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Acute Leukemias

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

Abstract

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=117; therapy-related: n=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 2 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=0.03), ASXL1mut on EFS (de novo AML: 20% vs 59.1%, P=0.011; total cohort: 28.6% vs 56.7%, P=0.021). Sex chromosome loss was favorable (2-year EFS: 66.9% vs 43.0%, P=0.031), whereas +8 was adverse on EFS (2-year EFS: 26.7% vs 55.9%, P=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.

Introduction

In patients with acute myeloid leukemia (AML), t(8;21)(q22;q22) with the resulting RUNX1-RUNX1T1 rearrangement is one of the most common cytogenetic abnormalities. It occurs in about 7–8% of adult de novo AML.1 According to the World Health Organization (WHO) classification, t(8;21)(q22;q22)/RUNX1-RUNX1T1 defines a distinct AML subtype. Although this AML subtype is generally associated with a favorable prognosis, about 30% of patients relapse, and, in this context, the frequency and impact of additional genetic lesions is incompletely understood as yet. On the molecular level, the RUNX1-RUNX1T1 fusion gene influences cell proliferation, differentiation and self-renewal capacity.2 Furthermore, RUNX1-RUNX1T1 interferes and represses the transcription factor CBF (core-binding factor), which has a key role during early hematopoiesis.3 However, targeting CBF, RUNX1-RUNX1T1 alone was shown not to be sufficient to induce leukemia, 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 Among them, the FLT3-ITD, one of the most frequent genetic alterations in AML,7 has been shown to cooperate with RUNX1-RUNX1T1 in inducing leukemia in a murine bone marrow transplantation model.8 Also, mutations in the KIT oncogene act as cooperative mutations9, 10 in leukemogenesis. The respective mutations were demonstrated to confer an independent negative impact on prognosis in patients with CBF leukemia harboring t(8;21)/RUNX1-RUNX1T111 or inv(16)/CBFB-MYH11.12 Rarely, other mutations such as RAS, a key player in AML cell proliferation, were found to be associated with RUNX1-RUNX1T1.8, 13, 14 These data support the hypothesis of an oncogenic cooperation in leukemogenesis between RUNX1-RUNX1T1 and additional molecular alterations4 and illustrate the need for evaluation of additional molecular markers at the time of diagnosis. Besides contributing to further risk stratification of AML patients, these data may provide a rationale for new therapies targeting pathways in t(8;21)/RUNX1-RUNX1T1-positive AML.15 For example, the addition of the second-generation tyrosine kinase inhibitor dasatinib to chemotherapy is currently being evaluated for patients with CBF leukemias.16 Aiming to further clarify the role of this and other additional genetic lesions in this AML subtype, we here performed comprehensive genetic analysis and studied the clinical outcome in a large cohort of 139 patients with t(8;21)/RUNX1-RUNX1T1-positive AML.

Patients and methods

A total of 139 patients diagnosed with t(8;21)/RUNX1-RUNX1T1-positive AML were included in the study. Patients were referred from different hematologic centers in Germany to the MLL Munich Leukemia Laboratory between August 2005 and November 2012. There were 65 female and 74 male patients (male/female ratio: 0.9). The median age was 53.3 years (range, 18.6–83.8 years). In all cases, the t(8;21)/RUNX1-RUNX1T1 was confirmed by chromosome banding analysis, fluorescence in situ hybridization (FISH) and reverse transcription-polymerase chain reaction in combination. French-American-British classification was available in 107 patients:17, 18 34 (31.8%) had AML M1 and 73 (68.2%) had AML M2. A total of 117 patients had de novo AML (84.2%), and 22 patients (15.8%) therapy-related AML (t-AML) following (radio-) chemotherapy for other malignancies (breast cancer, lung cancer, gastrointestinal tract cancer, multiple myeloma or Non-Hodgkin lymphoma) (Table 1). Clinical follow-up data were available for most of the patients. A total of 111 patients with follow-up data (79.9% from the total cohort; de novo AML: n=95, 85.6%, t-AML: n=16, 14.4%) received intensive treatment strategies19, 20 (like standard protocols including ‘7+3’ or combinations of chemotherapeutics such as TAD (6-thioguanine, cytarabine and daunorubicin) and HAM (high-dose cytarabine and mitoxantrone)) and were the basis for prognostic evaluation in this study. Twenty-three of those 111 patients (20.7%) received allogeneic hematopoietic stem cell transplantation. In 21 cases, paired samples from diagnosis and relapse were available and were analyzed for the molecular pattern.

