Andrew Feber, PhD, Liqiang Xi, MD, PhD, Arjun Pennathur, MD, William E

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MicroRNA Prognostic Signature for Nodal Metastases and Survival in Esophageal Adenocarcinoma Andrew Feber, PhD, Liqiang Xi, MD, PhD, Arjun Pennathur, MD, William E. Gooding, MS, Santhoshi Bandla, PhD, Maoxin Wu, MD, James D. Luketich, MD, Tony E. Godfrey, PhD, Virginia R. Litle, MD The Annals of Thoracic Surgery Volume 91, Issue 5, Pages 1523-1530 (May 2011) DOI: 10.1016/j.athoracsur.2011.01.056 Copyright © 2011 The Society of Thoracic Surgeons Terms and Conditions

Fig 1 The TGmirV1 array dynamic range, sensitivity, and specificity. (A) MicroRNA (miRNA) was labeled and hybridized at concentrations ranging from 125 ng to 1 μg. Labeled Cel-mir-2* and Cel-Lin-4** were used as hybridization controls (red lines). The signal to concentration relationship was analyzed for 50 randomly selected miRNAs, and the average slope for all miRNAs was 0.96 ± 0.01. (B) Array cross-hybridization and sequence specificity was assessed using the Let-7 family of miRNAs. Synthetic Let-7a was labeled and hybridized, and the percentage of cross-hybridization was assessed for all Let-7 family members. The Annals of Thoracic Surgery 2011 91, 1523-1530DOI: (10.1016/j.athoracsur.2011.01.056) Copyright © 2011 The Society of Thoracic Surgeons Terms and Conditions

Fig 2 Kaplan-Meier survival plots for known microRNAs (miRNAs) found to be individually associated with overall survival: (A) miR-143 (p = 0.0148); (B) miR-199a_3p (p = 0.0009); (C) miR-145 (p = 0.1176): (D) miR-100 (p = 0.0022); and (E) miR-199a_5p (p = 0.0129). Patients are split into high and low expression groups based on the median for each miRNA. (Solid lines = above median; dashed lines = below median.) The Annals of Thoracic Surgery 2011 91, 1523-1530DOI: (10.1016/j.athoracsur.2011.01.056) Copyright © 2011 The Society of Thoracic Surgeons Terms and Conditions

Fig 3 Kaplan-Meier survival plot for esophageal adenocarcinoma patients based on a microRNA (miRNA) signature. Patients were classified into high or low risk based on a median split of the risk score (log rank p = 0.005; hazard ratio 3.6). The Annals of Thoracic Surgery 2011 91, 1523-1530DOI: (10.1016/j.athoracsur.2011.01.056) Copyright © 2011 The Society of Thoracic Surgeons Terms and Conditions

Fig 4 (A) Class comparison analysis, showing the prediction of lymph node metastasis in esophageal adenocarcinoma patients for three microRNAs (miRNAs). (B) Using the three verified miRNAs (mir-99bR and mir-199a_3p and _5p) in a logistic regression model to predict lymph node metastasis, the area under the receiver operator characteristic curve was 0.788 (95% confidence interval: 0.651 to 0.924) and the cross-validated accuracy was 78%. The Annals of Thoracic Surgery 2011 91, 1523-1530DOI: (10.1016/j.athoracsur.2011.01.056) Copyright © 2011 The Society of Thoracic Surgeons Terms and Conditions