Where are we in 2013?

Transcription

1 The Use of Gene Profile Testing in the Adjuvant Therapy of Stages II & III Colon Cancer: Where are we in 2013? Howard S. Hochster, MD Professor of Medicine, Yale School of Medicine Associate Director, Yale Cancer Center Director, GI Oncology Translational Working Group

2 Disclosures Consultant and speaker for Genomic Health Investigator for Agendia Consultant for several pharmaceutical companies

3 Colon Cancer: Stage Drives Approach to Treatment Palliative Rx Surgery Alone No Adjuvant Rx Surgery + Adjuvant Rx Adjuvant Therapy? Annual incidence (all stages): ~110,000 cases Jemal A, Segal R, Ward E, Hao Y, Xu J, Thun MJ, Cancer statistics 2009.CA Cancer J Clin 2009, 59: Stage II: ~31,000 cases (60% Medicare)

4

5 AJCCv6 TNM v7 Effective Staging Jan Definitions 2010 Primary tumor (T) T is Carcinoma in situ T 1 Tumor invades submucosa T 2 Tumor invades muscularis propria T 3 Tumor invades through T4b: muscularis invasion propria of or organs subserosa T 4 Tumor directly invades other organs or structures Regional lymph nodes (N) N 0 No regional lymph node metastases N 1 Metastases in 1 3 regional lymph nodes N 2 Metastases in 4 or more regional lymph nodes Distant metastases (M) M 0 No distant metastases M 1 Distant metastases T4a: perf. visceral peritoneum N1a: 1 N+ N1b: 2-3 N+ N2a: 4-6 N+ N2b: >7 N+ AJCC = American Joint Committee on Cancer. National Comprehensive Cancer Network (NCCN), 2008; Greene et al., 2002.

6 5-Year Relative Survival By AJCC Stage Percentage of Patients (%) p < Stage I Stage Stage Stage Stage Stage Stage IV IIA IIB IIIA IIIB IIIC (T 1 2 N 0 ) (T 3 N 0 ) (T 4 N 0 ) (T 1 2 N 1 ) (T 3 4 N 1 ) (T any N 2 ) (M 1 ) O Connell et al., 2004.

7 Stage II Colon Cancer: The Problem 1. No single, adequately powered and convincing trial for stage II only 2. Which patients need treatment? - Can we identify those at high risk?

8 CAN WE GO BEYOND 19 TH CENTURY TECHNOLOGY?

9 Adjuvant Therapy of Colon Cancer 5-FU/Lev superior to surgery alone 5-FU/LV superior to surgery alone 5-FU/LV superior to 5-FU/Lev 6- and 12-month treatment cycles equivalent Lev unnecessary High- and low-dose LV equivalent Monthly and weekly treatment equivalent Capecitabine as effective as bolus 5-FU/LV LV5FU2 and monthly bolus equivalent FOLFOX4 superior to 5-FU/LV Andre et at. N Engl J Med. 2004;350:2343. Andre et al. Proc Am Soc Clin Oncol. 2002;21:133a. Abstract 529. Francini et al. Gastroenterology. 1994;106:899. Haller et al. Proc Am Soc Clin Oncol. 1998;17:256a. Abstract 982. Moertel et al. Ann Intern Med. 1995;122:321. O Connell et al. J Clin Oncol. 1998;16:295. Wolmark et al. Proc Am Soc Clin Oncol. 1996;15:205. Abstract 460.

10 NSABP EARLY COLON CANCER TRIALS COMBINED ANALYSIS Treatment 1 Treatment 2 C-01 OP Vs MOF C-02 OP Vs PVI C-03 MOF Vs FU+LV C-04 FU+LEV Vs FU+LV N = 1924 N = 1896

11 COMBINED 5 YEAR SURVIVAL DUKES B DUKES C % Year 0 P = TRT 1 TRT 2 Pts.Deaths P < Pts. Deaths v 85% 59 v 66.5% 4 5

12 REDUCTION IN CUMULATIVE ODDS All Patients Dukes' B Dukes' C DEATH P value

13 13 Clinical Validation of the Pre specified Colon Cancer Assay: Stage II Colon Cancer Patients from QUASAR Parent QUASAR Trial Resected Stage II Colon Cancer Observation Adjuvant treatment with 5FU/LV Enrolled , primarily from UK Parent study demonstrated 3 4% absolute benefit of adjuvant 5FU/LV for stage II disease (approximate 20% relative risk reduction) QUASAR Collaborative Group, et al. Lancet. 2007;370:

