The Interlaboratory Robustness of Next-Generation Sequencing (IRON) Study Phase II: Deep-Sequencing Analyses of Hematological Malignancies Performed by an International Network Involving 26 Laboratories

• Published in: Blood 2012-11, Vol.120 (21), p.1399-1399
• Main Authors: Kohlmann, Alexander, Martinelli, Giovanni, Hofmann, Wolf-Karsten, Kronnie, Geertruy, Chiaretti, Sabina, Preudhomme, Claude, Tagliafico, Enrico, Hernandez, Jesus, Gabriel, Christian, Lion, Thomas, Vandenberghe, Peter, Polakova, Katerina Machova, Béné, Marie-Christine, Brueggemann, Monika, Cazzaniga, Giovanni, Yeoh, Allen, Lehmann, Soren, Ernst, Thomas, Leibundgut, Elisabeth Oppliger, Ozbek, Ugur, Mills, Ken I, Dugas, Martin, Thiede, Christian, Spinelli, Orietta, Foroni, Letizia, Jansen, Joop H., Hochhaus, Andreas, Haferlach, Torsten
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V120.21.1399.1399

Abstract 1399 Massively parallel pyrosequencing in picoliter-sized wells is an innovative technique and allows highly-sensitive deep-sequencing to detect molecular aberrations. As an international consortium we had investigated previously the robustness, precision, and reproducibility of 454 amplicon next-generation sequencing (NGS) across 10 laboratories from 8 countries (Kohlmann et al., Leukemia, 2011;25:1840–8). Aims: In Phase II of the study we now established distinct working groups for various hematological malignancies, i.e. acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphatic leukemia (CLL), chronic myelogenous leukemia (CML), myelodysplastic syndromes (MDS), and myeloproliferative neoplasms (MPN). 26 laboratories from 13 countries are currently part of the research consortium. Each working group selected gene targets and developed amplicons of interest to be studied in various hematological malignancies by deep-sequencing (454 Life Sciences, Branford, CT). In total, 74 genes were identified by the study centers to be of interest for mutational screenings in the respective scientific working groups. Overall, 1146 primer sequences resulting in 573 amplicons were designed and tested. Where appropriate, individual genes were combined into panels and validated designs were set up as standardized preconfigured oligonucleotide primer plates. So far, in AML 679 cases had been screened for CEBPA mutations. RUNX1 mutations were analyzed in 864 cases applying the deep-sequencing read counts to study the stability of such mutations at relapse and the utility of this marker to detect minimal residual disease. Analyses on DNMT3A (n=126) and BCOR (n=83) were focused to perform landscape analyses and to investigate the prognostic utility of these markers. Additionally, this working group is focusing on TET2, ASXL1, and TP53 (n=195) analyses. A novel prognostic model is being developed allowing to stratify AML into prognostic subgroups based on molecular markers only. In ALL, 236 pediatric and adult cases have been screened for TP53 mutations both at diagnosis and relapse of ALL. Pediatric and adult leukemia expert labs developed content to study the mutation incidence of other B and T lineage markers such as IKZF1, JAK2, IL7R, PAX5, LEF1, CRLF2, PHF6, WT1, JAK1, PTEN, AKT1, IL7R; NOTCH1, or FBXW7. Interestingly, the molecular landscape of CLL is changing rapidly. As such, a separate working group focused on analyses including NOTCH