Articles Acute Myeloid Leukemia

324 haematologica | 2015; 100(3)

Introduction

The additional sex combs-like 1 (ASXL1) gene on chromoso-mal band 20q111 is one of three human homologs of the addi-tional sex combs (Asx) gene of drosophila.2 Somatic ASXL1mutations were found in a broad variety of myeloid malig-nancies including chronic myelomonocytic leukemia(CMML, 45%), myelodysplastic syndromes (MDS, 16%),primary myelofibrosis (35%), and acute myeloid leukemia(AML, secondary AML 30%; de novo AML 6.5%).3 Althoughthe exact role of ASXL1 in normal hematopoiesis and thecontribution of mutated ASXL1 to the development ofhematopoietic malignancies have not been fully delineatedyet, there are emerging data suggesting that ASXL1 is a tumorsuppressor. An animal study has shown that Asxl1 deletion orhaploinsuffiency constitutes a sufficient condition for thedevelopment of myeloid neoplasia reminiscent of MDS andMDS/myeloproliferative neoplasms.4 In another study thetransplantation of bone marrow cells expressing oncogeneic

NRASG12D together with knocked-down Asxl1 into lethallyirradiated mice promoted myeloproliferation.5 The animalswith additional loss of Asxl1 showed accelerated myeloprolif-eration and shorter survival than animals expressing Asxl1.5 Infact, in vitro, ASXL1 mutations cause a loss of Polycomb-repressive complex 2 (PRC2)-mediated repression of leuke-mogeneic target genes including those from the posteriorHOXA cluster.5

The first studies on ASXL1 mutations in adult AML areremarkably consistent with respect to the increasing inci-dence of these mutations with age and their association withdistinct clinical and genetic features.6-9 In addition, ASXL1mutations in AML appear to have an adverse impact oninduction success and long-term outcome.6-10 However, thereare only limited data on the prognostic relevance of ASXL1mutations in younger patients with AML.10,11

In our study, representing the largest AML cohort (n=1696)studied for ASXL1 mutations, we focused on youngerpatients with AML (≤61 years) and assessed the incidence

©2015 Ferrata Storti Foundation. This is an open-access paper. doi:10.3324/haematol.2014.114157Manuscript received on July 20, 2014. Manuscript accepted on December 31, 2014.The online version of this article has a Supplementary AppendixCorrespondence: konstanze.doehner@uniklinik-ulm.de

We studied 1696 patients (18 to 61 years) with acute myeloid leukemia for ASXL1 mutations and identified thesemutations in 103 (6.1%) patients. ASXL1mutations were associated with older age (P

and clinical impact of these mutations in the context ofother clinical and genetic factors in a well-defined popula-tion of patients intensively treated in trials of the German-Austrian AML Study Group (AMLSG).

Methods

PatientsA total of 1696 younger AML patients (18 to 61 years) were

studied. Patients were enrolled in prospective treatment protocolsof the AMLSG), namely AML HD98A12 (n=733; NCT00146120),AMLSG 07-0413 (n=893; NCT00151242), and APL HD9514 (n=70)for the patients with acute promyelocytic leukemia (APL). Theclinical studies were approved by the local ethics review commit-tees and all patients gave informed consent for both treatment andcryopreservation of leukemia samples according to theDeclaration of Helsinki. The only criterion to include patients inour study was the availability of a pretreatment bone marrow orperipheral blood specimen for analysis of ASXL1 mutations.Cytogenetic and additional molecular analyses were performed aspreviously described.15-19

ASXL1 mutation analysisA detailed description of the ASXL1 mutation analysis is pro-

vided in the Online Supplementary Material. Briefly, genomicDNA was used as a template for polymerase chain reactions toamplify several fluorescently-labeled DNA fragments coveringthe entire exon 12 (AMLSG 07-04) or regions within exon 12(AML HD98A and APL HD95) identified as main mutation clus-ters in AML.6,20 Amplicons were screened for mutations by aGeneScan-based fragment analysis (Online Supplementary FiguresS1 and S2). Samples classified as mutated after the GeneScananalysis (Online Supplementary Figure S2) were further analyzedby direct sequencing to validate the mutation and to determinethe mutation type.

