Cyclin E1 Is Amplified and Overexpressed in Osteosarcoma William W. Lockwood, Deirdre Stack, Thomas Morris, David Grehan, Conor O'Keane, Greg L. Stewart, Joanna Cumiskey, Wan L. Lam, Jeremy A. Squire, David M. Thomas, Maureen J. O'Sullivan  The Journal of Molecular Diagnostics  Volume 13, Issue 3, Pages 289-296 (May 2011) DOI: 10.1016/j.jmoldx.2010.11.020 Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Regions showing significant amplification (A) and deletion (B) by aCGH analysis generated by Genomic Identification of Significant Targets in Cancer analysis. Segmented copy number data for 22 OS tumors were analyzed using the Genomic Identification of Significant Targets in Cancer algorithm as detailed in Materials and Methods. The resulting statistical significance of the aberrations detected across the sample set are displayed as false discovery rate q-values (t0 account for multiple-hypothesis testing), as indicated by the red (amplification) and blue (deletion) lines and plotted along the x axis. The significance threshold (q < 0.25) is indicated by the vertical green line, with altered regions passing this threshold deemed significant. Chromosome positions are indicated by the alternating white and black bars on the y axis, with the locations of the identified significant peak regions located in the right margin of each figure. The Journal of Molecular Diagnostics 2011 13, 289-296DOI: (10.1016/j.jmoldx.2010.11.020) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Genes in amplified regions that are overexpressed in mouse OS xenografts compared with normal tissues. Median normalized log2 expression levels for 214 of the 281 genes in regions of significant amplification across the OS tumors were compared between 8 OS and 19 normal tissue xenograft samples, as detailed in Materials and Methods. Genes determined to be overexpressed in the tumor samples (corrected P < 0.01, greater than twofold average expression) are depicted. The 8 samples on the left are OS xenografts and the 19 samples on the right are normal tissue controls, with the organ/tissue of origin indicated on each one individually. The genes differentially expressed are listed from top to bottom along the right edge of the heatmap. Red indicates high relative expression and green indicates low expression of genes. The Journal of Molecular Diagnostics 2011 13, 289-296DOI: (10.1016/j.jmoldx.2010.11.020) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Impact of copy number alterations on the mRNA expression of CCNE1 in OS cell lines. Relative expression values for samples with copy number loss, no copy number change, gain, and amplification are shown in a heatmap generated using Genesis software.18 Copy number status was determined as follows: loss, fewer than two copies; neutral, two copies; gain, more than two copies; and amplification, 5 or more copies. Expression data were normalized and scaled from 0 to 100. The Journal of Molecular Diagnostics 2011 13, 289-296DOI: (10.1016/j.jmoldx.2010.11.020) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 DNA copy number changes in cell cycle components across the 22 OS tumors. The copy number status of a selected panel of genes with well-characterized roles in cell cycle control was determined for each tumor sample, as detailed in Materials and Methods. The resulting alterations thought to promote cell cycle progression (gain of cyclins and CDKs, loss of Rb1 and CDKN2A) are indicated. Nineteen of 22 cases (86%) contained an alteration in at least one of these genes that could potentially result in increased cell cycle. Each vertical column represents a single OS sample, with the corresponding sample identification denoted at the top. Red cells indicate copy number gain; green cells, copy number loss; and black cells, no change. The Journal of Molecular Diagnostics 2011 13, 289-296DOI: (10.1016/j.jmoldx.2010.11.020) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 5 Supervised analysis of cell cycle–related gene expression in primary OS. Genes were selected manually for their role in cell cycle progression and as markers for proliferation. The genes differentially expressed are listed from top to bottom along the right edge of the heatmap. Red indicates overexpression and green indicates relative underexpression of genes. The Journal of Molecular Diagnostics 2011 13, 289-296DOI: (10.1016/j.jmoldx.2010.11.020) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 6 FISH analysis showing amplification of CCNE1 (A) and MYC (B) on touch imprints, with immunohistochemical analysis for CCNE1 (C) and MYC (D) as applied to OS TMA-A. The Journal of Molecular Diagnostics 2011 13, 289-296DOI: (10.1016/j.jmoldx.2010.11.020) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions