1. References 1.Salazar R, Roepman P, Capella G et al. Gene expression signature to improve prognosis prediction of stage II and III colorectal cancer. J Clin Oncol 2011; 29: 17-24. 2.Maak M, Simon I, Nitsche U et al. Independent Validation of a Prognostic Genomic Signature (ColoPrint) for Patients With Stage II Colon Cancer. Ann Surg 2013. 3.Tian S, Roepman P, Popovici V et al. A robust genomic signature for the detection of colorectal cancer patients with microsatellite instability phenotype and high mutation frequency. J Pathol 2012; 228: 586-595. Title of the Poster Presentation Goes Here Authors of the Poster Presentation Goes Here Institutional and/or Graduate School of Biomedical Sciences Affiliation Goes Here 36X60 Genomic Classifiers (ColoPrint / MSI-Print) to Predict Outcome and Chemotherapy Benefit in Stage II and III Colon Cancer Patients Scott Kopetz 1*, Zhi-Qin Jiang 1, Michael J Overman 1, Christa Dreezen 2, Sun Tian 2, Ying Li 2, Iris Simon 2, George J Chang 3, Dipen M Maru 4 Department of 1 Gastrointestinal Medical Oncology, 3 Surgical Oncology, 4 Pathology, University of Texas, MD Anderson Cancer Center. 2 Agendia NV, Amsterdam, Netherlands and Agendia Inc., Irvine CA Background: Although the benefit of chemotherapy in stage II and III colon cancer patients is significant, many patients might not need adjuvant chemotherapy because they have a good prognosis even without additional treatment. ColoPrint is a gene expression classifier that distinguish patients with low or high risk of disease recurrence. It was developed using whole genome expression data and has been validated in public datasets, independent European patient cohorts and technical studies. Methods: In this study, the commercial ColoPrint test was validated in stage II (n=96) and III patients (n=94) treated at the MD Anderson Cancer Center from 2001 to 2009. Frozen tissue specimen, clinical parameters and follow-up data (median follow-up 64 months) were available. The 64-gene MSI-signature developed to identify patients with deficient mismatch repair (dMMR) system was evaluated for its accuracy to identify MSI patients and also for prognosis. Results: In this cohort, ColoPrint classified 56% of stage II and III patients as being at low risk. The 3-year recurrence- Free-Survival (RFS) was 90.6% for Low Risk and 78.4% for High Risk patients with a HR of 2.33 (P=0.012). In uni-and multivariate analysis ColoPrint and stage were the only significant factors to predict outcome. The MSI-signature classified 47 patients (24.6%) as MSI-H and most MSI-H patients were ColoPrint low risk (81%). Patients who were ColoPrint low risk and MSI-H by signature had the best outcome with a 3-year RFS of 95%, while patients with ColoPrint high risk had a worse outcome independently of the MSI-status. Low Risk ColoPrint patients had a good outcome independent of stage or chemotherapy treatment (90.1% 3-year RFS for treated patients, 91.4% for untreated patients) while ColoPrint high risk patients treated with adjuvant chemotherapy had 3-year RFS of 84%, compared to 70.1% 3-year RFS in untreated patients (P=0.037). Conclusions: The combination of ColoPrint and MSI-Print improves the prognostic accuracy in stage II and stage III patients and may help the identification of patients at higher risk who are more likely to benefit from additional treatment. Gene expression signature Developed on tumor tissue from 188 patients with known outcome, using whole genome arrays. From the comprehensive pool of 33,834 gene probes, an optimal set of 18 nonredundant probes showed robust clinical outcome association. These 18 genes were used to construct a nearest centroid- based prognostic classifier (ColoPrint). A genomic signature for the detection of colorectal cancer patients with microsatellite instability phenotype and high mutation frequency. MSI-Print identifies not only patients with MSI-H as measured by traditional methods but all patients with hypermutation phenotype. The 64-gene MSI signature could be linked to a deficient mismatch repair (dMMR) phenotype, as both MSI and MSI-like patients showed a high mutation frequency. MSI-Print: Table 1. 