Anna Stengel

Claudia Haferlach

Luise Hartmann

Manja Meggendorfer

Niroshan Nadarajah

Torsten Haferlach

Wolfgang Kern

Haematologica

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Molecular characterization of acute myeloidleukemia patients who relapse more than 3 yearsafter diagnosis: an exome sequencing study of 31patientsAcute myeloid leukemia (AML) is the most commonacute leukemia in adults, with affected patients having amedian age of 65-70 years at diagnosis. Standardchemotherapy protocols result in remission in ~40-80%of patients. Relapse of the disease, however, is commonand associated with a dismal outcome. Higher incidencesof relapse have been observed in patients with FLT3internal tandem duplication (ITD), TP53 alterations andadverse-risk cytogenetics.1,2 AML is known to be a genet-ically heterogeneous disease,3,4 a fact which also plays animportant role in resistance to chemotherapy andrelapse.5 Therefore, clonal evolution from diagnosis torelapse has been extensively studied in AML in order toidentify mechanisms of therapy resistance.6,7 In the major-ity of patients, genetic changes between the two diseasestates can be observed and the stability of a gene muta-tion was found to depend on the molecular function.NPM1mutations, one of the most common alterations inAML, were found to be relatively stable,8 similar to muta-tions affecting epigenetic modifiers (DNMT3A, IDH1/2and TET2).7 By contrast, mutations that alter componentsof signaling pathways or transcription factors have beenshown to be less stable during disease progression.8However, gene mutations specifically driving AMLrelapse remain uncertain as previous studies did not iden-tify a general relapse mechanism. The majority of relaps-es occur within the first 2 years after diagnosis and it hasbeen suggested that patients who remain in remission for>3 years can be considered as cured.9 Nonetheless, laterelapse of AML can occur,10,11 although comprehensivegenetic data on a larger cohort of AML patients with laterelapses are scarce. Thus, we selected a total of 31 AML cases sent to ourlaboratory for diagnostic work-up from 2005-2015 whorelapsed >3 years after initial diagnosis to delineate thegenetic profile and gain knowledge about the molecularpathogenesis in these patients. All patients were treatedintensively with chemotherapy and went into remission.Moreover, four patients received an allogeneic stem celltransplant. Based on sample availability, whole exomesequencing was carried out for 31 patients at the timepoints of diagnosis and relapse as well as on matchedremission samples that were available for 19 patients.Remission samples after stem cell transplantation wereavailable for all transplanted patients and also sequenced.NPM1 mutations and FLT3-ITD were identified by rou-tine diagnostics as exome sequencing is not reliable forthe detection of these types of aberrations. As a controlcohort, we identified patients who relapsed within 1 year(n=371) after diagnosis for comparison of cytogenetic andmolecular risk factors. For exome sequencing and variantcalling, enrichment based library preparation was per-formed using the TruSeq DNA Library Preparation Kit(Illumina, San Diego, CA, USA) and the xGen ExomeResearch Panel (IDT, Integrated DNA Technologies,Coralville, IA, USA) and sequenced (2x151 bp) on aNovaSeq instrument (Illumina). Further data processing,stringent filtering and the patients’ characteristics arehaematologica 2020; 105:e157LETTERS TO THE EDITORFigure 1. Mutation landscape of acute myeloid leukemia patients who relapsed more than 3 years after diagnosis. Each column represents one analyzedpatient, each line one analyzed gene. NA: not available; R: detected in the remission sample only.described in the Online Supplementary Material, OnlineSupplementary Figure S1 and Online Supplementary TablesS1 and S2. Exome sequencing was performed on diagnostic (meancoverage: 127x) and relapse (mean coverage: 115x) sam-ples. Combining data at diagnosis and relapse, we identi-fied a total of 590 mutations (single nucleotide variants,n=524; insertions-deletions, n=66), affecting 487 differ-ent genes (Online Supplementary Table S3). Of these genes,52 were recurrently mutated. Highest mutation frequen-cies were observed in DNMT3A (10/31 patients, 32%),RUNX1 (9/31 patients, 29%), IDH2 (8/31 patients, 26%),SRSF2 (6/31 of patients, 19%), BCOR (5/31 patients,16%), and CDC42EP1, FLT3, IDH1, USF3, and VCX3B(4/31 patients, 13%). Overall, we identified 4-34 (medi-an: 19) mutations per patient. In more detail, we detecteda median of 13 mutations (range, 4-25) at diagnosis anda median of 16 (range, 1-29) at relapse. In 30/31 patients,we identified mutations that were present at both timepoints, highlighting the genetic relationship betweenboth disease states (Figure 1). However, clonal evolutionby means of gained or lost mutations at relapse wasnoticed in every patient: 1-22 changes in the mutationalpattern were observed per patient (median: 8), 0-19(median: 3) mutations per patient were lost at relapse and0-15 (median: 4) mutations were gained at relapse.    