Statistical analysis Statistical analyses of clinical outcome were performed accord-

ing to previous reports.16 The median follow-up for survival wascalculated according to the method of Korn.21 The definition ofcomplete remission (CR), event-free survival (EFS), relapse-freesurvival (RFS), and overall survival (OS) as well as cytogenetic cat-egorization into favorable-, intermediate-, and adverse-risk groupsfollowed recommended criteria.22 Pairwise comparisons betweenpatients’ characteristics (covariates) were performed using theMann-Whitney test for continuous variables and the Fisher exacttest for categorical variables. The Kaplan-Meier method was usedto estimate the distribution of EFS, RFS and OS.23 Estimation ofconfidence intervals for the survival curves was based on theGreenwood formula for standard error estimation. A logisticregression model was used to analyze associations between base-line characteristics and the achievement of CR. A Cox model wasused to identify prognostic variables.24 In addition to ASXL1muta-tion status, age, sex, hemoglobin level, logarithm of white bloodcell count, type of AML (de novo, secondary AML, therapy-relatedAML), percentage of peripheral blood and bone marrow blasts,cytogenetic risk group,22 and mutational status of NPM1, FLT3(ITD and TKD), CEBPA (CEBPA double-mutated, CEBPAdm), IDH1,IDH2 (IDH2R140, IDH2R172), RUNX1, MLL (PTD), and DNMT3Awere included as explanatory variables in the regression analyses,as indicated, without further selection (full models). The fullmodel presentation was chosen to allow estimation of the relativeimpact of the new marker (ASXL1 mutational status) in the con-cert of the already known prognostic and predictive markers. For

multivariable analyses a missing value imputation technique wasused as recommended for the situation termed missing at random.25

We estimated missing data for covariates by using 50 multipleimputations in chained equations that incorporated predictivemean matching.26 All statistical analyses were performed with thestatistical software environment R version 2.14.0, using the Rpackages rms version 3.3-1, survival version 2.36-8, and cmprskversion 2.2-2.33.

Results

Demographics, clinical baseline characteristics and outcomes of the entire study population

The median age at diagnosis in the entire study cohort(n=1696) was 48.3 years (range, 18-61 years). The patients’baseline characteristics are summarized in OnlineSupplementary Table S2. The CR rate was 73.1% (1230 of1683 patients). With a median follow-up for survival of 5.6years (95%-confidence interval, 5.4 to 5.9 years), the esti-mated 5-year rates for EFS, RFS and OS in the entirecohort were 28.2%, 42.6% and 40.2%, respectively(Online Supplementary Table S2). In total, 422 patientsunderwent allogeneic hematopoietic stem cell transplanta-tion in first CR.

Frequency and types of ASXL1 mutationsASXL1 mutations were detected in 103 (6.1%) of the

1696 patients. The types of mutation at the DNA and pro-tein levels are described in Online Supplementary Table S3.The most common mutation, found in 60% (62/103) ofthe mutated cases, was a duplication of guanine at cDNAposition 1934 (c.1934dupG). Other ASXL1 mutationsfound in more than one patient were c.1900_1922del(n=17) and c.1934delG (n=6). All frame shift mutationsresulted in premature stop codons with consecutive loss ofthe c-terminal plant-homeo-domain. The wild-type allelewas retained in all mutated samples. Of note, the majority(>90%) of the mutations clustered within or around aglycine-rich domain located between amino acids 642 and685.27

Clinical and genetic characteristics of acute myeloidleukemia with ASXL1 mutations Patients with ASXL1 mutations were older (P

other gene mutations (Table 2). RUNX1 (P

EFS and OS rates in patients with ASXL1 mutations were15.9% and 30.3%, respectively, whereas in patients withwild-type ASXL1 they were 29.0 % and 45.7%, respec-tively (Table 3; Figure 1). The adverse effect of ASXL1mutations on EFS and OS was also present in the subset ofcytogenetically normal-AML (data not shown). In the subsetof AML with t(8;21), none of the survival endpoints wasimpacted by the presence of ASXL1 mutations (EFS,P=0.77; RFS, P=0.74; OS, P=0.81). The one patient witht(15;17) and an ASXL1 mutation is still alive and in com-plete remission with a follow-up of 4 years. Among the 58patients with ASXL1mutations achieving a CR, 24 under-went allogeneic hematopoietic stem cell transplantation infirst CR; Mantel-Byar analyses of allogeneic hematopoiet-ic stem cell transplantation did not reveal a significantimpact on either RFS (P=0.87) or OS (P=0.66). We also conducted an exploratory analysis of the com-

posite ASXL1/RUNX1 genotypes. Patients with the geno-type ASXL1mutated/RUNX1mutated had a significantly worseEFS (P

