Abstract. The development of targeted therapies in cancer has accelerated the development of molecular diagnosis. This new cancer discipline is booming, with an increasing number of gene alterations to analyze in a growing number of patients. To deal with this fast-developing activity, current analysis techniques (Sanger sequencing, allelic discrimination and high resolution melting) take more and more time. In recent years, next generation sequencing (NGS) technologies have appeared and given new perspectives in oncology. In this study, we analyzed FFPE lung and colon carcinomas using the Truseq Cancer Panel, which analyzes the mutation hotspots of 48 genes. We also tested the use of whole-genome amplification before NGS analysis. NGS results were compared with the data obtained from routine diagnosis. All of the alterations routinely observed were identified by NGS. Moreover, NGS revealed mutations in the KRAS and EGFR genes in patients diagnosed as wild-type by routine techniques. NGS also identified concomitant mutations in EGFR and KRAS or BRAF mutations, and a 15-nt deletion in exon 19 of EGFR in colon carcinomas. The study of the other genes sequenced in the Panel revealed 14 genes altered by 27 different mutations and three SNP with a possible role in cancer susceptibility or in the response to treatment. In conclusion, this study showed that NGS analysis could be used for the analysis of gDNA extracted from FFPE tissues. However, given the high sensitivity of this technology, highthroughput clinical trials are needed to confirm its reliability for the molecular diagnosis of cancer.
IntroductionLung and colon cancers are among the main causes of death in developed countries. The life expectancy of patients is very limited, especially in metastatic disease. Nevertheless, in recent years, the development of targeted therapies (Tyrosine Kinase inhibitors or inhibitors of receptors which hyperactivate survival pathways) has shown great therapeutic promise. For example, patients with a mutated EGFR gene and wild-type KRAS gene lung tumor are eligible for gefitinib therapy (1). In colon cancer, patients with wild-type KRAS and BRAF could be treated with panitumumab or cetuximab (2). In skin cancer, vemurafenib (3) or imatinib (4) can be used to treat mutated BRAF (V600E) or c-KIT melanoma, respectively. As the efficacy of these targeted therapies depends on specific genetic abnormalities, molecular diagnosis has become essential for the treatment of cancers. Since 2008, molecular biology platforms have screened for genetic alterations in EGFR (exons 18-21), KRAS (codons 12 and 13) and BRAF (codon 600). At the beginning, the gold standard was Sanger sequencing, but the technique has low sensitivity and is expensive. With the increasing number of samples and gene alterations to screen for, alternon-amplified techniques were developed. For example, allelic discrimination (5) was developed to screen for a specific mutation quickly at a relatively low price. In parallel, screening technologies, such as High Resolut...
