REGULAR ARTICLE Prognostic impact of DNMT3A mutation in acute myeloid leukemia with mutated NPM1 Guadalupe O~ nate,1 Alex Bataller,2 Ana Garrido,1 Montserrat Hoyos,1 Montserrat Arnan,3 Susana Vives,4 Rosa Coll,5 Mar Tormo,6 Antonia Sampol,7 Lourdes Escoda,8 Olga Salamero,9 Antoni Garcia,10 Joan Bargay,11 Alba Aljarilla,1 Josep F. Nomdedeu,1 Jordi Esteve,2 Jorge Sierra,1, and Marta Pratcorona1,, for the CETLAM (Spanish Cooperative Group for the Diagnosis and Treatment of Acute Myeloid Leukemia and Myelodysplastic Syndromes) 1 Hospital de la Santa Creu i Sant Pau, Autonomous University of Barcelona, Barcelona, Spain; 2Hospital Clınic, Barcelona, Spain; 3Catalan Institute of Oncology (ICO), Hospital Duran i Reynals, Barcelona, Spain; 4ICO, Hospital Germans Trias i Pujol, Jos e Carreras Leukemia Research Institute, Badalona, Spain; 5ICO, Hospital Josep Trueta, Girona, Spain; Hospital Clınico Universitario, Instituto de Investigaci 6 on del Hospital Clınico de la Comunidad Valenciana, Valencia, Spain; 7Hospital Son Espases, Palma de Mallorca, Spain; 8ICO, Hospital Joan XXIII, Tarragona, Spain; 9Hospital Vall d’Hebron, Barcelona, Spain; 10Hospital Universitari Arnau de Vilanova, Lleida, Spain; and 11Hospital Son Llatzer, Palma de Mallorca, Spain Key Points The negative prognostic impact of internal tandem duplication of FLT3 (FLT3-ITD) in patients with acute myeloid leukemia with mutated NPM1 (AML-NPM1) is restricted to  Patients with those with a higher FLT3-ITD allelic ratio (FLT3high; $0.5) and considered negligible in DNMT3Amut have those with a wild-type (FLT3WT)/low ITD ratio (FLT3low). Because the comutation of worse NPM1 MRD DNMT3A (DNMT3Amut) has been suggested to negatively influence prognosis in AML- clearance, which can NPM1, we analyzed the impact of DNMT3Amut in FLT3-ITD subsets (absent, low, and high be counteracted by preemptive allogeneic ratios). A total of 164 patients diagnosed with AML-NPM1 included in 2 consecutive transplantation. CETLAM protocols and with DNMT3A and FLT3 status available were studied. Overall, DNMT3Amut status did not have a prognostic impact, with comparable overall survival  DNMT3Amut does not (P 5 .2). Prognostic stratification established by FLT3-ITD (FLT3WT 5 FLT3low . FLT3high) modify the prognostic was independent of DNMT3Amut status. Measurable residual disease (MRD) based on NPM1 value of the FLT3-ITD allelic ratio in AML- quantitative polymerase chain reaction was available for 94 patients. DNMT3Amut was NPM1. associated with a higher number of mutated NPM1 transcripts after induction (P 5 .012) and first consolidation (C1; P , .001). All DNMT3Amut patients were MRD1 after C1 (P , .001) and exhibited significant MRD persistence after C2 and C3 (MRD1 vs MRD2; P 5 .027 and P 5 .001, respectively). Finally, DNMT3Amut patients exhibited a trend toward greater risk of molecular relapse (P 5 .054). In conclusion, DNMT3Amut did not modify the overall prognosis exerted by FLT3-ITD in AML-NPM1 despite delayed MRD clearance, possibly because of MRD-driven preemptive intervention. Introduction In recent years, the role of molecular genetics has proven to be essential in deciphering the heterogene- ity of acute myeloid leukemia (AML)1,2 and defining genetic markers of prognostic significance that can guide risk-adapted treatment.3 AML with mutations in the nucleophosmin 1 gene (AML-NPM1) forms a specific category in the latest World Health Organization classification because of its singular characteristics.4 The cooccurrence of Submitted 30 December 2020; accepted 7 May 2021; prepublished online on The full-text version of this article contains a data supplement. Blood Advances First Edition 13 September 2021; final version published online 2 ß 2022 by The American Society of Hematology. Licensed under Creative Commons February 2022. DOI 10.1182/bloodadvances.2020004136. Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), M.P. and J.S. contributed equally to this work. permitting only noncommercial, nonderivative use with attribution. All other rights For original data, please contact mpratcorona@santpau.cat. reserved. 882 8 FEBRUARY 2022 • VOLUME 6, NUMBER 3 mutated NPM1 (NPM1mut) and the internal tandem duplication of Methods FLT3 (FLT3-ITD) in de novo AML with intermediate-risk cytogenetics results in a different prognostic impact depending on the FLT3 alle- Patients and samples lic burden.5-7 Previous studies have shown that patients with Patients with de novo AML and intermediate-risk cytogenetics NPM1mut and an FLT3-ITD low ratio (FLT3low; FLT3-ITD/FLT3WT according to the Medical Research Council,35 NPM1mut, and avail- ratio of ,0.5) had overall survival (OS) and risk of relapse (RR) simi- able bone marrow sample from diagnosis were selected. All patients lar to those of patients with NPM1mut and wild-type (WT) FLT3 were diagnosed between 2003 and 2017 and were included in (FLT3WT).8,9 Since 2012, these findings have been included in our the CETLAM intensive treatment protocols AML-03 and AML-12 latest protocol (CETLAM [Spanish Cooperative Group for the Diag- (registered at www.clinicaltrials.gov as #NCT01723657 and nosis and Treatment of Acute Myeloid Leukemia and Myelodysplas- #NCT04687098) provided they met the criteria for eligibility. The tic Syndromes] AML-12), and patients with FLT3low-NPM1mut AML present study was reviewed and approved by the ethics committee are not considered for allogenic hematopoietic stem cell transplanta-  etic of the Hospital de la Santa Creu i Sant Pau (Comite tion (alloHSCT) in first complete remission (CR1). However, a o Clınica). Informed consent for both bone marrow anal- d’Investigaci molecular-based measurable residual disease (MRD) monitoring ysis and treatment was obtained in all cases according to the Decla- protocol is strictly followed to allow early-intervention strategies. ration of Helsinki. The DNA (cytosine-5)-methyltransferase 3A (DNMT3A) gene is located on the short arm of chromosome 2 and encodes for a DNA Molecular studies methyltransferase that methylates unmodified DNA cytosine resi- Diagnostic bone marrow samples from all patients were analyzed dues modulating the expression of several genes.10,11 Almost all for DNMT3Amut as previously described.17 The mutational status of DNMT3A mutations are heterozygous, and more than two-thirds the FLT3 gene was also established. In mutated cases, the allelic cluster at the methyltransferase domain in codon R882, causing ratio was calculated by dividing the area under the curve of the loss of methylation activity by disturbing DNMT3A tetrameriza- FLT3-ITD peak and the area under the curve of the FLT3WT peak. tion.12-16 However, although a precise methylation pattern alteration Patients were stratified into 2 groups: those with a high ratio resulting from mutations in DNMT3A has not yet been estab- (FLT3high) if the ratio of FLT3-ITD/FLT3WT was $0.5 and those with lished,17-21 a new mechanism of leukemogenesis characterized by FLT3low if the ratio of FLT3-ITD/FLT3WT was ,0.5. the upregulation of the hepatic leukemia factor (a specific leukemic transcription factor) has been shown to be related to the cooccur- Monitoring of NPM1 MRD was performed on bone marrow samples rence of DNMT3A, NPM1, and FLT3 mutations.22 by quantitative reverse transcription polymerase chain reaction (sen- DNMT3A is considered a founder mutation in AML.23,24 It has been sitivity 1024 to 1026) as previously described.36 After each treat- associated with age-related clonal hematopoiesis, with increasing