A next-generation sequencing–based assay for minimal residual disease assessment in AML patients with FLT3-ITD mutations

Levis, Mark J.; Perl, Alexander E.; Altman, Jessica K.; Gocke, Christopher D.; Bahceci, Erkut; Hill, Jason E.; Liu, Chaofeng; Xie, Zhiyi; Carson, Andrew R.; McClain, Valerie; Stenzel, Timothy T.; Miller, Jeffrey E.

doi:10.1182/bloodadvances.2018015925

Cited by 110 publications

(117 citation statements)

References 35 publications

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“…Recent studies utilizing NGS for the detection of MRD in AML vary greatly in their design and technical aspects. Cohorts studied have included AML patients undergoing allogeneic haematopoietic cell transplantation (alloHCT) (Getta et al , ; Kim et al , ; Thol et al , ; Zhou et al , ; Press et al , ), receiving standard induction chemotherapy (Klco et al , ; Gaksch et al , ; Jongen‐Lavrencic et al , ; Morita et al , ; Onecha et al , ; Rothenberg‐Thurley et al , ; Thol et al , ; Wong et al , ), receiving novel therapies in clinical trials (Levis et al , ), or having only specific mutations (Thol et al , ; Kohlmann et al , ; Salipante et al , ; Levis et al , ; Patkar et al , ; Zhou et al , ; Patel et al , ). Also, since most studies to date have had access to diagnostic samples, de novo leukaemia‐associated mutation discovery using remission samples alone remains an important unmet challenge.…”

Section: Current State Of Ngs Mrd Detection In Amlmentioning

confidence: 99%

Next‐generation sequencing for measurable residual disease detection in acute myeloid leukaemia

2019

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Acute myeloid leukaemia (AML) is a blood cancer characterized by acquired genetic mutations. There is great interest in accurately establishing measurable residual disease (MRD) burden in AML patients in remission after treatment but at risk of relapse. However, inter-and intrapatient genetic diversity means that, unlike in the chronic myeloid and acute promyelocytic leukaemias, no single genetic abnormality is pathognomonic for all cases of AML MRD. Next-generation sequencing offers the opportunity to test broadly and deeply for potential genetic evidence of residual AML, and while not currently accepted for such use clinically, is likely to be increasingly used for AML MRD testing in the future.

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Section: Current State Of Ngs Mrd Detection In Amlmentioning

confidence: 99%

Next‐generation sequencing for measurable residual disease detection in acute myeloid leukaemia

2019

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“…Amplicon-based next generation sequencing faces additional design challenges that compromise adequate sequence coverage of the FLT3-internal tandem duplication/wild-type junctions in large FLT3internal tandem duplications, and the uniform amplicon start-stop positions can further complicate bioinformatics algorithms for indel detection. These factors account for the variable success of FLT3-internal tandem duplication detection that has been reported [33][34][35][36][37][38]: it has been detected by hybridization capture-based next generation sequencing at 102-185 bp and less impressively by amplicon-based chemistry at 60-126 bp. The 231 bp FLT3internal tandem duplication identified in our study is larger than previous reports, further supporting the superiority of hybridization capture-based chemistry in FLT3-internal tandem duplication detection by next generation sequencing.…”

Section: Discussionmentioning

confidence: 99%

Hybridization capture-based next generation sequencing reliably detects FLT3 mutations and classifies FLT3-internal tandem duplication allelic ratio in acute myeloid leukemia: a comparative study to standard fragment analysis

Devine

et al. 2020

Modern Pathology

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FLT3-internal tandem duplication occurs in 20-30% of acute myeloid leukemia and confers an adverse prognosis with its allelic ratio being a key risk stratifier. The US Food and Drug Administration recently approved FLT3 inhibitors midostaurin and gilteritinib in FLT3 mutation-positive acute myeloid leukemia. Historically, FLT3 was tested by fragment analysis, which has become the standard method endorsed by international guidelines. However, next generation sequencing is increasingly used at acute myeloid leukemia diagnosis given its ability to simultaneously evaluate multiple clinically informative markers. As FLT3-internal tandem duplication detection was known to be challenging by next generation sequencing and the results carry profound prognostic and therapeutic implications, it is important to thoroughly examine its performance in FLT3-internal tandem duplication detection and allelic ratio classification. In a comparative study with fragment analysis, we retrospectively reviewed our experience using a custom-designed, hybridization capture-based, targeted next generation sequencing panel. Among 7902 cases, FLT3-internal tandem duplication was detected in 335 with variable sizes (3-231 bp) and insertion sites. Fragment analysis was also performed in 402 cases, demonstrating 100% concordance in FLT3-internal tandem duplication detection. In 136 dual-tested, positive cases, 128/136 (94%) exhibited concordant high/low allelic ratio classifications. The remaining 6% showed borderline low allelic ratio by next generation sequencing. The two methods were concordant in FLT3-tyrosine kinase domain mutation detection at the hotspot D835/I836 targeted by fragment analysis. Furthermore, seven mutations which may benefit from FLT3 inhibitor therapy were detected by next generation sequencing, in regions not covered by fragment analysis. Our study demonstrates that using a hybridization capture-based chemistry and optimized bioinformatics pipeline, next generation sequencing can reliably detect FLT3-internal tandem duplication and classify its allelic ratio for acute myeloid leukemia risk stratification. Next generation sequencing also exhibits superior comprehensiveness in FLT3 mutation detection and may further improve personalized, targeted therapy in acute myeloid leukemia.

