RUNX1-RUNX1T1: Frequency and Impact on Clinical Outcome

ORIGINAL ARTICLE High number of additional genetic lesions in acute myeloid leukemia with t(8;21)/RUNX1-RUNX1T1: frequency and impact on clinical outcome

M-T Krauth1,2, C Eder1, T Alpermann1, U Bacher1, N Nadarajah1, W Kern1, C Haferlach1, T Haferlach1 and S Schnittger1

t(8;21)/RUNX1-RUNX1T1-positive acute myeloid leukemia (AML) is prognostically favorable; however, outcome is heterogeneous. We analyzed 139 patients with t(8;21)/RUNX1-RUNX1T1-positive AML (de novo: n ¼ 117; therapy-related: n ¼ 22) to determine frequency and prognostic impact of additional genetic abnormalities. All patients were investigated for mutations (mut) in ASXL1, FLT3, KIT, NPM1, MLL, IDH1, IDH2, KRAS, NRAS, CBL and JAK2. Sixty-nine of 139 cases (49.6%) had 1 mutation in addition to RUNX1- RUNX1T1, and 23/139 (16.5%) had X2 additional mutations. Most common were KITmut (23/139; 16.5%), NRASmut (18/139; 12.9%) and ASXL1mut (16/139; 11.5%). FLT3-ITD, FLT3-TKDmut, CBLmut, KRASmut, IDH2mut and JAK2mut were found in 2.9–5.0%. Additional chromosomal abnormalities (ACAs) were found in 97/139 (69.8%). Two-year overall survival (OS) was 73.4% in 111 intensively treated patients. KITD816mut negatively impacted on OS in de novo AML (2-year OS: 59.1% vs 82.0%, P ¼ 0.03), ASXL1mut on EFS (de novo AML: 20% vs 59.1%, P ¼ 0.011; total cohort: 28.6% vs 56.7%, P ¼ 0.021). Sex chromosome loss was favorable (2-year EFS: 66.9% vs 43.0%, P ¼ 0.031), whereas þ 8 was adverse on EFS (2-year EFS: 26.7% vs 55.9%, P ¼ 0.02). In conclusion, t(8;21)/RUNX1-RUNX1T1-positive AML shows a high frequency of additional genetic alterations. Investigation for KITD816 and ASXL1mut combined with investigation of ACAs is recommended in t(8;21)/RUNX1-RUNX1T1-positive AML because of the prognostic signiﬁcance of these parameters.

Leukemia (2014) 28, 1449–1458; doi:10.1038/leu.2014.4 Keywords: acute myeloid leukemia; t(8;21)/RUNX1-RUNX1T1; secondary mutations; KIT mutation; ASXL1 mutation; prognosis

INTRODUCTION mutations such as RAS, a key player in AML cell proliferation, were 8,13,14 In patients with acute myeloid leukemia (AML), t(8;21)(q22;q22) found to be associated with RUNX1-RUNX1T1. These data with the resulting RUNX1-RUNX1T1 rearrangement is one of the support the hypothesis of an oncogenic cooperation in most common cytogenetic abnormalities. It occurs in about leukemogenesis between RUNX1-RUNX1T1 and additional 4 7–8% of adult de novo AML.1 According to the World molecular alterations and illustrate the need for evaluation of Health Organization (WHO) classification, t(8;21)(q22;q22)/RUNX1- additional molecular markers at the time of diagnosis. Besides RUNX1T1 defines a distinct AML subtype. Although this AML contributing to further risk stratification of AML patients, these subtype is generally associated with a favorable prognosis, about data may provide a rationale for new therapies targeting 15 30% of patients relapse, and, in this context, the frequency and pathways in t(8;21)/RUNX1-RUNX1T1-positive AML. For impact of additional genetic lesions is incompletely understood as example, the addition of the second-generation tyrosine kinase yet. On the molecular level, the RUNX1-RUNX1T1 fusion gene inhibitor dasatinib to chemotherapy is currently being evaluated 16 influences cell proliferation, differentiation and self-renewal for patients with CBF leukemias. Aiming to further clarify the role capacity.2 Furthermore, RUNX1-RUNX1T1 interferes and represses of this and other additional genetic lesions in this AML subtype, the transcription factor CBF (core-binding factor), which has a key we here performed comprehensive genetic analysis and studied role during early hematopoiesis.3 However, targeting CBF, RUNX1- the clinical outcome in a large cohort of 139 patients with t(8;21)/ RUNX1T1 alone was shown not to be sufficient to induce leukemia, RUNX1-RUNX1T1-positive AML. but requires additional mutations to trigger leukemogenesis.4 RUNX1-RUNX1T1 collaborates with mutations of members of the class III transmembrane receptor tyrosine kinase subfamily.5,6 PATIENTS AND METHODS Among them, the FLT3-ITD, one of the most frequent genetic A total of 139 patients diagnosed with t(8;21)/RUNX1-RUNX1T1-positive alterations in AML,7 has been shown to cooperate with RUNX1- AML were included in the study. Patients were referred from different RUNX1T1 in inducing leukemia in a murine bone marrow hematologic centers in Germany to the MLL Munich Leukemia Laboratory between August 2005 and November 2012. There were 65 female and transplantation model.8 Also, mutations in the KIT oncogene act 9,10 74 male patients (male/female ratio: 0.9). The median age was 53.3 years as cooperative mutations in leukemogenesis. The respective (range, 18.6–83.8 years). In all cases, the t(8;21)/RUNX1-RUNX1T1 mutations were demonstrated to confer an independent negative was confirmed by chromosome banding analysis, fluorescence in situ impact on prognosis in patients with CBF leukemia harboring hybridization (FISH) and reverse transcription-polymerase chain reaction in t(8;21)/RUNX1-RUNX1T111 or inv(16)/CBFB-MYH11.12 Rarely, other combination. French-American-British classification was available in

1MLL Munich Leukemia Laboratory, Munich, Germany and 2Department of Internal Medicine I, Division of Hematology and Hemostaseology, Medical University Vienna, Vienna, Austria. Correspondence: Dr S Schnittger, MLL Munich Leukemia Laboratory, Max-Lebsche-Platz 31, 81377 Munich, Germany. E-mail: [email protected] Received 20 November 2013; revised 19 December 2013; accepted 30 December 2013; accepted article preview online 9 January 2014; advance online publication, 31 January 2014 Secondary mutations in AML with t(8;21)/RUNX1-RUNX1T1 M-T Krauth et al 1450 107 patients:17,18 34 (31.8%) had AML M1 and 73 (68.2%) had AML M2. 21 and in 1 case both the derivative chromosomes 8 and 21 were A total of 117 patients had de novo AML (84.2%), and 22 patients (15.8%) involved in additional rearrangements; in 1 case the RUNX1- therapy-related AML (t-AML) following (radio-) chemotherapy for other RUNX1T1 rearrangement was based on an insertion of chromo- malignancies (breast cancer, lung cancer, gastrointestinal tract cancer, some 8q material into the long arm of chromosome 21 at multiple myeloma or Non-Hodgkin lymphoma) (Table 1). Clinical follow-up chromosome band 21q22. According to interphase FISH (124/139 data were available for most of the patients. A total of 111 patients with samples could be analyzed quantitatively), the percentage follow-up data (79.9% from the total cohort; de novo AML: n ¼ 95, 85.6%, t-AML: n ¼ 16, 14.4%) received intensive treatment strategies19,20 (like of positive cells was in median 90% (range: 15–100%). standard protocols including ‘7 þ 3’ or combinations of chemotherapeutics Fifteen cases could not be analyzed quantitatively because of such as TAD (6-thioguanine, cytarabine and daunorubicin) and HAM (high- poor quality of bone marrow or peripheral blood samples. dose cytarabine and mitoxantrone)) and were the basis for prognostic Real-time polymerase chain reaction revealed heterogeneous evaluation in this study. Twenty-three of those 111 patients (20.7%) %RUNX1-RUNX1T1/ABL1 expressions with a median of 46.8% received allogeneic hematopoietic stem cell transplantation. In 21 cases, (range: 9.2–451.2%). This expression level does not correlate with paired samples from diagnosis and relapse were available and were % t(8;21)-positive metaphases or interphases, indicating that analyzed for the molecular pattern. patients have individual RUNX1-RUNX1T1 expression levels per All patients gave their informed consent for genetic analysis and for the use of the laboratory results for scientiﬁc studies. The study was approved cell. At relapse, the median %RUNX1-RUNX1T1/ABL1 expression by the Internal Review Board of the MLL Munich Leukemia Laboratory and was 34.4% (range: 0.2–300.7%) and did not differ signiﬁcantly adhered to the tenets of the Declaration of Helsinki.