ment cycle, absolute transcript reduction was estimated, and its frequency in healthy elderly individuals,25 although a recent study logarithm (log10) reduction from diagnosis was also explored. found a correlation of DNMT3A mutations with younger age in Based on the latest ELN MRD working party recommendations,37 NPM1mut AML.26 Patients with AML and mutated DNMT3A MRD positivity was considered when NPM1 transcripts were ampli- (DNMT3Amut) are frequently older and present with higher white fied in at least 2 of 3 replicates with cycle threshol values of #40 at blood cell (WBC) counts and higher platelet counts compared with a cycling threshold of 0.1. Molecular relapse was confirmed if the WT DNMT3A (DNMT3AWT).13,17,27 DNMT3A is the third most fre- MRD level (in a patient previously MRD2) increased $1 log10 quently mutated gene in AML patients included in intensive chemo- between 2 consecutive positive samples, and molecular progression therapy trials. It is predominantly observed in AML-NPM1 (73%) was confirmed if copy numbers increased $1 log10 between 2 and less frequently in patients with mutations in chromatin remodel- positive samples in a patient with MRD1. ing genes or genes involved in spliceosome function. Interestingly, a recurrent association of NPM1mut/DNMT3Amut/FLT3-ITD has been Statistical analysis observed in 6% of AML cases.28,29 The prognostic significance of Analysis of the relationship between categorical variables was mutations in DNMT3A has been controversial; some studies have performed using the x2 or Fisher’s exact test. Differences between found no significant influence on survival outcomes,17 whereas groups for continuous variables were established through the inde- others have suggested that the cooccurrence of NPM1mut/ pendent samples t test or Mann-Whitney U test. All tests were 2 DNMT3Amut/FLT3-ITD in AML patients is associated with adverse sided and considered significant where P , .05. OS was calcu- outcomes.7,13,30-33 lated from diagnosis to death, whereas leukemia-free survival (LFS) In 2013, the German AML Study Group described the clinical impact was calculated from CR to death or relapse; both functions were of DNMT3Amut in younger adults with AML.34 In a univariable explor- estimated with the Kaplan-Meier method. Unless specified other- atory subset analysis, the group showed a significant prognostic wise, all survival results reported reflect 5-year estimates. A log-rank impact of DNMT3Amut in unfavorable European LeukemiaNet (ELN) test was run to determine differences in the survival distribution, AML, whereas no impact was observed in favorable ELN AML. In with a significance threshold of P # .05. RR was estimated using 2016, the proposed AML gene classification by Papaemmanuil et the cumulative incidence method, defining relapse as the main event al28 showed a deleterious effect of DNMT3Amut when specifically and death without relapse as the competitive event. Molecular LFS associated with FLT3-ITD independently of its allelic ratio. (molLFS) was estimated from CR to molecular failure, overt hemato- We analyzed whether this triple-gene alteration led to an unfavorable logical relapse, or death. All statistical analyses were performed with prognosis in AML-NPM1 patients, with particular attention to those SPSS software (version 26; IBM, Armonk, NY) and R statistics (ver- harboring FLT3low. sion 3.6.1; R Foundation for Statistical Computing, Vienna, Austria). 