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“…Disease relapse can occur in patients who received transplantation. Recently, it has been shown that the FLT3-ITD mutation could be a good marker in identifying MRD, in addition tobeing a beneficial guide for using FLT3 inhibitors in AML patients (22)(23)(24). In a recent study, Wan and colleagues showed the predictive value of pretransplant FLT3-ITD levels in 84 patients who underwent allo-HCT during their first remission (25).…”

Section: Discussionmentioning

confidence: 99%

Comparison of Delta- PCR and Conventional Fragment Analysis for the Detection of FLT3-ITD Mutations in Paired Diagnosis-Relapse Samples of Patients with Acute Myeloid Leukemia

Arab

Rostami

Ghandforoush

et al. 2019

J Adv Med Biomed Res

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10.30699/jambs.27.123.38 Background & Objective: FLT3-ITD mutation detection has been an integral part of diagnostic work ups focused on acute myeloid leukemia. However, some studies have indicated that the mutation is unstable during the various stages of the disease. The purpose of this study was to evaluate the stability of this marker in paired diagnosis-relapse samples using Delta-PCR method. Materials & Methods: In this retrospective study, paired diagnosis-relapse bone marrow or peripheral blood samples from 180 adult AML patients were analyzed for FLT3-ITD mutations using conventional fragment analysis and Delta-PCR methods. A dilutional experiment of DNA derived from a FLT3-ITD mutated patient in normal peripheral blood was performed in order to evaluate the sensitivity of each method. Results: All samples were analyzed using both conventional fragment analysis and Delta-PCR methods. FLT3-ITD mutations were detected in 24 diagnostic samples (13.3%) and 28 relapse samples (15.5 %) through conventional fragment analysis. Three out of four patients who were FLT3-ITD positive in the relapse samples had a mutation in the diagnostic samples using the Delta-PCR method. On the other hand, at the time of diagnosis and relapse, the mutation test results were incompatible in only 3.6% of patients based on the results of the Delta-PCR method compared to 14.2 based on conventional fragment analysis. Our findings revealed that the sensitivity of Delta-PCR as related to FLT3-ITD detection was 0.2 %. Compared to the conventional fragment analysis, with a sensitivity of 2%, Delta-PCR shows greater sensitivity and specificity. Conclusion: The conventional testing of the FLT3-ITD mutation by fragment analysis did not detect a significant proportion (11%) of FLT3-ITD positive samples in AML patients. Delta PCR increased the sensitivity and specificity relative to the conventional method. The detection of FLT3-ITD mutation through Delta PCR is important in order to detect minor clones at diagnosis or during the monitoring of AML patients.

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A next-generation sequencing–based assay for minimal residual disease assessment in AML patients with FLT3-ITD mutations

Abstract: Key Points• A sensitive and specific assay was developed for detection of MRD in patients with AML who harbor FLT3-ITD mutations.• This standardized assay is readily available and may be used to guide therapy decisions in patients with AML.

Cited by 110 publications

References 35 publications

Next‐generation sequencing for measurable residual disease detection in acute myeloid leukaemia

Next‐generation sequencing for measurable residual disease detection in acute myeloid leukaemia

Hybridization capture-based next generation sequencing reliably detects FLT3 mutations and classifies FLT3-internal tandem duplication allelic ratio in acute myeloid leukemia: a comparative study to standard fragment analysis

Comparison of Delta- PCR and Conventional Fragment Analysis for the Detection of FLT3-ITD Mutations in Paired Diagnosis-Relapse Samples of Patients with Acute Myeloid Leukemia

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