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IDH1/2 mutant allele burden in AML
abnormalities as targets for MRD assessment in AML, such as mutations in isocitrate dehydrogenase (IDH) 1 and 2.
IDH1/2 mutations affecting IDH1R132, IDH2R140, and IDH2R172 residues are single-nucleotide mutations that collectively occur in 15-20% of AML and represent driver mutations in leukemogenesis.9 Mutant IDH1/2 enzymes have neomorphic activity and catalyze the reduction of a- ketoglutarate to an oncometabolite, the R-enantiomer of 2-hydroxyglutarate (2-HG), which promotes DNA and histone hypermethylation, altered gene expression, and impaired hematopoietic differentiation.10,11 Quantification of single-nucleotide mutations by qPCR can be challeng- ing because of problems with background amplification from the wild-type allele. Recently, the development of digital PCR has enabled absolute quantification of various genomic targets with high precision and sensitivity and has, therefore, turned out to be a promising technique for MRD monitoring, especially for gene mutations.7,12
The clinical significance of residual IDH1/2 mutations in bone marrow in complete remission after chemotherapy is currently unknown. In this study, we employed digital PCR assays to quantify IDH1/2 mutant allele fraction at AML diagnosis and during follow-up in a large cohort of AML patients intensively treated in the Acute French Leukemia Association (ALFA) trials to investigate whether IDH1/2 mutations are suitable MRD markers that could predict clinical outcome in AML patients and provide fur- ther information for risk-adapted therapy.
Methods
Patients and treatment
This study was performed in 103 adult patients (18-70 years) with previously untreated primary IDH1/2 mutated AML and enrolled on the prospective ALFA-0701 (Eudra-CT 2007-002933-
36; ClinicalTrials.gov NCT00927498 or ALFA-0702 (Eudra-CT 2008-000668-18; ClinicalTrials.gov NCT00932412) trials. Treatment schemes have been previously reported for both tri- als.13,14 These studies were approved by the ethics committee of Saint-Germain en Laye and Sud Est IV, France, respectively, and the institutional review board of the French Regulatory Agency. Bone marrow or peripheral blood samples collected at the time of diagnosis of AML and during follow-up were obtained from the tissue bank Tumorothèque du Centre de Référence Régional en Cancérologie de Lille (CRRC)” and approval for this study was obtained from the institutional review board of CHRU of Lille (CSTMT089). All patients provided written informed consent to both treatment and genetic analysis before inclusion in the study, in accordance with the declaration of Helsinki. Among all patients included in the ALFA-0701 (n=278) or ALFA-0702 (n=704) trials, we selected patients meeting the following criteria: (i) the pres- ence of an IDH1R132 or an IDH2R140/R172 mutation at AML diagnosis (n=160), (ii) achievement of complete remission after induction therapy (n=130), and (iii) one or more bone marrow fol- low-up sample available for IDH1/2 variant allele fraction (IDH1/2-VAF) assessment (n=103) (Figure 1).
Molecular analysis
Droplet DigitalTM PCR (ddPCR) assays were used to quantify the IDH1/2 mutant allele and its wild-type counterpart in diagnos- tic and follow-up samples. During complete remission, only bone marrow samples were analyzed for IDH1/2-VAF assessment. IDH1/2-VAF was quantified on genomic DNA using Bio-RadTM reagents, primers and probes (HEX-labeled wild-type allele; FAM- labeled mutant alleles). All samples were tested in duplicate wells, using 90 ng of DNA per well. The PCR product from each well was then subjected to the QX100 droplet reader (Bio-RadTM), which measures the fluorescence of each droplet individually using a two-color detection system. Raw data were analyzed using QuantaSoft software, version 1.7.4.0917 (Bio-RadTM). Representative two-dimensional plots of droplet fluorescence for
haematologica | 2018; 103(5)
Figure 1. Patient flow chart. VAF, variant allele fraction.
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