When comparing the matched diagnostic and relapsesamples for all 31 patients, of the 590 mutations identi-fied, 306 mutations were present at both disease timepoints, while 114 mutations were detected only in thediagnostic specimen and 170 mutations were exclusivelyidentified in the relapse sample. We queried the molecu-lar function (8 distinct groups) of the genes affected usingthe PANTHER gene list analysis online tool(http://www.pantherdb.org/). The frequency of the affectedfunction was compared between all mutated genes andgenes mutated either exclusively at diagnosis or relapse.This comparison of mutations revealed a slight increasein mutations affecting transcription regulation (8% vs.11%; P=not significant) and decrease of mutations affect-ing signal transduction (10% vs. 3%, P=not significant)towards disease progression (Online Supplementary FigureS2). Next, we limited our analyses to genes known to berecurrently mutated in myeloid neoplasms and to molec-ular functions relevant to the pathogenesis of AML.12Overall, 11/22 (50%) of mutations lost at relapse affectedsignaling pathways, followed by transcription factors(n=6), splicing factors and epigenetic modifiers (n=2)(Figure 2). In contrast, 20 mutations were gained duringdisease progression: 12/20 (60%) affected transcriptionfactors with known roles in hematopoiesis (RUNX1, n=6;ETV6 and BCOR, n= 2; WT1 and BCORL1, n=1). Themajority of these mutations are predicted to result intruncation of the coding protein. In addition, four muta-tions acquired in the relapse clone affected tumor sup-pressor genes (NF1, n=2; TP53, n=1, PHF6, n=1). Fourpatients (AML-4, AML-13, AML-28 and AML-30)received an allogeneic stem cell transplant before relapse.In these patients, of the 15 mutations gained at relapse,six affected transcription (co)factors (DEPDC1, PLAG1,SPI1, ETV6, RUNX1 and BCOR)  and three affected pro-teins involved in cell cycle regulation or apoptoticprocesses (PPP1R10, G2E3 and MAPK4). However, wedid not observe mutations that can be linked to immune-escape mechanisms.   Data from conventional cytogenetic analyses wereavailable for 29 patients at the time point of diagnosisand 29 patients at the time point of relapse, comparisonof the disease states was possible for 27 patients.Cytogenetic changes between diagnosis and relapse wereobserved in 13/27 (48%) patients analyzed at both timepoints. Evolution from a normal to aberrant karyotypewas evident in 7/13 of these patients. A complex kary-otype different from the one detected initially at diagno-sis was observed in 2/13 (15%) relapsed patients and inone patient with a complex karyotype at diagnosis, fur-ther additional aberrations were detected at relapse(Online Supplementary Table S4). Furthermore, we identi-fied emergence of cytogenetically independent clonesbetween diagnosis and relapse in 3/13 (23%) cases. We also performed exome sequencing of remissionsamples which were available for 19 patients (mean cov-haematologica 2020; 105:e158LETTERS TO THE EDITORFigure 2. Mutation frequency based on functional groups proposed to be relevant in acute myeloid leukemogenesis. Stability of the mutations between diag-nostic and matched relapse samples depended on the affected gene. While mutations in epigenetic modifiers were mostly present at both time points (indi-cated in red), mutations in transcription factors and tumor suppressor genes were often gained at relapse (dark gray), while mutations affecting signaling path-ways were often lost during disease progression (light gray); erage: 118x). In order to avoid confounding by privategermline variants, we filtered for genes known to berecurrently affected in AML patients and identified a totalof nine persisting mutations (DNMT3A, n=3; TET2, n=3;SRSF2, n=2 and IDH1, n=1) in 6/19 (32%) patients eval-uated in remission. Importantly, the remission sampleswere negative for all other mutations identified at diag-nosis, ruling out refractory AML, but rather suggestingthe persistence of a preleukemic clone. Age at diagnosisof patients with or without persisting mutations wascomparable (median: 52.3 vs. 57.3 years; P=0.8).Moreover, time from diagnosis to relapse did not differbetween the two groups of patients (median: 47 vs. 47months; P=0.8). An analysis of mutations occurring afterchemotherapy is described in the Online SupplementaryData and Online Supplementary Figure S3. Furthermore,four case reports are presented in the OnlineSupplementary Data to further explain the clonal evolu-tion of molecular and cytogenetic alterations in AMLpatients with late relapses (Online Supplementary FigureS4).We were able to show that mutations in epigeneticmodifiers were mostly stable between diagnosis