Lu Chen, PhD, Brienne E. Engel, PhD, Eric A. Welsh, PhD, Sean J

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A Sensitive NanoString-Based Assay to Score STK11 (LKB1) Pathway Disruption in Lung Adenocarcinoma Lu Chen, PhD, Brienne E. Engel, PhD, Eric A. Welsh, PhD, Sean J. Yoder, Stephen G. Brantley, MD, Dung-Tsa Chen, PhD, Amer A. Beg, PhD, Chunxia Cao, PhD, Frederic J. Kaye, MD, Eric B. Haura, MD, Matthew B. Schabath, PhD, W. Douglas Cress, PhD Journal of Thoracic Oncology Volume 11, Issue 6, Pages 838-849 (June 2016) DOI: 10.1016/j.jtho.2016.02.009 Copyright © 2016 International Association for the Study of Lung Cancer Terms and Conditions

Figure 1 Serine/threonine kinase 11 (STK11) protein expression in cell lines and tumor samples. (A) Cell lines and (B) patient tumors with previously determined serine/threonine kinase 11 gene (STK11) mutational status16 were subjected to Western blotting for STK11. Samples specifically highlighted in the text are boxed. Repeated cell lines were obtained from different laboratory sources. The three STK11 isoforms are indicated. (C) Empty pNTAPb vector (Vector), a vector expressing a negative control affinity-tagged Mef2a protein (Mef2a control), wild-type (WT) STK11, and each of the three indicated STK11 variants were transfected into H1299 cells, immunoprecipitated using streptavidin beads, and blotted for binding to mouse protein 25 (MO25) and STE20-related adaptor protein α (STRAD). (D) A549 cells were transfected with the indicated combination of plasmids, extracted and blotted for adenosine monophosphate–activated kinase (AMPK) phosphorylated at T172, total AMPK, and β-actin. The first lane (–) represents empty vectors (pcDNA3 and pNTAPb), the second lane (+) represents cells transfected with pcDNA3 WT STK11 and empty pNTAPb, and the lanes labeled D194Y received pNTAPb-D194Y alone (–) or pNTAPb-D194Yplus WT in 1:1 (+), 1:5 (++), and 1:10 (+++) ratios. Lanes labeled F354L received pNTAPb-F354L alone (–) or pNTAPb-F354L plus WT in 1:1 (+), 1:5 (++), and 1:10 (+++) ratios. Journal of Thoracic Oncology 2016 11, 838-849DOI: (10.1016/j.jtho.2016.02.009) Copyright © 2016 International Association for the Study of Lung Cancer Terms and Conditions

Figure 2 Validation of the serine/threonine kinase 11 (STK11) mutation signature in two large patient-derived data sets. (A, B) PCA was performed on the indicated patient-derived data sets using the refined STK11 signature. Summary signature scores for first the first and second principal components for each individual patient are plotted. Patients with wild type (WT) STK11 are plotted with green symbols, splice-site variants are plotted in blue, exon mutations are plotted with yellow symbols and The Cancer Genome Atlas (TCGA) samples of unknown mutation status are plotted in red. (C, D) ROC curves for the TCGA and Moffitt lung adenocarcinoma overall survival (MLOS) cohorts. Journal of Thoracic Oncology 2016 11, 838-849DOI: (10.1016/j.jtho.2016.02.009) Copyright © 2016 International Association for the Study of Lung Cancer Terms and Conditions

Figure 3 A NanoString-based serine/threonine kinase 11 gene (STK11) assay applied to clinical samples. (A) Principal component analysis using 122 NanoString codesets was performed on 140 RNA samples isolated from formalin-fixed, plasma-embedded (FFPE) blocks, and the loading coefficients from the first (p[1]) and second (p[2]) principal components from human tumors were plotted. The signal variation covered by each principal component is indicated as a percentage of total variation. Blue symbols represent probesets with a negative loading coefficient in cell lines and red symbols represent those with positive values in cell lines. (B) Summary signature scores for first (PC 1) and second (PC 2) principal components for each individual patient are plotted. See Materials and Methods for details of the analysis. Patients with wild-type (WT) mutations are plotted with green symbols, splice site variants are plotted in blue, and exon mutations are plotted with yellow symbols. (C–F) Waterfall plots of the NanoString-based STK11 signature score. The number of WT and mutant (MUT) samples are indicated for each gene. p Values indicating correlation by the Wilcoxon rank sum test demonstrated significant correlation with STK11 and epidermal growth factor receptor gene (EGFR) (mutually exclusive) mutation status. (C) STK11, (D) Kirsten rat sarcoma viral oncogene homolog gene (KRAS), (E) tumor protein p53 gene (TP53), and (F) EGFR. Journal of Thoracic Oncology 2016 11, 838-849DOI: (10.1016/j.jtho.2016.02.009) Copyright © 2016 International Association for the Study of Lung Cancer Terms and Conditions