Table 1 Characteristics of 139 patients with t(8;21)/RUNX1-RUNX1T1-positive AML

All patients gave their informed consent for genetic analysis and for the use of the laboratory results for scientific studies. The study was approved by the Internal Review Board of the MLL Munich Leukemia Laboratory and adhered to the tenets of the Declaration of Helsinki.

Cytomorphology, cytogenetics and immunophenotyping

Cytomorphologic assessment was based on May–Grünwald–Giemsa stains, myeloperoxidase reaction and nonspecific esterase using α-naphthyl acetate following French-American-British and WHO classifications.17, 18, 21 Chromosome banding analysis combined with FISH was performed in all patients following standard methods.22, 23 Interphase FISH with probes for RUNX1 and RUNX1T1 was performed with commercially available probes (Abbott, Wiesbaden, Germany; Metasystems, Altlussheim, Germany). Karyotypes were described according to the International System for Human Cytogenetic Nomenclature.24 Immunophenotyping was performed in 56/139 cases (40.3%) as described previously.25, 26

Molecular analysis

At diagnosis, in 127/139 (91.4%) cases bone marrow and in 12/139 (8.6%) cases peripheral blood was used for the molecular analysis. Isolation of mononuclear cells, DNA extraction and mRNA extraction as well as random-primed cDNA synthesis followed previous descriptions.7 Quantitative real-time polymerase chain reaction for RUNX1-RUNX1T1 expression was carried out at the time of diagnosis and during follow-up, as has been published previously.27 ABL1 was used as a reference gene and expression levels were calculated as %RUNX1-RUNX1T1/ABL1. Investigations for ASXL1,28 FLT3-ITD,7 FLT3-TKD,29 KIT (D816, exon 8, exons 9–11),11, 30 NPM1,31, 32 MLL-PTD,33 IDH1 and IDH2,34 KRAS, NRAS,35 CBL36 and JAK2 mutations37 were performed in all patients.

Definition of clinical end points and statistical analysis

Survival curves were calculated for overall survival (OS), event-free survival (EFS) and OS with patients censored on the day of allogeneic stem cell transplantation (OSalloSCT) according to Kaplan–Meier and compared using log-rank test. OS was the time from diagnosis to death or last follow-up. EFS was the time from diagnosis to treatment failure, relapse, death or last follow-up in complete remission. Relapse was defined according to the International Working Group Criteria.38 Median follow-up was calculated taking the respective last observations in surviving cases into account and censoring non-surviving cases at the time of death. Differences were considered significant at P 0.05. Dichotomous variables were compared between different groups using the χ2-test and continuous variables by Student’s t-test. All reported P-values are two-sided. SPSS (version 19.0.0) software (IBM Corporation, Armonk, NY, USA) was used for statistical analysis.

Results

Characterization of t(8;21)/RUNX1-RUNX1T1

Cytogenetic data were available in all 139 patients. At diagnosis, the percentage of metaphases with t(8;21)(q22;q22), assessed by chromosome banding analysis, was in median 95% (range: 14.3–100%). One hundred and twenty-two of 139 cases harbored a standard t(8;21)(q22;q22), and 17/139 (12.2%) showed variant forms of t(8;21)(q22;q22): 12 cases showed a variant translocation involving one additional chromosome, 1 case involved 3 additional chromosomes; in 2 cases the derivative chromosome 21 and in 1 case both the derivative chromosomes 8 and 21 were involved in additional rearrangements; in 1 case the RUNX1-RUNX1T1 rearrangement was based on an insertion of chromosome 8q material into the long arm of chromosome 21 at chromosome band 21q22. According to interphase FISH (124/139 samples could be analyzed quantitatively), the percentage of positive cells was in median 90% (range: 15–100%). Fifteen cases could not be analyzed quantitatively because of poor quality of bone marrow or peripheral blood samples. Real-time polymerase chain reaction revealed heterogeneous %RUNX1-RUNX1T1/ABL1 expressions with a median of 46.8% (range: 9.2–451.2%). This expression level does not correlate with % t(8;21)-positive metaphases or interphases, indicating that patients have individual RUNX1-RUNX1T1 expression levels per cell. At relapse, the median %RUNX1-RUNX1T1/ABL1 expression was 34.4% (range: 0.2–300.7%) and did not differ significantly from diagnostic samples (P=0.225), as measured in 21 paired samples.

Additional cytogenetic alterations

Ninety-seven of 139 patients (69.8%) had at least one additional chromosomal abnormality (ACA) besides t(8;21)(q22;q22); 39.6% (55/139) had one ACA, and 42 (30.2%) patients had two or more ACAs. Most frequent was the loss of either X or Y chromosomes (n=65, 46.8%), followed by 9q deletion (del(9q); n=21, 15.1%) and trisomy 8 (+8; n=8, 5.8%) (Table 1).