190 stage II and III colon cancer patients characteristics ColoPrint: Fig. 1. Probability of recurrence or death between ColoPrint high-risk and low-risk group. Fig. 3. Probability of recurrence or death between ColoPrint high-risk and low-risk group with or without adjuvant chemotherapy. Fig. 2. Probability of recurrence or death between ColoPrint high-risk and low-risk group in stage II or stage III patients. Fig. 5 Probability of recurrence or death between ColoPrint high-risk and low-risk group combined with MSI-print. Table 2. Multivariate cox regression analysis for stage II and III colon cancer. Abstract #378 Coloprint and MSI-Print can identify high-risk patients group among stage II and III colon cancer patients after surgery. This may help to select high risk patients who would derive the most benefit from additional treatment. Fig. 4. Probability of recurrence or death between MSI-Print high and MSI-Print low group. Table 3. ColoPrint risk groups vs. ASCO clinical risk groups Background Benefit of chemotherapy in stage II and III colon cancer patients is significant However, many low-risk patients might not need adjuvant chemotherapy How to identify low-risk and high-risk patients still remains unclear Stage II and III colon cancer patients from MD Anderson Cancer Center Staging pTNM by AJCC 6 th edition Validation cohort: N=190 Frozen tumor and normal tissue available Surgery time: 2001-2009 Median follow-up time: 64 months RNA extracted and microarray performed by Agilent platform as previously described MSI by IHC or PCR has not been performed P<0.001 Methods *Corresponding author : Scott Kopetz, M.D., Ph.D: skopetz@mdanderson.org Results 56% of patients were considered low-risk ColoPrint is prognostic for recurrence (Fig.1) -- 3-year RFS of 90.6% and 5-year RFS of 87.1% for low risk group, 78.4% and 71.5% for high risk group. When separated by stage, ColoPrint remained prognostic (Fig. 2.) -- 3-year RFS of 95.5% in low risk and 88.4% in high risk group for stage II -- 3-year RFS of 84.3% in low risk and 64.2% in high risk group for stage III Results In univariate analysis, significant prognostic factors were ColoPrint and stage. In multivariate analysis, both ColoPrint (HR 2.55 for high risk) and stage (HR 3.48 for stage III) remained significant (Table 2.) ColoPrint is independent of traditional ASCO high risk features, with 50% of cases discordant based on presence/absence of high risk features and ColoPrint results (Table 3.) Conclusions 25% of patients were characterized as MSI-Print high which was previously defined to incorporate both MSI- high tumors and additional tumors which share similar gene expression features In this population, MSI-Print did not provide independent prognostic ability, but provided additional information when combined with ColoPrint. (Fig. 4 and Fig. 5) --Most MSI-Print high patients were ColoPrint low risk. These patients had the best outcome with RFS of 95% at 3 years. ColoPrint high riskColoPrint low risk ASCO high risk clinical features 34%41% ASCO low risk clinical features 9%16% Variables Total N=190 ColoPrint Low risk N=107 ColoPrint High risk N=83 P-value n (%) Male gender97(51)54(50)43(52)0.855 Median age62 590.487 Right-sided tumor96(51)59(55)37(45)0.053 TNM Stage0.756 II96(51)53(50)43(52) III94(49)54(50)40(48) pT stage0.252 T10(0)0 0 T28(4)4 4(5) T3152(80)82(77)70(84) T430(16)21(20)9(11) pN stage0.540 N096(51)53(50)43(52) N160(32)33(31)27(33) N234(18)21(20)13(16) Differentiation0.009 Well to Moderate156(82)81(76)75(90) Poor34(18)26(24)8(10) Obstruction & Perforation0.695 Yes9(5)4(4)5(6) No158(83)90(84)68(82) Unknown23(12)13(12)10(12) Lymphvascular/ Perineural invasion 0.245 Yes106(56)54(50)52(63) No68(36)43(40)25(30) Both16(8)10(9)6(7) Median LN assessed21(11)22(21)20(24)0.493 Adjuvant chemotherapy120(63)71(66)49(59)0.300 Median OS months70(37)75(70)64(77)0.445 Months to recurrence61(32)69(64)52(62)0.037 MSI-Print high47(25)38(36)9(11)<0.001 CovariatesHR95% CIP-value ColoPrint (high-risk vs. low-risk) 2.551.17-5.520.018 TNM Stage (stage III vs. stage II) 3.481.48-8.190.004 Time (months) MSI-Print low MSI-Print high Time (months) CP low risk, MSI-Print low CP low risk, MSI-Print high CP high risk, MSI-Print low CP high risk, MSI-Print high Time (months) P=0.037 P=0.309 P=0.059 P=0.012