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Haematologica. 2019 Feb;104(2):288-296. doi: 10.3324/haematol.2018.194712. Epub 2018 Aug 9.

A novel deep targeted sequencing method for minimal residual disease monitoring in acute myeloid leukemia.

Author information

1
Hematology Department, Hospital Universitario 12 de Octubre, Madrid.
2
Hematological Malignancies Clinical Research Unit, CNIO, Madrid.
3
Centro de Investigación Biomédica en Red Cáncer (CIBERONC), Madrid.
4
Complutense University, Madrid.
5
Hematology Department, Hospital Santa Creu i Sant Pau, Barcelona.
6
Hematology Department, Hospital Universitario Sanchinarro, Madrid.
7
Hematology Department, Hospital Universitario Ramon y Cajal, Madrid.
8
Hematology Department, Hospital Universitario La Fe, Valencia.
9
Department of Hematology, Hospital Universitario de Getafe, Madrid.
10
Hematology Department, Hospital Universitario Principe de Asturias, Madrid.
11
Hematology Department, Hospital Clínico San Carlos, IdISSC, UCM, Madrid.
12
Hematology Department, Hospital Universitario de la Princesa, Madrid.
13
Hematology Department, Hospital Universitario Severo Ochoa, Madrid.
14
Hematology Department, Hospital Universitario Fundación Alcorcón, Madrid, Spain.
15
Hematology Department, Hospital Universitario 12 de Octubre, Madrid rosam.ayala@salud.madrid.org.

Abstract

A high proportion of patients with acute myeloid leukemia who achieve minimal residual disease negative status ultimately relapse because a fraction of pathological clones remains undetected by standard methods. We designed and validated a high-throughput sequencing method for minimal residual disease assessment of cell clonotypes with mutations of NPM1, IDH1/2 and/or FLT3-single nucleotide variants. For clinical validation, 106 follow-up samples from 63 patients in complete remission were studied by sequencing, evaluating the level of mutations detected at diagnosis. The predictive value of minimal residual disease status by sequencing, multiparameter flow cytometry, or quantitative polymerase chain reaction analysis was determined by survival analysis. The sequencing method achieved a sensitivity of 10-4 for single nucleotide variants and 10-5 for insertions/deletions and could be used in acute myeloid leukemia patients who carry any mutation (86% in our diagnostic data set). Sequencing-determined minimal residual disease positive status was associated with lower disease-free survival (hazard ratio 3.4, P=0.005) and lower overall survival (hazard ratio 4.2, P<0.001). Multivariate analysis showed that minimal residual disease positive status determined by sequencing was an independent factor associated with risk of death (hazard ratio 4.54, P=0.005) and the only independent factor conferring risk of relapse (hazard ratio 3.76, P=0.012). This sequencing-based method simplifies and standardizes minimal residual disease evaluation, with high applicability in acute myeloid leukemia. It is also an improvement upon flow cytometry- and quantitative polymerase chain reaction-based prediction of outcomes of patients with acute myeloid leukemia and could be incorporated in clinical settings and clinical trials.

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