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C.Y. Ok et al.
together with mutations of other genes, at frequencies that depend on the IDH allele, suggesting that additional genomic insults are needed for AML to develop fully. For example, IDH2 R140 mutations are strongly associated with NPM1 mutations.10
IDH1 and IDH2 encode NADP+-dependent isocitrate dehydrogenases, converting isocitrate to α-ketoglutarate, while reducing NADP+ to NADPH with the production of CO2. IDH1 is present in the cytoplasm and peroxisome, whereas IDH2 resides in mitochondria and is a compo- nent of the Krebs’ cycle.14 IDH1 R132 mutation and IDH2 R140/R172 mutations reduce α-ketoglutarate to the oncometabolite D-2-hydroxyglutarate (also known as R- 2-hydroxyglutarate).14,15 D-2-hydroxyglutarate has struc- tural similarities to α-ketoglutarate and can competitively inhibit enzymes dependent on α-ketoglutarate, such as the TET enzyme family and histone lysine demethylases, and indeed IDH1/2 mutations in AML are associated with global DNA hypermethylation and impaired hematopoi- etic differentiation.16,17
Persistent IDH1 or IDH2 mutations have been observed in AML patients at the time of clinical and morphological remission.18,19 Debarri et al. reported that persistent IDH1/2 mutations in AML at the time of remission could predict relapse.19 However, their study cohort was small with only eight patients in complete remission with persistent IDH1/2 mutations, precluding a definitive conclusion. In this study, we explored the utility of mutant IDH1 and IDH2 as minimal residual disease markers in predicting relapse in a large cohort of AML patients.
Methods
Patients
We searched the database of The University of Texas MD Anderson Cancer Center from November 1, 2012 to December 31, 2017 and identified 80 newly diagnosed AML patients with IDH1 R132 or IDH2 R140/R172 mutations who achieved com- plete remission (CR) or CR with incomplete hematologic recovery (CRi), according to the 2017 European LeukemiaNet (ELN) recom- mendations for the diagnosis and management of AML,20 in bone marrow at any time-point of their treatment. To investigate the effect of predominant and well-established mutant IDH1/2 clones in AML, only cases with a mutant allelic frequency (MAF) ≥10% in a pre-treatment sample were included. All cases were collected consecutively and classified according to the 2017 World Health Organization (WHO) classification system.21 Patients with thera- py-related AML were excluded from this study. Clinical, laborato- ry and cytogenetic data were collected from the patients’ electron- ic medical records. This study was approved by the Institutional Review Board at The University of Texas MD Anderson Cancer Center (Houston, TX, USA) and was conducted in accordance with the Declaration of Helsinki.22
IDH1/2 sequencing
IDH1/2 sequencing was performed on all patients as a part of
clinically validated next-generation sequencing-based (NGS) assay (a 53-gene panel, a 28-gene panel or an 81-gene panel) as described previously.23 The limit of detection was 1% for the NGS assay. A sequencing library was prepared using 250 ng of genomic DNA and respective sequencing libraries were subjected to a MiSeq sequencer (Illumina Inc.). NGS data were analyzed using MiSeq Reporter (TruSeq) or SureCall (Haloplex). The Integrative Genomics Viewer (IGV, Broad Institute) was used to visualize read
alignment and confirm variant calls.24 A custom-developed, in- house software package (OncoSeek) was used to annotate sequence variants and to interface the data with the IGV. Nomenclature of genetic variants was designated following the Human Genome Variation Society recommendations.25
FLT3 analysis
The presence of internal tandem duplications or point muta- tions at codon 835 or 836 in FLT3 was determined as described previously.26
Cytogenetic analysis
Conventional chromosome analysis (karyotyping) was per- formed on G-banded metaphase cells prepared from unstimulated 24-hour and 48-hour bone marrow cultures as described previous- ly.27 Twenty metaphases were analyzed in most cases, but fewer than 20 metaphases were analyzed in some cases when inade- quate metaphases were available for complete analysis. The results were reported using the current International System for Human Cytogenetic Nomenclature.28 Cytogenetic risk stratifica- tion was assessed in each patient using the United Kingdom Medical Research Council (UKMRC) system.29
Statistical analysis
A Fisher exact test was used when comparing categorical vari- ables. Mann-Whitney and Kruskal-Wallis tests were used when comparing numerical variables in two groups or three or more groups, respectively. The cumulative incidence rate of relapse was determined using the competing risk method. The association between an IDH1/2 mutation and the cumulative incidence out- come was determined using a proportional subdistribution haz- ards regression model (Fine and Gray regression model).30 Differences in the cumulative incidence among patients with dif- ferent mutations were assessed using the Gray test.31 Time to relapse was calculated from the date of morphological remission to the date of relapse. All variables with a P value <0.05 (two- tailed) were considered to be statistically significant. Statistical analyses were performed using GraphPad Prism 6 (GraphPad Software, Inc., La Jolla, CA, USA) and SAS 9.4 for Windows (SAS Institute Inc., Cary, NC, USA).
Results
Patients
The study group included 80 patients (37 men and 43 women) with a median age of 59 years (range, 31 to 90) at diagnosis. The median hemoglobin concentration, white blood cell count and platelet count were 9.0 g/dL (range, 6.3 to 13.9), 7.9x109/L (range, 0.4 to 263.1x109/L) and 58x109/L (range, 1 to 1,069x109/L), respectively (Table 1). The median bone marrow blast count was 62% (range, 21 to 95%). Among 76 patients with cytogenetic information available, 88% (n=67) and 12% (n=9) had intermediate and adverse cytogenetic risk, respectively. There were no patients with favorable cytogenetic risk. A diploid kary- otype was seen in 52 (68%) patients. Various frontline therapies were administered to this cohort of patients, but no patients received an IDH inhibitor as frontline therapy. All patients younger than 60 years of age (n=41) were treated with intensive chemotherapy including 7+3 (idaru- bicin and cytarabine), CIA (clofarabine, idarubicin and cytarabine), FIA (fludarabine, idarubicin and cytarabine), or CLIA (cladribine, idarubicin and cytarabine with or without sorafenib). The patients over 60 years old (n=39)
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haematologica | 2019; 104(2)