14 Quasar: Demographics & treatment Patient Population stage II 92% colon 71% median f/u 4.6 yrs 5-FU + low-dose folinic acid 1318 (81%) (11% also received levamisole) 5-FU + high-dose folinic acid 304 (19%) (47% also received levamisole) Total allocated chemotherapy 1622 Allocated to observation only 1617

15 Quasar Dukes B Survival N = 2980 dths 5yr surv p Chemo None

16 16 The challenge: Which stage II colon cancer patients should receive adjuvant chemotherapy? It is unclear which 75 80% of patients are cured with surgery alone Absolute chemotherapy benefit is small Chemo has significant toxicity and impacts quality of life Median age 71 years old; comorbidities and competing causes of mortality Selection of patients for chemotherapy is subjectively based on: Risk assessment with a limited set of clinical/pathologic markers Patient age, comorbidities, patient preference

17 Mismatch Repair Deficiency (MMR D): Unique Biological Subgroup of Colon Cancer 17 IHC for MMR protein status MLH1+ MLH1 MSH2 MSH2+ Thus, IHC for MMR proteins and PCR for MSI detect two manifestations of the same tumor biology: MMR D is synonymous with MSI H MMR P is synonymous with MSI L/MSS Imai K, et al. Carcinogenesis. 2008;29: Umetani N, et al. Ann Surg Oncol. 2000;7: Rosen DG, et al. Mod Pathol. 2006;19: PCR on tumor DNA for MSI (microsatellite instability)

18 MMR D Identifies Resected Colon Cancer Patients With Low Recurrence Risk 18 Overall Survival (%) Pooled Analysis of Stage II and III colon cancer patients (surgery alone) No adjuvant chemotherapy, n = 287 MMR-D MMR-P P = Years after Randomization Multiple studies have consistently demonstrated that the ~15% of colon cancer patients with MMR D tumors have markedly lower recurrence risk, particularly for the stage II colon cancer patient. Adapted from Ribic CM, et al. N Engl J Med. 2003;349:

19 MMR D Has Consistently Been Shown to Be a Favorable Prognostic Marker 19 Source Stage / Treatment Endpoint MMR D vs MMR P HR (95% CI); p value Ribic et al 1 II/III Surgery alone Overall survival 0.31 ( ) p=0.004 Sargent et al 2 II/III Surgery alone Disease free survival Overall survival 0.46 ( ); p= ( ); p=0.06 Gray et al 3 (QUASAR) II Surgery alone Recurrence free interval 0.31 ( ) p<0.001 Roth et al 4 (PETACC 3) II 5FU ±irinotecan Relapse free survival 0.30 p=0.004 The ~15% of stage II colon cancer patients with MMR deficient tumors have been found consistently to have a lower risk of recurrence and/or death 1. Ribic CM, et al. N Engl J Med. 2003;349: Sargent DJ, et al. J Clin Oncol. 2010;28: Gray R, et al. J Clin Oncol. In press. 4. Roth AD, et al. J Clin Oncol. 2009;27: abstract 288.

20 DFS/OS in Stage II dmmr Patients (N=102) 5-yr DFS N = 47 N = 55 5-yr OS Untreated 87% Treated 72% HR: 2.80 ( ) p=0.05 Untreated 93% Treated 75% HR: 3.15 ( ) p=0.03 Sargent ASCO 2008

21 21 High Tumor Grade Consistently Found NOT to be a Marker of High Recurrence Risk in Stage II Colon Cancer 1. O Connell MJ, et al. J Clin Oncol. 2010;28: Gray R, et al. J Clin Oncol. In press. 3. Roth AD et al. J Clin Oncol. 2010;28: Venook AP, et al. J Clin Oncol. 2011;29: abstract Quah HM, et al. Dis Colon Rectum. 2008;51:

22 Recurrence Risk Assessment in Stage II Colon Cancer: The Clinical Need 22 The majority (>70%) of stage II colon cancer patients are standard risk (T3, MMR proficient) For the standard risk patient, conventional markers such as grade and LVI, are not standardized or validated, and they do not provide reliable, accurate determinations of recurrence risk