8 FEBRUARY 2022 • VOLUME 6, NUMBER 3 PROGNOSTIC IMPACT OF DNMT3A IN AML-NPM1 883 Table 1. Patient characteristics according to DNMT3A mut status Prognostic impact of DNMT3Amut No. (%) In the entire cohort of AML-NPM1 patients, OS, LFS, and RR were DNMT3AWT DNMT3Amut 59% 6 4%, 60% 6 5%, and 27% 6 7%, respectively. FLT3-ITD (n 5 85) (n 5 79) P allelic ratio confirmed its prognostic impact, with a similar outcome Female sex 51 (60) 36 (46) .08 for patients with FLT3WT or FLT3low and a significantly worst prog- Age, y .7 nosis for cases with FLT3high. OS was 67% 6 6% vs 62% 6 9% Median 53 53 vs 40% 6 8% (P 5 .002; supplemental Figure 1), respectively; RR was 18% 6 9% vs 27% 6 16% vs 41% 6 17% (P 5 .008), Range 18-71 25-72 respectively; and LFS was 71% 6 5% vs 56% 6 9% vs 40% 6 WBC, 3 109/L ,.001 9% (P 5 .002), respectively. In contrast, DNMT3Amut did not exert Median 16 50 a significant effect on overall outcomes (Figure 1), with OS in Range 0.55-408 1.3-384 DNMT3AWT vs DNMT3Amut patients of 62% 6 6% vs 56% 6 6% BM blasts, % .7 (P 5 .2), respectively; LFS of 65% 6 6% vs 54% 6 6% (P 5 .1), Median 73 73 respectively; and RR of 22% 6 11% vs 31% 6 11% (P 5 .2). The outcomes of DNMT3A subsets among the entire AML-12 Range 20-100 20-100 cohort are available in supplemental Figure 2. Platelet count, 3 109/L .4 Median 64 70 Additionally, the individual effect of R882 DNMT3Amut was analyzed separately and did not show any prognostic impact (P 5 .4; supple- Range 8-488 12-625 mental Figure 3). Multivariate analysis performed for OS included FLT3 mutational status .6 age, protocol, WBC count, DNMT3Amut status, and FLT3-ITD sub- WT 46 (54) 40 (50) sets, the latter being the only statistically significant variable (data not FLT3low 19 (22) 15 (19) shown). high FLT3 19 (22) 23 (30) The effect of the cooccurrence of DNMT3Amut and FLT3-ITD was Treatment protocol analyzed separately. In the DNMT3Amut cohort, whereas OS AML-03 26 (31) 22 (28) between FLT3WT and FLT3low was similar, patients with FLT3high AML-12 59 (69) 57 (72) showed particularly worse outcomes (FLT3WT vs FLT3low OS, 68% Postinduction CR 75 (88) 69 (87) .7 6 8% vs 56% 6 13%, respectively; P 5 .3; FLT3WT/low vs FLT3high OS, 65% 6 7% vs 28% 6 13%, respectively; P 5 .015). This was No. of cycles to achieve CR (1 vs 2) 67 vs 8 61 vs 8 .86 further validated in terms of LFS (FLT3WT vs FLT3low, 68% 6 8% vs No. of patients undergoing alloHSCT in CR1 16 (19) 22 (28) .9 50% 6 13%, respectively; P 5 .1; FLT3WT/low vs FLT3high, 62% 6 BM, bone marrow. 7 vs 20% 6 16%, respectively; P 5 .014) and RR in the same groups (23% 6 15% vs 29% 6 23%, respectively; P 5 .4; 25% 6 13% vs 48% 6 20%, respectively; P 5 .017; Figure 2). Results In patients without DNMT3Amut, the allelic ratio of FLT3-ITD main- tained its prognostic value, with similar outcomes between FLT3WT Patient characteristics according to and FLT3low patients. Interestingly, in this group of patients, the dele- DNMT3Amut status terious effect of FLT3high was mildly modulated (FLT3WT vs FLT3low A total of 164 patients with AML-NPM1 were included. Clinical and OS, 66% 6 8% vs 66% 6 11%, respectively; P 5 .5; FLT3WT/low genetic characteristics at diagnosis are described in Table 1. vs FLT3high OS, 66% 6 7% vs 47% 6 12%, respectively; P 5 Patients were included in protocols AML-03 (n 5 48) and AML-12 .028). Similar findings were seen in terms of LFS (FLT3WT vs (n 5 116). DNMT3Amut was found in 79 patients (48%); in 62 FLT3low, 74% 6 8% vs 61% 6 13%, respectively; P 5 .2; cases (38%), mutations were in codon R882 or were insertions/ FLT3WT/low vs FLT3high, 70% 6 7% vs 52% 6 13%, respectively; deletions, whereas 17 (10%) were different missense mutations. P 5 .083) and RR (14% 6 11% vs 26% 6 21%, respectively; P 5 Seventy-six patients (46%) harbored FLT3-ITD, 42 of whom had .2; 17% 6 12% vs 33% 6 23%, respectively; P 5 .1; Figure 3). FLT3high (55%). According to DNMT3Amut status, patient character- The direct comparison of DNMT3Amut status according to each istics were comparable, except for a higher WBC presentation FLT3 status (WT, low, and high) was not statistically different (sup- among DNMT3Amut patients. Of note, the proportion of FLT3-ITD plemental Figure 4), although the general analysis of DNMT3A allelic burden subsets (ie, FLT3WT, FLT3low, and FLT3high) was inde- depending on FLT3WT or FLT3mut showed statistical differences. pendent of DNMT3Amut (P 5 .6). This might be due to the low number of patients analyzed when Eighty-eight percent of patients achieved CR after 1 or 2 cycles of dividing into the 3 groups. induction therapy (n 5 128 and 16, respectively); 6% (n 5 10) had refractory disease, and 10 patients died during induction. As consol- MRD kinetics according to DNMT3Amut status idation therapy, 58 patients received intensive treatment consisting Patients included in the CETLAM AML-12 trial with AML-NPM1 of 2 to 3 high-dose cytarabine cycles. AlloHSCT was performed in allocated to the favorable ELN category (FLT3WT or FLT3low) were 65 patients (CR1, n 5 44; CR2, n 5 15; with refractory disease, not intended to undergo alloHSCT in CR1 unless a molecular failure n 5 4); 14 patients underwent autologous transplantation. The was identified. Therefore, patients were closely monitored for NPM1 median follow-up time was 30 months. MRD at specific time points (postinduction, after each consolidation 884 ~ ONATE et al 8 FEBRUARY 2022 • VOLUME 6, NUMBER 3 A 1.0 B 1.0 0.8 0.8 Cumulative survival Cumulative survival 0.6 0.6 0.4 0.4 0.2 0.2 p = 0.235 p = 0.126 0.0 0.0 0 50 100 150 0 50 100 150 Number at risk OS months Number at risk LFS months DNMT3Awt 85 28 4 0 DNMT3Awt 75 24 3 0 DNMT3Amut 79 18 6 1 DNMT3Amut 69 13 5 1 Figure 1. Overall impact of DNMT3Amut on AML-NPM1. OS (A) and LFS (B) of patients according to DNMT3Amut status. cycle, and every 3 months for 3 years after treatment completion) to FLT3WT or FLT3low, n 5 63 and 22, respectively), the median ensure rapid detection of molecular relapse and subsequent therapy molLFS was not reached at a mean follow-up of 30 months (supple- initation.38 Patients with FLT3high underwent alloHSCT after the first mental Figure 6). When stratified by DNMT3Amut status, patients consolidation cycle (C1). with the WT form exhibited a trend toward a long-term sustained molecular CR (molLFS, 63% 6 9% vs 50% 6 9% in DNMT3AWT In 94 patients with available MRD data, we further investigated (n 5 42) vs DNMT3Amut (n 5 35), respectively; P 5 .054; Figure 5). whether DNMT3Amut status influenced NPM1 MRD. Although the probability of achieving hematological CR was not affected by Eleven patients in the favorable-risk group harbored NPM1mut/ DNMT3A (75 vs 69 patients in with DNMT3AWT and DNMT3Amut, FLT3low/DNMT3Amut; of these, only 5 experienced a molecular or respectively; P 5 .46), NPM1 MRD kinetics differed according to hematological relapse and underwent alloHSCT. In total, 23 patients DNMT3Amut status. Patients with DNMT3Amut showed a higher num- (27%) in this favorable subgroup underwent alloHSCT because of ber of absolute NPM1mut transcripts after induction (P 5 .012) and molecular or hematological relapse. C1 (P , .001; Figure 4A). Similar findings were observed after C2 Overall, these findings suggest a deleterious effect of DNMT3Amut and C3 among patients not intended to undergo alloHSCT in CR1. on NPM1 MRD that should be validated in larger studies. Therefore, we explored the relationship between DNMT3A subsets, posttreatment molecular MRD status (positive vs negative), and Discussion MRD response depth (log10 reduction; Figure 4B-G). After induc- tion, all but 1 patient remained MRD1. Although there were no sta- Several studies have been published attempting to elucidate the tistical differences in the number of log10 reductions, a trend prognostic impact of DNMT3Amut, but many have