Cytomorphology, cytogenetics and immunophenotyping Cytomorphologic assessment was based on May–Gru¨nwald–Giemsa stains, Table 1. Characteristics of 139 patients with t(8;21)/RUNX1-RUNX1T1- myeloperoxidase reaction and nonspecific esterase using a-naphthyl positive AML acetate following French-American-British and WHO classifications.17,18,21 Chromosome banding analysis combined with FISH was performed in all Features Results patients following standard methods.22,23 Interphase FISH with probes for RUNX1 and RUNX1T1 was performed with commercially available probes Gender, female/male (ratio) 65/74 (0.9) (Abbott, Wiesbaden, Germany; Metasystems, Altlussheim, Germany). Median age (years) (range) 53.3 (18.6–83.8) Karyotypes were described according to the International System for 24 Blood counts, median values (range) Human Cytogenetic Nomenclature. Immunophenotyping was performed 9 in 56/139 cases (40.3%) as described previously.25,26 WBC count ( Â 10 /l) 7.1 (1.4–170.0) Platelet count ( Â 109/l) 35.5 (5.0–271.0) Hemoglobin level (g/dl) 8.8 (3.7–19.0) Molecular analysis At diagnosis, in 127/139 (91.4%) cases bone marrow and in 12/139 (8.6%) Etiology (n ¼ 139) cases peripheral blood was used for the molecular analysis. Isolation of De novo AML 117 (84.2%) mononuclear cells, DNA extraction and mRNA extraction as well as Therapy-related AML 22 (15.8%) random-primed cDNA synthesis followed previous descriptions.7 Quantitative real-time polymerase chain reaction for RUNX1-RUNX1T1 FAB classification (n ¼ 107) expression was carried out at the time of diagnosis and during follow-up, M1 34 (31.8%) as has been published previously.27 ABL1 was used as a reference gene M2 73 (68.2%) and expression levels were calculated as %RUNX1-RUNX1T1/ABL1. Investigations for ASXL1,28 FLT3-ITD,7 FLT3-TKD,29 KIT (D816, exon 8, Number of cytogenetic aberrations in addition to t(8;21)(q22;q22) exons 9–11),11,30 NPM1,31,32 MLL-PTD,33 IDH1 and IDH2,34 KRAS, NRAS,35 Sole t(8;21)(q22;q22) 42 (30.2%) CBL36 and JAK2 mutations37 were performed in all patients. At least 1 ACA 97 (69.8%) X2 ACAs 42 (30.2%) Definition of clinical end points and statistical analysis Specification of cytogenetic aberrations in addition to t(8;21)(q22;q22) Survival curves were calculated for overall survival (OS), event-free survival Loss of sex chromosomes (either X or Y) 65 (46.8%) (EFS) and OS with patients censored on the day of allogeneic stem cell alloSCT Del(9q) 21 (15.1%) transplantation (OS ) according to Kaplan–Meier and compared using Trisomy 8 8 (5.8%) log-rank test. OS was the time from diagnosis to death or last follow-up. Other 44 (31.7%) EFS was the time from diagnosis to treatment failure, relapse, death or last follow-up in complete remission. Relapse was defined according to the 38 Number of molecular mutations in addition to RUNX1-RUNX1T1 International Working Group Criteria. Median follow-up was calculated Sole RUNX1-RUNX1T1 70 (50.4%) taking the respective last observations in surviving cases into account and At least 1 additional molecular mutation 69 (49.6%) censoring non-surviving cases at the time of death. Differences were X2 Additional mutations 23 (16.5%) considered significant at P p0.05. Dichotomous variables were compared 2 between different groups using the w -test and continuous variables by Specification of molecular mutations in addition to RUNX1-RUNX1T1 Student’s t-test. All reported P-values are two-sided. SPSS (version 19.0.0) KIT (D816: n ¼ 18; exon 8: n ¼ 4; exon 11: n ¼ 1) 23 (16.5%) software (IBM Corporation, Armonk, NY, USA) was used for statistical NRAS 18 (12.9%) analysis. ASXL1 16 (11.5%) FLT3-ITD 7 (5.0%) FLT3-TKD 6 (4.3%) RESULTS CBL 6 (4.3%) Characterization of t(8;21)/RUNX1-RUNX1T1 KRAS 6 (4.3%) Cytogenetic data were available in all 139 patients. At diagnosis, IDH2R140 5 (3.6%) the percentage of metaphases with t(8;21)(q22;q22), assessed JAK2V617F 4 (2.9%) IDH1R132 1 (0.7%) by chromosome banding analysis, was in median 95% (range: NPM1 Not found 14.3–100%). One hundred and twenty-two of 139 cases harbored MLL-PTD Not found a standard t(8;21)(q22;q22), and 17/139 (12.2%) showed variant IDH2R172 Not found forms of t(8;21)(q22;q22): 12 cases showed a variant translocation involving one additional chromosome, 1 case involved 3 Abbreviations: ACA, additional cytogenetic aberration; AML, acute myeloid additional chromosomes; in 2 cases the derivative chromosome leukemia; FAB, French-American-British; WBC, white blood cell count.

