Longitudinal detection of <i>DNMT3A</i><sup>R882H</sup> transcripts in patients with acute myeloid leukemia
Authors: Tiziana Ottone, Valentina Alfonso, Licia Iaccarino, Syed Khizer Hasan, Melissa Mancini, Mariadomenica Divona, Serena Lavorgna, Laura Cicconi, Paola Panetta, Luca Maurillo, Maria Ilaria Del Principe, Maria Irno Consalvo, Luca Franceschini, Daniela F. Angelini, Luca Battistini, Gisella Guerrera, Marco De Bardi, Emiliano Fabiani, Giulia Falconi, William Arcese, Sergio Amadori, Francesco Buccisano, Adriano Venditti, Maria Teresa Voso, Francesco Lo‐Coco
Journal: American Journal of Hematology (2018)

DNMT3A gene mutations have been reported in about 22% of acute myeloid leukemia (AML) and often occur in association with other mutations such as FLT3 and NPM1 (60% of cases).6 Recent studies reported that DNMT3A-mutated cells are detectable in patients with AML in long-lasting complete remission (CR), questioning whether quantification of DNMT3A levels may be used to monitor minimal residual disease (MRD).2 To address this issue, we performed sequential monitoring of DNMT3AR882H and NPM1-mutation (mutation A) levels in AML patients and compared the results to those of multiparametric-flow-cytometry (MPFC). A total of 556 patients with de novo AML (median: 49 years, range 16–89 years) were enrolled in clinical trials conducted by the GIMEMA group. Patients received intensive chemotherapy according to EORTC/GIMEMA protocols. Routine morphologic, MPFC, and genetic analyses were carried out in all AML cases at diagnosis at the University of Rome Tor Vergata. DNMT3AR882H, NPM1, and FLT3-ITD-TKD mutations were identified by DNA sequencing, while quantitative assessment of DNMT3AR882H, NPM1mutA, and patient-specific FLT3-ITD was performed on bone marrow (BM) samples harvested at diagnosis, during follow-up, and at the time of relapse, as reported elsewhere.1, 3, 4 The DNMT3AR882H molecular status was also studied in peripheral blood (PB) of 100 chronic myeloid leukemia (CML) patients and in 56 healthy individuals by a qRT-PCR assay.1 According to the declaration of Helsinki, all individuals gave informed consent to the study, which was approved by the Institutional Review Board. Early myeloid progenitors, mature lymphoid, and myeloid cells were sorted from several BM/PB samples of AML, CML, and healthy individuals using a high-speed cell-sorter (Moflo, Beckman Coulter), achieving a purity of over 90%. Statistical analysis was performed with the GraphPad Prism version 5.0 (LaJolla, CA). P values <.05 were considered statistically significant. Differences between categorical variables were evaluated by the Pearson chi-square test, whereas differences between continuous variables were analysed by the unpaired t-test with 95% confidence intervals. At the time of diagnosis, DNMT3A, NPM1, FLT3-ITD, and TKD gene mutations were identified in 56 (10%), 177 (32%), 116 (21%), and 24 (4%) of 556 de novo AMLs. (Supporting Information Table 1). Karyotypic analysis was available for n = 38 (73%) DNMT3AR882H-mutated patients: 37 had a normal and 1 had a complex karyotype. NPM1mutA transcript levels (Supporting Information Table 2) were significantly different when comparing diagnosis, post-induction, and post-consolidation therapy time-points in 51 patients (Figure 1A), and were independent from DNMT3AR882 and FLT3 status. DNMT3AR882H expression levels, analyzed longitudinally in 21 patients (Figure 1B) significantly decreased only after induction, but not after consolidation therapy, regardless of the presence of FLT3 mutations. Comparison of the kinetics of DNMT3AR882H and NPM1mutA in 10 patients at the time of relapse, at a median of 6.5 months from diagnosis, showed that DNMT3AR882H levels did not significantly increase at relapse, unlike NPM1mutA transcripts, which as expected followed the disease course (Figure 1C,D). In nine FLT3-ITD+ve AML, the kinetics of DNMT3AR882H paralleled that of NPM1mutA in three patients only, while DNMT3AR882H expression levels were constant, independent from NPM1mutA in all six FLT3-wt patients (data not shown). Furthermore, DNMT3AR882H levels were unrelated to the BM-blast proportion at diagnosis assessed by immunophenotyping5 (Supporting Information Figure 1A), conversely, NPM1mutA transcript levels showed a significant correlation with BM-blast counts (Supporting Information Figure 1B). Similarly, there were no differences in DNMT3AR882H expression between MPFC MRDpos and MRDneg follow-up samples (n = 47) from 15 