Oncogene Mutations, Copy Number Gains and Mutant Allele Specific Imbalance (MASI) Frequently Occur Together in Tumor Cells Junichi Soh 1,5. , Naoki Okumura 1. , William W. Lockwood 6 , Hiromasa Yamamoto 5 , Hisayuki Shigematsu 5 , Wei Zhang 1 , Raj Chari 6 , David S. Shames 1,11 , Ximing Tang 7 , Calum MacAulay 6 , Marileila Varella-Garcia 9 , To ˜ nu Vooder 10 , Ignacio I. Wistuba 7,8 , Stephen Lam 6 , Rolf Brekken 1 , Shinichi Toyooka 5 , John D. Minna 1,3,4 , Wan L. Lam 6 , Adi F. Gazdar 1,2 * 1 Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America, 2 Department of Pathology, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America, 3 Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America, 4 Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America, 5 Department of Cancer and Thoracic Surgery, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan, 6 Departments of Cancer Genetics and Developmental Biology, and Cancer Image, British Columbia Cancer Research Centre, Vancouver, British Columbia, Canada, 7 Department of Thoracic/Head and Neck, University of Texas MD Anderson Cancer Center, Houston, Texas, United States of America, 8 Department of Pathology, University of Texas MD Anderson Cancer Center, Houston, Texas, United States of America, 9 Department of Medicine, University of Colorado Health Sciences Center, Aurora, Colorado, United States of America, 10 Department of Biotechnology, Institute of Molecular and Cell Biology, Tartu University Hospital, Tartu University, Tartu, Estonia, 11 Oncology Diagnostics, Genentech Inc., South San Francisco, California, United States of America Abstract Background: Activating mutations in one allele of an oncogene (heterozygous mutations) are widely believed to be sufficient for tumorigenesis. However, mutant allele specific imbalance (MASI) has been observed in tumors and cell lines harboring mutations of oncogenes. Methodology/Principal Findings: We determined 1) mutational status, 2) copy number gains (CNGs) and 3) relative ratio between mutant and wild type alleles of KRAS, BRAF, PIK3CA and EGFR genes by direct sequencing and quantitative PCR assay in over 400 human tumors, cell lines, and xenografts of lung, colorectal, and pancreatic cancers. Examination of a public database indicated that homozygous mutations of five oncogenes were frequent (20%) in 833 cell lines of 12 tumor types. Our data indicated two major forms of MASI: 1) MASI with CNG, either complete or partial; and 2) MASI without CNG (uniparental disomy; UPD), due to complete loss of wild type allele. MASI was a frequent event in mutant EGFR (75%) and was due mainly to CNGs, while MASI, also frequent in mutant KRAS (58%), was mainly due to UPD. Mutant: wild type allelic ratios at the genomic level were precisely maintained after transcription. KRAS mutations or CNGs were significantly associated with increased ras GTPase activity, as measured by ELISA, and the two molecular changes were synergistic. Of 237 lung adenocarcinoma tumors, the small number with both KRAS mutation and CNG were associated with shortened survival. Conclusions: MASI is frequently present in mutant EGFR and KRAS tumor cells, and is associated with increased mutant allele transcription and gene activity. The frequent finding of mutations, CNGs and MASI occurring together in tumor cells indicates that these three genetic alterations, acting together, may have a greater role in the development or maintenance of the malignant phenotype than any individual alteration. Citation: Soh J, Okumura N, Lockwood WW, Yamamoto H, Shigematsu H, et al. (2009) Oncogene Mutations, Copy Number Gains and Mutant Allele Specific Imbalance (MASI) Frequently Occur Together in Tumor Cells. PLoS ONE 4(10): e7464. doi:10.1371/journal.pone.0007464 Editor: Irene Oi-Lin Ng, The University of Hong Kong, Hong Kong Received June 22, 2009; Accepted September 14, 2009; Published October 14, 2009 Copyright: ß 2009 Soh et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The Specialized Program of Research Excellence in Lung Cancer (P50CA70907, http://grants.nih.gov/grants/guide/), the Early Detection Research Network, National Cancer Institute, Bethesda, Maryland (U01CA084971, http://edrn.nci.nih.gov/) and the Canary Foundation, Palo Alto, CA. Funds from these grants were utilized for salary support and for performance of assays. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Dr. Gazdar is a paid consultant/lecturer for AstraZeneca PLC. Dr. Garcia recieves Research Funding .10,000 from AstraZeneca, Genentech and OSI; Honorarium ,10,000 from Roche. Dr. Minna receives research support from AstraZeneca PLC. David Shames is an employee of Genentech Inc. * E-mail: Adi.Gazdar@UTSouthwestern.edu . These authors contributed equally to this work. Introduction Oncogenes may be activated by mutation, structural rearrange- ment or gene copy number gains (CNGs) [1,2]. While activating somatic mutations in one allele of an oncogene (heterozygous mutation, ‘‘one hit’’) is generally believed to be sufficient to confer a selective growth advantage on the cell [1], mutant allele specific imbalance (MASI, Fig. 1a) has been observed in tumors and cell PLoS ONE | www.plosone.org 1 October 2009 | Volume 4 | Issue 10 | e7464