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Clinical Trial
. 2018 Apr 24;2(8):825-831.
doi: 10.1182/bloodadvances.2018015925.

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

Mark J Levis  1 , Alexander E Perl  2 , Jessica K Altman  3 , Christopher D Gocke  1 , Erkut Bahceci  4 , Jason Hill  4 , Chaofeng Liu  4 , Zhiyi Xie  5 , Andrew R Carson  5 , Valerie McClain  5 , Timothy T Stenzel  5 , Jeffrey E Miller  5
Affiliations
Clinical Trial

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

Mark J Levis et al. Blood Adv. .

Abstract

Internal tandem duplications in fms-like tyrosine kinase 3 (FLT3-ITDs) are common in acute myeloid leukemia (AML) and confer a poor prognosis. A sensitive and specific assay for the detection of minimal residual disease (MRD) in FLT3-ITD mutated AML could guide therapy decisions. Existing assays for MRD in FLT3-ITD AML have not been particularly useful because of limited sensitivity. We developed a sensitive and specific MRD assay for FLT3-ITD mutations using next-generation sequencing. The initial validation of this assay was performed by spiking fixed amounts of mutant DNA into wild-type DNA to establish a sensitivity of detection equivalent to ≥1 FLT3-ITD-containing cell in 10 000, with a minimum input of 100 000 cell equivalents of DNA. We subsequently validated the assay in bone marrow samples from patients with FLT3-ITD AML in remission. Finally, we analyzed bone marrow samples from 80 patients with FLT3-ITD relapsed/refractory AML participating in a trial of a novel FLT3 inhibitor, gilteritinib, and demonstrated a relationship between the mutation burden, as detected by the assay, and overall survival. This novel MRD assay is specific and 2 orders of magnitude more sensitive than currently available polymerase chain reaction- or next-generation sequencing-based FLT3-ITD assays. The assay is being prospectively validated in ongoing randomized clinical trials.

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Conflict of interest statement

Conflict-of-interest disclosure: M.J.L. has received consultancy fees and research funding from Astellas and Novartis, honoraria from Daiichi-Sankyo and Novartis, and research funding from Fujifilm and Millenium Pharmaceuticals/Takeda Pharmaceuticals. A.E.P. has received consultancy fees from Astellas, Daiichi-Sankyo, Novartis, Pfizer, and Arog Pharmaceuticals and has participated in advisory boards for Actinium Pharmaceuticals and Asana Biosciences. J.K.A has received consultancy fees from Astellas, Syros, Bristol-Myers Squibb, Celgene, Ceplene, Novartis, and Janssen Pharmaceuticals and has served as an educational speaker for the American Society of Hematology and the National Comprehensive Cancer Network. J.H., C.L., and E.B. are employees of Astellas Pharma, Inc. J.H. has ownership of Ligacept, LLC and patents (US7852995B2 [issued] and W02013163419A1 [pending]). A.R.C., J.E.M., T.T.S., V.M., and Z.X. are employees of Invivoscribe, Inc. C.D.G. declares no competing financial interests.

Figures

None
Graphical abstract
Figure 1.
Figure 1.
Linearity of NGS-MRD assay. The NGS-MRD assay was performed using mutant DNA spiked into wild-type DNA. Two cell lines, each expressing different ITD mutations, were used (cell line A [MV4-11], 30 bp and cell line B [PL-21], 126 bp). The results of the assay were plotted after linear conversion as detected vs expected. Results of regression analysis are shown.
Figure 2.
Figure 2.
OS for subjects achieving remission in the CHRYSALIS study (ASP2215-CL-0101) according to detectable MRD. Kaplan-Meier analysis of survival for subjects treated with FLT3-inhibitory doses of gilteritinib (ASP2215) who achieved a CR, CRi, or CRp, according to the presence or absence of MRD detected by the NGS-MRD assay after treatment. MRD is defined as FLT3-ITD VAF ≥10−4. CI, confidence interval; NA, not achieved.
Figure 3.
Figure 3.
OS for subjects in the CHRYSALIS study (ASP2215-CL-0101) according to molecular response. Kaplan-Meier analysis of survival for subjects treated with FLT3-inhibitory doses of gilteritinib (ASP2215), according to ITD VAF detected by the NGS-MRD assay after treatment.
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References

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