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C. Sargas et al.
Introduction
Acute myeloid leukemia (AML) is a heterogeneous dis- ease characterized by a wide spectrum of molecular alter- ations that lead malignant transformation of normal hematopoietic cells.1 The relevance of chromosomal alter- ations and gene variants for diagnosis, risk stratification and choice of targeted therapies (i.e, FLT3 and IDH1/2 inhibitors) has remarkably increased the complexity of rou- tine molecular diagnostic strategies.2–5 Next-generation sequencing (NGS) has been established as a new molecular diagnostic tool rapidly adopted by clinical laboratories, being able to simultaneously assess different genetic alter- ations such as rearrangements, single nucleotide variants (SNV), insertions-deletions (indels) and copy number varia- tions (CNV) in a wide variety of genes.6 NGS gene panels have been preferentially adopted rather than whole genome or exome sequencing due to an easier results inter- pretation, lower cost and less time consumption, as well as higher read deep needed for low frequency variant detec- tion. Compared to NGS, conventional single-gene approaches by polymerase chain reaction (PCR)7 are labori- ous, time-consuming and less efficient to detect minor clones, but they are still needed as rapid-screening tests for druggable variants.8
The new scenario for AML molecular diagnosis, requiring rapid screening by conventional PCR and comprehensive characterization by NGS, is a great challenge for molecular biology laboratories. For this purpose, the PETHEMA (Programa Español de Tratamientos en Hematología) group established a nationwide network involving seven central laboratories aimed to deliver molecular results to clinics for newly diagnosed and relapsed/refractory AML patients. The first step was to ensure appropriate logistic support, including geographical localization of highly skilled central laboratories strategically distributed according to popula- tion density and distance. The second step was to harmo- nize NGS and PCR techniques methodology and result reporting across the seven central laboratories, establishing consensus panel genes, quality metrics cutoffs and variant reporting criteria.
In this work, we performed the first analysis of a NGS- AML study (clinicaltrials gov. Identifier: NCT03311815), reporting the technical cross-validation results for NGS panel genes during the standardization process and the clin- ical validation in 823 samples of 751 patients with newly diagnosed or refractory/relapse AML.
Methods
Study design and reference laboratories
This was a prospective, multi-center, non-interventional study, performed in seven Spanish PETHEMA central laboratories: Hospital Universitario La Fe (HULF, Valencia), Hospital Universitario de Salamanca (HUS, Salamanca), Hospital Universitario 12 de Octubre (H12O, Madrid), Hospital Universitario Virgen del Rocío (HUVR, Sevilla), Hospital Universitario Reina Sofía (HURS, Córdoba), Hospital Universitario de Gran Canaria Dr. Negrín (HUDN, Las Palmas de Gran Canaria) and CIMA LAB Diagnostics (UNAV, Pamplona) (see the Online Supplementary Appendix for further details).
Inclusion criteria
relapsed/refractory AML (excluding acute promyelocytic leukemia) according to the World Health Organization criteria (2008), regardless of the treatment received, were eligible for the NGS-AML study. The Institutional Ethics Committee for Clinical Research of each institution approved this study. Written informed consent in accordance with the recommendations of the Declaration of Human Rights, the Conference of Helsinki, and institutional regulations were obtained from all patients.
Cross-validation
The first cross-validation round was developed to evaluate the starting situation of reference laboratories (see the Online Supplementary Appendix). For this purpose, four samples harboring 24 variants were distributed from HULF (coordinator center) and each laboratory carried out NGS analysis according to their already implemented protocols. Reports were sent to the coordinator cen- ter to analyze the results.
Taking into account the obtained results, the collaborative group established a set of relevant AML genes and minimum quality metrics criteria. Then, a second cross-validation round was designed to strengthen the established quality parameters, the con- sensus recommendations, and variant reporting for NGS analysis among the seven reference laboratories. Variant detection, variant allele frequency (VAF), dispersion among centers and variant reporting (clinically and non-clinically relevant variants) were assessed in six samples with 30 variants (five with a lower VAF than 5%). Reports were sent to the coordinator center to analyze the results.
Consensus genes establishment
Thrirty genes were established as key genes for AML pathogen- esis: ABL1, ASXL1, BRAF, CALR, CBL, CEBPA, CSF3R, DNMT3A, ETV6, EZH2, FLT3, GATA2, HRAS, IDH1, IDH2, JAK2, KIT, KRAS, MPL, NPM1, NRAS, PTPN11, RUNX1, SETBP1, SF3B1, SRSF2, TET2, TP53, U2AF1 and WT1. ASXL1, CEBPA, FLT3, IDH1, IDH2, NPM1, RUNX1, and TP53 were mandatory for their implication in clinical guidelines, targeted therapy and risk stratification. The remaining genes were recommended for NGS panels, according to laboratory features and sequencing panel options.
Sequencing platforms and panels
The sequencing platform and panel were selected by each labo- ratory using the following criteria: i) to include all eight mandatory genes and, ii) to include the maximum number of other 22 relevant genes (sequencing platforms and panels data are shown in the Online Supplementary Appendix and Online Supplementary Table S1).
Clinical validation
NGS was performed according to already implemented proto- cols and the consensus parameters established in both cross-vali- dation rounds. Samples meeting the quality metrics criteria estab- lished in previous standardization rounds were considered in the clinical validation.
Statisticalanalyses
All statistics were performed using SPSS version 22 (IBM, Armonk, NY, USA) and GraphPad Prism 4 (GraphPad, La Jolla, CA, USA) software programs. A P-value (P) <0.05 was considered sta- tistically significant (see the Online Supplementary Appendix).
Results
Cross-validation
All adult patients (≥18 years) with newly diagnosed or
In the first cross-validation round (pre-standardization),
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