1. Validation of a 12-gene colon cancer Recurrence Score in stage II colon cancer patients from CALGB 9581 A.P. Venook 1, D. Niedzwiecki 2, M. Lopatin 3, M. Lee 3, P. N. Friedman 4, W. Frankel 5, K. Clark-Langone 3, C. Yoshizawa 3, C. Millward 3, S. Shak 3, R. M. Goldberg 6, N. N. Mahmoud 7, R. L. Schilsky 8, M. M. Bertagnolli 9 1. University of California, San Francisco, San Francisco, CA; 2. Duke University, Durham, NC; 3. Genomic Health, Redwood City, CA; 4. Cancer and Leukemia Group B, Chicago, IL; 5. The Ohio State University, Columbus, OH; 6. University of North Carolina at Chapel Hill, Chapel Hill, NC; 7. University of Pennsylvania, Philadelphia, PA; 8. The University of Chicago, Chicago, IL; 9. Brigham and Women's Hospital, Boston, MA

2. Background

3. The Need for Validated Markers of Recurrence Risk in Stage II Colon Cancer Decision to use chemotherapy in stage II colon cancer is challenging −Balance between toxicity, risk of recurrence and expected absolute benefit of treatment Current guidelines 1,2 recommend consideration of adjuvant chemotherapy for “higher risk” stage II patients based on clinical and pathologic factors −Evidence for these conventional risk factors is limited −For 75% of stage II patients who have T3N0, Mismatch Repair- Intact (MMR-I) tumors, there are no informative markers to ascertain risk There is a need for standardized, validated markers of recurrence risk to inform adjuvant decision-making in stage II colon cancer, particularly for patients with T3N0 MMR-I tumors

4. Genomic Markers for Risk Assessment in Stage II Colon Cancer Many studies support the concept that genomic markers may reveal underlying biology in colon cancer to potentially inform an individualized approach to therapy The 12-gene colon cancer Recurrence Score (RS) (Genomic Health, Inc., Redwood City, CA) was developed using data from 1,851 stage II patients in 4 large, independent studies conducted with the NSABP and Cleveland Clinic 3 In a prospectively-designed study in 1,436 stage II colon cancer patients from the QUASAR clinical trial, the continuous 12-gene RS, T stage, and MMR status were validated as a predictor of recurrence risk in stage II colon cancer patients following surgery 4

5. The 12-Gene Oncotype DX ® Colon Cancer Recurrence Score ® CELL CYCLE Ki-67 C-MYC MYBL2 REFERENCE ATP5E GPX1 PGK1 UBB VDAC2 GADD45B Recurrence Score STROMAL FAP INHBA BGN RS = 0.15 x Stromal Group - 0.30 x Cell Cycle Group + 0.15 x GADD45B

6. CALGB 9581: A Unique Opportunity to Test Markers of Recurrence Risk in Stage II Colon Cancer Phase III intergroup study –Randomized to observation vs adjuvant edrecolomab (MoAb 17- 1A, anti-EpCAM) –Mostly stage II patients without high risk features Excluded pT4b, N1 or greater, metastatic disease, obstruction or perforation, positive resection margins –Enrolled 1,738 patients between 1997 and 2002 –Results: no significant differences between arms for all endpoints examined 5 (ASCO 2004) This data set represents a rare opportunity to validate the 12-gene RS in a well-defined stage II colon cancer patient population wherein there was no treatment effect and there was, on average, a low recurrence risk

7. Design and Methods for CALGB 150705, B4* study * CALGB protocol which directs analyses described here

8. Study Objectives Prospectively-designed study with pre-specified endpoints, analytical methods and analysis plan (A “Prospective-Retrospective” Study 6 ) Primary Objective: ‒ Determine whether there is a significant relationship between the continuous 12-gene RS and recurrence risk Secondary Objectives −Determine whether there is a significant relationship between individual clinical/pathologic covariates and recurrence risk −Determine whether the RS provides significant information beyond number of nodes examined, pathologic T stage, tumor grade, mismatch repair (MMR) status and lymphatic and/or vascular invasion −Compare recurrence risk between high and low RS groups based on pre-specified percentile cut-points