23 Development and Validation of the Oncotype DX Colon Cancer Assay 23 Colon Cancer Technical Feasibility Development Studies Surgery Alone NSABP C 01/C 02 (n = 270) Cleveland Clinic (n = 765) Development Studies Surgery + 5FU/LV NSABP C 04 (n = 308) NSABP C 06 (n = 508) Selection of Final Gene List & Algorithm Standardization and Validation of Analytical Methods Clinical Validation Study Stage II Colon Cancer QUASAR (N = 1436) Confirmation Study Stage II Colon Cancer CALGB 9581 (N = 690)

24 STROMAL FAP INHBA BGN 7 Genes The 12 Gene Oncotype DX Colon Cancer Recurrence Score Recurrence Score GADD45B CELL CYCLE Ki 67 C MYC MYBL2 Recurrence Score = 0.15 Stromal Group 0.30 Cell Cycle Group GADD45B Reference Genes ATP5E GPX1 PGK1 UBB VDAC2 24 O Connell MJ, et al. J Clin Oncol. 2010;28: Kerr D, et al. J Clin Oncol. 2009;27: abstract 4000.

25 QUASAR: Evaluable Stage II Colon Cancer Patients 25 Parent QUASAR Trial N = 3239 Patients with collected blocks n = 2197 (68%) Confirmed stage II colon cancer n = 1490 (69%) Final evaluable populations n = cases of stage III and rectal cancer 54 excluded (3.6%): 29 synchronous tumors 08 insufficient tissue 07 identifier queries 06 RNA quality/quantity 04 ineligible histology Kerr D, et al. J Clin Oncol. 2009;27: abstract Gray R, et al. J Clin Oncol. In press.

26 QUASAR: Demographics of 1,436 Evaluable Patients 26 Characteristic Value Surgery alone (N = 711) n (%) Age, years <60 60 to < (35.3) 308 (43.3) 152 (21.4) Surgery + 5FU/LV (N =725) n (%) 269 (37.1) 317 (43.7) 139 (19.2) Gender Female 302 (42.5) 295 (40.7) T stage T4 108 (15.3) 113 (15.7) # nodes examined < (62.9) 244 (37.1) 409 (61.0) 262 (39.0) LVI Present 90 (12.7) 110 (15.2) Tumor grade High 222 (31.2) 219 (30.2) Tumor type Mucinous 144 (20.3) 169 (23.3) MMR Deficient 89 (13.6) 92 (14.1) Location Right 273 (46.9) 278 (46.2) Kerr D, et al. J Clin Oncol. 2009;27: abstract Gray R, et al. J Clin Oncol. In press.

27 QUASAR Results: Colon Cancer Recurrence Score Predicts Recurrence Following Surgery 27 Prospectively defined Primary Analysis in Stage II Colon Cancer (n = 711) 35% Risk of Recurrence at 3 years 30% 25% 20% 15% 10% 5% p= % Kerr D, et al. J Clin Oncol. 2009;27: abstract Gray R, et al. J Clin Oncol. In press Recurrence Score

28 QUASAR Results: Recurrence Risk in Pre specified Recurrence Risk Groups 28 Recurrence Risk Group Range of RS Proportion of patients 1.0 Low < % 0.8 Intermediat e % High % Comparison of high vs. low recurrence risk groups using Cox model: HR = 1.47 (p=0.046) Proportion Event Free Recurrence Risk Group Low Intermediate High Kaplan-Meier Estimates (95% CI) of Recurrence Risk at 3 years 12% ( 9% -16%) 18% (13%-24%) 22% (16%-29%) Kerr D, et al. J Clin Oncol. 2009;27: abstract Gray R, et al. J Clin Oncol. In press. Years n = 711

29 QUASAR Results: Clinical/Pathological Covariates and Recurrence 29 Pre specified Multivariate Analysis, Surgery Alone Patients (n = 605) Variable Categories HR 95% CI P value Recurrence Score Continuous per 25 units 1.61 (1.13, 2.29) Mismatch Repair 13% deficient vs. 87% proficient 0.32 (0.15, 0.69) <0.001 T stage 15% T4 vs. 85% T (1.23, 2.75) Tumor grade 29% high vs. 71% low 0.62 (0.40, 0.96) # of nodes examined 62% <12 vs. 38% (1.01, 2.14) LVI 13% present vs. 87% absent 1.40 (0.88, 2.23) In these multivariate analyses, Recurrence Score, MMR status, and T stage were found to be the most significant independent predictors of recurrence risk. Kerr D, et al. J Clin Oncol. 2009;27: abstract Gray R, et al. J Clin Oncol. In press.

