DNMT3A Mutations in AML Patients: Prognostic Impact and Comparative Analysis of Mutations Burden in Diagnostic Samples, after Standard Therapy, and after Allogeneic Stem Cell Transplantation
Authors: Blau Olga , Behrenbeck Franziska , Suckert Nikola , Fransecky Lars R. , Baldus Claudia D. , Arnold Renate , Pezzutto Antonio , Dörken Bernd , Blau Igor Wolfgang 
Published: October 2019

Abstract
DNA (cytosine-5)-methyltransferase 3 alpha (DNMT3A) mutations is one of the most frequent somatic mutations in acute myeloid leukemia (AML). Although DNMT3A mutations have been studied extensively, influence of mutations on AML prognosis and their role on the pathogenesis of AML remains not completely clear. Little is known about the significance of mutations burden in the time of diagnosis on the clinical and prognostic features of AML. Recently discovered persistence of DNMT3A mutations in complete remission after standard therapy indicates the presence of mutation in early pre-leukemic stem cells. In this aspect, it would be essential to analyze the existence of DNMT3A-positive cells after allogeneic stem cell transplantation (alloSCT).
Using quantitative PCR, we analyzed mutations burden in diagnostic samples obtained from 65 AML patients with DNMT3A mutation. Then follow up samples were analyzed repeatedly, after induction, consolidation therapy, and after alloSCT with a mean follow-up of 33 months. Using well-established markers of minimal residual disease and donor engraftment, we monitored DNMT3A stability during complete molecular remission (CMR). In addition, we analyzed the combination of DNMT3A mutation with others mutations, such as FLT3, NPM1, IDH1, IDH,and others, at the time of diagnosis, as well as in relapse of AML. Finally, we analyzed the prognostic impact of DNMT3A mutations depending on the distinct combination of mutations with other genetic markers. For this, 150 matched-pairs AML patients without DNMT3A mutation were analyzed.
We have found considerable variation of mutations burden (from 1% till 90%) in diagnostic samples. Although, the prognostic impact of mutations burden in diagnostic samples have not been verify, we have found a significant associations of higher mutations burden with M4/M5 FAB variant of AML (p=0,04) and presence of NPM1 mutation (p=0,023).
Persistence of DNMT3A mutations in CMR after standard chemotherapy was found in all patients. The loss of correlation between DNMT3A and others could be explained by sub-clone architecture and clonal evolution of AML. After alloSCT, DNMT3A mutations were not discovered in patients with CMR and complete donor chimerism. This data suggests the removal of leukemic stem cells after alloSCT and indicate the importance of alloSCT for high risk AML patients. In relapse of leukemia, all samples showed an increasing of DNMT3A mutated alleles.
In addition, matched-pairs analysis suggests that DNMT3A, particularly when accompanied by FLT3-ITD, is a significant prognostic factor for inferior outcome, even after alloSCT.
We conclude that long-term quantitative monitoring of DNMT3A mutations could provide additional prognostic information and could explain the role of mutations in development and progression of AML.

Disclosures
Pezzutto: Cellgene, Novartis, Roche, Gilead: Membership on an entity's Board of Directors or advisory committees.