Integration of Technical, Bioinformatic, and Variant Assessment Approaches in the Validation of a Targeted Next-Generation Sequencing Panel for Myeloid Malignancies

Abstract

Context.-Detection of variants in hematologic malignancies is increasingly important because of a growing number of variants impacting diagnosis, prognosis, and treatment response, and as potential therapeutic targets. The use of next-generation sequencing technologies to detect variants in hematologic malignancies in a clinical diagnostic laboratory setting allows for efficient identification of routinely tested markers in multiple genes simultaneously, as well as the identification of novel and rare variants in other clinically relevant genes.Objective.-To apply a systematic approach to evaluate and validate a commercially available next-generation sequencing panel (TruSight Myeloid Sequencing Panel, Illumina, San Diego, California) targeting 54 genes. In this manuscript, we focused on the parameters that were used to evaluate assay performance characteristics.Data Sources.-Analytical validation was performed using samples containing known variants that had been identified previously. Cases were selected from different disease types, with variants in a range of genes. Panel performance characteristics were assessed and genomic regions requiring additional analysis or wet-bench approaches identified.Conclusions.-We validated the performance characteristics of a myeloid next-generation sequencing panel for detection of variants. The TruSight Myeloid Sequencing Panel covers more than 95% of target regions with depth greater than 5003. However, because of unique variant types such as large insertions or deletions or genomic regions of high GC content, variants in CEBPA, FLT3, and CALR required supplementation with non-next-generation sequencing assays or with informatics approaches to address deficiencies in performance. The use of multiple bioinformatics approaches (2 variant callers and informatics scripts) allows for maximizing calling of true positives, while identifying limitations in using either method alone.

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

“…All panels showed mean coverage over 1000x. However, we observed that TSMP did not cover CEBPA gene as homogeneously as the other panels; this might be because TSMP is an amplicon-based panel, and CEBPA is a one-exon gene lying within a CpG Island [20]. Therefore, PCR-based library preparation struggles to amplify (and capture) this gene, challenging the detection of variants in CEBPA gene (S3 Fig i.…”

“…FLT3 exons 14 and 15 were assessed by PCR and capillary electrophoresis using 5ng of genomic DNA per samples to detect the presence of internal tandem duplications (ITD) [19]. The ratio of FLT3-ITD to wild-type FLT3 was quantified by the Applied Biosystems sequencing software GeneScan 1 as described previously [20]. FLT3 exon 20 was tested by PCR and RFLP analysis for presence of mutations in codons p.Asp835/p.Ile836 [21].…”

CEBPAmutational analysis in acute myeloid leukaemia by a laboratory-developed next-generation sequencing assay

“…We observed TAD2 polymorphism VAFs ranging from 11.0% to 35.1% by CEBNX. This finding appears to be incongruous with the expected VAF of 50%, given the heterozygous status of this widely known polymorphism 16. We believe that this is due to the presence of fragmented libraries that therefore pose challenges for accurate VAF determination of duplication variants.…”

Laser‐driven radiation: Biomarkers for molecular imaging of high dose‐rate effects

“…This radiation‐monitoring approach may allow, in the case of high dose‐rate radiation, for fine tuning of the doses and of the sequences of radiation pulses for personalized therapy, as well as for the development of cotherapies to increase treatment efficacy and to limit radiotherapy side effects …”

“…This radiation-monitoring approach may allow, in the case of high dose-rate radiation, for fine tuning of the doses and of the sequences of radiation pulses for personalized therapy, as well as for the development of cotherapies to increase treatment efficacy and to limit radiotherapy side effects. 12 Molecular imaging techniques are used to monitor functional and metabolic biomarkers for better orienting and monitoring personalized radiotherapeutic strategies. 13 Reliable biomarkers that mirror the effects of radiotherapy in terms of efficacy, side effects, and underlying mechanisms are a prerequisite.…”