30 Recurrence Score Guideposts for Clinical Decisions: T3, MMR P Patients with RS Risk of Recurrence at 3 years 45% 40% 35% 30% 25% 20% 15% 10% 5% 0% Recurrence Score T4 and MMR proficient (13%) T3 and MMR proficient (74%) T3 and MMR deficient (11%) This population of patients with high Recurrence Score disease (~25% of total) has recurrence risk that overlaps with T4 patients and would be expected to have >3% benefit with adjuvant 5FU.

31 Summary: QUASAR Validation Study 31 Recurrence Score independently and quantitatively predicts individual recurrence risk and provides additional clinical value beyond other available measures. These results support a new paradigm for quantitative assessment of recurrence risk in stage II colon cancer, emphasizing the role of three measures: Recurrence Score, MMR, and T stage. The continuous Recurrence Score will have the greatest clinical utility for T3, MMR proficient patients, who constitute the majority of stage II colon cancer (~70% of patients).

32 32 CALGB 9581 Parent Trial Randomized Phase III Clinical Trial in Stage II Colon Cancer Low/standard risk, resected Stage II colon cancer (excluded T4b, obstruction, perforation, positive margins) Observation MAb 17 1A (edrecolomab) 1738 patients enrolled Negative results for MAb 17 1A Targeted and enrolled primarily low risk stage II patients (excluded pt4b, obstruction/perforation, positive margins) Niedzwiecki D, et al. J Clin Oncol. 2011;29. Oncol. 2011; 29:3146.

33 CALGB 9581: Unique Opportunity to Study Low Risk Stage II Colon Cancer 33 Characteristic CALGB 9581 % of cohort QUASAR % of cohort T4 6% 15% MMR D 22% 14% Age >70 yrs 35% 20% <12 Nodes examined 47% 62% LVI 11% 14% High tumor grade 32% 31% Recurrence risk (5 yr) 14.6% 21.7% Compared to QUASAR, the CALGB 9581 population had: More patients with age >70 years Fewer patients with T4 tumors More patients with MMR deficiency Overall lower recurrence risk

34 CALGB 9581 Primary Analysis: Association of Continuous RS with Recurrence Risk 34 Variable HR 95% CI P value RS per 25 units 1.52 (1.09, 2.12) The continuous RS was significantly associated with the risk of recurrence Strength of association consistent with QUASAR Venook AP, et al. J Clin Oncol. 2011;29: abstract 3518.

35 35 Conclusions Results confirm the 12 gene RS previously validated in QUASAR RS is significantly associated with risk of recurrence beyond known prognostic factors RS improves ability to discriminate higher vs. lower recurrence risk particularly in standard risk patients with T3 MMR P tumors Implications for clinical practice: For patients with T3, MMR P tumors, a high RS reveals a more aggressive underlying biology for which adjuvant therapy may be more appropriately considered Venook AP, et al. J Clin Oncol. 2011;29: abstract 3518.

36 Other Approaches to Stage II MSI and 18q-

37 E5202: Targeting the Tumor Stage II Colon Cancer High Risk mfolfox6 18q- MSS mfolfox6 + bevacizumab Low Risk OBSERVATION

38 OXALIPLATIN ADJUVANT TRIALS and Results in Stage III CRC MOSAIC C-07 ASCO 2012 Data on Oncotype DX Colon

39 MOSAIC: Treatment arms FOLFOX4: LV5FU2 + Oxaliplatin 85mg/m² D1 5FU bolus D2 5FU bolus LV 5-FU infusion* LV 5-FU infusion* OXA R D1 5FU bolus D2 5FU bolus LV5FU2 LV 5-FU infusion* LV 5-FU infusion* Every 2 weeks, 6 months of treatment (12 cycles) *Baxter LV5 infusors

40 NSABP C-07 FU 500 LV 500 Rest R N=2409 FU 500 LV 500 OHP 85 2hr Rest x3 Week (primary endpoint 3-yr DFS; 89% power to detect 5.4% difference)