9. Inclusion Criteria −Patients with stage II colon cancer who were enrolled in CALGB 9581 Exclusion Criteria −No tumor block from initial diagnosis −No patient consent for tumor tissue research −Standard Oncotype DX Colon assay Clinical Laboratory exclusion criteria Inadequate tumor (<5% tumor present) Tumor types other than adenocarcinoma NOS and mucinous carcinoma Insufficient RNA (<5 ng/µL or 300 ng) for RT-PCR analysis. Failure of assay to meet pre-specified quality control (QC) specifications Methods: Study Population

10. Expression levels for each of the 12 RS genes were quantitated by RT-PCR from 30 μm of manually microdissected, fixed, paraffin embedded primary colon cancer tissue −The TaqMan assay was used to analyze the expression levels of 7 cancer-related genes and 5 reference genes −RT-PCR was performed in triplicate qPCR wells with an equivalent of 2 ng RNA input per 10 μL-reaction volume The 12-gene RS was calculated using the same pre-specified gene list and algorithm as previously tested in QUASAR Methods: Quantitative Gene Expression Analysis

11. Colon cancer defined as tumors with gross distal margins above the peritoneal reflection MMR protein status (D= Deficient; I= Intact) was assessed centrally by IHC for MLH1 and MSH2 T stage, number of lymph nodes assessed, tumor location, lymphatic and/or vascular invasion (LVI), and evidence of bowel obstruction and/or perforation were obtained from the CALGB clinical database Tumor grade and tumor type were assessed centrally according to the criteria specified by College of American Pathologists Consensus Statement 7. All tumors classified as mucinous carcinoma were characterized as high tumor grade All centrally-performed pathology and assay procedures were pre- specified and conducted without knowledge of the clinical characteristics or outcomes Methods: Pathology Analysis

12. Cohort sampling study design 8 included all patients with available tissue and recurrence and a random sample of patients without recurrence (3:1 ratio) ‒ Sampling stratified on treatment ‒ No matching performed Primary endpoint: Recurrence Free Interval (RFI) ‒ Time (in years) from randomization to first colon cancer recurrence or death due to recurrence of colon cancer ‒ Deaths due to other causes were censored ‒ Second primary cancers were ignored Weighted Cox proportional hazards models were fit to RFI and Wald test statistics were constructed using a weighted partial likelihood estimate and robust variance estimate 9 Methods: Statistical Analysis

13. Results

14. Parent CALGB 9581 Trial 1738 patients enrolled 1672 stage II colon cancer (261 recurrences) Patients with Available Tissue 1137 stage II colon cancer (187 recurrences) CALGB/GHI study 728 patient samples processed (both study arms pooled) All available recurrences + random sample non-recurrences Final Study Population 690 pts 162 recurrences 528 non-recurrences Derivation of Study Population 38 cases (5%) excluded: 17 insufficient tumor 6 ineligible tumor type 8 RNA quantity 7 RNA quality

15. Demographics and Clinical Characteristics CharacteristicValue CALGB-GHI cohort Not in CALGB-GHI cohort Parent trial N=690%*N=982%*N=1672% Age<60 21431.332332.953732.1 60-70 23133.631131.654232.4 >70 24535.034835.559335.5 GenderMale 36051.951352.487352.2 RaceWhite 63092.089491.4152491.5 Year of Surgery≤1998 13419.514014.327416.4 1999-2000 34350.254055.288352.8 ≥2001 21330.330230.551530.8 Treatment ArmObservation 34350.449950.484250.4 Mab 17-1A 34749.648349.683049.6 * Unweighted * Weighted based on cohort sampling design