andrelapse, suggesting that these mutations are early eventsin leukemogenesis. By contrast, mutations affecting sig-naling pathways were frequently lost while the majorityof mutations in transcription factors and tumor suppres-sor genes were gained in the relapse clone of our patients.Prognostic markers have been established to identifyAML patients with dismal outcome.3 We compared thefrequency of some of these markers in our cohort ofpatients with late relapses to the frequency in patientswho relapsed within the first year after diagnosis tocheck for a relationship between high-risk markers andtime to relapse. Although we observed a trend towardsan enrichment of high-risk parameters in patients withearly relapse, this trend was not statistically significant.Similarly, in a different study no significant differences inage, AML status, white blood cell count, karyotype,FLT3-ITD mutation, CEBPA mutation and induction reg-imen were observed between patients with early or laterelapse,13 indicating that the dynamics of relapse might beinfluenced by other factors. In summary, we describe theclonal evolution in a large cohort of AML patients whoexperienced a late relapse of their disease. Our data indi-cate that the relapse clone is characterized by gain ofmutations that mainly affect transcription factors andtumor suppressor genes, suggesting an important role inthe pathogenesis. This knowledge might help to identifytailored therapy approaches to reduce the risk of AMLrelapse.Luise Hartmann, Claudia Haferlach, Manja Meggendorfer,Niroshan Nadarajah, Wolfgang Kern, Torsten Haferlach and  Anna StengelMLL Munich Leukemia Laboratory, Munich, GermanyAcknowledgments: the authors would like to thank all co-workers at the MLL Munich Leukemia Laboratory for approaching togethermany aspects in the field of leukemia diagnostics and research withtheir dedicated work. The authors would also like to thank all physicians for providing samples and caring for patients as well as collecting data. Correspondence: ANNA STENGEL - anna.stengel@mll.comdoi:10.3324/haematol.2019.227702Information on authorship, contributions, and financial & other dis-closures was provided by the authors and is available with the onlineversion of this article at www.haematologica.org.References1. Döhner H, Estey E, Grimwade D, et al. Diagnosis and managementof AML in adults: 2017 ELN recommendations from an internationalexpert panel. Blood. 2017;129(4):424-447. 2. Rücker FG, Schlenk RF, Bullinger L, et al. TP53 alterations in acutemyeloid leukemia with complex karyotype correlate with specificcopy number alterations, monosomal karyotype, and dismal out-come. Blood. 2012;119(9):2114-2121.3. Papaemmanuil E, Döhner H, Campbell PJ. Genomic classification inacute myeloid leukemia. N Engl J Med. 2016;375(9):900-901. 4. Grimwade D, Ivey A, Huntly BJ. Molecular landscape of acutemyeloid leukemia in younger adults and its clinical relevance. Blood.2016;127(1):29-41.5. Landau DA, Carter SL, Getz G, Wu CJ. Clonal evolution in hemato-logical malignancies and therapeutic implications. Leukemia. 2014;28(1):34-43. 6. Li S, Garrett-Bakelman FE, Chung SS, et al. Distinct evolution anddynamics of epigenetic and genetic heterogeneity in acute myeloidleukemia. Nat Med. 2016;22(7):792-799.7. Greif PA, Hartmann L, Vosberg S, et al. Evolution of cytogeneticallynormal acute myeloid leukemia during therapy and relapse: anexome aequencing study of 50 patients. Clin Cancer Res. 2018;24(7):1716-1726.8. Krönke J, Bullinger L, Teleanu V, et al. Clonal evolution in relapsedNPM1-mutated acute myeloid leukemia. Blood. 2013;122(1):100-108.9. Yanada M, Garcia-Manero G, Borthakur G, Ravandi F, Kantarjian H,Estey E. Potential cure of acute myeloid leukemia: analysis of 1069consecutive patients in first complete remission. Cancer.2007;110(12):2756-2760.10. Verma D, Kantarjian H, Faderl S, et al. Late relapses in acute myeloidleukemia: analysis of characteristics and outcome. Leuk Lymphoma.2010;51(5):778-782.11. Watts JM, Wang XV, Litzow MR, et al. Younger adults with acutemyeloid leukemia in remission for ≥ 3 years have a high likelihoodof cure: the ECOG experience in over 1200 patients. Leuk Res. 2014;38(8):901-906. 12. Döhner H, Weisdorf DJ, Bloomfield CD. Acute myeloid leukemia. NEngl J Med. 2015;373(12):1136-1152.13. Bertoli S, Tavitian S, Berard E, et al. More than 10% of NPM1-mutat-ed AML relapses occur after 5 years from complete remission. Blood.2018;132(Suppl 1):2802.haematologica 2020; 105:e159LETTERS TO THE EDITOR

Molecular characterization of acute myeloid leukemia patients who relapse more than 3 years after diagnosis: an exome sequencing study of 31 patients

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Molecular characterization of acute myeloid leukemia patients who relapse more than 3 years after diagnosis: an exome sequencing study of 31 patients

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