Leukemia (2014) 1449 – 1458 & 2014 Macmillan Publishers Limited Secondary mutations in AML with t(8;21)/RUNX1-RUNX1T1 M-T Krauth et al 1451 from diagnostic samples (P ¼ 0.225), as measured in 21 paired 11.1%), D816F (n ¼ 1, 5.6%) and D816T (n ¼ 1, 5.6%). One samples. patient exhibited both D816H and D816F mutations. Among exon 8, four different amino-acid changes were detected in one patient each: p.Thr417_Asp419delinsIle, p.Tyr418_Asp419insArg- Additional cytogenetic alterations PhePhe, p.Asp419del and p.Asp419_Arg420delinsGlu. In exon 11, Ninety-seven of 139 patients (69.8%) had at least one additional p.Pro577_Tyr578ins7AS amino-acid change was detected. chromosomal abnormality (ACA) besides t(8;21)(q22;q22); 39.6% Frequencies of other mutations were as follows: FLT3-ITD, (55/139) had one ACA, and 42 (30.2%) patients had two or more 7/139 (5.0%), FLT3-TKD, CBL and KRAS, each 6/139 (4.3%). ACAs. Most frequent was the loss of either X or Y chromosomes Concerning IDH gene alterations, an IDH1R132 mutation was (n ¼ 65, 46.8%), followed by 9q deletion (del(9q); n ¼ 21, 15.1%) detected in 1/139 (0.7%) and IDH2R140 in 5/139 (3.6%), whereas and trisomy 8 ( þ 8; n ¼ 8, 5.8%) (Table 1). IDH2R172 mutations were not found. JAK2 mutations were Cytogenetic data were then separately calculated for detectable in 4/139 (2.9%). Notably, NPM1mut and MLL-PTD were patients with de novo AML and t-AML. Eighty-one of 117 not found and thus were mutually exclusive of RUNX1-RUNX1T1 (69.2%) patients with de novo AML had ACAs. Again, the most (Table 1 and Figure 1). frequent ACA was the loss of a sex chromosome (n ¼ 55, 47.0%; Taken together, RAS pathway-activating mutations including in more detail: 55.7% À Yinmalesand34.0% À X in females). NRAS, KRAS, FLT3-ITD, FLT3-TKD, CBL and JAK2 were found in 43 9q deletion was found in 13.7% (n ¼ 16), and þ 8in4.3%(n ¼ 5) (30.9%) of all patients: 39 patients had one RAS pathway- of patients (Table 2). activating mutation and 4 patients had two RAS pathway- In t-AML, 72.7% (16/22) cases had ACAs, 45.5% (n ¼ 10) had activating mutations (combinations were: NRAS and KRAS, n ¼ 2; loss of a sex chromosome (all males showed À Y, and 36.8% À X FLT3-TKD and NRAS, n ¼ 1; FLT3-ITD and FLT3-TKD: n ¼ 1) in females). Again, the second most frequent ACA was del(9q), (Figure 1). which occurred in 22.7% (n ¼ 5) of patients; 13.6% (3/22) of t-AML patients showed þ 8 (Table 2). Overall, the frequency of ACAs did Comparison of additional mutation frequency in de novo AML and not differ signiﬁcantly between patients with de novo AML and t-AML t-AML, showing only a trend toward higher frequency of À Y, del(9q) and þ 8 in patients with t-AML (Table 2 and Figure 1). The overall frequency of additional molecular mutations did not differ signiﬁcantly between de novo AML and t-AML: in 117 patients with de novo AML, 58 (49.6%) had at least one additional Frequency and characterization of additional mutations in the molecular mutation. In t-AML this frequency was 11/22 (50.0%). total cohort of patients In de novo AML, most frequent were KIT mutations (D816, exon Overall, additional molecular mutations were detected in 69/139 8 or exon 11; 17.1%), followed by NRAS (13.7%) and ASXL1 (11.1%). (49.6%) patients. Twenty-three (16.5%) had two or more additional To a lower extent, FLT3-ITD mutations were found in 6% of mutations (2 mutations: n ¼ 19, 13.7%; 3 mutations: n ¼ 2, 1.4%). patients, KRAS and CBL in 4.3%, FLT3-TKD and IDH2R140 in 3.4% Most common were KIT mutations (n ¼ 23; 16.5%), followed by each and JAK2 in 2.6% of cases. IDH1R132 was detectable only in NRAS (n ¼ 18; 12.9%) and ASXL1 mutations (n ¼ 16; 11.5%). KIT 0.9% of patients. In t-AML, most frequent were KIT mutations mutations were distributed in detail as follows: D816 point (D816V, exon 8 or exon 11) and ASXL1 mutations (both 13.6%), mutations in 18 cases (78.3%), exon 8 in 4 cases (17.4%) and exon followed by NRAS and FLT3-TKD mutations (both 9.1%). KRAS, CBL, 11 in 1 case (4.3%). Among KITD816, the following mutations were IDH2R140 and JAK2 were each found in 4.5% of cases. FLT3-ITD found: D816V (n ¼ 11, 61.1%), D816H (n ¼ 4, 22.2%), D816E (n ¼ 2, and IDH1R132 mutations were not found in the t-AML cohort.

Table 2. Differences in chromosomal aberration and mutation frequencies between de novo AML and therapy-related RUNX1-RUNX1T1-positive AML

Total cohort (n ¼ 139) De novo AML (n ¼ 117) t-AML (n ¼ 22) P-value

Mutations in addition to RUNX1-RUNX1T1 Sole RUNX1-RUNX1T1 70 (50.4%) 59 (50.4%) 11 (50%) NS At least 1 additional molecular mutation 69 (49.6%) 58 (49.6%) 11 (50%) NS X2 Additional mutations 23 (16.5%) 20 (17.1%) 3 (13.6%) NS KIT (D816, exon 8, exon 11) 23 (16.5%) 20 (17.1%) 3 (13.6%) NS NRAS 18 (12.9%) 16 (13.7%) 2 (9.1%) NS ASXL1 16 (11.5%) 13 (11.1%) 3 (13.6%) NS FLT3-ITD 7 (5.0%) 7 (6.0%) 0 (0.0%) FLT3-TKD 6 (4.3%) 4 (3.4%) 2 (9.1%) NS CBL 6 (4.3%) 5 (4.3%) 1 (4.5%) NS KRAS 6 (4.3%) 5 (4.3%) 1 (4.5%) NS IDH2R140 5 (3.6%) 4 (3.4%) 1 (4.5%) NS JAK2V617F 4 (2.9%) 3 (2.6%) 1 (4.5%) NS IDH1R132 1 (0.7%) 1 (0.9%) 0 (0.0%) NPM1, MLL-PTD, IDH2R172 Not found Not found Not found

Cytogenetic aberrations in addition to t(8;21)(q22;q22) Sole t(8;21)(q22;q22) 42 (30.2%) 36 (30.8%) 6 (27.3%) NS At least 1 ACA 97 (69.8%) 81 (69.2%) 16 (72.7%) NS X2 ACAs 42 (30.2%) 34 (29.1%) 8 (36.4%) NS Loss of sex chromosomes (either X or Y) 65 (46.8%) 55 (47.0%) 10 (45.5%) NS Del(9q) 21 (15.1%) 16 (13.7%) 5 (22.7%) NS Trisomy 8 8 (5.9%) 5 (4.3%) 3 (13.6%) NS Abbreviations: ACA, additional cytogenetic aberration; AML, acute myeloid leukemia; NS, not signiﬁcant; t-AML, therapy-related AML.

& 2014 Macmillan Publishers Limited Leukemia (2014) 1449 – 1458 Secondary mutations in AML with t(8;21)/RUNX1-RUNX1T1 M-T Krauth et al 1452 In summary, with exception of FLT3-ITD, which was only present in However, KIT mutations (D816, exon 8 and exon 11) were found to de novo AML (6%, 7/117 patients), there was no signiﬁcant be mutually exclusive of CBL, FLT3-TKD, IDH1R132 and IDH2R140 difference in frequencies of single mutations between de novo mutations. NRAS mutations were mutually exclusive of CBL, AML and t-AML (Table 2 and Figure 1). JAK2 or IDH1R132 mutations and ASXL1 mutations were found to appear mutually exclusive of FLT3-ITD, as well as of FLT3-TKD Correlation studies mutations. Associations between molecular mutations. In general, signiﬁcant associations between different molecular mutations were not Associations of molecular and cytogenetic alterations. We aimed found, which was probably due to the relatively small cohorts of to correlate mutations and cytogenetic changes, and found that patients when divided into different subgroups (also see Figure 1). trisomy 8 was mutually exclusive of FLT3-ITD, FLT3-TKD, NRAS and

KIT NRAS ASXL1 FLT3-ITD FLT3-TKD CBL KRAS IDH2R140 JAK2V617F IDH1R132 Loss of sex chromosomes Deletion (9q) Trisomy 8 Entity

de novo AML t-AML

Figure 1. Pattern of molecular and cytogenetic lesions in patients with AML, in addition to t(8;21)/RUNX1-RUNX1T1. Distribution and frequencies are given for all analyzed gene mutations and the most frequent cytogenetic aberrations. The patient cohort is further annotated according to biological origin of the disease (de novo vs t-AML). The boxes represent single patient cases. Cases with additional molecular mutations or cases with ACAs are illustrated in red, and wild-type cases in gray. Cases that are classiﬁed as de novo AML are illustrated in dark blue, and t-AML in light blue.

