Assessment of the clinical utility of four NGS panels in myeloid malignancies. Suggestions for NGS panel choice or design

Abstract

The diagnosis of myeloid neoplasms (MN) has significantly evolved through the last few decades. Next Generation Sequencing (NGS) is gradually becoming an essential tool to help clinicians with disease management. To this end, most specialized genetic laboratories have implemented NGS panels targeting a number of different genes relevant to MN. The aim of the present study is to evaluate the performance of four different targeted NGS gene panels based on their technical features and clinical utility. A total of 32 patient bone marrow samples were accrued and sequenced with 3 commercially available panels and 1 custom panel. Variants were classified by two geneticists based on their clinical relevance in MN. There was a difference in panel's depth of coverage. We found 11 discordant clinically relevant variants between panels, with a trend to miss long insertions. Our data show that there is a high risk of finding different mutations depending on the panel of choice, due both to the panel design and the data analysis method. Of note, CEBPA, CALR and FLT3 genes, remains challenging the use of NGS for diagnosis of MN in compliance with current guidelines. Therefore, conventional molecular testing might need to be kept in place for the correct diagnosis of MN for now. OPEN ACCESS Citation: Aguilera-Diaz A, Vazquez I, Ariceta B, Mañú A, Blasco-Iturri Z, Palomino-Echeverría S, et al. (2020) Assessment of the clinical utility of four NGS panels in myeloid malignancies. Suggestions for NGS panel choice or design. PLoS ONE 15(1): e0227986. https://doi.org/10.

Clinical performance and utility of a comprehensive next-generation sequencing DNA panel for the simultaneous analysis of variants, TMB and MSI for myeloid neoplasms

“…Also, the two FlT3 ITD variants in the Seraseq Myeloid Mutation DNA were not detected. The same difficulty has been documented for the large deletion of 52 bp in the CALR gene [10,22,23]. The CALR 52 bp deletion was not detected by the panel in the three replicates of Seraseq Myeloid Mutation DNA.…”

“…The list of genes altered in myeloid neoplasms is rapidly increasing, as is evident from the above mentioned profiles. Currently, the NGS panels used in clinical laboratories are limited in coverage (typically~54 genes) which leads to incomplete definition of the mutation profile, often excluding important known hot spots thus impeding identification of a complete personalized diagnosis [ 9 , 10 ].…”

“…Recent reports have highlighted that the sequencing of CEBPA , CALR , and FLT3 genes remains a challenge on NGS platforms. The difficulty of detecting long FLT3 ITD variants on NGS platforms has been reported previously, which might be because the chemistries employ short read sequencing (read length 50-300bp) that makes them prone to lose structural variants such as long indels [ 10 , 21 , 22 ]. In alignment with these reports, three Flt3 ITD confirmed by Sanger sequencing in three clinical samples were not detected in this current study.…”

“…However, many laboratories have used relatively small targeted panels that screen prominent mutation hotspots in ≤ 50 genes. Although this approach is cost- and time- effective with minimal data analysis and reporting complexity, it yields an incomplete mutational profile, omitting several important known hotspot mutations [ 9 , 10 ]. Molecular profiling by NGS methodology has already introduced a paradigm shift in detecting pathogenic variants, however, recognizing the extensive molecular heterogeneity of these neoplasms, there is a dire need to investigate these tumors on a high-throughput NGS platform for comprehensive genetic screening.…”

Impact of Integrated Genetic Information on Diagnosis and Prognostication for Myeloproliferative Neoplasms in the Next-Generation Sequencing Era

“…We used a customized myeloid panel, which included disease-associated mutations and prognostic factors. Various commercial and customized NGS assays have been introduced in clinic, and they cover most significant genes for diagnosis and risk stratification in this study [ 47 , 48 ]. Bioinformatics were also incorporated in commercial NGS assays and increase the liability of analyzed results.…”