41 C-07 DFS Ev # 3yr DFS FLOX % FULV % p < HR: 0.79 [ ] % risk reduction

42 C-07 and Mosaic OXALIPATIN benefit 3y DFS Δ HR C % 4.9 % 0.79 Mosaic 77.9 % 5.1 % 0.77

43 Validation of the 12-gene colon cancer Recurrence Score (RS) in NSABP C-07 as a predictor of recurrence in stage II and III colon cancer patients treated with 5FU/LV (FU) and 5FU/LV + oxaliplatin (FU+Ox) O Connell MJ, 1 Lee M, 2 Lopatin M, 2 Yothers G, 1 Clark-Langone K, 2 Millward C, 2 Paik S, 1 Sharif S, 1 Shak S, 2 Wolmark N 1 1 National Surgical Adjuvant Breast and Bowel Project, Pittsburgh, PA; 2 Genomic Health, Inc., Redwood City, CA

44 Development and Validation of the 12-Gene Colon Cancer Recurrence Score Colon Cancer Technical Feasibility Development Studies (Surgery) NSABP C 01/C 02 (n = 270) Cleveland Clinic (n = 765) Development Studies (5FU/LV) NSABP C 04 (n = 308) NSABP C 06 (n = 508) Selection of Final Gene List & Algorithm Standardization and Validation of Analytical Methods Clinical Validation Study Stage II Colon Cancer QUASAR (N = 1436) Confirmation Study Stage II Colon Cancer CALGB 9581 (N = 690) Clinical Validation Study Stage II/III Colon Cancer 5FU vs 5FU+Oxaliplatin NSABP C 07 (N = 892)

45 Demographic and Clinical Patient Characteristics Variable Value FU FU+Oxali All N=449 N=443 N=892 Race White 89% 88% 88% Gender Female 43% 42% 42% Age Average, years years 16% 18% 17% Stage II 29% 30% 30% III A/B 46% 45% 46% III C 24% 25% 25% T stage T4 in stage II 5% 8% 6% T3 T4 in stage III 85% 84% 85% MMR status MMR D in stage II* 21% 16% 18% Nodes examined MMR D in stage III* 8% 10% 9% 12 in Stage II 12 in Stage III 64% 59% 60% 63% * In patients with known MMR status. MMR status was available for 80% of all patients. 62% 61% Treatment arms are well balanced with respect to clinical and pathologic covariates

46 Demographics and Clinical Characteristics The demographic and clinical characteristics of patients in this study were similar to those of patients in the parent C- 07 trial Patient characteristics in this study were also similar to those of C- 07 patients not included in this study Median FU 8.6 yrs; 865 (97%) of 892 evaluable patients had complete follow-up at 3 years and 840 (94%) had complete follow-up at 5 years. Recurrences 31 (12%) of 264 stage II patients 214 (34%) of 628 stage III patients

47 Oxaliplatin Effect Similar to the Parent Study 1.0 Proportion Recurrence Free Solid: 5FU Dashed: 5FU+Ox HR = 0.80 for 5FU+Ox vs. 5FU Stage II Stage III Years Similar to the parent study, most of the oxaliplatin effect was observed in stage III patients, though the interaction of treatment and stage was not significant (p=0.36)

48 Pre-Specified Primary Analysis: Recurrence Score Predicts Recurrence Risk in Stage II & III Colon Cancer Patients in NSABP C-07 (n=892) Variable Value HR HR 95% CI p value Stage <0.001 (by nodal status) Stage III A/B vs. II 2.53 (1.70,3.78) Stage III C vs. II 5.29 (3.54,7.90) Treatment FU+Oxali vs. FU 0.76 (0.59,0.98) RS per 25 units 1.96 (1.50,2.55) <0.001 Continuous RS is significantly associated with risk of recurrence controlling for effects of treatment and stage (by nodal status) Interaction of RS and nodal status not significant (p=0.90) Interaction of RS and treatment not significant (p=0.48) Interaction of RS and age not significant (p=0.76)