Table 3. Changes in detected molecular mutations between diagnosis and relapse in patients with t(8;21)/RUNX1-RUNX1T1-positive AML

Pat. Sex/age AML subtype Mutation(s) at Mutation(s) stable at Mutation(s) gained at Mutation(s) lost at no. (years) (FAB) diagnosis relapse relapse relapse

1 M/39 AML-M2 KRAS JAK2V617F KRAS NRAS KITD816 NRAS 2 M/71 AML-M2 No mutation FLT3-ITD 3 F/23 AML-M2 FLT3-ITD FLT3-ITD 4 M/19 AML-M2 ASXL1 ASXL1 IDH2R140 IDH2R140 5 M/42 AML-M1 No mutation No mutation 6 F/34 AML-M2 ASXL1 ASXL1 KITD816 KITD816 7 F/53 AML-M2 FLT3-ITD FLT3-ITD 8 M/67 AML-M2 KITD816 KITexons 9–11 KITD816 ASXL1 9 F/50 AML-M1 FLT3-TKD FLT3-TKD KITD816 10 F/53 AML-M2 No mutation KITD816 11 M/50 AML-M2 No mutation NA 12 M/52 AML-M1 No mutation No mutation 13 F/56 AML-M1 No mutation No mutation 14 F/55 t-AML ASXL1 ASXL1 15 F/71 t-AML No mutation ASXL1 KITD816 CBL 16 F/64 AML-M2 ASXL1 ASXL1 KITD816 TP53 17 M/21 AML-M2 ASXL1 IDH1R132 ASXL1 18 F/71 AML-M1 FLT3-ITD FLT3-ITD FLT3-TKD FLT3-TKD 19 M/47 AML-M1 IDH2R140 IDH2R140 IDH1R132 20 F/53 AML-M1 ASXL1 ASXL1 NRAS p.Gln61His NRAS p.Gly12Asp NRAS p.Gln61His IDH2R140 IDH2R140 21 M/59 AML-M1 No mutation No mutation Abbreviations: AML, acute myeloid leukemia; F, female; FAB, French-American-British; M, male; NA, not analyzed (no material available); t-AML, therapy-related AML.

Leukemia (2014) 1449 – 1458 & 2014 Macmillan Publishers Limited Secondary mutations in AML with t(8;21)/RUNX1-RUNX1T1 M-T Krauth et al 1453 IDHR140 mutations. Interestingly, loss of X chromosome never emerged in 1/21 patients (4.8%) each. Loss of a mutation at occurred together with FLT3-ITD or IDH2R140 mutations. However, relapse was observed in KIT, ASXL1 and NRAS (each n ¼ 2, 9.5%), we could not identify other significant associations between as well as in KRAS, FLT3-ITD and FLT3-TKD (each n ¼ 1, 4.8%) molecular and cytogenetic alterations (Figure 1). (Table 3 and Figure 2). Concerning cytogenetic alterations at relapse, seven patients (33.3%) showed a change of their initial Association of biological characteristics and of RUNX1-RUNX1T1 cytogenetic pattern (gain of chromosomal aberrations: n ¼ 5, expression levels with genetic alterations. We evaluated correla- 23.8%; loss of chromosomal aberrations: n ¼ 2, 9.5%) (Table 4). tions of mutations and ACAs with demographic parameters (age, gender) and peripheral blood counts (leukocytes, hemoglobin, platelets). No significant correlations of genetic lesions and these Survival analysis parameters were found (data not shown). Only patients who received intensive treatment (n ¼ 111/139, We also aimed to analyze if RUNX1-RUNX1T1 expression levels at 79.9%) were included into prognostic analyses (de novo AML, diagnosis differ in patients with de novo or t-AML, and further, if n ¼ 95; t-AML, n ¼ 16). For these patients, the median follow-up there were differences in patients with or without additional was 26.9 months with a 2-year survival rate (OS) of 73.4%. The EFS mutations or ACAs. The median %RUNX1-RUNX1T1/ABL1 expres- rate after 2 years was 54.6%, and the OS with patients censored sion level was 46.8% (range: 9.2–451.2%). We found no significant at the day of allogeneic transplantation (OSalloTX) was 73.0% differences in expression levels when comparing de novo and (Table 5). t-AML patients, with or without additional genetic lesions. Also, The 2-year survival rate was slightly worse in patients with when analyzing expression levels in different patient subgroups t-AML than in those with de novo AML (46.8% vs 78.4%, P ¼ 0.061). (de novo and t-AML) and selected mutations (KIT, NRAS and ASXL1) Further the effect of different ACAs and mutations on survival was or ACAs (loss of sex chromosomes, del(9q) and þ 8), no significant analyzed. Within the total cohort, 55/111 patients with at least one differences were found. additional molecular mutation (49.5%) were compared with those with sole RUNX1-RUNX1T1. EFS after 2 years was significantly worse in patients with X1 molecular mutation (42.0% vs 66.7%, Genetic alterations at relapse P ¼ 0.012) (Table 5 and Figure 3a), but no difference in OS or In 21 cases, paired samples from diagnosis and relapse were OSalloTX was found between the two groups (72.4% vs 74.9% and available and compared for the pattern of molecular mutations. 72.0% vs 75.0%, after 2 years). When analyzing the prognostic In all cases, t(8;21)(q22;q22)/RUNX1-RUNX1T1 remained stable at impact of distinct mutations, ASXL1mut had a negative impact on the time of relapse. In 14/21 (66.7%) patients, the initial molecular EFS (28.6% vs 56.7%, P ¼ 0.021), but not on OS or OSalloTX (Table 5 mutation pattern changed at relapse. Mutations commonly gained and Figure 3b). Also, ACAs that were detected in 76/111 of these at relapse were KIT (6/21, 28.6%), followed by ASXL1 and IDH1R132 patients (68.5%) had no significant impact on survival after 2 years (each n ¼ 2, 9.5%). FLT3-ITD, CBL, NRAS and JAK2 mutations (76.7% vs 62.9%, P ¼ NS) (Figure 3c). Interestingly, when analyzing

Case No. #1 #2 #3 #4 #5 #6 #7 #8 #9 #10 #12 #13 #14 #15 #16 #17 #18 #19 #20 #21 D ASXL1 R

D CBL R

D FLT3-ITD R

D FLT3-TKD R

D IDH1R132 R

D IDH2R140 R

D JAK2V617F R

D KIT R

D KRAS R

D NRAS R Figure 2. Pattern of gained and/or lost molecular mutations in t(8;21)/RUNX1-RUNX1T1-positive AML at diagnosis (D) and in case of relapse (R). Red boxes indicate the presence, and gray boxes the absence of mutations.

& 2014 Macmillan Publishers Limited Leukemia (2014) 1449 – 1458 Secondary mutations in AML with t(8;21)/RUNX1-RUNX1T1 M-T Krauth et al 1454

- different ACAs separately, patients with loss of sex chromosomes (either X or Y) had a better EFS after 2 years (66.9% vs 43.0%,

RUNX1 P ¼ 0.031), whereas patients with trisomy 8 revealed shorter EFS (26.7% vs 55.9%, P ¼ 0.02) (Table 5 and Figures 3d and e). FISH: a

[16] Next, the analysis was restricted to de novo AML. Like in the total cohort, EFS after 2 years was significantly worse in patients with X1 molecular mutation (43.1% vs 70.1%, P 0.015). Also, [12]/46,XY[1] ¼ separate analysis for KIT, NRAS and ASXL1 mutations were t(8;21)(q22;q22)[6]/46,XY[1] performed. ASXL1 and NRAS mutation status did not impact [16]/46,XX[4] significantly on the OS. Furthermore, for cumulating KIT mutations t(14;14)(q24;q32) (KITD816, exon 8 and exons 9–11), no significant impact on survival was found (68.9% vs 80.9%, P NS) (Figure 3f). However,

3q22), ¼ 46,XX[11]

/ when restricting the analysis to KITD816 mutations, OS was

[3] signiﬁcantly worse in the mutated patients (59.1% vs 82.0%, 19 P ¼ 0.03; Figure 3g), while mutations in exon 8 or exon 11 had no þ signiﬁcant impact on survival (Table 5). There was also a trend to a 13, alloTX

þ worse EFS and OS after 2 years for patients harboring the KITD816 mutation (EFS: 40.9% vs 59.5%, P ¼ 0.074; OSalloTX: 64.2% hybridization; M, male; NA, not analyzed. Y,t(2;5)(q21;q23),del(5)(q31q34), vs 82.3%, P 0.052) (Table 5). However, like for the total cohort, in

À ¼ patients with de novo AML ASXL1 mutations had a signiﬁcant in situ 45,X, e

22;q22)inv(21)(p11q22)[3]/46,XY[17] negative impact on EFS (20.0% vs 59.1%, P ¼ 0.011) (Table 5; t(8;21)(q22;q22), der(16)inv(16)(p13q12)t(16;17)(q24;q22) Figure 3h) and patients with loss of sex chromosomes had a better 6,

