Kunal Mehta, BSE, Erika Moravcikova, PhD, David McFall, AB, James D

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The Mesenchymal State Predicts Poor Disease-Free Survival in Resectable Non-Small Cell Lung Cancer Kunal Mehta, BSE, Erika Moravcikova, PhD, David McFall, AB, James D. Luketich, MD, Arjun Pennathur, MD, Albert D. Donnenberg, PhD, Vera S. Donnenberg, PhD, FCP The Annals of Thoracic Surgery Volume 104, Issue 1, Pages 321-328 (July 2017) DOI: 10.1016/j.athoracsur.2017.01.091 Copyright © 2017 The Society of Thoracic Surgeons Terms and Conditions

Fig 1 Cluster of differentiation (CD)44+/CD90+/cytokeratin+ (CTK) cells in normal lung, and primary and metastatic non-small cell lung cancer. (Center Panel) Notched box plot shows the prevalence of CD44+/CD90+ cells among cytokeratin+ cells. The top and bottom borders of the box indicate 75th and 25th percentiles, respectively; the notches indicate the 95% confidence intervals about the median values; and the whiskers mark the range, exclusive of outliers. Note that CD44/CD90 expression in primary tumors (T) was bimodal, with most comparable to that seen in adjacent normal (NL) parenchyma, but with a smaller group having expression comparable to that observed in malignant pleural effusions (MPE). (Side Panels) Examples of CD44/CD90 expression in normal lung, primary tumors, and MPE. Cells were gated on singlets/DNA≥2N/CD45–/CD14–/cytokeratin+. (ECD = energy coupled dye; PE = phycoerythrin.) The Annals of Thoracic Surgery 2017 104, 321-328DOI: (10.1016/j.athoracsur.2017.01.091) Copyright © 2017 The Society of Thoracic Surgeons Terms and Conditions

Fig 2 Cluster of differentiation (CD)90+ tumor cells visualized by immunofluorescence. Archived formalin-fixed paraffin-embedded sections from primary non-small cell lung cancer tissue from 2 patients in the present study cohort were stained with hematoxylin and eosin and also analyzed by immunofluorescence for CD90. CD90+ cells are shown in red. All sections were counterstained with 4,6-diamidino-2-phenylindole, dihydrochloride to show DNA-containing nuclei (blue). The yellow borders indicate the position of a zoomed image. The scale bars in the unzoomed images represents 1 mm; the scale bars in the zoomed images represent 100 micrometers. (a) Rapid progressor: A 66-year-old man with adenocarcinoma of the lung, American Joint Committee on Cancer (AJCC) stage IIIA. (b) Nonprogressor: A 77-year-old woman with adenocarcinoma of the lung, AJCC stage IB. The Annals of Thoracic Surgery 2017 104, 321-328DOI: (10.1016/j.athoracsur.2017.01.091) Copyright © 2017 The Society of Thoracic Surgeons Terms and Conditions

Fig 3 (Left Panel) Relapse-free survival based on cluster of differentiation (CD)44/CD90 expression and (Right Panel) American Joint Committee on Cancer (AJCC) stage (<II, ≥II). The cohort with high CD44/CD90 coexpression (red curve) experienced significantly reduced relapse-free survival compared to the cohort with low CD44/CD90 co-expression (p = 0.00006 by Mantel test). AJCC stage was less predictive of relapse-free survival (p = 0.03). The Annals of Thoracic Surgery 2017 104, 321-328DOI: (10.1016/j.athoracsur.2017.01.091) Copyright © 2017 The Society of Thoracic Surgeons Terms and Conditions

Fig 4 Mesenchymal gene expression in three non-small cell lung cancer (NSCLC) explant cultures. Patient samples are designated malignant pleural effusion (PE). For each analyte, β-actin normalized 2–ΔCT was standardized where negative values are more epithelial (shown in blue) and positive values are more mesenchymal (shown in red). H1299 serves as an epithelial control, and human foreskin fibroblasts serve as a mesenchymal control. (CD = cluster of differentiation; CT = cycle threshold; ECAD = e-cadherin; EMT = epithelial-mesenchymal transition; EpCAM = epithelial cell adhesion molecule; KRT19 = keratin 19; VIM = vimentin.) The Annals of Thoracic Surgery 2017 104, 321-328DOI: (10.1016/j.athoracsur.2017.01.091) Copyright © 2017 The Society of Thoracic Surgeons Terms and Conditions