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
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 
Published: October 2019

Abstract
Abstract 1399

Introduction:
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).

Results:
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 NOTCH1, SF3B3, MYD88, XPO1, FBXW7 and BIRC3. 541 cases were screened already to investigate the range of mutational burden of NOTCH1 mutations for their prognostic relevance in a large unselected cohort of adult CLL. In MDS, RUNX1 mutation analyses were performed in 898 cases. Further, the prognostic relevance of TP53 mutations in MDS with isolated deletions of chromosome 5q was studied in a cohort including 105 MDS 5q- cases. Additional content was developed targeting genes of the cellular splicing component, e.g. SF3B1, SRSF2, SF1, U2AF1, ZRSR2. In BCR-ABL-negative MPN, 10 genes of interest (JAK2, MPL, EZH2, IDH1, IDH2, TET2, CBL, IKZF1, SH2B3, ASXL1) have been analyzed in a cohort of 170 cases searching for novel somatic mutations addressing their relevance for disease progression and leukemia transformation. Moreover, an assay was developed and applied to 10 CMML cases allowing the simultaneous analysis of 25 leukemia-associated target genes in a single sequencing using just 20 ng of starting DNA. A group of laboratories focused on ultra-deep sequencing analyses of the BCR-ABL tyrosine kinase domain. Analyses were performed so far on 106 cases to study the dynamics of expansion of mutated clones under various tyrosine kinase inhibitors.

Conclusion:
A comprehensive molecular characterization of hematological malignancies today requires high diagnostic sensitivity and specificity. As part of the IRON-II study, a network of laboratories studied a variety of hematological diseases applying standardized amplicon-based deep-sequencing assays. Distinct working groups have been forged to address scientific questions and in total 4013 cases had been analyzed thus far.

Disclosures:
Kohlmann: Roche Diagnostics: Honoraria; MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership; Roche Diagnostics: Research Funding.