þ EFS after 2 years (69.6% vs 43.5%, P ¼ 0.030) (Table 5). Y,del(2)(q21q33),t(8;21)(q22;q22)[9]/46,XY[8] 3, der(9)t(8;9;21)(q22;q13;q22), der(12)t(12;17)(q24;q12) À þ þ ), DISCUSSION 50,XX, der(21)t(8;21)(q22;q22)[19]/46,XY[1] ,t(8;21)(q22;q22)[3]/ ,t(8;21)(q22;q22),

þ t(8;21)/RUNX1-RUNX1T1-positive AML is firmly established as a distinct biological and clinically relevant AML subentity according to the WHO classification.39 However, prognosis of patients with this favorable genetic alteration is heterogeneous, with relapses 8,t(8;21)(q22;q22)[7]/46,XX[13] a þ del(5)(q31q34) 8,der(8)t(8;21)(q22;q22),der(21)t(8;21)(q22;p11)del(21)(q22)[5]/46,XX[15] being described in roughly 30% of patients in the literature. We Y,t(8;21)(q22;q22)[3]/45,X, X,t(8;21)(q22;q22)[17]/46,XX[3] Y,t(8;9;21)(q22;q13;q22 X, Y,t(8;21)(q22;q22)[20] Y, þ t(3;17)(q21;q12) À À À À À À aimed to deepen insights into the cooperating genetic events and their clinical impact by comprehensive investigation of a large 46,XX,t(8;15;21)(q22;q26;q22)[5]/46,XX[15] cohort of 139 t(8;21)/RUNX1-RUNX1T1-positive AML patients. First, in our cohort, the favorable prognosis of the respective AML subtype was confirmed. Second, a high rate of ACAs in 69.8% of cases, and also of additional molecular mutations in 49.6%, were detected, giving further evidence to the assumption that the [12] t(8;21)/RUNX1-RUNX1T1 requires cooperation partners to induce AML.4 Molecular mutations in addition to the t(8;21)/RUNX1- RUNX1T1 could be subdivided into three main categories: (i) mutations in KIT in 16.5% of cases; (ii) other mutations in genes mediating cell proliferation (NRAS, KRAS, FLT3-ITD, FLT3-TKD, CBL and JAK2), which were found in 30.9%; (iii) mutations of ASXL1 in 11.5% of cases. The frequencies of KIT mutations and of the [13] 46,XY [20] der(15)t(8;15)(q22;q26) Y,del(2)(q21q33),t(8;21)(q22;q22)[3] 45,X, additional molecular mutations listed in the above second þ À category were similar to previously published cohorts.4,11,15 In the murine model, Wang et al.40 had been able to demonstrate

der(21)t(8;21)(q22;q22)[19]/46,XY[1] 47,XY,t(8;21)(q22;q22), that KIT mutations cooperate with RUNX1-RUNX1T1 to induce AML. þ del(9)(q13q34) In addition, we were able to demonstrate that mutations of ASXL1, which are found in a variety of AML subtypes and have been mainly analyzed for the normal karyotype subgroup,28 also 8,t(8;21)(q22;q22)[13]/46,XX[6] 46,X, occur in t(8;21)/RUNX1-RUNX1T1-positive AML. Moreover, ASXL1 t(8;15;21)(q22;q26;q22), þ 8,der(8)t(8;21)(q22;q22),der(21)t(8;21)(q22;p11)del(21)(q22)[20] 47,XX, X, Y,t(8;21)(q22;q22)[9]/45,X, X,t(8;21)(q22;q22)[13]/46,XX[7] 45,X, Y,t(8;9;21)(q22;q13;q22)[18]/46,XY[2] 45,X, Y,t(8;21)(q22;q22)[17]/46,XY[5] 45,X, Y,t(8;21)(q22;q22)[12]/46,XY[8] 45,X, X,t(8;21)(q22;q22)[20] NA X, mutations mediate an adverse prognostic impact on EFS in t(8;21)/ þ À À À À À À À À RUNX1-RUNX1T1-positive AML which is probably corresponding to Cytogenetic aberrations at diagnosis Cytogenetic aberrations at relapse 46,X, their unfavorable impact in normal or intermediate karyotype AML,28 respectively. Additional KITD816 mutations were adverse for the OS in our cohort and conﬁrm the previous data.11,41,42

FAB However, in our cohort, this effect was seen only if we restrict the subtype analysis to patients with de novo AML. In contrast, none of the remaining additional mutations, which were either involved in the RAS pathway or cell proliferation from the above second category, were prognostically relevant in our cohort. Furthermore, (years) ACAs were prognostically relevant, as loss of sex chromosomes Cytogenetic aberrations at diagnosis and at the time of relapse rearrangement in 4/100 interphase nuclei and 3/157 metaphases; ACAs gained or lost(either are shown in bold. X or Y) was associated with a significantly better EFS, and an additional trisomy 8 was prognostically adverse with regard to the EFS. In contrast to our finding regarding a positive impact of 21 M/59 AML M1 46,XY,t(8;21)(q22;q22)[9]/46,XY[11] 46,XY,t(8;21)(q22;q22)[14]/46,XY[6] 45 M/19 M/42 AML M2 AML M1 46,XY,t(8;21)(q22;q22),del(9)(q13q33)[21] 45,X, NA 78 F/53 M/67 AML M2 AML M2 46,XX,t(8;21)(q22;q22)[20] 46,XY,t(8;21)(q22;q22), NA 6 F/34 AML M2 47,XX, 1718 M/21 F/71 AML M2 AML M1 46,XY,t(6;21;8)(q15;q22;q22)[20]/46,XY[2] 46,XX,t(8;21)(q22;q22)[19]/46,XX[1] 46,XY,t(6;21;8)(q15;q22;q22)[11]/46,XY[11] 46,XX, 19 M/47 AML M1 46,XY,der(8)t(8;21)(q22;q22),der(21)t(8;21)(q22;q22)inv(21)(p11q22)[20] 46,XY,der(8)t(8;21)(q22;q22),der(21)t(8;21)(q 20 F/53 AML M1 46,XX,t(8;21)(q22;q22)[20] 46,XX,t(8;21)(q22;q22)[8]/ 1516 F/71 F/64 t-AML AML M2 46,XX,t(8;21)(q22;q22)[9]/46,XX[11] 45,X, 46,XX,t(8;21)(q22;q22)[9]/46,XX[11] 1 M/39 AML M2 45,X, 3 F/23 AML M2 46,XX,t(8;21)(q22;q22),del(11)(q13q22)[21] 46,XX,t(8;21)(q22;q22),del(11)(q13q22)[4]/46,XX,t(8;21)(q22;q22),del(11)(q1 2 M/71 AML M2 45,X, 11 M/50 AML M2 45,X, Pat. no. Sex/age 910 F/50 F/53 AML M1 AML M2 46,XX,t(8;21)(q22;q22)[16]/46,XX[4] 45,X, NA 12 M/52 AML M1 47,XY,t(8;21)(q22;q22), 14 F/55 t-AML 46,X, 13 F/56 AML M1 46,XX,t(8;15;21)(q22;q26;q22)[8]/ RUNX1T1 Table 4. Abbreviations: ACA, additional chromosomal aberration; AML, acute myeloid leukemia; F, female; FAB, French-American-British; FISH, fluorescenc sex chromosome loss, Schlenk et al.43 had shown an adverse

Leukemia (2014) 1449 – 1458 & 2014 Macmillan Publishers Limited Secondary mutations in AML with t(8;21)/RUNX1-RUNX1T1 M-T Krauth et al 1455 Table 5. 2-Year survival data of the total cohort and de novo AML

Parameter Mutation status n OS (%) at P-value EFS (%) at P-value OSalloSCT (%) at P-value 2 years 2 years 2 years

