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Deep sequencing method for MRD monitoring in AML
hematopoietic stem cell transplantation in first complete remission.1 The definition of complete remission for AML includes criteria for the identification of patients with poor prognosis using cytomorphological methods,2 but these studies do not have a good predictive value because most of the patients in complete remission relapse within 3 years of diagnosis.3
Assessment of minimal residual disease (MRD) is critical in monitoring patients in morphological remission, to inform decisions about further therapy.1 Indeed, several studies have reported MRD status as a stronger predictor of relapse, because patients who are MRD negative have a better prognosis than those who are MRD positive.4,5 In support of this, recent non-randomized studies from prospective multicenter trials suggested better outcomes when leukemia therapy was selected based on the results of MRD assessment.6-8
AML is, nevertheless, a biologically complex and het- erogeneous disease, which makes MRD testing more chal- lenging in this condition than in other hematologic neo- plasms such as acute lymphoblastic leukemia or multiple myeloma. The detection of very low levels of MRD by conventional methods such as quantitative (q) polymerase chain reaction (PCR) or multiparameter flow cytometry (MFC) provides powerful independent prognostic infor- mation. Unfortunately, as described for cytomorphologi- cal complete remission, many patients who achieve MRD negative status relapse as a result of the progression of undetected leukemic cells. The most common method for MRD detection is MFC, which has intermediate applica- bility (70–80%) and limited sensitivity.9,10 However, there is no consensus on multi-antibody panels with regards to inter-laboratory performance, and the technique requires a high level of expertise. The other principal method for monitoring MRD, qPCR, has good sensitivity (10-4-10-6), but its applicability is limited to the approximately 40% of patients who present with molecular alterations (RUNX1- RUNX1T1, CBFβ-MYH11 or NPM1) at diagnosis.11
For the above reasons, new methods with higher sensi- tivity, specificity, applicability and performance are need- ed for MRD assessment in AML. Against this background, next-generation sequencing (NGS) and digital PCR (dPCR) have recently emerged as potentially promising platforms for the assessment of MRD.12 Here, we optimized and clinically validated a new deep targeted NGS-based method, supported with dPCR technical validation, for the detection and quantification of MRD [both small insertion/deletions (indels) and single nucleotide variants (SNV)] in AML patients, in an attempt to improve and/or complement the current techniques for MRD evaluation, and to establish its potential as a predictor of patients’ out- come.
Methods
More detailed information can be found in the Online Supplementary Data (1–6).
Patients and samples
One hundred and ninety patients with de novo or secondary non–M3 AML were included in mutational profile screening at diagnosis. We performed a new selection for retrospective MRD assessment using the following criteria: presence of the NPM1 type A mutation, or SNV in FLT3, IDH1 and/or IDH2 at diagno-
sis, and availability of at least one follow-up genomic (g)-DNA sample.
The MRD approach was applied to 51 (48%) follow-up sam- ples taken after induction therapy and 55 (52%) taken after con- solidation, corresponding to 63 patients diagnosed between 2006 and 2016 (for selection criteria see Online Supplement 6 and Supplementary Table S1). Patients were treated according to PETHEMA (Programa Español de Tratamientos en Hematología) or CETLAM (Grupo cooperativo de Estudio y Tratamiento de Leucemias Agudas y Mielodisplasias) protocols. The study was conducted according to Spanish law 14/2007 on biomedical research, and was approved by the Research Ethics Board of each participating institution. All patients provided informed consent. The main clinical characteristics of the patients are summarized in Table 1. All patients achieved complete remission according to cytomor- phological criteria after induction therapy (<5% of bone marrow blasts).
To construct calibration curves, commercial (Horizon Discovery, UK) reference standard gDNA was used for somatic SNV in IDH1 (R132C) and IDH2 (R172K). As a further source of gDNA, we used the OCI-AML3 cell line (ACC 582, DSMZ, Germany) with the NPM1 type A mutation (c.863_864insCCTG) to examine indels. As OCI-AML3 cells also present a SNV in DNMT3A (R882C), this was included only for technical optimization.
Deep targeted sequencing workflow
The sequencing workflow included a first study at diagnosis and a second study at follow-up. Mutational profile screening at diagnosis was done with a customized NGS myeloid panel of 32 genes frequently mutated in myeloid diseases,13 (Online Supplementary Table S2) and NPM1 analysis was carried out with qPCR.14
The specific mutations detected at diagnosis were studied at fol- low-up. We first tested a variety of experimental steps to define optimal conditions (Online Supplement 1). We established an opti- mal protocol (Figure 1) that included DNA amplification, library preparation and sequencing as experimental steps (Online Supplement 2).
Libraries were sequenced on the Ion Proton System platform (Life Technologies, Thermo Fisher Scientific Inc.) with an estimat- ed depth ≥1,000,000 of reads, generating .fastq files. These files were analyzed using a customized bioinformatic pipeline; which leads from the .fastq file and a .csv file that contains information about name identifier, run and barcode identifier, chromosomal position and the variant detected in the diagnosis to be evaluated in the follow-up sample. Through Ensembl Perl API,15 the aligned mutated sequence and the aligned wild-type (wt) sequence are presented in FASTA format (sequences of 40 bp). Finally, we obtained a .csv file containing the name identifier, run and barcode identifier, chromosomal position, the variant, the specific target sequence in FASTA format (mutated forward, mutated reverse, wt forward and wt reverse), the counts of each and the ratio (mutat- ed/wt) in absolute values.
Results
A high percentage of acute myeloid leukemia patients could benefit from deep sequencing minimal residual disease assessment
In total, 211 (80%) SNV and 46 (20%) indels were detected in the 190 patients analyzed at diagnosis using the customized NGS panel. We detected one variant (SNV or indel) in 48 (25%) cases, two or more variants in 116
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