The knowledge of molecular alterations involved in colorectal carcinoma (CRC) and non-small-cell lung carcinoma (NSCLC) has significantly increased in the past few years. Molecular subgroups of tumors carrying mutations in druggable genes have been identified in CRC and NSCLC. In this respect, the availability of approaches that are compatible with small amounts of input DNA from FFPE tissue and that allow a rapid screening of a large number of somatic mutations is definitely needed. Ion AmpliSeq™ technology introduces a groundbreaking workflow enabling the rapid sequencing of hundreds of mutations with low allele frequency using as little as 10ng of DNA per reaction. The OncoNetwork Consortium is a European collaborative effort of 8 Cancer Translational Research Institutions to evaluate a custom panel targeting hotspots mutations in 22 genes implicated in CRC and NSCLC. In particular, 7 labs will test the gene panel in 3 phases using 180 retrospective FFPE samples previously tested with orthogonal technologies, and 1 lab will perform confirmatory testing on novel SNPs. According to the established workflow, 10 ng of genomic DNA are used with a single primer pool that includes 86 pairs of primers spanning 15.7kb of targets. Individual libraries are barcoded during the ligation step, quantified and pooled 5 by 5 for amplification on spheres using the Ion OneTouch™ 200 Template Kit v2. Spheres are loaded on Ion 316 chip for sequencing with the Ion PGM™ Sequencer using the Ion PGM™ 200 Sequencing Kit. Results are analyzed using the Ion Reporter™ Software v1.2. In the first phase of the project, 7 labs received the same 5 blind, control FFPE samples. The average coverage obtained for the genes included in the panel ranged between 1.000x and 6.000x. The percentage of reads on target was 88-93% for all samples except for a sample showing a mean of 77%, with an average per base accuracy >98% for all the samples. Data analysis confirmed that all laboratories identified the expected mutations, when using a mapping quality value (MQV) of 4 as first filter. In the second phase of the project, each lab submitted to the consortium 10 previously tested CRC or NSCLC FFPE samples and received back 10 blind samples for sequencing. These samples were expected to contain 5 to 10 variants including point mutations and indels. The six labs that completed this phase were able to detect all the expected variants. Therefore, a 100% true positive rate was found in controls and blind study. The third phase of the project is ongoing and involves the assessment in each participating lab of 10 NSCLC and 5 CRC samples with a low percentage of tumor cells. In conclusion, the preliminary results suggest that the Onconetwork Ion AmpliSeq Colon and Lung Cancer panel is able to detect with high sensitivity and reproducibility the most frequent mutations of CRC and NSCLC by using a just 10 ng of DNA from FFPE samples.

Citation Format: Nicola Normanno, Rosella Petraroli, Alain Rico, Henriette Kurth, Bastiaan Tops, Eliana Amato, Andrea Mafficini, Delphine Le Corre, Anna Maria Rachiglio, Anne Reiman, Orla Sheils, Christoph Noppen, Nora Rieber, Jonathan Mangion, Chrysanthi Ainali, Ludovic Lacroix, Ian Cree, Marjolijn Ligtenberg, Andy Felton, Aldo Scarpa, Pierre Laurent Puig. The OncoNetwork Consortium: A European Collaborative Research study on the development of an Ion AmpliSeq gene panel targeting hot spots in colon and lung cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 36. doi:10.1158/1538-7445.AM2013-36