Total cohort 111 73.4 54.6 73.0 Additional mutation 0 56 74.9 0.241 66.7 0.012 75.0 0.321 X1 55 72.4 42.0 72.0 KIT (total) Wt 90 74.0 0.615 55.5 0.769 72.9 0.822 Mut 21 71.2 51.0 76.4 KITexon 8 Wt 108 72.6 NA 54.5 0.543 71.9 0.321 Mut 3 100 50.0 100 KITexons 9–11 Wt 110 73.1 NA 54.0 0.434 72.6 0.554 Mut 1 100 100 100 KITD816 Wt 94 75.3 0.184 56.1 0.308 74.5 0.243 Mut 17 62.9 46.2 68.1 NRAS Wt 97 72.9 0.889 54.0 0.931 72.9 0.606 Mut 14 79.5 57.7 79.5 ASXL1 Wt 100 72.6 0.755 56.7 0.021 72.3 0.980 Mut 11 83.3 28.6 80.0 ACA 0 35 62.9 0.350 40.9 0.272 63.5 0.440 X1 76 76.7 59.3 76.1 Loss of sex chromosomes Absent 60 71.0 0.272 43.0 0.031 71.8 0.508 Present 51 76.3 66.9 75.2 Del(9q) Absent 96 69.1 0.177 52.9 0.203 69.6 0.213 Present 15 100 65.6 100 Trisomy 8 Absent 106 74.4 0.150 55.9 0.020 74.4 0.232 Present 5 53.3 26.7 40.0

De novo AML 95 78.4 56.6 79.3 Additional mutation 0 48 80.6 0.157 70.1 0.015 82.2 0.245 X1 47 76.5 43.1 77.2 KIT (total) Wt 76 80.9 0.216 59.0 0.358 81.0 0.368 Mut 19 68.9 47.1 74.3 KITexon 8 Wt 92 77.7 NA 56.6 0.602 78.4 0.377 Mut 3 100 50.0 100 KITexons 9–11 Wt 94 78.1 NA 55.9 0.448 79.0 0.604 Mut 1 100 100 100 KITD816 Wt 80 82.0 0.030 59.5 0.074 82.3 0.052 Mut 15 59.1 40.9 64.2 NRAS Wt 82 78.4 0.965 56.1 0.773 79.9 0.431 Mut 13 79.5 57.7 79.5 ASXL1 Wt 87 77.0 0.737 59.1 0.011 78.0 0.857 Mut 8 100 20.0 100 ACA 0 32 64.3 0.205 41.6 0.265 68.0 0.304 X1 63 83.3 61.9 83.6 Loss of sex chromosomes Absent 51 76.4 0.302 43.5 0.030 77.5 0.481 Present 44 80.9 69.6 81.8 Del(9q) Absent 83 75.1 0.408 56.5 0.532 76.4 0.353 Present 12 100 58.3 100 Trisomy 8 Absent 93 78.0 NA 57.3 0.153 79.1 0.633 Present 2 100 100 100 Abbreviations: ACA, additional chromosomal aberration; AML, acute myeloid leukemia; EFS, event-free survival; mut, mutation; NA, not analyzed; OS, overall survival; OSalloSCT, overall survival with patients censored on the day of allogeneic stem cell transplantation; wt, wild type. Significant P-values are shown in bold. prognostic impact of loss of Y chromosome in male patients, There was a trend toward a positive correlation between with t(8;21)/RUNX1-RUNX1T1-positive AML receiving different KIT and mutations with a function for the RAS pathway postremission therapeutic strategies. This discrepancy may (FLT3-ITD, NRAS and JAK2), which is suggestive of leukemo- slightly varying therapies or different age distributions, as they geneic cooperation of the respective gene mutations within included only young AML patients. t(8;21)/RUNX1-RUNX1T1-positive AML, whereas KIT mutations In accordance with previous studies,44,45 in our cohort, patients were under-represented in patients with an additional ASXL1 with t-AML showed worse OS as compared to de novo mutation. t(8;21)/RUNX1-RUNX1T1-positive AML, but there were no With regard to changes of mutation patterns between diagnosis significant differences in the frequencies of ACAs or in the and relapse, the t(8;21)/RUNX1-RUNX1T1 showed 100% stability. frequencies of the specific additional molecular mutations However, the additional molecular mutations behaved highly between both subgroups. KIT mutations were also detected at dynamic at relapse, as 66.7% of analyzed patients gained or lost similar frequencies in de novo AML and t-AML. Therefore, the molecular mutations. Most frequent gains were represented by KIT prognostic difference between de novo AML and t-AML with mutations, and most frequent losses were KIT and ASXL1 t(8;21)/RUNX1-RUNX1T1 cannot be explained by the so far revealed mutations. Gains or, less frequently, losses of chromosomal underlying genetic features. alterations were also observed in 33.3% of cases.

& 2014 Macmillan Publishers Limited Leukemia (2014) 1449 – 1458 Secondary mutations in AML with t(8;21)/RUNX1-RUNX1T1 M-T Krauth et al 1456 100% 100% 100%

80% 80% 80% 1 ACA, n=45 sole RUNX1-RUNX1T1, n=56 60% 60% 60% ≥2 ACAs, n=31 ASXL1wt, n=100

sole t(8;21)(q22;q22), n=35 40% 1 additional mutation, n=37 40% 40%

ASXL1mut, n=11 Overall Survival Event-free survival 20% ≥2 additional mutations, n=18 Event-free survival 20% 20%

p=0.038 p=0.021 p=n.s 0% 0% 0% 0 12 24 36 48 60 72 84 96 0 12 24 36 48 60 72 84 96 0 12 24 36 48 60 72 84 96 Months Months Months

100% 100% 100% 80% 80% 80% no additional KITmut, n=76 60% 60% loss of sex chromosomes, n=51 60% no additional +8, n=106 additional KITmut, n=19 40% 40% 40% no loss of sex chromosomes, n=60

additional +8, n=5 Overall Survival Event-free survival Event-free survival 20% 20% 20%

p=0.031 p=0.020 p=n.s 0% 0% 0% 0 12 24 36 48 60 72 84 96 0 12 24 36 48 60 72 84 96 0 12 24 36 48 60 72 84 Months Months Months

100% 100%

80% no additional 80% KITD816mut, n=80

60% 60% ASXL1wt, n=87

40% 40%

ASXL1mut, n=8 Overall Survival additional KITD816mut, n=15

20% Event-free survival 20%

p=0.030 p=0.011 0% 0% 0 12 24 36 48 60 72 84 0 12 24 36 48 60 72 84 96 Months Months Figure 3. Two-year survival data of AML patients with t(8;21)/RUNX1-RUNX1T1, who received intensive treatment according to additional (a) molecular lesions (EFS in the total cohort, n ¼ 111: sole RUNX1-RUNX1T1: 66.7%, 1 additional mutation: 43.7%, X2 additional mutations: 37.3%, P ¼ 0.038), (b) ASXL1 mutation status (EFS in the total cohort, n ¼ 111: ASXL1mut 28.6% vs ASXL1wt 56.7%, P ¼ 0.021), (c) cytogenetic aberrations (OS in the total cohort, n ¼ 111: sole t(8;21)(q22;q22): 62.9%, 1 ACA: 75.3%, X2 ACAs: 79.8%, P ¼ NS), (d) present or absent loss of sex chromosomes (EFS in the total cohort, n ¼ 111: 66.9% vs 43.0%, P ¼ 0.031) and (e) with or without þ 8 (EFS in the total cohort, n ¼ 111: 26.7% vs 55.9%, P ¼ 0.020). (f) De novo AML patients (n ¼ 95), with or without additional KIT mutations (D816, exon 8 and exon 11) (OS, KITmut: 68.9% vs KITwt: 80.9%, P ¼ NS), (g) with or without additional KITD816 mutation (OS, KITD816mut: 59.1% vs KITD816wt: 82.0%, P ¼ 0.03) and (h) with (mut) or without (wt) additional ASXL1 mutation (EFS, ASXL1mut 20.0% vs ASXL1wt 59.1%, P ¼ 0.011).