49 5-year Recurrence Risk in 5FU treated arm Cox Regression Analysis (n=892) Stage II Stage IIIA/B Stage IIIC RS Group* % pts Average Risk 95% CI % pts Average Risk 95% CI % pts Average Risk 95% CI Low 39% 9% (6-13%) 41% 21% (16-26%) 33% 40% (32-48%) Intermediate 36% 13% (8-17%) 34% 29% (24-34%) 37% 51% (43-59%) High 25% 18% (12-25%) 25% 38% (30-46%) 30% 64% (55-74%) * Pre-specified RS Groups: Low (RS<30), Intermediate (30 RS<41), High (RS 41). Recurrence risk is significantly higher in High vs. Low RS group: HR = 2.11, p<0.001

50 Contribution of RS Beyond Clinical and Pathologic Covariates Pre-specified Multivariate Analysis (n=892) Variable Value HR HR 95% CI P value Stage <0.001 (by nodal status) Stage III A/B vs. II 0.97 (0.55,1.71) Stage III C vs. II 2.07 (1.16,3.68) Treatment FU+Oxali vs. FU 0.82 (0.64,1.06) 0.12 MMR MMR D vs. MMR P 0.27 (0.12,0.62) <0.001 T stage T4 st II & T3 T4 st III vs. T3 st II & T1 T2 st III 3.04 (1.84,5.02) <0.001 Nodes examined <12 vs (1.17,1.95) Tumor grade High vs. Low 1.36 (1.02,1.82) RS per 25 units 1.57 (1.19,2.08) RS is significantly associated with risk of recurrence after controlling for effects of T and N stage, MMR status, number of nodes examined, grade and treatment

51 Primary Analysis: Recurrence Score Predicts Recurrence Risk in Stage II & III Colon Cancer Patients in NSABP C-07 (n=892) Solid: 5FU Dashed: p< FU+Ox Stage III C Stage III A/B Stage II Solid: 5FU Dashed: 5FU+Ox With similar relative benefit of oxaliplatin added to adjuvant 5FU across the range of RS, absolute benefit of oxaliplatin increases with increasing RS, most apparently in stage II & stage IIIA/B pts

52 Study Design A prospective multi center study enrolled 221 patients with stage IIA (T3N0) colon cancer across 17 academic and community sites within the Mayo Clinic Cancer Research Consortium network. Study Schema Primary analysis focused on patients with MMR Proficient tumors only

53 Treatment Recommendations Pre and Post Assay T3 MMR P Patients Post Assay Pre Assay Observation 5 FU monotherapy 5 FU+ Oxaliplatin Total Observation 54 (38.3%) 8 (5.7%) 6 (4.3%) 68 (48%) 5 FU monotherapy 24 (17.0%) 8 (5.7%) 2 (1.4%) 34 (24%) 5 FU+ Oxaliplatin 21 (14.9%) 2 (1.4%) 16 (11.3%) 39 (28%) Total 99 (70%) 18 (13%) 24 (17%) 141 (100%) 63 (44.7%) of 141 recommendations changed 47 (33.3%) patients were recommended LESS treatment 16 (11.3%) patients were recommended MORE treatment

54 Overall change in Treatment Recommendations T3 MMR P Patients Recommendation Pre Assay Post Assay Observation 68 (48%) 99 (70%) Any Chemo 73 (52%) 42 (30%) 5 FU monotherapy 34 (24%) 18 (13%) 5 FU+ Oxaliplatin 39 (28%) 24 (17%) Total 141 (100%) 141 (100%) Recommendations for chemotherapy were reduced by 22% Recommendations for 5 FU + oxaliplatin were reduced by 11%

55 Decision Algorithm in Adjuvant Therapy Resected Colon Ca Stage II Stage III Low-Risk yes T4 and/or <12 LNs no dmmr yes High-Risk FOLFOX * no No therapy! Intermed. Risk * 5-FU/LV or Capecitabine? Oncotype DX Colon? *pts not considered candidates for oxaliplatin

56 Development and Validation of the Molecular Subtype Signature AGENDIA RNA profile Development Cohort Validation Cohort N Hospital NKI, LUMC, Slotervaart Vall d Hebron, ICO Barcelona, Munich Stage Gender Subtype I II III IV F M A B C 24 (13%) 100 (53%) 56 (30%) 8 (4%) 104 (55%) 84 (45%) 65 (35%) 98 (52%) 25 (13%) (59%) 223 (41%) (42%) 317 (58%) 117 (22%) 336 (62%) 90 (16%) Tabernero, et al, GI Ca Symp, 2013