16. CharacteristicValue CALGB-GHI cohort Not in CALGB-GHI cohort Parent trial N=690%*N=982%*N=1672% T stageT4 415.9353.7764.6 Nodes examined<12 examined 32747.142744.275445.1 Lymphovascular Invasion (LVI) Present 7810.911211.519011.4 Tumor LocationRight 36052.849250.185251.0 MMRDeficient 13721.54822.918521.4 Obstruction or Perforation Present 111.7192.1301.8 Central Mucinous Histology Present 12418.4** Central Tumor grade High 22032.4** Demographics and Clinical Characteristics, continued * Weighted based on cohort sampling design ** No comparable variable in parent trial

17. Demographics and Clinical Characteristics, continued Compared to most adjuvant colon cancer populations, patients enrolled in the CALGB 9581 study were generally older and at lower risk of recurrence (recurrence risk of 14.6% at 5 years) Consistent with the expected pattern of patient selection for a clinical trial with no conventional chemotherapy arm Smaller proportion of T4 and higher proportion of MMR-D consistent with lower recurrence risk Based on 690 evaluable patients −Median age: 66 years (interquartile range 57-73), 35% >70 years old −Median number of nodes examined: 12 (interquartile range 8-18) −<6% with T4 tumor stage −>20% with MMR-D tumors −Median FU: 8.0 yrs The distribution of clinical characteristics for patients in the CALGB- GHI cohort was similar to those not in CALGB-GHI cohort (p=ns) with the exception of T stage and nodes examined (p<0.05), and those differences were relatively small in magnitude

18. Distribution of Recurrence Score VariableRS Mean32.7 SD11.5 Min2 P2524.7 Median31.4 P7539 Max78

19. Primary Analysis: Association of Continuous RS with Recurrence Risk VariableHR HR 95% CI P value RS per 25 units1.52(1.09,2.12)0.013 The continuous RS was significantly associated with the risk of recurrence * Only 2 RS values were above 70 *

20. RFI and Clinical and Pathologic Covariates: Univariable Analysis VariableValueHR 95% CIP value MMR (deficient vs intact)21% MMR-D0.62(0.39,0.99)0.044 T Stage (T4 vs T3)6% T41.19(0.60,2.37)0.616 Number of Nodes Examined (<12 vs ≥12) 47% <12 nodes1.17(0.85,1.62)0.342 Number of Nodes Examinedcontinuous per 1 node0.98(0.96,1.00)0.062 Tumor Grade (high vs low)32% high grade0.74(0.52,1.07)0.114 Lymphovascular Invasion (present vs not) 11% LVI present1.56(0.98,2.50)0.062 Mucinous Histology18% mucinous0.73(0.46,1.16)0.179 Tumor Location (right-sided vs other)53% right-sided0.79(0.57,1.10)0.158 Age (≥ 70 years vs <70 years)35% ≥ 70 years1.21(0.87,1.69)0.259 Agecontinuous per 1 year1.01(1.00,1.03)0.145 Gender (male vs female)52% male1.14(0.82,1.58)0.422

21. RFI and Clinical and Pathologic Covariates: Univariable Analysis, continued MMR status was the only covariate significantly associated with recurrence risk ‒ MMR-D associated with lower risk of recurrence as expected LVI and number of nodes examined as a continuous variable were of marginal significance, with the effects in the directions consistent with published studies. T4 stage was not a significant predictor of recurrence in this cohort ‒ Protocol-specified exclusion of the most advanced T4 cases (T4 tumors which invaded adjacent organs or structures - T4b in AJCC 7 th edition) and low overall frequency of T4 patients in the CALGB 9581 study may have contributed to this result

22. Contribution of RS to Prediction of Recurrence Risk Beyond Clinical and Pathologic Covariates Contribution of RS to prediction of recurrence risk beyond clinical and pathological characteristics was examined in two-variable models of RS and any of the following covariates: age, gender, race, T stage, MMR status, grade, nodes examined, mucinous histology, tumor location and LVI. RS was significantly associated with recurrence risk after all individual clinical and pathological covariates included in two-variable models

