1. Microsatellite Instability Predicts Improved Response to Adjuvant Therapy With Irinotecan, Fluorouracil, and Leucovorin in Stage III Colon Cancer In association with This program is supported by an educational grant from Bertagnolli MM, Niedzwiecki D, Compton CC, et al. J Clin Oncol. 2009;27:1814-1821.

2. clinicaloptions.com/oncology MSI Predicts Improved Response to Adjuvant IFL Therapy in Stage III CRC clinicaloptions.com/oncology Background  Microsatellite DNA comprises di-, tri-, and tetra- dinucleotide repeat sequences; expansion or shortening known as microsatellite instability (MSI) –MSI can result in inactivation of tumor suppressor genes [1,2] –High levels of MSI (MSI-H) seen in 15% to 25% of sporadic CRC cases [3]  MLH1 and MSH2: MMR proteins most commonly lost in sporadic CRC [3] –DNA mismatch repair deficiency (MMR-D) results in MSI-H [4] 1. Parsons R, et al. Cancer Res. 1995;55:48-50. 2. Rampino N, et al. Science. 1997;275:967-969. 3. Peltomaki P. J Clin Oncol. 2003;21:1174-1179. 3. Kuismanen SA, et l. Am J Pathol.2000;157: 1052-1053. 4. Shia J, et al. Virchows Arch.2004;445:431-441.

3. clinicaloptions.com/oncology MSI Predicts Improved Response to Adjuvant IFL Therapy in Stage III CRC clinicaloptions.com/oncology Background  MSI-H/MMR-D tumors more commonly right colon and poorly differentiated –Have better treatment outcomes than tumors with chromosomal instability (80% to 85% of sporadic CRC tumors) [1,2] 1. Gryfe R, et al. N Engl J Med. 2000; 342:69-77. 2. Popat S, et al. J Clin Oncol.2005;23:609-618.

4. clinicaloptions.com/oncology MSI Predicts Improved Response to Adjuvant IFL Therapy in Stage III CRC clinicaloptions.com/oncology Study Objectives  Determine the relationship between MSI status assigned by genotyping with MMR protein IHC  Determine whether tumor MMR/MSI status was associated with different OS or DFS  Compare the addition of irinotecan to FU/LV to FU/LV alone –Irinotecan inhibits the catalytic function of topoisomerase-I

5. clinicaloptions.com/oncology MSI Predicts Improved Response to Adjuvant IFL Therapy in Stage III CRC clinicaloptions.com/oncology Schematic of Study Design Adjuvant IFL Arm Irinotecan 125 mg/m 2 + LV 20 mg/m 2 + FU 500 mg/m 2 Cycle = 4 wks treatment + 2 wks rest Treatment for 5 cycles (30 wks) (n = 635) Adjuvant FU/LV Arm (Roswell Park regimen) LV 500 mg/m 2 wkly + FU 500 mg/m 2 wkly Cycle = 6 wks treatment + 2 wks rest Treatment for 4 cycles (32 wks) (n = 629) Patients with histologically confirmed St III CRC (N = 1264) R A N D O M I Z E D 1:1

6. clinicaloptions.com/oncology MSI Predicts Improved Response to Adjuvant IFL Therapy in Stage III CRC clinicaloptions.com/oncology Mircosatellite Analysis  Tumor MLH1 and MSH2 protein levels were determined by IHC –< 10% tumor cells staining = “negative”  Tumor was judged MMR-deficient (MMR-D) if either MLH1 or MSH2 negative  Tumor was MMR-Intact (MMR-I) if both MLH1 and MSH2 positive  MSI determined by PCR –MSI-H, MSI-L, or MSI-S

7. clinicaloptions.com/oncology MSI Predicts Improved Response to Adjuvant IFL Therapy in Stage III CRC clinicaloptions.com/oncology Baseline Characteristics CharacteristicAll Patients (N = 1264) MMR-D Patients (n = 96) MMR-I Patients (n = 606) P, MMR-D vs MMR-I Treatment, %  FU/LV49.847.949.8NS  FLI49.852.150.2NS Median age, yrs616661NS Male sex, %55.544.856.3.039 Extracellular mucin, %9.928.16.8<.0001 Proximal site, %56.491.751.3<.0001 Poorly differentiated, %24.149.021.0<.0001 Node ratio.005  Median0.230.180.23  Mean0.300.250.32

8. clinicaloptions.com/oncology MSI Predicts Improved Response to Adjuvant IFL Therapy in Stage III CRC clinicaloptions.com/oncology Description of Current Analysis  Primary endpoint: OS –Interval from study entry to death from any cause  Secondary endpoint: DFS –Interval from study entry to documented progression or death from any cause  Median follow-up: > 6 yrs

9. clinicaloptions.com/oncology MSI Predicts Improved Response to Adjuvant IFL Therapy in Stage III CRC clinicaloptions.com/oncology Microsatellite Validation  MMR protein immunohistochemistry is a good predictor of MSI status Immunohistochemistry Result MMR-DMMR-I Genotyping result, n  MSI-H9617  MSI-L or stable4606 Agreement, % (n)97 (702)  0.88 (95% CI: 0.84-0.93)

