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Next-generation sequencing-based multigene mutational screening for acute myeloid leukemia using MiSeq: applicability for diagnostics and disease monitoring.

ABSTRACT: Routine molecular testing in acute myeloid leukemia involves screening several genes of therapeutic and prognostic significance for mutations. A comprehensive analysis using single-gene assays requires large amounts of DNA, is cumbersome and timely consolidation of results for clinical reporting is challenging. High throughput, next-generation sequencing platforms widely used in research have not been tested vigorously for clinical application. Here we describe the clinical application of MiSeq, a next-generation sequencing platform to screen mutational hotspots in 54 cancer-related genes including genes relevant in acute myeloid leukemia (NRAS, KRAS, FLT3, NPM1, DNMT3A, IDH1/2, JAK2, KIT and EZH2). We sequenced 63 samples from patients with acute myeloid leukemia/myelodysplastic syndrome using MiSeq and compared the results with those obtained using another next-generation sequencing platform, Ion-Torrent Personal Genome Machine and other conventional testing platforms. MiSeq detected a total of 100 single nucleotide variants and 23 NPM1 insertions that were confirmed by Ion Torrent or conventional platforms, indicating complete concordance. FLT3-internal tandem duplications (n=10) were not detected; however, re-analysis of the MiSeq output by Pindel, an indel detection algorithm, did detect them. Dilution studies of cancer cell-line DNA showed that the quantitative accuracy of mutation detection was up to an allelic frequency of 1.5% with a high level of inter- and intra-run assay reproducibility, suggesting potential utility for monitoring response to therapy, clonal heterogeneity and evolution. Examples demonstrating the advantages of MiSeq over conventional platforms for disease monitoring are provided. Easy work-flow, high throughput multiplexing capability, 4-day turnaround time and simultaneous assessment of routinely tested and emerging markers make MiSeq highly applicable for clinical molecular testing in acute myeloid leukemia.

SUBMITTER: Luthra R

PROVIDER: S-EPMC3943309 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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Next-generation sequencing-based multigene mutational screening for acute myeloid leukemia using MiSeq: applicability for diagnostics and disease monitoring.

Luthra Rajyalakshmi R Patel Keyur P KP Reddy Neelima G NG Haghshenas Varan V Routbort Mark J MJ Harmon Michael A MA Barkoh Bedia A BA Kanagal-Shamanna Rashmi R Ravandi Farhad F Cortes Jorge E JE Kantarjian Hagop M HM Medeiros L Jeffrey LJ Singh Rajesh R RR

Haematologica 20131018 3

Routine molecular testing in acute myeloid leukemia involves screening several genes of therapeutic and prognostic significance for mutations. A comprehensive analysis using single-gene assays requires large amounts of DNA, is cumbersome and timely consolidation of results for clinical reporting is challenging. High throughput, next-generation sequencing platforms widely used in research have not been tested vigorously for clinical application. Here we describe the clinical application of MiSeq, ...[more]

PMID: 24142997

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Project description:Minimal residual disease (MRD) in acute myeloid leukemia (AML) is typically measured using multiparameter flow cytometry (MFC). Detection of leukemia mutations using multigene next-generation sequencing (NGS) can potentially be used to measure residual disease. We used a targeted 28-gene NGS panel to detect mutations and different-from-normal 10-color MFC to measure MRD in AML patients before allogeneic hematopoietic stem cell transplantation (HCT). Residual disease was defined when any abnormal blast population was detected using MFC and when any leukemia allele was detected with a variant allele frequency (VAF) ≥ 5% using NGS. We tracked the clearance of leukemia alleles between AML diagnosis and immediately before HCT and found that mutations in DNMT3A, TET2, and JAK2 were less likely to be cleared than NPM1, IDH 1/2, and FLT3-ITD. Despite varying sensitivities, the concordance rate of residual disease detection before HCT using the 2 assays was 44 of 62 (71%) evaluable cases. Discordance could be explained by residual mutations in DNMT3A and TET2 that were not detected by MFC and presence of residual leukemia mutations with VAF below the established thresholds for mutation calling. Presence of flow MRD and residual mutations immediately before HCT using the 2 assays was associated with relapse risk (MFC: hazard ratio, 4.62; 95% confidence interval [CI], 1.32 to 16.09; P = .016 and NGS: hazard ratio, 4.35; 95% CI, 1.63 to 11.6; P = .003) and survival (MFC: hazard ratio, 2.44; 95% CI, 1 to 5.97; P = .05 and NGS: hazard ratio, 2.1; 95% CI, .97 to 4.55; P = .059) after HCT. Residual disease detected concurrently by MFC and NGS conferred the highest relapse risk compared with patients who were either negative by both assays or had discordant status (overall, P = .008). Although MFC is universally applicable, a multigene NGS approach to measuring residual disease in AML provides additional information on differential clearance of disease alleles and can assess clonal architecture before transplantation.

Dataset Information

Next-generation sequencing-based multigene mutational screening for acute myeloid leukemia using MiSeq: applicability for diagnostics and disease monitoring.

Publications

Next-generation sequencing-based multigene mutational screening for acute myeloid leukemia using MiSeq: applicability for diagnostics and disease monitoring.

Similar Datasets

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