Monitoring of Minimal Residual Disease (MRD) of DNMT3A Mutations (DNMT3A^mut) in Acute Myeloid Leukemia (AML): A Study of the AML Study Group (AMLSG)

Background: The DNA methyltransferase 3A (DNMT3A) is one of the most frequent mutated genes in AML with a hot spot mutation at codon R882 in 80% of the DNMT3A^mut cases. In most of the studies DNMT3A^mut predicts for poor overall (OS) and relapse-free survival (RFS). Recently, DNMT3A^mut have been associated with age-related clonal hematopoiesis, and they have been identified in early preleukemic stem cells. These findings suggest that DNMT3A^mut represents an early event in leukemogenesis and may be part of the leukemia founder clone in most AMLs harboring a DNMT3A^mut. We thought to address the question whether MRD monitoring in DNMT3A^mut patients (pts) can be used for prognostic classification and risk stratification in these pts.

Aims: We monitored MRD for the most common DNMT3A^mut (DNMT3A^mut -R882H, n=126 and -R882C, n=55) in a large cohort of adult AML pts entered on three AMLSG treatment trials [AML HD98A (n=14; NCT00146120), AMLSG 07-04 (n=86; NCT00151242), AMLSG 09-09 (n=81; NCT00893399)].

Methods: DNMT3A^mut MRD monitoring was performed using a cDNA-based RQ-PCR-assay by TaqMan technology with a sensitivity between 10^-3 and 10^-4. MRD levels are reported as normalized values of DNMT3A^mut transcripts per 10⁴ABL1 transcripts (DNMT3A^mut/10⁴ABL1).

Results: In total, 1,494 samples [bone marrow (BM), n=798; peripheral blood (PB), n=696] from 181 DNMT3A^mut pts were analysed [diagnosis, n=287; during therapy, n=840; follow-up, n=367]. Median age of the patients was 50 years (range, 22 to 78); median BM DNMT3A^mut transcript level (TL) at the time of diagnosis was 12690 (range, 1396-54280). There was no significant association of TL with presenting clinical characteristics, such as age, white blood cell count, platelet count, BM and PB blasts, lactate dehydrogenase, or with mutations in NPM1, FLT3 [ITD and TKD], CEBPA and cytogenetics. DNMT3A^mut TL as log 10 transformed continuous variable and stratified by study did not impact OS (p=0.29), RFS (p=0.17) and EFS (p=0.28). Comparing TL after double induction (DI) did not show a significant difference between 13 patients without complete remission (CR) and 117 in CR (12983 and 12595, respectively; p=0.52). In Cox regression analyses, BM DNMT3A^mut TL as log 10 transformed continuous variable during therapy did not impact the clinical endpoints death and relapse. In general, DNMT3A^mut TL during therapy (after induction I, induction II, consolidation I and II) were significantly higher in BM than in PB (p=0.01; p=0.05; p=0.004; p=0.008, respectively). We observed the greatest TL reduction (one log) after induction I, whereas subsequent cycles of therapy did not significantly influence TL. To evaluate the impact of DNMT3A^mut MRD monitoring with regard to the clinical endpoints OS, cumulative incidence of relapse (CIR) and remission duration (RD) after DI and after end of therapy (ET) we used different statistical approaches; all survival analyses were stratified by study. After DI and ET, only 8/90 and 4/88 BM samples became MRD negative. At these two time-points MRD positivity did not significantly impact OS (p=0.99; p=0.74), CIR (p=0.73; p=0.15) and RD (p=0.83; p=0.16). Next, we investigated the MRD DNMT3A^mut log10-reduction (compared to levels at diagnosis) after DI and ET using the median as a cut-off. Again, we could not detect a significant correlation for pts with a higher TL reduction compared with pts with a lower TL reduction for OS and RD after DI and ET (p=0.83; p=0.30; p=0.04; p=0.21, respectively). Lastly, we evaluated the BM DNMT3A^mut TL as 4 increasing equally sized intervals according to the quartiles of the distribution. There was no prognostic impact after DI on OS and RD (p=0.53; p=0.89) and ET (p=0.76; p=0.53). When combining PB and BM samples for the analyses we could not find significant changes in the results.

Conclusion: In our study most pts had persistent DNMT3A^mut TL with only a minority achieving MRD negativity, a finding that supports the presence of persistent clonal hematopoiesis in hematologic remission. Using different explorative approaches, DNMT3A^mut TL did not impact clinical outcome neither during therapy nor during follow-up.