Classification of the Four Main Types of Lung Cancer Using a MicroRNA-Based Diagnostic Assay  Shlomit Gilad, Gila Lithwick-Yanai, Iris Barshack, Sima Benjamin, Irit Krivitsky, Tina Bocker Edmonston, Marluce Bibbo, Craig Thurm, Laurie Horowitz, Yajue Huang, Meora Feinmesser, J. Steve Hou, Brianna St. Cyr, Ilanit Burnstein, Hadas Gibori, Nir Dromi, Mats Sanden, Michal Kushnir, Ranit Aharonov  The Journal of Molecular Diagnostics  Volume 14, Issue 5, Pages 510-517 (September 2012) DOI: 10.1016/j.jmoldx.2012.03.004 Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Differential expression of miRNAs between several lung cancer types. Examples of differential expression for three pairs of miRNAs between three different lung cancer comparisons are shown: NSCLC versus neuroendocrine lung cancer using hsa-miR-7 and hsa-miR-375 (A and B), squamous versus nonsquamous NSCLC using hsa-miR-205 and hsa-miR-375 (C and D), and small versus carcinoid lung cancer using hsa-miR-7 and hsa-miR-375 (E and F). Left column: separations using data from the custom microarray experiments (normalized fluorescence units) performed during the discovery phase. Right column: the same separations as seen in the set of PCR experiments (inverted normalized signals) used to determine the final probe list in the assay development phase. The Journal of Molecular Diagnostics 2012 14, 510-517DOI: (10.1016/j.jmoldx.2012.03.004) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Comparison of hsa-miR-375 signals in two laboratories. Raw CTs observed in the two laboratories (RGL-US and RG-IL) in the interlaboratory validation study are plotted. The signals shown are from samples used to test the concordance of the cDNA preparation and RT-qPCR amplification stages. The Pearson correlation for the CTs shown herein in the two laboratories is 0.995. The Journal of Molecular Diagnostics 2012 14, 510-517DOI: (10.1016/j.jmoldx.2012.03.004) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Assay results for the validation set. The assay results for all the validation samples (A) as well as for the subsets of pathologic samples (B) and cytologic samples (C) are shown. On the y axis, the “Reference diagnosis” represents the classification given by the two independent pathologists, and on the x axis, the “Classifier answer” is the classification given by the test. Values in the matrix represent the number of samples with a specific “Reference diagnosis” that were given a specific “Classifier answer” by the assay. The tables show, for each histologic class, the total number of samples (No.), the sensitivity, the specificity, and the PPV. The 95% CIs are given in square brackets. The PPV was calculated using the validation set distribution and the estimated prevalence in the test population (in parentheses). See Materials and Methods for explanations regarding PPV calculation. The Journal of Molecular Diagnostics 2012 14, 510-517DOI: (10.1016/j.jmoldx.2012.03.004) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions