Prognostic Impact of Bcl-2 Depends on Tumor Histology and Expression of MALAT-1 lncRNA in Non–Small-Cell Lung Cancer Lars Henning Schmidt, Dennis Görlich,

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Prognostic Impact of Bcl-2 Depends on Tumor Histology and Expression of MALAT-1 lncRNA in Non–Small-Cell Lung Cancer Lars Henning Schmidt, Dennis Görlich, Tilmann Spieker, Christian Rohde, Martin Schuler, Michael Mohr, Julia Humberg, Tim Sauer, Nils H. Thoenissen, Andreas Huge, Reinhard Voss, Alessandro Marra, Andreas Faldum, Carsten Müller-Tidow, Wolfgang E. Berdel, Rainer Wiewrodt Journal of Thoracic Oncology Volume 9, Issue 9, Pages 1294-1304 (September 2014) DOI: 10.1097/JTO.0000000000000243 Copyright © 2014 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 1 Immunohistochemical staining results for Bcl-xl, Bcl-2, Mcl-1, and pp32/PHAPI. All tested Bcl-2 family proteins Bcl-2 (A), Bcl-xl (B), and Mcl-1 (C) displayed a cytoplasmatic expression pattern, pp32/PHAPI (D) was also found in the nuclei. All images at x20, inlay x40. Journal of Thoracic Oncology 2014 9, 1294-1304DOI: (10.1097/JTO.0000000000000243) Copyright © 2014 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 2 Kaplan-Meier survival analysis for all tested apoptotic factors (n = 383). The blue line indicates negative antigenic expression, and the red line stands for a positive immunohistochemical signal. The p values of the Log rank test are displayed in the corresponding charts. In the total study collective no prognostic effect was found for Bcl-2 (A; p = 0.218, log rank test), for Bcl-xl (B; p = 0.590, log rank test), for Mcl-1 (C; p = 0.270, log rank test) nor for pp32/PHAPI (D; p = 0.385, log rank test). Journal of Thoracic Oncology 2014 9, 1294-1304DOI: (10.1097/JTO.0000000000000243) Copyright © 2014 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 3 The prognostic effect of Bcl-2 depends on tumor histology and lymph node stage (Kaplan-Meier survival analysis, n = 383). The blue line indicates negative antigenic expression, and the red line stands for a positive immunohistochemical signal. The p values of the Log rank test are displayed in the corresponding charts. Depending on histology a prognostic effect was shown for Bcl-2 in non-adenocarcinoma (A; p = 0.036) but not for adenocarcinoma (B; p = 0.993). Moreover, in non-adenocarcinoma survival rates increased for lymph node negative patients (C; p = 0.049) in contrast to lymph node positive patients (D; p = 0.151). Journal of Thoracic Oncology 2014 9, 1294-1304DOI: (10.1097/JTO.0000000000000243) Copyright © 2014 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 4 Univariate prognostic analysis for Bcl-2 and MALAT-1. The blue line in the Kaplan-Meier charts for overall survival (n = 383) indicates negative antigenic expression, and the red line stands for a positive immunohistochemical signal. The p values of the Log rank test are displayed in the corresponding charts. In univariate analysis, no prognostic effect was found neither for weak MALAT-1 expression (A; p = 0.136) nor for strong MALAT-1 expression (B; p = 0.734). Journal of Thoracic Oncology 2014 9, 1294-1304DOI: (10.1097/JTO.0000000000000243) Copyright © 2014 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 5 Bcl-2 and its direct interaction partners on protein level. Protein-protein interaction network of Bcl-2 according to the IntAct database. Edges indicate direct protein-protein interactions. The effect of MALAT1 (fold change [logFC] of strong MALAT-1 versus weak MALAT-1) on the expression of the interaction partners of Bcl-2 is color coded. Green nodes indicate downregulated genes, red nodes upregulated genes. Bcl-2 shows the strongest change in expression (logFC = 0.46), whereas NLRP1 displays the lowest logFC among the downregulated genes (logFC = −1.04). Among the upregulated factors, we found Bcl-2, Bcl2l1, BID, and BAX whereas BIK, Bcl2l11, TP53, and MAPK1 were downregulated by MALAT-1. Moreover, interactions among the Bcl-2 interacting proteins are demonstrated by crosslinks. Journal of Thoracic Oncology 2014 9, 1294-1304DOI: (10.1097/JTO.0000000000000243) Copyright © 2014 International Association for the Study of Lung Cancer Terms and Conditions