Cytogenetic data were then separately calculated for patients with de novo AML and t-AML. Eighty-one of 117 (69.2%) patients with de novo AML had ACAs. Again, the most frequent ACA was the loss of a sex chromosome (n=55, 47.0%; in more detail: 55.7% −Y in males and 34.0% −X in females). 9q deletion was found in 13.7% (n=16), and +8 in 4.3% (n=5) of patients (Table 2).

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

In t-AML, 72.7% (16/22) cases had ACAs, 45.5% (n=10) had loss of a sex chromosome (all males showed −Y, and 36.8% −X in females). Again, the second most frequent ACA was del(9q), which occurred in 22.7% (n=5) of patients; 13.6% (3/22) of t-AML patients showed +8 (Table 2). Overall, the frequency of ACAs did not differ significantly between patients with de novo AML and t-AML, showing only a trend toward higher frequency of −Y, del(9q) and +8 in patients with t-AML (Table 2 and Figure 1).

Figure 1
figure1

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.

Frequency and characterization of additional mutations in the total cohort of patients

Overall, additional molecular mutations were detected in 69/139 (49.6%) patients. Twenty-three (16.5%) had two or more additional mutations (2 mutations: n=19, 13.7%; 3 mutations: n=2, 1.4%). Most common were KIT mutations (n=23; 16.5%), followed by NRAS (n=18; 12.9%) and ASXL1 mutations (n=16; 11.5%). KIT mutations were distributed in detail as follows: D816 point mutations in 18 cases (78.3%), exon 8 in 4 cases (17.4%) and exon 11 in 1 case (4.3%). Among KITD816, the following mutations were found: D816V (n=11, 61.1%), D816H (n=4, 22.2%), D816E (n=2, 11.1%), D816F (n=1, 5.6%) and D816T (n=1, 5.6%). One 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_Asp419insArgPhePhe, p.Asp419del and p.Asp419_Arg420delinsGlu. In exon 11, p.Pro577_Tyr578ins7AS amino-acid change was detected.

Frequencies of other mutations were as follows: FLT3-ITD, 7/139 (5.0%), FLT3-TKD, CBL and KRAS, each 6/139 (4.3%). Concerning IDH gene alterations, an IDH1R132 mutation was detected in 1/139 (0.7%) and IDH2R140 in 5/139 (3.6%), whereas IDH2R172 mutations were not found. JAK2 mutations were detectable in 4/139 (2.9%). Notably, NPM1mut and MLL-PTD were not found and thus were mutually exclusive of RUNX1-RUNX1T1 (Table 1 and Figure 1).

Taken together, RAS pathway-activating mutations including NRAS, KRAS, FLT3-ITD, FLT3-TKD, CBL and JAK2 were found in 43 (30.9%) of all patients: 39 patients had one RAS pathway-activating mutation and 4 patients had two RAS pathway-activating mutations (combinations were: NRAS and KRAS, n=2; FLT3-TKD and NRAS, n=1; FLT3-ITD and FLT3-TKD: n=1) (Figure 1).

Comparison of additional mutation frequency in de novo AML and t-AML

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 molecular mutation. In t-AML this frequency was 11/22 (50.0%).

In de novo AML, most frequent were KIT mutations (D816, exon 8 or exon 11; 17.1%), followed by NRAS (13.7%) and ASXL1 (11.1%). To a lower extent, FLT3-ITD mutations were found in 6% of patients, KRAS and CBL in 4.3%, FLT3-TKD and IDH2R140 in 3.4% each and JAK2 in 2.6% of cases. IDH1R132 was detectable only in 0.9% of patients. In t-AML, most frequent were KIT mutations (D816V, exon 8 or exon 11) and ASXL1 mutations (both 13.6%), followed by NRAS and FLT3-TKD mutations (both 9.1%). KRAS, CBL, IDH2R140 and JAK2 were each found in 4.5% of cases. FLT3-ITD and IDH1R132 mutations were not found in the t-AML cohort. In summary, with exception of FLT3-ITD, which was only present in de novo AML (6%, 7/117 patients), there was no significant difference in frequencies of single mutations between de novo AML and t-AML (Table 2 and Figure 1).

Correlation studies

Associations between molecular mutations

In general, significant associations between different molecular mutations were not found, which was probably due to the relatively small cohorts of patients when divided into different subgroups (also see Figure 1). However, KIT mutations (D816, exon 8 and exon 11) were found to be mutually exclusive of CBL, FLT3-TKD, IDH1R132 and IDH2R140 mutations. NRAS mutations were mutually exclusive of CBL, JAK2 or IDH1R132 mutations and ASXL1 mutations were found to appear mutually exclusive of FLT3-ITD, as well as of FLT3-TKD mutations.

