Abstract 2232: Next generation sequencing of the EGFR signaling pathway in colon cancer tumors from Chilean patients
Authors: Alvarez Karin , Orellana Paulina , Villarroel Cynthia , Encina Gonzalo , Simian Daniela , Estay Camila , Kobayashi Maki , Kawashi Hiroshi , Kronberg Udo , Lopez Francisco , Carvallo Pilar 
Published: May 2015

Abstract
Introduction: The Epidermal Growth Factor Receptor (EGFR) signaling pathway regulates key cell functions like proliferation and survival. This pathway presents two signaling cascades, RAS/RAF/MAPK and PIK3/PTEN/AKT/mTOR. Mutations in different components of the EGFR pathway have been described in different cancers. This pathway is a therapeutic target for monoclonal antibodies and tyrosine kinase inhibitors based therapies. Additionally, in advanced colorectal cancer patients, somatic KRAS mutations are response predictors for biological therapy with anti-EGFR antibody. The sensitivity of the mutations detection methods is key to a proper application of personalized therapeutic strategies. Traditionally, somatic mutations are detected by Sanger sequencing and real-time PCR. We determined the presence of mutations in specific genes of the EGFR pathway in colon cancer tumors from Chilean patients. Methods: DNA was extracted from twenty-eight fresh frozen tumor samples. DNA libraries were created using SureSelect (Agilent) designed for the analysis of EGFR, KRAS, BRAF, MAP2K1, PIK3CA, PIK3R1 and PTEN genes. Next generation sequencing was performed in MiSeq sequencer (Illumina) at Sistemas Genómicos (Spain). Results: Sequencing coverage varied between 100 and 250 readings between tumors, which assures the real presence of the changes. This technique allows detecting mutations that are present in a smaller percentage of tumoral cells, as low as 20% in the sample. Pathogenic mutations were identified in 19 out of the 28 tumors. Mutations were found in KRAS (p.Gly12Asp, p.Gly12Arg, p.Gly12Val and p.Gly12Cys), BRAF (p.Val600Glu and p.Gly606Glu), PIK3CA (p.Glu542Lys, p.Glu545Lys, p.Glu545Gln, p.Ala1035Val, p.Asp1045Val and p.His1047Arg) and PTEN (p.Lys267ArgfsX9). Five tumors presented mutations in both branches of the EGFR signaling pathway (KRAS-PIK3CA, BRAF-PIK3CA and BRAF-PTEN). Three allelic variants were identified in MAP2K1 and PIK3R1. Mutations that were identified in KRAS, BRAF and PIK3CA genes corresponded to gain of function mutations while a change found in PTEN gene corresponded to a loss of function mutation. Finally, mutations in PIK3CA and KRAS of 6 tumors were not previously detected by Sanger sequencing. Discussion: Mutations were found in 68% of analyzed tumors. Eighteen percent of the tumors presented mutations in two genes. Forty-six percent of mutations were identified in KRAS gene. Three allelic variants of unknown significance were detected which have not been described in cancer mutations nor in SPNs databases. Therefore, it is necessary to perform functional studies to determine the effect on protein function. Next generation sequencing allowed us to obtain more reliable and reproducible results, and was able to detect mutations present in a low percentage of tumoral cells in the sample, maybe due to tumoral heterogeneity or contamination with normal cells. FONDECYT 1111020
Citation Format: Karin Alvarez, Paulina Orellana, Cynthia Villarroel, Gonzalo Encina, Daniela Simian, Camila Estay, Maki Kobayashi, Hiroshi Kawashi, Udo Kronberg, Francisco Lopez, Pilar Carvallo. Next generation sequencing of the EGFR signaling pathway in colon cancer tumors from Chilean patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2232. doi:10.1158/1538-7445.AM2014-2232