had contradictory toward a more profound molecular response ($4 log10) was results. This may be due to differences in the biological characteris- observed in the DNMT3AWT group (DNMT3AWT vs DNMT3Amut, tics of the patients included (age, cytogenetics, availability of molec- 39% vs 15%, respectively; P not significant). ular studies), the treatment protocols, or other factors.39 Of note, even those studies comparing the impact of DNMT3Amut status After C1, none of the DNMT3Amut patients achieved MRD2 status, based on NPM1mut and FLT3mut status have shown contradictory compared with 32% of DNMT3AWT patients (P 5 .001). Of note, results.13,17,34 The aim of this study was not to analyze the impact patients without DNMT3Amut presented a deeper MRD reduction of DNMT3Amut on AML outcomes, but rather to analyze its effect in ($4 log10 reduction in 77% of DNMT3AWT vs 46% of DNMT3Amut the particular subset of patients with NPM1mut and FLT3-ITD, after patients; P 5 .033). The relationship between DNMT3A and NPM1 the publication of a large study showing that DNMT3Amut have a MRD was also sustained after C2 and C3 (Figure 4B-G; supplemen- deleterious effect on outcomes when cooccurring in this sub- tal Figure 5). Additionally, when considering the triple-mutated group group.28 Our group described the effect of the FLT3-ITD ratio in (NPM1, FLT3-ITD, and DNMT3A), all patients remained MRD1 after 2012, and it was incorporated into the new treatment protocol. induction, C1, and C2 regardless of FLT3-ITD allelic ratio. Consequently, patients with NPM1mut and FLT3low did not undergo Finally, the potential influence of DNMT3Amut status on molecular fail- alloHSCT in CR1. Therefore, we had a long follow-up in this group ure was explored. Among 85 cases included in the AML-12 protocol of patients treated following the ELN 2017 recommendations in not initially considered for alloHSCT in CR1 (AML-NPM1 with which to analyze the possible effect of DNMT3Amut. 8 FEBRUARY 2022 • VOLUME 6, NUMBER 3 PROGNOSTIC IMPACT OF DNMT3A IN AML-NPM1 885 A DNMT3Awt B DNMT3Amut 1.0 1.0 0.8 0.8 Cumulative survival Cumulative survival 0.6 0.6 0.4 0.4 0.2 0.2 p = 0.045 p = 0.117 0.0 0.0 0 20 40 60 80 100 120 0 20 40 60 80 100 120 OS (months) OS (months) Number at risk Number at risk FLT3wt 38 26 17 8 5 2 FLT3wt 46 37 25 14 6 3 FLT3-ITD 39 17 5 2 2 2 FLT3-ITD 39 23 14 6 3 1 C 1.0 D 1.0 0.8 0.8 Cumulative survival Cumulative survival 0.6 * 0.6 * ** 0.4 0.4 ** * FLT3wt vs low p = 0.5 0.2 ** FLT3wt/low vs high p = 0.028 0.2 * FLT3wt vs low p = 0.3 ** FLT3wt/low vs high p = 0.015 p = 0.081 p = 0.037 0.0 0.0 0 20 40 60 80 100 120 0 50 100 150 OS (months) OS (months) Number at risk Number at risk FLT3wt 46 37 25 14 6 3 FLT3wt 40 14 4 1 FLT3low 19 12 7 3 1 1 FLT3low 15 2 1 0 FLT3high 19 10 6 3 2 0 FLT3high 23 2 1 0 Figure 2. DNMT3A influence over FLT3-ITD allelic ratio subgroups. OS of DNMT3AWT (A,C) and DNMT3Amut (B,D) patients in different FLT3 subsets. A DNMT3Awt B DNMT3Amut Cumulative incidence of relapse 1.0 1.0 Cumulative incidence of relapse * wt vs low p = 0.2 * wt vs low p = 0.4 0.8 0.8 ** wt/low vs high p = 0.017 ** wt/low vs high p = 0.1 0.6 0.6 0.4 0.4 ** 0.2 ** 0.2 * * 0.0 p = 0.2 p = 0.047 0.0 0 20 40 60 80 100 0 50 100 150 Months Months Number of patients at risk Number of patients at risk FLT3wt 42 34 22 14 6 3 FLT3wt 34 11 3 1 FLT3low 17 10 5 2 0 0 FLT3low 15 1 1 0 FLT3high 15 8 4 2 2 0 FLT3high 19 1 1 0 Figure 3. Cumulative incidence of relapse in AML-NPM1 patients based on DNMT3Amut status. DNMT3AWT (A) and DNMT3Amut (B) patients according to FLT3-ITD subgroup. 