10. clinicaloptions.com/oncology MSI Predicts Improved Response to Adjuvant IFL Therapy in Stage III CRC clinicaloptions.com/oncology Survival Outcomes FU/LV Arm (n = 629) IFL Arm (n = 635) P Value OS, %.484  MedianNot reached  5-yr survival7270 DFS, %.222  MedianNot reached  5-yr survival6259

11. clinicaloptions.com/oncology MSI Predicts Improved Response to Adjuvant IFL Therapy in Stage III CRC clinicaloptions.com/oncology OS by Treatment: FU/LV vs IFL  OS outcomes after IFL and FU/LV treatments were statistically indistinguishable across all patients  Kaplan-Meier analysis 5-year survival probability –FU/LV: 0.72 –IFL: 0.70  Median OS not reached  Median follow-up: > 6.0 yrs

12. clinicaloptions.com/oncology MSI Predicts Improved Response to Adjuvant IFL Therapy in Stage III CRC clinicaloptions.com/oncology DFS by Treatment: FU/LV vs IFL  DFS across all patients was statistically indistinguishable regardless of treatment  5-year survival probability –FU/LV: 0.62 –IFL: 0.59  Median DFS not reached  Median follow-up: > 6.0 yrs

13. clinicaloptions.com/oncology MSI Predicts Improved Response to Adjuvant IFL Therapy in Stage III CRC clinicaloptions.com/oncology OS by MMR Status: MMR-I vs MMR-D  Across both treatments, the OS outcomes of patients with MMR-D and MMR-I tumors were statistically indistinguishable  HR: 0.86 (95% CI: 0.57-1.29; log-rank =.44)  5-yr OS probability –MMR-D/MSI-H: 0.73 –MMR-I/MSI-L: 0.71

14. clinicaloptions.com/oncology MSI Predicts Improved Response to Adjuvant IFL Therapy in Stage III CRC clinicaloptions.com/oncology DFS by MMR Status: MMR-I vs MMR-D  Across both treatments, the DFS outcomes of patients with MMR-D and MMR-I tumors were also statistically indistinguishable  HR: 0.61 (95% CI: 0.57-0.64; log-rank P =.15)  5-yr DFS probability –MMR-D/MSI-H: 0.67 –MMR-I/MSI-L: 0.60

15. clinicaloptions.com/oncology MSI Predicts Improved Response to Adjuvant IFL Therapy in Stage III CRC clinicaloptions.com/oncology OS by Treatment and MMR Status  When OS outcomes were examined by both treatment and MSI status, no statistically significant differences between groups emerged  No significant differences between groups in OS  MMR-D/MSI-H patients treated with IFL 1.0 0.8 0.6 0.4 0.2 0 OS (Probability) 02468 Yrs X 2 = 1.94 P =.585 FU/LV, MSI-LS FU/LV, MSI-H IFL MSI-LS IFL MSI-H Reprinted with permission. © 2008 American Society of Clinical Oncology. All rights reserved. Bertagnolli M, et al. J Clin Oncol. 2009 Vol. 29(11), 1814-1817.

16. clinicaloptions.com/oncology MSI Predicts Improved Response to Adjuvant IFL Therapy in Stage III CRC clinicaloptions.com/oncology DFS by Treatment and MMR Status  5-yr DFS outcomes by both treatment and MSI status showed IFL-treated patients with MMR-D/MSI-H tumors did better than MMR-I/MSH-L or S patients  5-yr DFS for patients treated with IFL –MMR-D/MSI-H HR: 0.76 –MMR-I/MSI-L HR: 0.59  P =.03 1.0 0.8 0.6 0.4 0.2 0 DFS (Probability) 02468 Yrs X 2 = 4.99 P =.173 FU/LV, MSI-LS FU/LV, MSI-H IFL MSI-LS IFL MSI-H Reprinted with permission. © 2008 American Society of Clinical Oncology. All rights reserved. Bertagnolli M, et al. J Clin Oncol. 2009 Vol. 29(11), 1814-1817.

17. clinicaloptions.com/oncology MSI Predicts Improved Response to Adjuvant IFL Therapy in Stage III CRC clinicaloptions.com/oncology DFS of MMR-D/MSI-H Patients: FU/LV vs IFL  Trend toward improved DFS for MMR-D/MSI-H patients on IFL vs those on FU/LV, suggesting the possibility of an interaction of irinotecan with MSI status  5-yr DFS for MMR-D/MSI-H patients –IFL treatment: 0.76 –FU/LV treatment: 0.57  P =.07 1.0 0.8 0.6 0.4 0.2 0 02468 Yrs FU/LV IFL X 2 = 3.24 P =.072 DFS Reprinted with permission. © 2008 American Society of Clinical Oncology. All rights reserved. Bertagnolli M, et al. J Clin Oncol. 2009 Vol. 29(11), 1814-1817.

18. clinicaloptions.com/oncology MSI Predicts Improved Response to Adjuvant IFL Therapy in Stage III CRC clinicaloptions.com/oncology Summary of Key Conclusions  MMR-D/MSI-H colon cancer may be a clinically distinct subset of stage III CRC  MSI can be accurately assessed using IHC for MMR proteins MLH1 and MSH2  Improved 5-yr DFS was seen for MMR-D disease treated with the IFL regimen compared with FU/LV –FU/LV alone may not be the optimal regimen for adjuvant treatment of stage III colon cancers that demonstrate MSI