However, the importance of cytogenetic alterations as evaluated in patients with CBF leukemias.16 ASXL1 mutations prognostic factors for clinical outcome in AML patients led to should be further studied in this AML subtype, aiming to comparison of different postinduction treatment strategies. evaluate whether the presence of these adverse marker in When comparing chemotherapy and allogeneic hematopoietic addition to a t(8;21)/RUNX1-RUNX1T1 may justify an up-front stem cell transplantation within distinct cytogenetic AML intensification of therapy. subentities, early hematopoietic stem cell transplantation In conclusion, t(8;21)/RUNX1-RUNX1T1-positive AML shows high during first complete remission showed no significant benefit frequencies of additional cytogenetic and molecular lesions. Loss of in patients with good-risk AML, for example with t(8;21)/RUNX1- sex chromosomes is prognostically favorable, whereas an additional RUNX1T1,ornormalkaryotype.46,47 This was also confirmed in þ 8 is adverse. On the molecular level, mutations with an activating larger studies with patients with CBF leukemias.48,49 At present, function for the RAS pathway, KIT and ASXL1 mutations are most tyrosine kinase inhibitors in addition to chemotherapy are frequent. KITD816 and ASXL1 mutations had adverse prognostical

Leukemia (2014) 1449 – 1458 & 2014 Macmillan Publishers Limited Secondary mutations in AML with t(8;21)/RUNX1-RUNX1T1 M-T Krauth et al 1457 impact. Our data underscore that screening for the respective 15 Boissel N, Leroy H, Brethon B, Philippe N, de Botton S, Auvrignon A et al. mutations should be included in all patients at diagnosis of AML with Incidence and prognostic impact of c-Kit, FLT3, and Ras gene mutations in core t(8;21)/RUNX1-RUNX1T1 to improve risk stratification and probably binding factor acute myeloid leukemia (CBF-AML). Leukemia 2006; 20: might further personalize future therapies. 965–970. 16 Schlenk R. Dasatinib (Sprycelt) in patients with newly diagnosed core binding factor (CBF) acute myeloid leukemia (AML). ClinicalTrials gov 2013, NCT00850382. CONFLICT OF INTEREST 17 Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA, Gralnick HR et al. Proposals for the classification of the acute leukaemias. French-American-British SS, WK, CH and TH are part owners of the MLL Munich Leukemia Laboratory GmbH. (FAB) co-operative group. Br J Haematol 1976; 33: 451–458. MTK, CE, TA, UB and NN are employed by the MLL Munich Leukemia Laboratory 18 Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA, Gralnick HR et al. GmbH. Proposed revised criteria for the classification of acute myeloid leukemia. A report of the French-American-British Cooperative Group. Ann Intern Med 1985; 103: 620–625. AUTHOR CONTRIBUTIONS 19 Buchner T, Berdel WE, Schoch C, Haferlach T, Serve HL, Kienast J et al. Double SS and MTK were the principal investigators of this study, analyzed the data and induction containing either two courses or one course of high-dose cytarabine wrote the manuscript. CE did analysis of molecular mutations. CH was plus mitoxantrone and postremission therapy by either autologous stem-cell responsible for chromosome banding analysis. WK was responsible for transplantation or by prolonged maintenance for acute myeloid leukemia. J Clin immunophenotyping and was involved in the statistical analysis. TH was Oncol 2006; 24: 2480–2489. responsible for cytomorphologic analysis, and MTK and UB contributed to 20 Bu¨chner T, Schlenk RF, Schaich M, Dohner K, Krahl R, Krauter J et al. Acute myeloid leukemia (AML): different treatment strategies versus a common standard arm— cytomorphologic analysis. TA collected and analyzed clinical data. UB combined prospective analysis by the German AML Intergroup. J Clin Oncol 2012; contributed writing of the manuscript. NN contributed to statistics and 30: 3604–3610. graphics. All authors read and contributed to the final version of the 21 Arber DA, Brunning RD, Le Beau MM, Falini B, Vardiman J, Porwit A et al. manuscript. Acute myeloid leukemia with recurrent genetic abnormalities. In: Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H et al. (eds). WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. International Agency for REFERENCES Research on Cancer (IARC): Lyon, France, 2008, pp 110–123. 1 Grimwade D, Hills RK, Moorman AV, Walker H, Chatters S, Goldstone AH et al. 22 Schoch C, Schnittger S, Bursch S, Gerstner D, Hochhaus A, Berger U et al. Refinement of cytogenetic classification in acute myeloid leukemia: determina- Comparison of chromosome banding analysis, interphase- and hypermeta- tion of prognostic significance of rare recurring chromosomal abnormalities phase-FISH, qualitative and quantitative PCR for diagnosis and for follow-up among 5876 younger adult patients treated in the United Kingdom Medical in chronic myeloid leukemia: a study on 350 cases. Leukemia 2002; 16: Research Council trials. Blood 2010; 116: 354–365. 53–59. 2 Mulloy JC, Cammenga J, MacKenzie KL, Berguido FJ, Moore MAS, Nimer SD. 23 Haferlach C, Rieder H, Lillington DM, Dastugue N, Hagemeijer A, Harbott J et al. The AML1-ETO fusion protein promotes the expansion of human hematopoietic Proposals for standardized protocols for cytogenetic analyses of acute leukemias, stem cells. Blood 2002; 99: 15–23. chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproli- 3 Speck NA, Gilliland DG. Core-binding factors in haematopoiesis and leukaemia. ferative disorders, and myelodysplastic syndromes. Genes Chromosomes Cancer Nat Rev Cancer 2002; 2: 502–513. 2007; 46: 494–499. 4 Kuchenbauer F, Schnittger S, Look T, Gilliland G, Tenen D, Haferlach T et al. 24 Shaffer LG, Tommerup N. ISCN 2013: An International System for Human Identification of additional cytogenetic and molecular genetic abnormalities in Cytogenetic Nomenclature. Karger: Basel, Switzerland and New York, NY, USA, 2013. acute myeloid leukaemia with t(8;21)/AML1-ETO. Br J Haematol 2006; 134: 25 Kern W, Voskova D, Schoch C, Hiddemann W, Schnittger S, Haferlach T. 616–619. Determination of relapse risk based on assessment of minimal residual disease 5 Yuan Y, Zhou L, Miyamoto T, Iwasaki H, Harakawa N, Hetherington CJ et al. during complete remission by multiparameter flow cytometry in unselected AML1-ETO expression is directly involved in the development of acute myeloid patients with acute myeloid leukemia. Blood 2004; 104: 3078–3085. leukemia in the presence of additional mutations. Proc Natl Acad Sci USA 2001; 98: 26 Kern W, Bacher U, Haferlach C, Schnittger S, Haferlach T. The role of 10398–10403. multiparameter flow cytometry for disease monitoring in AML. Best Pract Res Clin 6 Higuchi M, O’Brien D, Kumaravelu P, Lenny N, Yeoh EJ, Downing JR. Expression of Haematol 2010; 23: 379–390. a conditional AML1-ETO oncogene bypasses embryonic lethality and establishes a 27 Schnittger S, Weisser M, Schoch C, Hiddemann W, Haferlach T, Kern W. New score murine model of human t(8;21) acute myeloid leukemia. Cancer Cell 2002; 1: predicting for prognosis in PML-RARA þ , AML1-ETO þ , or CBFBMYH11 þ acute 63–74. myeloid leukemia based on quantification of fusion transcripts. Blood 2003; 102: 7 Schnittger S, Schoch C, Dugas M, Kern W, Staib P, Wuchter C et al. Analysis of FLT3 2746–2755. length mutations in 1003 patients with acute myeloid leukemia: correlation to 28 Schnittger S, Eder C, Jeromin S, Alpermann T, Fasan A, Grossmann V et al. ASXL1 cytogenetics, FAB subtype, and prognosis in the AMLCG study and usefulness as a exon 12 mutations are frequent in AML with intermediate risk karyotype and are marker for the detection of minimal residual disease. Blood 2002; 100: 59–66. independently associated with an adverse outcome. Leukemia 2013; 27: 82–91. 8 Schessl C, Rawat VP, Cusan M, Deshpande A, Kohl TM, Rosten PM et al. 29 Bacher U, Haferlach C, Kern W, Haferlach T, Schnittger S. Prognostic relevance of The AML1-ETO fusion gene and the FLT3 length mutation collaborate in inducing FLT3-TKD mutations in AML: the combination matters—an analysis of 3082 acute leukemia in mice. J Clin Invest 2005; 115: 2159–2168. patients. Blood 2008; 111: 2527–2537. 9 Beghini A, Peterlongo P, Ripamonti CB, Larizza L, Cairoli R, Morra E et al. C-kit 30 Kohl TM, Schnittger S, Ellwart JW, Hiddemann W, Spiekermann K. KIT exon 8 mutations in core binding factor leukemias. Blood 2000; 95: 726–727. mutations associated with core-binding factor (CBF)-acute myeloid leukemia 10 Wang YY, Zhou GB, Yin T, Chen B, Shi JY, Liang WX et al. AML1-ETO and (AML) cause hyperactivation of the receptor in response to stem cell factor. Blood C-KIT mutation/overexpression in t(8;21) leukemia: implication in stepwise 2005; 105: 3319–3321. leukemogenesis and response to Gleevec. Proc Natl Acad Sci USA 2005; 102: 31 Schnittger S, Kern W, Tschulik C, Weiss T, Dicker F, Falini B et al. Minimal residual 1104–1109. disease levels assessed by NPM1 mutation-specific RQ-PCR provide important 11 Schnittger S, Kohl TM, Haferlach T, Kern W, Hiddemann W, Spiekermann K et al. prognostic information in AML. Blood 2009; 114: 2220–2231. KIT-D816 mutations in AML1-ETO-positive AML are associated with impaired 32 Schnittger S, Schoch C, Kern W, Mecucci C, Tschulik C, Martelli MF et al. event-free and overall survival. Blood 2006; 107: 1791–1799. Nucleophosmin gene mutations are predictors of favorable prognosis in acute 12 Paschka P, Du J, Schlenk RF, Gaidzik VI, Bullinger L, Corbacioglu A et al. myelogenous leukemia with a normal karyotype. Blood 2005; 106: 3733–3739. Secondary genetic lesions in acute myeloid leukemia with inv(16) or t(16;16): a study 33 Weisser M, Kern W, Schoch C, Hiddemann W, Haferlach T, Schnittger S. Risk of the German–Austrian AML Study Group (AMLSG). Blood 2013; 121: 170–177. assessment by monitoring expression levels of partial tandem duplications in the 13 Bowen DT, Frew ME, Hills R, Gale RE, Wheatley K, Groves MJ et al. RAS mutation in MLL gene in acute myeloid leukemia during therapy. Haematologica 2005; 90: acute myeloid leukemia is associated with distinct cytogenetic subgroups but 881–889. does not influence outcome in patients younger than 60 years. Blood 2005; 106: 34 Schnittger S, Haferlach C, Ulke M, Alpermann T, Kern W, Haferlach T. 2113–2119. IDH1 mutations are detected in 6.6% of 1414 AML patients and are 14 Kiyoi H, Naoe T, Nakano Y, Yokota S, Minami S, Miyawaki S et al. Prognostic associated with intermediate risk karyotype and unfavorable prognosis in implication of FLT3 and N-RAS gene mutations in acute myeloid leukemia. Blood adults younger than 60 years and unmutated NPM1 status. Blood 2010; 116: 1999; 93: 3074–3080. 5486–5496.