886 ~ ONATE et al 8 FEBRUARY 2022 • VOLUME 6, NUMBER 3 A 1,000E8 p = 0.81 64 10.000.000 16 NPM1/ABL (logarithm scale) 1.000.000 p = 0.012 p = 0.024 100.000 17 p <0.001 * 38 DNMT3A 10.000 p = 0.003 Wild type 65 Mutated 6 1.000 77 100 62 84 8 14 * 10 * * 162 85 1 –1 Diagnose post-induction post-C1 post-C2 post-C3 Treatment timepoints B Post-induction C Post-consolidation 1 D Post-consolidation 2 n = 49 n = 38 n = 39 100 100 100 n = 27 p = n.s. p <0.001 p = 0.027 Percentage of patients Percentage of patients Percentage of patients 80 80 n = 34 80 n = 28 60 60 60 40 40 40 n = 11 n = 11 20 20 20 n=2 n=1 0 0 0 Wild type Mutated Wild type Mutated Wild type Mutated DNMT3A DNMT3A DNMT3A NPM1 MRD NPM1 MRD NPM1 MRD Negative Positive Negative Positive Negative Positive E F G 100 100 100 n = 30 n = 34 p = n.s. p = 0.033 p = 0.055 Percentage of patients Percentage of patients Percentage of patients 80 80 80 n = 17 n = 18 60 60 60 n = 19 n = 14 n = 12 n = 13 n = 10 n = 12 n=7 40 40 40 n=8 n=7 n=6 n=4 n=6 n=5 n=4 n=3 n=3 n=2 20 20 20 n=1 n=2 n=1 0 0 0 Wild type Mutated Wild type Mutated Wild type Mutated DNMT3A DNMT3A DNMT3A NPM1 log reduction NPM1 log reduction NPM1 log reduction <1 1log 2log 3log >4log <1log 1log 2log 3log >4log <1log 1log 2log 3log >4log Figure 4. NPM1 MRD distribution at relevant clinical time points according to DNMT3Amut status. (A) NPM1 absolute transcript distribution in logarithmic scale. (B-G) MRD response, with MRD1 and MRD2 rates (B-D) and corresponding equivalent log10 reductions (E-G) at postinduction (B,E), post-C1 (C,F), and post-C2 (D,G). In our study, survival analysis showed that DNMT3Amut did not have evaluation time points, all of them support the prognostic impact of an impact in this particular group and that patients with NPM1mut MRD1 persistence, with a higher incidence of relapse and shorter and FLT3low had similar outcomes to patients with NPM1mut and OS.38,40-43 The largest study performed44 evaluated the impact of FLT3WT regardless of DNMT3Amut status. However, because an MRD1 in peripheral blood after the second chemotherapy cycle; it effect of DNMT3Amut on NPM1 MRD clearance was demonstrated, found the same impact on prognosis as previously reported, but the we investigated the influence of an early intervention planned in the authors also reported that MRD persistence was the only indepen- treatment protocol when molecular relapse was detected. In the last dent prognostic factor for death in multivariate analysis. The ELN few years, several publications analyzing the prognostic value of recommendations37 also state that in AML-NPM1, rising MRD levels MRD follow-up based on NPM1 transcript levels have been pub- or the failure to achieve MRD2 CR is associated with disease lished. Although there is no consensus regarding the cutoff level or relapse and consequently advise that a change in therapy should be 8 FEBRUARY 2022 • VOLUME 6, NUMBER 3 PROGNOSTIC IMPACT OF DNMT3A IN AML-NPM1 887 DNMT3A influence in FLT3-ITDwt/low patients A 1.0 B 1.0 0.8 0.8 Cumulative survival Cumulative survival 0.6 0.6 0.4 0.4 0.2 0.2 p = 0.238 p = 0.0542 0.0 0.0 0 20 40 60 80 0 20 40 60 80 OS months molLFS Number at risk Number at risk DNMT3Awt 42 30 21 9 0 DNMT3Awt 42 30 19 7 0 DNMT3Amut 35 24 11 2 0 DNMT3Amut 35 18 8 2 0 Figure 5. DNMT3A influence in the favorable FLT3-ITD subgroup. Distribution of patients with FLT3WT or FLT3low according to OS (A) and molLFS (B). considered. Following the same reasoning, it was recently published follow-up is recommended to detect a molecular relapse and pro- by our group that an MRD ratio (NPM1mut/ABL1X100) of $0.05 (in ceed to a therapeutic intervention. bone marrow) after the C1 was associated with significantly lower molLFS and that an early intervention resulted in a favorable out- Acknowledgments comes.38 Consequently, using the MRD level to guide postremis- This work was supported in part by the Biomedical Research Insti- sion therapy can be considered a good strategy.  