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Clonal Evolution Patterns in Acute Myeloid Leukemia with NPM1 Mutation

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Clonal Evolution Patterns in Acute Myeloid Leukemia with NPM1 Mutation ARTICLE https://doi.org/10.1038/s41467-019-09745-2 OPEN Clonal evolution patterns in acute myeloid leukemia with NPM1 mutation Sibylle Cocciardi1, Anna Dolnik1, Silke Kapp-Schwoerer1, Frank G. Rücker1, Susanne Lux1, Tamara J. Blätte1, Sabrina Skambraks1, Jan Krönke1, Florian H. Heidel2,3, Tina M. Schnöder2,3, Andrea Corbacioglu1, Verena I. Gaidzik1, Peter Paschka1, Veronica Teleanu1, Gudrun Göhring4, Felicitas Thol5, Michael Heuser5, Arnold Ganser5, Daniela Weber1, Eric Sträng6, Hans A. Kestler 6, Hartmut Döhner1, Lars Bullinger1,7,8 & Konstanze Döhner1,8 1234567890():,; Mutations in the nucleophosmin 1 (NPM1) gene are considered founder mutations in the pathogenesis of acute myeloid leukemia (AML). To characterize the genetic composition of NPM1 mutated (NPM1mut) AML, we assess mutation status of five recurrently mutated oncogenes in 129 paired NPM1mut samples obtained at diagnosis and relapse. We find a substantial shift in the genetic pattern from diagnosis to relapse including NPM1mut loss (n = 11). To better understand these NPM1mut loss cases, we perform whole exome sequencing (WES) and RNA-Seq. At the time of relapse, NPM1mut loss patients (pts) feature distinct mutational patterns that share almost no somatic mutation with the corresponding diagnosis sample and impact different signaling pathways. In contrast, profiles of pts with persistent NPM1mut are reflected by a high overlap of mutations between diagnosis and relapse. Our findings confirm that relapse often originates from persistent leukemic clones, though NPM1mut loss cases suggest a second “de novo” or treatment-associated AML (tAML) as alternative cause of relapse. 1 Department of Internal Medicine III, University Hospital of Ulm, Ulm 89081, Germany. 2 Department of Internal Medicine II, Hematology and Oncology, Friedrich-Schiller-University Medical Center, Jena 07743, Germany. 3 Leibniz-Institute on Aging, Fritz-Lipmann-Institute, Jena 07745, Germany. 4 Institute of Cell & Molecular Pathology, Hannover Medical School, Hannover 30625, Germany. 5 Department of Haematology, Haemostasis, Oncology, and Stem Cell Transplantation, Hannover Medical School, Hannover 30625, Germany. 6 Institute of Medical Systems Biology, Ulm University, Ulm 30625, Germany. 7 Department of Hematology, Oncology and Tumorimmunology, Charité University Medicine, Berlin 13353, Germany. 8These authors contributed equally: Lars Bullinger, Konstanze Döhner. Correspondence and requests for materials should be addressed to L.B. (email: [email protected])orto K.D. (email: [email protected]) NATURE COMMUNICATIONS | (2019) 10:2031 | https://doi.org/10.1038/s41467-019-09745-2 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09745-2 ne of the most common mutations (mut) in AML to salvage therapy. To date, it is still unclear whether in these involves the NPM1 gene, which is present in about one cases clonal evolution resulted in loss of NPM1mut or whether the O 1 mut third of AML pts . NPM1 AML is characterized by initial leukemia clone was cured and a new leukemia developed distinct biological and clinical features and pts with the NPM1mut on the basis of dominant clonal hematopoiesis with persisting and no FLT3 internal tandem duplication (ITD) or low FLT3- preleukemic mutations. This new leukemia could then also be ITD levels have a good response to induction chemotherapy and considered as therapy-related AML (tAML) that evolved from a a favorable prognosis. Based on these findings NPM1mut AML common pre-leukemic clone. was included as an entity in the World Health Organization To gain further insight into the genomic evolution of NPM1mut classification 2016 and the NPM1mut/FLT3-ITD genotypes stra- AML, we have extended our previous cohort and assess mutational tified by the ITD allelic ratio were integrated in the risk stratifi- status of five genes by conventional mutation analysis in paired cation of the European LeukemiaNet (ELN) recommendations, samples obtained at diagnosis and relapse from 129 NPM1mut pts. subdividing AML in subsets with highly distinct prognosis2–4. For a subgroup of pts a more detailed analysis comprising the For a long time NPM1mut was considered a founder event, mutational status of nine genes and comprehensive SNP microarray because it is usually maintained at relapse. The consideration of profiling is performed. Finally, for in-depth analysis we sequence NPM1mut as a founder event in AML is further supported by the the exome of ten pts with persistent NPM1mut and ten pts with loss distinct morphological and clinical presentation associated with of NPM1mut and perform RNA-Seq analysis for selected NPM1mut this subtype of AML. However, recent studies have