Inversely to DNMT3A, IDH1/IDH2 Are Good Targets for Monitoring Minimal Residual Disease (MRD) in Acute Myeloid Leukemia (AML): A Pilot Study of the ALFA Group
Authors: Debarri Houria , Lebon Delphine , Roumier Christophe , Cheok Meyling , Marceau-Renaut Alice , Nibourel Olivier , Geffroy Sandrine , Helevaut Nathalie , Rousselot Philippe , Marolleau Jean-Pierre , Gardin Claude , Caillot Denis , Figeac Martin , Berthon Céline , Quesnel Bruno , Boissel Nicolas , Castaigne Sylvie , Dombret Hervé , Renneville Aline , Preudhomme Claude 
Published: October 2019

Abstract
Background: Acute myeloid leukemia (AML) is a heterogeneous disease even within the same genetic subgroup, e.g. within the NPM1 mutated subgroup, some patients have additional mutations in FLT3, IDH1/2, DNMT3A or TET2. Recent reports have shown that minimal residual disease (MRD) in AML, during or after treatment has prognostic impact. However, the clinical assessment of MRD in AML faces many questions. Firstly, which of the potential molecular and/or cellular markers should be included? Secondly, what type of biological sample should be analyzed? Thirdly, what is the sensitivity threshold to set and what are the relevant time frames for the MRD evaluation. The current technology development next-generation sequencing (NGS), opens new perspectives for monitoring MRD in AML, with the opportunity to expand and multiplex various markers. In this context, we tested the NGS technology for MRD monitoring IDH1/2 and DNMT3A in AML cases with mutated NPM1. We validated our results against MRD evaluated by RTqPCR.
Patients and methods: In this study, we included 94 patients (pts) from the ALFA 0701 trial. NPM1 monitoring by RTqPCR was performed as previously described (Lambert et al, Oncotarget july 2014). IDH1/2 and DNMT3A monitoring was done by NGS using the Ion Torrent Proton instrument. In order to obtain very high coverage (approximately 2 Millions reads by sample), 24 samples were analyzed per run. Bioinformatic analysis was performed according to our previous work (Boyer et al, Am JHematol 2013).
Results. 94 samples from 31 NPM1 mutated pts were analyzed. (17pts were positive for IDH1/2 and 15 were positive for DNMT3A). Sequencing data showed an excellent depth with a median of 2012459 reads (range 102,657 to 5,160,118 reads) and 966,298 reads (range 565,152 to 2,700,349 reads) for IDH1/2 and DNMT3A, respectively. Unfortunately, despite of this excellent coverage, a median of 520 reads were found positive in the negative controls due to a multi steps cross contaminations (OT2, Clonal amplification, purification). Thus, limiting the sensitivity to 0.07% (0.001-0.097%, p <0.001, Fisher’s exact test) and 0.1% (0.011-0.426%, p<0.0001, Fisher’s exact test) for IDH1/2 and DNMT3A, respectively.
The correlation with NPM1 MRD was 78% (r = 0.68183, p <0.0001) and 78.6% (r = 0.55514, p <0.0001) for IDH1/2 and DNMT3A, respectively.
For the 17 IDH1/2 positive patients, we found concordant MRD results between IDH1/2 mutation and NPM1 RTqPCR in 13 cases: (4 pos/pos), which was associated to disease relapse, (9 neg/neg), which was associated to maintenance of CR1. For the four remaining patients, we have observed a discrepancy between some NPM1 results and IDH1/2 results. Indeed, these 4 patients presented one or more MRD negative points using the NPM1 marker, while rates of the IDH1/2 marker ranged from 0.1% to 47%. Three of them, have relapsed after 504,395,158 days, and all of them with the same NPM1 mutation detected at diagnosis. The one patient, who has not relapsed, did evolve to a myelodysplastic syndrome which was NPM1 negative.
For the 15 DNMT3A positive patients, we found co-occurrence of the NPM1 mutation and IDH1/2 mutations in 9 cases (8 pos/pos), which was associated to disease relapse and we found concordant MRD results between DNMT3A mutation and NPM1 RTqPCR, (1 neg/neg), which was associated to maintenance of CR1. For the six remaining patients, we have observed a discrepancy between NPM1 results and DNMT3A results. Indeed, those 6 patients presented a great majority of negative results using MRD monitoring by NPM1, while rates of MRD by DNMT3A ranged from 5% to 45%. All of these 6 pts are still in first CR after a median follow up of 4 years. Bone marrow and PBMC were analyzed for 3 of them. A positive result was observed in all cell fractions except for the T cells compartment.
Conclusion. Monitoring of MRD by NGS mutation detection in two genes IDH1/2 is feasible and valuable, because it enabled us to predict relapse in additional patients, with an area under the curve of 0.7971 (95% CI: 0.6693 - 0.9250), and 100% if we include the patient who evolved to MDS. Furthermore, this category of patients may benefit from new targeted therapies such as inhibitors of mutated IDH proteins. On the contrary, DNMT3A is not a good marker for MRD monitoring, because we detected persistence of a preleukemic clone in 40% of patients remaining in CR, reflecting the molecular heterogeneity clonal hematopoiesis.

Disclosures
No relevant conflicts of interest to declare.