage II colon cancer patients not receive adjuvant chemotherapy given that such tumors have a favorable prognosis and exhibit resistance to 5-FU Msi status validated as a prognostic marker in untreated patients No suggestion of benefit from 5-FU based treatment in dMMR patients MSI-H MSS HR (95% CI) P value 5Y DFS rate 70% 67% 0.79 (0.49-1.25) p=0.30 MSI-H MSS HR (95% CI) P value 5Y RFS rate 83% 66% 0.50 (0.30-0.83) p=0.0077 18

Predictive effects of MSI-H All patients treated with 5FU (w/o irinotecan) Study predictive effects for irinotecan Data shown limited to stage III for comparison to (1), not different for stage II (1) J Clin Oncol.27:1814-21, 2009 19

Results: Predictive effects of MSI Population Stage III rate of 5-Year RFS 95% CI N All patients analyzed 62% (0.59 - 0.66) N=842 Prognostic analysis All MSI-H tumors 72% (0.60-0.86) N= 103  All MSS tumors 59% (0.54-0.64) N= 739 Predictive analysis within treatment arm FU/LV /Irinotecan All MSI-H tumors 69% (0.57-0.83) N= 49 All MSS tumors 64% (0.59-0.69) N=372 Predictive analysis within tumor subtype FU/LV N= 54 Dirk, add p values for blue lines?. Were not in email attach 20

Purple and red (1) J Clin Oncol.27:1814-21, 2009 Population Stage III Rate of 5-Year RFS Rate of 5 -Year DFS (1) 95% CI N All patients analyzed 62% 61% (0.59 - 0.66) (0.57 - 0.64 ) N=842 N= 702 Prognostic analysis All patients with MSI-H tumors 72% 67% (0.60-0.86) (0.57-076) N= 103 N= 96  All patients with MSS tumors 59% 60% (0.54-0.64) (0.56-0.64) N= 739 N= 606 Predictive analysis within treatment arm FU/LV /Irinotecan 69% 76% (0.57-0.83) (0.64-0.88) N= 49 N= 50 All patients with MSS tumors 64% (0.59-0.69) (0.53-0.64) N=372 N= 304 Predictive analysis within tumor subtype FU/LV 57% (0.42_0.71) N= 54 N= 46 P=0.03 P=0.07 Purple and red (1) J Clin Oncol.27:1814-21, 2009 21

Take home message treatment effects In contrast to previous reports (a) in stage II+III and in stage II alone the prognostic effect of MSI on RFS and OS remained significant even in pts treated with 5FU (b) There is no evidence for an effect of the addition of irinotecan. Read monica, did she find an effect in stage III?

The prognostic effect is weaker in stage III Conclusions Microsatellite instability is a strong prognostic factor for RFS and OS in stage II The prognostic effect is weaker in stage III conclusions are limited by sample size, the unbalance of MSI in the stages and multiple testing. Taken together this may suggest stage specific biological effects of MSI.

retained for RFS and OS despite treatment with 5FU Conclusions(2) Prognostic effect retained for RFS and OS despite treatment with 5FU Predictive: no evidence for an effect of the addition of irinotecan in MSI-H Discordances with previous studies Biomarker studies may be confounded small size studies, retrospective studies, pooled analysis Biology: Confounded by molecular heterogeneity 24