Figure 3 A NanoString-based serine/threonine kinase 11 gene (STK11) assay applied to clinical samples. (A) Principal component analysis using 122 NanoString codesets was performed on 140 RNA samples isolated from formalin-fixed, plasma-embedded (FFPE) blocks, and the loading coefficients from the first (p[1]) and second (p[2]) principal components from human tumors were plotted. The signal variation covered by each principal component is indicated as a percentage of total variation. Blue symbols represent probesets with a negative loading coefficient in cell lines and red symbols represent those with positive values in cell lines. (B) Summary signature scores for first (PC 1) and second (PC 2) principal components for each individual patient are plotted. See Materials and Methods for details of the analysis. Patients with wild-type (WT) mutations are plotted with green symbols, splice site variants are plotted in blue, and exon mutations are plotted with yellow symbols. (C–F) Waterfall plots of the NanoString-based STK11 signature score. The number of WT and mutant (MUT) samples are indicated for each gene. p Values indicating correlation by the Wilcoxon rank sum test demonstrated significant correlation with STK11 and epidermal growth factor receptor gene (EGFR) (mutually exclusive) mutation status. (C) STK11, (D) Kirsten rat sarcoma viral oncogene homolog gene (KRAS), (E) tumor protein p53 gene (TP53), and (F) EGFR. Journal of Thoracic Oncology 2016 11, 838-849DOI: (10.1016/j.jtho.2016.02.009) Copyright © 2016 International Association for the Study of Lung Cancer Terms and Conditions

Figure 4 Direct comparison between serine/threonine kinase 11 (STK11) immunohistochemical (IHC) staining and NanoString. STK11 IHC staining was performed on a tissue microarray representing cell lines, normal tissues, and lung adenocarcinoma tumors (Moffitt LUAD, Complete cohort). Scale bar represents 200 μM. (A) STK11 IHC staining in representative cores: (i) core 231, H1299 cells with WT STK11 and expression of the 52-kDa isoform (see Fig. 1A); (ii) core 207, A549 cells with mutant STK11 and lacking expression of any STK11 isoform (see Fig. 1A); (iii) core 141, normal alveolar tissue, +4 staining; (iv) core 78, normal bronchial tissue, +4 staining; (v) core 1, carcinoma with WT STK11, +4 staining; (vi) core 139, carcinoma with mutant STK11, +4 staining; (vii) core 29, carcinoma with WT STK11, 0 staining, negative; and (viii) core 66, carcinoma with mutant STK11, 0 staining, negative. (B) Receiver operating characteristic (ROC) curves comparing STK11 NanoString with IHC staining (area under the curve [AUC] was 0.8377 for NanoString versus 0.6789 for IHC staining). Supplementary Table 6 identifies cores by STK11 mutation status. Journal of Thoracic Oncology 2016 11, 838-849DOI: (10.1016/j.jtho.2016.02.009) Copyright © 2016 International Association for the Study of Lung Cancer Terms and Conditions

Supplementary Figure 1 Journal of Thoracic Oncology 2016 11, 838-849DOI: (10.1016/j.jtho.2016.02.009) Copyright © 2016 International Association for the Study of Lung Cancer Terms and Conditions

Supplementary Figure 2 Journal of Thoracic Oncology 2016 11, 838-849DOI: (10.1016/j.jtho.2016.02.009) Copyright © 2016 International Association for the Study of Lung Cancer Terms and Conditions