Carreras Leukemia Research Insti- tute (IIB Sant-Pau) and the Jose Interestingly, in the present study, a trend toward worse molLFS was tute as well as grants from the Catalan Government (PERIS observed in patients with DNMT3Amut, but without an impact on OS. SLT002/16/0043 and AGAUR 2017 SGR 139) and the Instituto When only patients in the favorable ELN 2017 risk group were consid- de Salud Carlos III, Ministerio de Economıa y Competitividad, ered, we found that 27% of patients met either cytological or molecu- Spain (PI17/01246, PI20/01621 and CM20/00061). lar relapse criteria. Of those, 70% underwent alloHSCT in CR1 G.O. is a PhD candidate at the Autonomous University of Bar- (in molecular relapse) or CR2. As a result, the effect of this strategy celona, and this work is submitted in partial fulfillment of the might counteract the negative effect on OS seen in the DNMT3Amut requirement for a PhD. subgroup. This intervention might be the most important difference between the treatment protocols for our patients and those included Authorship in the Papaemmanuil et al28 study, which considered alloHSCT only in Contribution: G.O. and A.A. performed research. M.P. and G.O. patients at high cytogenetic risk, whereas intermediate-risk patients designed research, analyzed data, and wrote the paper; J.S., J.F.N., underwent alloHSCT only when a sibling donor was available.45-47 and J.E. supervised research and wrote the paper; M.A., S.V., R.C., M.T., A.S., L.E., O.S., A.G., and J.B. collected and provided the clini- Considering these findings, close MRD monitoring in DNMT3Amut cal data; A.B., A.G., and M.H. analyzed data; and all authors AML-NPM1 patients, along with early intervention strategies when a reviewed the final manuscript. molecular relapse is detected, could be an appropriate approach, with a possible impact on OS. Conflict-of-interest disclosure: J.E. reports an advisory role and mut high trial investigation for Novartis, Daiichi Sankyo, Astellas, Celgene, Patients with DNMT3A and FLT3 had poorer outcomes than Jazz Pharmaceuticals, Roche, Boehringer Ingelheim, and Janssen. patients in the favorable ELN group (ie, FLT3WT or FLT3low). None- J.S. reports personal fees from AbbVie, Vyxeos, Gilead, CSL Beh- theless, DNMT3Amut status did not seem to affect patients with ring, Astellas, and Gilead; grants and personal fees from Novartis FLT3high, although a deleterious effect of this triple-mutation status and Daiichi-Sankyo; and grants from Amgen. The remaining authors (NPM1mut/FLT3high/DNMT3Amut vs NPM1mut/FLT3high/DNMT3AWT) declare no competing financial interests. cannot be definitively excluded because of the small size of the sub- groups analyzed. These findings may show a dosage effect on the ORCID profiles: G.O., 0000-0003-2180-2371; A.B., 0000-0002- interaction between FLT3 and DNMT3Amut in AML-NPM1, highlight- 6085-2745; R.C., 0000-0003-0560-1254; A.S., 0000-0001-7465- ing the relevance of considering not only the presence of every single 6203; J.E., 0000-0002-8056-648X; M.P., 0000-0001-6375-596X. mutation but also the interaction among them. Correspondence: Marta Pratcorona, Department of Hematology, In conclusion, patients with NPM1-AML with FLT3low and Hospital de la Santa Creu i Sant Pau, Sant Quintı 89 08046 Barce- DNMT3Amut can be classified as favorable risk, but closer MRD lona, Spain; e-mail: mpratcorona@santpau.cat. 888 ~ ONATE et al 8 FEBRUARY 2022 • VOLUME 6, NUMBER 3 References 1. Bullinger L, D€ ohner K, Bair E, et al. Use of gene-expression profiling to identify prognostic subclasses in adult acute myeloid leukemia. N Engl J Med. 2004;350(16):1605-1616. 2. Patel JP, G€ onen M, Figueroa ME, et al. 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