patients in CR as defined by cytomorphological criteria (Supporting Information Figure 2). To trace the DNMT3AR882H mutated cell subsets, we sorted different BM-cell fractions of three AML patients (UPN 241, 243, and 278) (Figure 1E). In cells from UPN 241 and UPN 278 sorted at relapse, using the CD34/CD25/CD123/CD99-positive leukemia associated immunophenotype (LAIP),3 NPM1mutA and FLT3-ITD levels were similar in all cell fractions. On the contrary, DNMT3AR882H expression levels were higher in total BM-MNC, than in the LAIP-positive cells in both patients, indicating the presence of the mutation in non-leukemic cells. We then evaluated myeloid precursors, monocytes and T-lymphocytes purified from the BM of UPN 278 (MRDpos) and UPN 243 (MRDneg), at 7- and 9-month follow-up. All analyzed cell populations, including T-lymphocytes, harbored the DNMT3AR882H mutation. By contrast, in UPN 243, NPM1mutA transcripts were almost absent in all cell fractions at the time of CR. These data show that DNMT3AR882H may persist in myeloid and lymphoid BM-cell populations independent of disease status, while NPM1mutA and FLT3-ITD mutations are only detectable in active disease phases. NPM1mutA, DNMT3AR882H, and FLT3-ITD expression levels in AML samples and in sorted cell populations. A, NPM1mutA levels in 51 patients. B, DNMT3AR882H levels in 21 patients. C, NPM1mutA and D, DNMT3AR882H expression level in 9 AML patients at diagnosis, after induction and consolidation treatment, and at relapse. P values were calculated by a paired Student's t-test. E, BM cell subpopulations were sorted in three AML patients (UPN 241, UPN 278, UPN 243) at diagnosis and during follow-up. The antibody combination used for cell sorting is indicated, and reflects the patient-specific LAIP. FU: follow-up In CML patients, the DNMT3AR882H mutation was identified in four cases (4%) only during follow-up, while it was undetectable at the time of diagnosis. DNMT3AR882H transcript levels were constantly low in all available samples (Supporting Information Figure 3), without correlation with BCR/ABL1 levels. Granulocytes and T-lymphocytes sorted from UPN 30 at 46 months from diagnosis, tested DNMT3AR882Hpositive, albeit at low levels (Supporting Information Figure 4A). Finally, DNMT3AR882H was identified in 3 of 56 (5%) elderly healthy individuals as marker of clonal hematopoiesis of indetermined potential (CHIP) and was present also in sorted T-lymphocytes (Supporting Information Figure 4B). Interestingly, as shown in Supporting Information Figure 2, DNMT3AR882H expression levels were similarly low in samples from CML patients, healthy individuals (P = .13), and AML follow-up samples. We show here that, different from NPM1mutA, which is a reliable MRD marker, DNMT3AR882H expression levels do not reflect disease burden in AML, but the mutation is likely present in pre-leukemic clonal myeloid and lymphoid populations persisting at the time of CR. DNMT3AR882H signals persisting after chemotherapy may be part of CHIP, which has been identified in the PB of elderly normal individuals and CML patients, as susceptibility factor for hematological malignancies. This hypothesis was confirmed by constantly low DNMT3AR882H levels in sorted LAIP-positive leukemic blasts, myeloid precursors, monocytes, and T-lymphocytes from AML patients and normal elderly individuals. In summary, our data indicates that DNMT3AR882H quantification is an unreliable tool for MRD monitoring since it does not reflect disease status in AML, and may rather represent the CHIP substrate. This study was supported by Associazione Italiana per la Ricerca sul Cancro (IG N. 5916 to F.L.C. and N. 16952 to M.T.V.), by Veronesi Foundation (to T.O.) and by Associazione italiana contro le Leucemie-Linfomi e Mieloma (to L.I.). All authors declared no conflicts of interest. T.O., V.A., L.I., S.K.H., M.M., M.D., S.L., L.C., P.P., E.F., G.F. collected and analyzed the data; T.O., V.A., M.T.V., and F.L.C. wrote the paper; M.B., T.O., V.A., and L.I. designed the study; L.M., M.I.D.P., M.I.C., L.F., D.F.A., L.B., G.G., M.D.B. analyzed the MPFC data; T.O., V.A., W.A., S.A., F.B., A.V., M.T.V., and F.L.C. analyzed clinical data and revised the manuscript. Additional Supporting Information may be found online in the supporting information tab for this article. Supporting Information Supporting Information Supporting Information Supporting Information Supporting Information Supporting Information Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.