23. Contribution of RS to Prediction of Recurrence Risk Beyond Clinical and Pathologic Covariates VariableHR HR 95% CIP value MMR (deficient vs intact)0.70(0.42,1.17)0.17 T Stage (T4 vs T3)0.93(0.44,1.97)0.85 Number of Nodes Examined ( =12)1.14(0.81,1.60)0.46 Tumor Grade (high vs low)0.78(0.51,1.18)0.24 LVI (present vs not)1.39(0.85,2.26)0.19 RS per 25 units1.68(1.18,2.38)0.004 Pre-specified Multivariable Cox Regression of RS and covariates on RFI (n=656, 95% of all pts) RS was significantly associated with recurrence risk (p=0.004) after MMR status, T stage, number of nodes examined, grade and LVI were included in the model

24. Contribution of RS to Prediction of Recurrence Risk Beyond MMR and T-Stage VariableHR HR 95% CIP value MMR-I T4 vs MMR-I T31.14(0.53,2.44)0.73 MMR-D T3 vs MMR-I T30.62(0.39,0.98)0.043 RS per 25 units1.60(1.13,2.27)0.008 RS was significantly associated with recurrence risk (p=0.008) after MMR status, T stage and interaction of MMR and T stage were included in the model In T3 patients, MMR-D was associated with significantly lower recurrence risk (p=0.04) * Multivariate Cox model included continuous RS and indicator variables comparing groups of patients defined by T stage and MMR status to a reference group of MMR-I T3 patients. There were no events in 7 T4 MMR-D patients in our study providing insufficient data to estimate effect in these patients. Pre-specified Multivariable Cox Regression* of RS and covariates on RFI (n=656, 95% of all pts)

25. Recurrence Risk in RS Groups* * Groups based on pre-specified percentile cutpoints (cutpoint equivalent for RS: 39) ** Weighted based on cohort sampling design In the overall study population The estimates of recurrence risk at 5 years (95% CI) were 13% (10%, 17%), 13% (10%, 18%) and 18% (14%, 24%) for low, intermediate and high RS groups, respectively The hazard ratio for the comparison of high vs. low RS groups was 1.38, 95% CI (0.93, 2.05) In the T3 MMR-I population Among T3 MMR-I patients, the RS identified 22% of patients with an average risk of recurrence at 5 years above 20% RS Group*% of patients ** Average 5-Year Recurrence Risk (95% CI) Low 44 13% (10%, 16%) Intermediate 33 16% (13%, 19%) High 22 21% (16%, 26%)

26. Summary In this prospectively-designed study, the continuous 12-gene RS was significantly associated with the risk of recurrence in univariable and multivariable analyses in a large set of well- defined stage II colon cancer patients reflective of contemporary standards for staging In the 9581 population, which had low recurrence risk on average (15% at 5 years), none of the conventional clinical/pathologic risk factors, other than MMR, was significantly associated with recurrence –MMR-D was associated with lower risk of recurrence, consistent with prior studies The continuous RS predicted risk of recurrence beyond other covariates such as T stage, MMR, number of nodes examined, grade and LVI Among T3 MMR-I patients, the RS identified 22% of patients with an average risk of recurrence at 5 years above 20% improving the ability to discriminate higher from lower recurrence risk stage II colon cancer patients beyond known prognostic factors Summary

27. The results of this study confirm the performance of the 12- gene RS previously validated in the QUASAR study: –RS is significantly associated with risk of recurrence beyond the known prognostic factors in stage II colon cancer –RS improves the ability to discriminate higher from lower recurrence risk patients, particularly for standard risk patients with T3 MMR-I tumors Implications for clinical practice: for patients with T3 MMR-I tumors, a high Recurrence Score can reveal an underlying biology indicative of more aggressive disease for which adjuvant therapy may be more appropriately considered Conclusions

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