& 2014 Macmillan Publishers Limited Leukemia (2014) 1449 – 1458 Secondary mutations in AML with t(8;21)/RUNX1-RUNX1T1 M-T Krauth et al 1458 35 Bacher U, Haferlach T, Schoch C, Kern W, Schnittger S. Implications of NRAS 43 Schlenk RF, Benner A, Krauter J, Buchner T, Sauerland C, Ehninger G et al. mutations in AML: a study of 2502 patients. Blood 2006; 107: 3847–3853. Individual patient data-based meta-analysis of patients aged 16 to 60 years with 36 Schnittger S, Bacher U, Alpermann T, Reiter A, Ulke M, Dicker F et al. core binding factor acute myeloid leukemia: a survey of the German Acute Use of CBL exon 8 and 9 mutations in diagnosis of myeloproliferative neoplasms Myeloid Leukemia Intergroup. J Clin Oncol 2004; 22: 3741–3750. and myelodysplastic/myeloproliferative disorders: an analysis of 636 cases. 44 Gustafson SA, Lin P, Chen SS, Chen L, Abruzzo LV, Luthra R et al. Therapy-related Haematologica 2012; 97: 1890–1894. acute myeloid leukemia with t(8;21) (q22;q22) shares many features with de novo 37 Schnittger S, Bacher U, Kern W, Schroder M, Haferlach T, Schoch C. Report on two acute myeloid leukemia with t(8;21)(q22;q22) but does not have a favorable novel nucleotide exchanges in the JAK2 pseudokinase domain: D620E and E627E. outcome. Am J Clin Pathol 2009; 131: 647–655. Leukemia 2006; 20: 2195–2197. 45 Kayser S, Dohner K, Krauter J, Kohne CH, Horst HA, Held G et al. The impact of 38 Cheson BD, Bennett JM, Kopecky KJ, Buchner T, Willman CL, Estey EH et al. therapy-related acute myeloid leukemia (AML) on outcome in 2853 adult patients Revised recommendations of the international working group for diagnosis, with newly diagnosed AML. Blood 2011; 117: 2137–2145. standardization of response criteria, treatment outcomes, and reporting standards 46 Burnett AK, Wheatley K, Goldstone AH, Stevens RF, Hann IM, Rees JH et al. for therapeutic trials in acute myeloid leukemia. J Clin Oncol 2003; 21: 4642–4649. The value of allogeneic bone marrow transplant in patients with acute myeloid 39 Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H et al. leukaemia at differing risk of relapse: results of the UK MRC AML 10 trial. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. 4th edn. Br J Haematol 2002; 118: 385–400. International Agency for Research on Cancer (IARC): Lyon, France, 2008. 47 Suciu S, Mandelli F, de Witte T, Zittoun R, Gallo E, Labar B et al. Allogeneic 40 Wang YY, Zhao LJ, Wu CF, Liu P, Shi L, Liang Y et al. C-KIT mutation cooperates compared with autologous stem cell transplantation in the treatment of patients with full-length AML1-ETO to induce acute myeloid leukemia in mice. Proc Natl younger than 46 years with acute myeloid leukemia (AML) in first complete Acad Sci USA 2011; 108: 2450–2455. remission (CR1): an intention-to-treat analysis of the EORTC/GIMEMAAML-10 trial. 41 Paschka P, Marcucci G, Ruppert AS, Mrozek K, Chen H, Kittles RA et al. Blood 2003; 102: 1232–1240. Adverse prognostic significance of KIT mutations in adult acute myeloid leukemia 48 Nguyen S, Leblanc T, Fenaux P, Witz F, Blaise D, Pigneux A et al. A white blood cell with inv(16) and t(8;21): a Cancer and Leukemia Group B Study. J Clin Oncol 2006; index as the main prognostic factor in t(8;21) acute myeloid leukemia (AML): 24: 3904–3911. a survey of 161 cases from the French AML Intergroup. Blood 2002; 99: 42 Kim HJ, Ahn HK, Jung CW, Moon JH, Park CH, Lee KO et al. KIT D816 mutation 3517–3523. associates with adverse outcomes in core binding factor acute myeloid leukemia, 49 Delaunay J, Vey N, Leblanc T, Fenaux P, Rigal-Huguet F, Witz F et al. Prognosis of especially in the subgroup with RUNX1/RUNX1T1 rearrangement. Ann Hematol inv(16)/t(16;16) acute myeloid leukemia (AML): a survey of 110 cases from the 2013; 92: 163–171. French AML Intergroup. Blood 2003; 102: 462–469.

Leukemia (2014) 1449 – 1458 & 2014 Macmillan Publishers Limited