Residual disease detection using targeted parallel sequencing predicts relapse in cytogenetically normal acute myeloid leukemia

Abstract

Despite achieving complete remission after intensive therapy, most patients with cytogenetically normal (CN) AML relapse due to the persistence of submicroscopic residual disease. In this pilot study, we hypothesized that detection of leukemia-specific mutations following consolidation treatment using a targeted parallel sequencing approach predicts relapse. We included 34 AML patients of whom diagnostic material and remission bone marrow slides after at least one cycle of consolidation were available. Isolated DNA was screened for mutations in 19 genes using an Ion Torrent sequencing platform. Furthermore, the variant allelic frequency of distinct mutations was validated by digital PCR and sequencing using a barcoding approach. Twenty-seven out of 34 patients could be analyzed for mutation clearance. We identified 68 somatic mutations at diagnosis (median, 3 mutations per patient; range 1-5) and 22 of these were still detected in 16 patients after consolidation therapy with a reliable sensitivity of 0.5% (median, 1 mutation; range 0-3). The most frequent noncleared mutations were found in DNMT3A. However, as persistence of these mutations has recently been shown to be without any impact on relapse risk, we performed survival and relapse risk analysis excluding DNMT3A mutations. Importantly, persistence of non-DNMT3A mutations was associated with a higher risk of AML relapse (7/8 pts versus 6/19 pts; P = .013) and with a shorter relapse-free survival (333 days vs. not reached; log-rank P = .0219). Detection of residual disease by routine targeted parallel sequencing proved feasible and effective as persistence of somatic mutations other than DNMT3A were prognostic for relapse in CN AML.

Accurate and Sensitive Analysis of Minimal Residual Disease in Acute Myeloid Leukemia Using Deep Sequencing of Single Nucleotide Variations

“…15 Recently, clearance of mutations <0.5% and 0.01% (using deep sequencing and droplet digital PCR, respectively) was shown to be associated with decreased risk of relapse. 17,18 Morita et al 47 reported significantly better 2-year overall survival for AML patients with mutation clearance VAF <1% as well as complete mutation clearance in complete remission, but not if the VAF of residual mutations was <2.5%. The combined use of MFC-MRD and mutation clearance in complete remission was shown by Jongen-Lavrencic et al 48 to confer additive prognostic value for relapse rate and overall survival compared with either method alone.…”

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

“…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.…”

Sensitive and broadly applicable residual disease detection in acute myeloid leukemia using flow cytometry‐based leukemic cell enrichment followed by mutational profiling

“…were analyzed by dPCR as described. 20 In brief, after DNA extraction using the QIAamp DNA Micro Kit (Qiagen, Hilden, Germany) dPCR was performed in duplicates using QuantStudio 3D Master Mix v2 and a QuantStudio 3D Digital PCR System (Applied Biosystems).…”

“…Sequencing of other mutations was done using an Ion Torrent platform as described. 20 as competing risk. 9 The Fine and Gray model was used for univariate and multivariate assessment of risk factors for relapse using cmprisk 2.2-8.…”