57 Whole Genome Array Development Set (stage I IV) (n=188) Netherlands Cancer Institute, Leiden Medical Center, Slotervaart Molecular classification based on nearest centroid single sample predictor (SSP) 3 gene expression profiles (A, B & C) Validation Set (stage II III) (n=543) Technical University Munich, Institut Catala d Oncologia & Vall d Hebron Hospital Barcelona, Medical University Vienna, University of Ferrara Development Validation Analysis of mutations in BRAF(V600), KRAS (codons 12, 13 & 61) and PIK3CA (exons 9 & 20) All samples Analysis of 615 (incl. kinome) by NGS 73 samples MSI analysis by IHC All samples MSI/dMMR gene expression pattern (64 gene signature) All samples Analysis of Epithelial and Mesenchymal genes All samples OS and Distant Metastasis free survival (DMS) All samples Effect of adjuvant treatment Stage III samples, validation cohort (n=123) All sets Tabernero, et al, GI Ca Symp, 2013

58 Unsupervised hierarchical clustering of whole genome reveals 3 distinct patient groups Gene profiles were developed to identify these subgroups A-Type B-Type C-Type Tabernero, et al, GI Ca Symp, 2013

59 Unsupervised hierarchical clustering of whole genome reveals 3 distinct patient groups Gene profiles were developed to identify these subgroups A-Type (32 genes) B-Type (53 genes) C-Type (102 genes) Tabernero, et al, GI Ca Symp, 2013

60 Subtypes are significantly associated with prognosis DM Risk Death C-Type B-Type A-Type Risk of Distant Metastasis Risk of Death Tabernero, et al, GI Ca Symp, 2013

61 Subtypes are significantly correlated with benefit from adjuvant 5-FU-based treatment Tabernero, et al, GI Ca Symp, 2013

62 Colon molecular subtype model Subtype Clinical features Biological features Clinical utility No adjuvant or 5FU Chemotherapy New targeted therapy? (companion Dx) Tabernero, et al, GI Ca Symp, 2013

63 CONCLUSIONS (1) Early Stage CRC different molecular phenotypes and behaviors Need for large prospective trials but likely impractical ONCOTYPE DX COLON Test is an independent predictor of Risk of Recurrence and benefit in stage II & III colon cancer DEVELOPED & VERIFIED IN MULTIPLE LARGE PROSPECTIVE CLINICAL TRIALS INCLUDING STAGE II & III Can extrapolate potential benefit of therapy with FU/LV

64 CONCLUSIONS (2) May be helpful in situations with greater uncertainty (< 12 LNs, for example) Helpful in discussing quantitated risks and benefits with patients ONCOTYPE DX COLON Test is an independent predictor of benefit for use of 5FU and FU/Oxaliplatin in Stage II & III colon cancer Some stage IIIA/B patients have little benefit Some stage II patients have considerable benefit Agendia RNA profile (fresh tissue) suggests 3 subtypes with differential sensitivity New era of understanding and directing therapy more specifically BEYOND THE MICROSCOPE

65

66 But, is it worth it? What is the benefit required to receive oxaliplatin? Surveys by Neil Love 150 Medical Oncologists 95 Patients (Proc ASCO 2008, 2010)

67 Predictions of Patient Trade Offs (Medical Oncologists and Clinical Investigators) % 90% 80% 70% 60% 50% 40% 30% 20% PT CI MO 19% 35% 36% 17% 14% 10% 28% 29% 57% 59% 52% 68% 48% 50% 77% 72% 83% 88% 10% MO CI MO CI MO CI 0% 1% 50% 49% 1% 20% 19% 3% 20% 17% 5% 50% 45% 5% 20% 15% 10% 50% 40% Reduction in Risk of Recurrence Survey of 150 patients with colorectal cancer: 2006 Survey of 150 practicing oncologists: 2006

68 Recurrence Score and Alternative endpoints Disease Free Survival Variable HR HR 95% CI P value RS per 25 units 1.60 (1.28,1.99) <0.001 Overall Survival Variable HR HR 95% CI P value RS per 25 units 1.89 (1.46,2.44) <0.001 RS is significantly associated with DFS and OS after controlling for nodal status and treatment