of the cases,9 whereas a study from the Dutch-BelgianHematology-Oncology Cooperative Group (HOVON) on882 unselected AML patients with a median age

with RUNX116,9,32 and IDH29,11 mutations, whereas theywere only rarely detected in patients with FLT3-ITD6-9 orNPM1 mutations.6-9,11,28 In contrast to two other studies,7,9we and others6,11 did not observe any significant associa-tion of ASXL1mutations with CEBPA or FLT3-TKD muta-tions. However, the association of ASXL1 mutations withCEBPAmutations was only reported in older patients withcytogenetically normal AML7 and for FLT3-TKD muta-tions in an AML cohort with a relatively high median ageof 67 years.9 Thus, the selection of the AML cases and agedifference might in part contribute to the differencesamong the results of the studies. In our study, 31 (30%) ofthe 103 ASXL1 mutated cases had a concurrent geneticabnormality involving the RUNX1 gene, i.e., a somaticRUNX1 mutation or t(8;21)(q22;q22); RUNX1-RUNX1T1.Similar data can be obtained from the publication byChou et al. comprising 54 ASXL1 mutated cases.6The presence of ASXL1mutations in patients with a clin-

ically preleukemic condition such as MDS or CMML andthe association of ASXL1 mutations with secondary AMLsuggest that these mutations represent a relatively earlyevent in leukemogenesis. One recent study backtrackedASXL1mutations in four patients with secondary AML anddemonstrated that these mutations were already present inall patients at the stage of the preceding MDS.9 Other stud-ies provide additional evidence that ASXL1mutations favorthe development of AML from MDS or CMML. Thol et al.reported that the presence of ASXL1 frameshift mutationsin patients with lower-risk MDS, i.e., those with anInternational Prognostic Scoring System classification oflow or intermediate-1, was associated with a shorter timeto AML progression,33 and a more recent study byPapaemmanuil et al. of 595 MDS cases found an inferiorleukemia-free survival in patients with ASXL1 mutations(n=81).30 A study by Gelsi-Boyer et al. of 51 CMML patientsidentified ASXL1mutations as an unfavorable factor for theprogression to AML.34 Of note, in that study only CMMLpatients with an ASXL1mutation (11/25; 44%) did progressto AML, whereas none of the patients with wild-typeASXL1 (n=26) developed AML.34Chou et al. found in a group of 360 unselected AML

cases that ASXL1mutations had an unfavorable impact onCR and OS in univariable, but not in multivariable analy-ses.6 In a subsequent CALGB study in older patients withcytogenetically normal AML, ASXL1 mutations wereassociated with inferior CR, EFS, DFS, and OS in univari-able analyses,7 but on multivariable analyses ASXL1muta-tions were revealed as a relevant factor for CR, EFS, DFS,and OS only in cytogenetically normal AML classified asfavorable-risk (n=220) according to the EuropeanLeukemiaNet (ELN) recommendations.7 A HOVON studyof 807 AML patients

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7. Metzeler KH, Becker H, Maharry K, et al.ASXL1 mutations identify a high-risk sub-group of older patients with primary cyto-genetically normal AML within the ELNfavorable genetic category. Blood. 2011;118(26):6920-6929.

8. Pratcorona M, Abbas S, Sanders MA, et al.Acquired mutations in ASXL1 in acutemyeloid leukemia: prevalence and prognosticvalue. Haematologica. 2012;97(3):388-392.

9. Schnittger S, Eder C, Jeromin S, et al. ASXL1exon 12 mutations are frequent in AMLwith intermediate risk karyotype and areindependently associated with an adverseoutcome. Leukemia. 2013;27(1):82-91.

10. Patel JP, Gonen M, Figueroa ME, et al.Prognostic relevance of integrated geneticprofiling in acute myeloid leukemia. N EnglJ Med. 2012;366(12):1079-1089.

11. El-Sharkawi D, Ali A, Evans CM, et al.ASXL1 mutations are infrequent in youngpatients with primary acute myeloidleukemia and their detection has a limitedrole in therapeutic risk stratification. LeukLymphoma. 2014;55(6):1326-1331.

12. Schlenk RF, Döhner K, Mack S, et al.Prospective evaluation of allogeneichematopoietic stem-cell transplantationfrom matched related and matched unrelat-ed donors in younger adults with high-riskacute myeloid leukemia: German-Austriantrial AMLHD98A. J Clin Oncol. 2010;28(30):4642-4648.

