Validation of a Next-Generation Sequencing Panel for AML Routine Diagnosis
Authors: Alonso* Carmen M , Llop* Marta , Ibáñez Mariam , Cervera Jose , Such Esperanza , Villamon Eva , Palanca Sarai , de Juan Inmaculada , Murria Rosa , de Unamuno Blanca , Pedrola Laia , Martin Ivan , Montesinos Pau , Bolufer Pascual , Sanz Miguel A. , Barragan Eva 
Published: October 2019

Abstract
Numerous recurrently mutated genes have been described in the last years for acute myeloid leukemia (AML). Nevertheless, only a few of them (NPM1, CEBPA, FLT3), and usually individually analyzed, have been systematically incorporated into clinical laboratories to stratify prognosis and guide risk-adapted therapy. So far, technology made impossible their simultaneous study, however, development of high throughput techniques as "next generation sequencing" (NGS) allows a parallel assay. Recently, our group designed a NGS panel (custom panel) interrogating the complete coding sequence of 87 genes potentially involved in AML. Interactome analysis revealed that 13 genes were highly implicated in AML pathogenesis. Although NGS has been broadly utilized in the investigational scope, its application to the routine diagnosis is still far from become a standard. The aim of this study is to validate the clinical applicability to routine laboratories of a hotspot NGS panel that includes these 13 genes and other potentially actionable targets.
We included 62 "de novo" AML patients in which we tested the Ion Ampliseq AML community panel (Life Technologies) (IAAC panel) in the Ion PGM/Proton platforms. This panel includes hotspots of ASXL1 (exon 12), BRAF (V600E), CBL (exons 8-9), FLT3 (codons 676, 830-850), IDH1 (exon 4), IDH2 (exon 4), JAK 2 (exon 14), KIT (exons 8, 10, 11 and 17), KRAS (exons 2-4), NRAS (exons 2-4), PTPN11 (exons 3,7,8,13), RUNX1 (exons 3-8) y WT1 (exons 7 and 9), and the entire coding sequence of CEBPA, DNMT3A, GATA, TET2 and TP53. The design does not include the FLT3-ITD region. This panel requires only a total of 40 ng of DNA, far less than the custom panel (250 ng), which is very convenient in a clinical laboratory, where the input sample can be limited.
The hotspot NGS panel detected 153 variants in 62 patients (2.47 mutations/patient). Mean read depth and uniformity were 1580 and 94.89%, respectively. The IAAC panel detected 25 NPM1 mutations, 20 TET2 and DNMT3A mutations, 14 in CEBPA, 12 in TP53, 11 in RUNX1, 8 in FLT3, 7 in IDH2, 6 in ASXL1, GATA2 and NRAS, 5 in PTPN11 and KRAS, 3 in WT1, 2 in CBL and 1 mutation in IDH1, KIT, and BRAF. Only 4 patients (6%) remained wild-type for these genes after the analysis with the IAAC panel.
These samples had previously been analyzed by conventional molecular biology techniques (CMBT) for FLT3-D835, NPM1-T288, DNMT3A-R882 and CEBPA. The IAAC panel found 100% of these previously known mutations plus 5 extra mutations that were negative by CMBT. These mutations were reconfirmed by Sanger sequencing. Therefore, the IAAC panel detects more mutations than CMBT.
To further assess the suitability of the hotspot panel, we selected a subset of 25 patients that had been also analyzed by the custom panel (Sure Desing Tool (Agilent) and an Illumina platform). This allowed us to analyze the complete coding sequence of the genes included in the IAAC panel and thus look for mutations outside the hotspots. A total of 53 variants were found with the custom panel. Fifty of these variants (95%) were also detected with the IAAC panel. The remaining 3 variants (5%) were located outside the hotspot regions. Additionally, the IAAC panel detected 13 variants in the overlapping regions that were not found with the custom panel. This could be explained because when focusing on recurrent regions of particular genes, it is possible to increase the mean read depth and therefore reach higher sensibility, which can be achieved with the IAAC panel but not with the custom panel.
In conclusion, the Ion Ampliseq AML community panel detects mutations currently analyzed in most of clinical laboratories with validated prognostic relevance (NPM1 and CEBPA) in one assay, with low sample input requirement and with 100% sensibility compared with CMBT. In addition, this panel is able to find out alterations in these genes that are lost by CMBT. Moreover, other mutations with probable diagnostic or prognostic value and/or potential therapeutic targets are also studied and identified with high sensibility. Only a few changes are excluded of the covered regions. Therefore, IAAC panel is useful for routine diagnosis; however, detection of FLT3 internal tandem duplications is not possible, which limits its clinical utility.

Disclosures
No relevant conflicts of interest to declare.