shown that persistent and loss cases. Findings are confirmed by targeted deep- NPM1mut occurs rather late, due to its absence in preleukemic sequencing and flow cytometry based single cell protein expression hematopoietic stem cells (HSCs)5,6. Moreover, in ~10% of analysis. relapsed pts NPM1mut is lost while further chromosomal and 7–9 molecular changes are acquired . Results The recently identified preleukemic mutations in DNMT3A, Genomic characterization of clonal evolution in NPM1mut TET2, ASXL1, IDH1,andIDH2 often persist at remission due to AML. Paired samples at diagnosis and relapse from 129 NPM1mut persistent clonal hematopoiesis5,10. Preleukemic mutations were AML pts were assessed for clonal evolution-associated mutations also found to be present in non-leukemic T cells of AML patients, at in the most recurrently mutated genes (FLT3, DNMT3A, IDH1, thetimeofdiagnosisandtheyhavebeenidentified in individuals IDH2, NRAS) by conventional mutation analysis as previously without hematologic malignancy or who were unselected for cancer reported7. At diagnosis, 83 pts (64%) harbored concurrent or hematologic phenotypes in an age related-manner11. Age-related DNMT3Amut, 40 pts (31%) FLT3-ITD, 22 pts (17%) FLT3-tyr- clonal hematopoiesis is a common condition that is associated with – osine kinase domain (TKD)mut, 23 pts (18%) NRASmut, 29 pts an increased risk to develop hematologic cancer12 15.Indeed,co- (23%) and 24 pts (19%) IDH1mut and IDH2mut, respectively occurring mutations in DNA methylation or hydroxymethylation (Fig. 1). In addition, we screened a subgroup of pts for MLL- genes (DNMT3A, IDH1, IDH2R140,andTET2)arefrequent partial tandem duplications (PTD) and mutations in ASXL1, amongst NPM1mut leukemias and found in ~73% of pts6. TP53 and RUNX1. None of the pts analyzed had a MLL-PTD, In our previously investigated cohort of 53 NPM1mut AML pts, ASXL1mut, TP53mut or RUNX1mut at the time of diagnosis we also described this frequent co-occurrence of preleukemic (Supplementary Fig. 1 and Supplementary Table 1). mutations7. Here, the majority of relapsed leukemias showed At relapse, a shift in the mutation pattern was found in 76 pts clonal evolution with a clear relationship of relapse and diagnostic (59%, Supplementary Table 2). While NPM1mut was lost in 11 pts leukemia clones by acquisition of additional genetic lesions, or (9%), DNMT3Amut persisted in 79 of 83 pts (95%) and thus was the more commonly the relapse clone arose from a common ances- most stable mutation at relapse. Sixteen of 40 pts (40%) had the tral clone, which was also shown by other studies7,16,17. Inter- identical FLT3-ITD, whereas 14 pts (35%) showed a distinct FLT3- estingly, in our study we identified five pts with NPM1mut loss at ITD clone indicated by a change of the ITD length, and 10 pts relapse showing a shift in genetic lesions between the diagnosis (25%) lost the FLT3-ITD at relapse. Gain of a FLT3-ITD clone was and relapse sample; clinically, these patients had a significantly detected in 23 of 128 cases (18%). Similarly, FLT3-TKDmut and longer time to relapse compared to NPM1mut persistent pts NRASmut were rather unstable and lost in 16 of 22 pts (73%) and 15 (33.7 months versus 8.6 months, p = 0.03), and none responded of 23 pts (65%), respectively. On the contrary, IDH1mut and Stability D NPM1 R 91% D DNMT3A R 95% D FLT3-ITD 40% X X X X X X X X X X X X X X R D FLT3-TKD R 27% D NRAS 35% X X R D IDH1 R 86% D IDH2 R 88% Fig. 1 Incidence of mutations in 129 paired (diagnosis/relapse) NPM1mut pts. Colored bars indicate the presence of a mutation, white bars represent wild- type, data not available is indicated by a gray bar. Light and dark green bars illustrate heterozygous and homozygous FLT3-ITD mutations, respectively. Bars marked by X illustrate different mutation types found in the diagnosis and relapse sample. Stability was calculated by the number of mutations that persisted at relapse divided by all mutations present at diagnosis. D, diagnosis; R, relapse 2 NATURE COMMUNICATIONS | (2019) 10:2031 | https://doi.org/10.1038/s41467-019-09745-2 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09745-2 ARTICLE 36.3 36.2 * Loss 36.1 25 35 34.3 24 34.2 Gain 34.1 23 33 22 32 21 UPD 16 26 31 25 15 14 24 16 15.3 15.3 22 13 23 22 15.2 15.2 25 ** 12 15.1 15.1 24 21 23 11.2 21 14 14 22 13 13 22 11.2 12 13 21 12 12 14 12 21.3 23 12 13 21.2 15 14.1 13 21.1 22 12 14.2 13 11.2 14 12 21 14.3 12 13 12 21 11.2 12 21 21 13 12 12 11.2 11.2 22 22 11.2 22 13 12 14 * 23 23 11.2 23 13.1 24 14 11.2 24 13.2 25 15 15 12 25 24 13.3 16 26 21 13 21 22 21 31 27 21 21.1 31 22 21.2 23 23 22 28 21.3 32 24 31.1 22 32 31 22 33 25 31.2 31 23 34 26.1 31.3 41 32 24 32 23 35 26.2 32 26.3 33 33 42 36 33 25 27 34 24.1 * 43 28 34 34 26 35 24.2 37 35 35 27 44 29 36 24.3 1 2 3 4 5 6 7 8 24 15 15 23 14 13 22 13 21 12 12 14 13 13 12 13 11.2 13 11.2 11.2 12 12 12 .
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