Volume 138, Issue 3, Pages 958-968 (March 2010) Experimentally Derived Metastasis Gene Expression Profile Predicts Recurrence and Death in Patients With Colon Cancer  J. Joshua Smith, Natasha G. Deane, Fei Wu, Nipun B. Merchant, Bing Zhang, Aixiang Jiang, Pengcheng Lu, J. Chad Johnson, Carl Schmidt, Christina E. Bailey, Steven Eschrich, Christian Kis, Shawn Levy, M. Kay Washington, Martin J. Heslin, Robert J. Coffey, Timothy J. Yeatman, Yu Shyr, R. Daniel Beauchamp  Gastroenterology  Volume 138, Issue 3, Pages 958-968 (March 2010) DOI: 10.1053/j.gastro.2009.11.005 Copyright © 2010 AGA Institute Terms and Conditions

Figure 1 Cell culture and mouse model: murine model of metastasis, in vivo monitoring, and ex vivo proof of metastases. (A) MC-38 parental cells (heterogeneous, blue and red) were subjected to 6 sequential passages through matrigel-coated transwells (enrichment of invasive subpopulations of MC-38 cells [red]) called “MC-38inv.” After in vivo passage, a stabilized cell line (pink cells) called “MC-38met” was established. (B) MC-38inv cells were tested alongside MC-38 parental cells for the ability to form lung metastasis in a tail vein assay. The figure shows representative tumor progression in live mice by bioluminescent imaging (days 1–21) and at the time of autopsy (day 21). Gastroenterology 2010 138, 958-968DOI: (10.1053/j.gastro.2009.11.005) Copyright © 2010 AGA Institute Terms and Conditions

Figure 2 (A) Recurrence classifier development. VMC 2-step schematic for enrichment and establishment of the 34-gene recurrence classifier for colon cancer. Mouse genes were mapped to human orthologs, and 300 differentially expressed genes (MC-38 parental versus MC-38met) were identified. These 300 genes were next refined with 19 high-risk patients from the VMC training dataset for concordance. This analysis showed 34 genes with concordant expression among the 19 high-risk patients and the MC-38met cells. The 34-gene recurrence classifier was then applied to the independent MCC database to determine whether it could be used to discriminate patients on the basis of outcomes. (B) Functional genomic cluster analysis of the 34-gene recurrence classifier. Mean-centered gene expression data (rows) clustered with individual VMC patients (columns) results in 2 distinct patient groups (cluster 1, pink; cluster 2, green). The 19 VMC high-risk patients used in the concordance analysis are marked with a red asterisk. Gastroenterology 2010 138, 958-968DOI: (10.1053/j.gastro.2009.11.005) Copyright © 2010 AGA Institute Terms and Conditions

Figure 3 The 34-gene recurrence classifier as tested in the MCC dataset across all stages. Kaplan–Meier estimates of overall and disease-specific survival in the MCC test set. Expression data for probes corresponding to the 34-gene recurrence classifier were used to build the Cox proportional hazard model from patient data in the Vanderbilt dataset. Plots represent survival analyses in the MCC patient dataset, based on β and Wald statistics (see Supplementary Methods) from the Vanderbilt dataset. (A) Overall and (B) disease-specific survival analyses were performed. Gastroenterology 2010 138, 958-968DOI: (10.1053/j.gastro.2009.11.005) Copyright © 2010 AGA Institute Terms and Conditions

Figure 4 Kaplan–Meier estimates from 114 patients with colon cancer (stages II and III) under study at MCC were analyzed with the 34-gene–based metastasis score. Lower-than-median metastasis score is denoted in black and higher-than-median metastasis score is noted in red. A low score was associated with better disease-specific and disease-free survival in patients with stage II (A cancer-related death, n = 57 patients; high scores [9 of 9 total deaths] and B disease-free survival; n = 55; high scores [10 of 11 total events]). Similarly, a low score was associated with better disease-specific and disease-free survival in patients with stage III colon cancer (C cancer-related death, n = 57 patients, high scores [14 of 17 total deaths] and D disease-free survival, n = 56, high scores [16 of 20 total events]). Five-year mortality and recurrence rates are shown for patients with stage II and III colon cancer (A to D). Gastroenterology 2010 138, 958-968DOI: (10.1053/j.gastro.2009.11.005) Copyright © 2010 AGA Institute Terms and Conditions