13. Schlenk RF, Döhner K, Krauter J, et al. All-trans retinoic acid improves outcome inyounger adult patients with nucleophosmin-1 mutated acute myeloid leukemia – resultsof the AMLSG 07-04 randomized treatmenttrial. Blood. 2011;118(21):38-39 (abstract #80).

14. Schlenk RF, Germing U, Hartmann F, et al.High-dose cytarabine and mitoxantrone inconsolidation therapy for acute promyelocyt-ic leukemia. Leukemia. 2005;19(6):978-983.

15. Gaidzik VI, Bullinger L, Schlenk RF, et al.RUNX1 mutations in acute myeloidleukemia: results from a comprehensive

genetic and clinical analysis from the AMLstudy group. J Clin Oncol. 2011;29(10):1364-1372.

16. Gaidzik VI, Schlenk RF, Paschka P, et al.Clinical impact of DNMT3A mutations inyounger adult patients with acute myeloidleukemia: results of the AML Study Group(AMLSG). Blood. 2013;121(23):4769-4777.

17. Schlenk RF, Döhner K, Krauter J, et al.Mutations and treatment outcome in cyto-genetically normal acute myeloid leukemia.N Engl J Med. 2008;358(18):1909-1918.

18. Paschka P, Schlenk RF, Gaidzik VI, et al.IDH1 and IDH2 mutations are frequentgenetic alterations in acute myeloidleukemia and confer adverse prognosis incytogenetically normal acute myeloidleukemia with NPM1 mutation withoutFLT3 internal tandem duplication. J ClinOncol. 2010;28(22):3636-3643.

19. Schlenk RF, Taskesen E, van Norden Y, et al.The value of allogeneic and autologoushematopoietic stem cell transplantation inprognostically favorable acute myeloidleukemia with double mutant CEBPA.Blood. 2013;122(9):1576-1582.

20. Gelsi-Boyer V, Trouplin V, Adelaide J, et al.Mutations of polycomb-associated geneASXL1 in myelodysplastic syndromes andchronic myelomonocytic leukaemia. Br JHaematol. 2009;145(6):788-800.

21. Korn EL. Censoring distributions as a meas-ure of follow-up in survival analysis. StatMed. 1986;5(3):255-260.

22. Döhner H, Estey EH, Amadori S, et al.Diagnosis and management of acutemyeloid leukemia in adults: recommenda-tions from an international expert panel, onbehalf of the European LeukemiaNet.Blood. 2010;115(3):453-474.

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27. Boultwood J, Perry J, Pellagatti A, et al.Frequent mutation of the polycomb-associ-ated gene ASXL1 in the myelodysplasticsyndromes and in acute myeloid leukemia.Leukemia. 2010;24(5):1062-1065.

28. Carbuccia N, Trouplin V, Gelsi-Boyer V, etal. Mutual exclusion of ASXL1 and NPM1mutations in a series of acute myeloidleukemias. Leukemia. 2010;24(2):469-473.

29. Bejar R, Stevenson K, Abdel-Wahab O, etal. Clinical effect of point mutations inmyelodysplastic syndromes. N Engl J Med.2011;364(26):2496-2506.

30. Papaemmanuil E, Gerstung M, Malcovati L,et al. Clinical and biological implications ofdriver mutations in myelodysplastic syn-dromes. Blood. 2013;122(22):3616-3627.

31. Devillier R, Gelsi-Boyer V, Brecqueville M,et al. Acute myeloid leukemia withmyelodysplasia-related changes are charac-terized by a specific molecular pattern withhigh frequency of ASXL1 mutations. Am JHematol. 2012;87(7):659-662.

32. Mendler JH, Maharry K, Radmacher MD,et al. RUNX1 mutations are associatedwith poor outcome in younger and olderpatients with cytogenetically normal acutemyeloid leukemia and with distinct geneand microRNA expression signatures. J ClinOncol. 2012;30(25):3109-3118.

33. Thol F, Friesen I, Damm F, et al. Prognosticsignificance of ASXL1 mutations in patientswith myelodysplastic syndromes. J ClinOncol. 2011;29(18):2499-2506.

34. Gelsi-Boyer V, Trouplin V, Roquain J, et al.ASXL1 mutation is associated with poorprognosis and acute transformation inchronic myelomonocytic leukaemia. Br JHaematol. 2010;151(4):365-375.

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330 haematologica | 2015; 100(3)