Associations of molecular and cytogenetic alterations

We aimed to correlate mutations and cytogenetic changes, and found that trisomy 8 was mutually exclusive of FLT3-ITD, FLT3-TKD, NRAS and IDHR140 mutations. Interestingly, loss of X chromosome never occurred together with FLT3-ITD or IDH2R140 mutations. However, we could not identify other significant associations between molecular and cytogenetic alterations (Figure 1).

Association of biological characteristics and of RUNX1-RUNX1T1 expression levels with genetic alterations

We evaluated correlations of mutations and ACAs with demographic parameters (age, gender) and peripheral blood counts (leukocytes, hemoglobin, platelets). No significant correlations of genetic lesions and these parameters were found (data not shown).

We also aimed to analyze if RUNX1-RUNX1T1 expression levels at diagnosis differ in patients with de novo or t-AML, and further, if there were differences in patients with or without additional mutations or ACAs. The median %RUNX1-RUNX1T1/ABL1 expression level was 46.8% (range: 9.2–451.2%). We found no significant differences in expression levels when comparing de novo and t-AML patients, with or without additional genetic lesions. Also, when analyzing expression levels in different patient subgroups (de novo and t-AML) and selected mutations (KIT, NRAS and ASXL1) or ACAs (loss of sex chromosomes, del(9q) and +8), no significant differences were found.

Genetic alterations at relapse

In 21 cases, paired samples from diagnosis and relapse were available and compared for the pattern of molecular mutations. In all cases, t(8;21)(q22;q22)/RUNX1-RUNX1T1 remained stable at the time of relapse. In 14/21 (66.7%) patients, the initial molecular mutation pattern changed at relapse. Mutations commonly gained at relapse were KIT (6/21, 28.6%), followed by ASXL1 and IDH1R132 (each n=2, 9.5%). FLT3-ITD, CBL, NRAS and JAK2 mutations emerged in 1/21 patients (4.8%) each. Loss of a mutation at relapse was observed in KIT, ASXL1 and NRAS (each n=2, 9.5%), as well as in KRAS, FLT3-ITD and FLT3-TKD (each n=1, 4.8%) (Table 3 and Figure 2). Concerning cytogenetic alterations at relapse, seven patients (33.3%) showed a change of their initial cytogenetic pattern (gain of chromosomal aberrations: n=5, 23.8%; loss of chromosomal aberrations: n=2, 9.5%) (Table 4).

Table 3 Changes in detected molecular mutations between diagnosis and relapse in patients with t(8;21)/RUNX1-RUNX1T1-positive AML
Figure 2
figure2

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.

Table 4 Cytogenetic aberrations at diagnosis and at the time of relapse

Survival analysis

Only patients who received intensive treatment (n=111/139, 79.9%) were included into prognostic analyses (de novo AML, n=95; t-AML, n=16). For these patients, the median follow-up was 26.9 months with a 2-year survival rate (OS) of 73.4%. The EFS rate after 2 years was 54.6%, and the OS with patients censored at the day of allogeneic transplantation (OSalloTX) was 73.0% (Table 5).

Table 5 2-Year survival data of the total cohort and de novo AML

The 2-year survival rate was slightly worse in patients with t-AML than in those with de novo AML (46.8% vs 78.4%, P=0.061). Further the effect of different ACAs and mutations on survival was analyzed. Within the total cohort, 55/111 patients with at least one additional molecular mutation (49.5%) were compared with those with sole RUNX1-RUNX1T1. EFS after 2 years was significantly worse in patients with 1 molecular mutation (42.0% vs 66.7%, P=0.012) (Table 5 and Figure 3a), but no difference in OS or OSalloTX was found between the two groups (72.4% vs 74.9% and 72.0% vs 75.0%, after 2 years). When analyzing the prognostic impact of distinct mutations, ASXL1mut had a negative impact on EFS (28.6% vs 56.7%, P=0.021), but not on OS or OSalloTX (Table 5 and Figure 3b). Also, ACAs that were detected in 76/111 of these patients (68.5%) had no significant impact on survival after 2 years (76.7% vs 62.9%, P=NS) (Figure 3c). Interestingly, when analyzing 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%, P=0.031), whereas patients with trisomy 8 revealed shorter EFS (26.7% vs 55.9%, P=0.02) (Table 5 and Figures 3d and e).

Figure 3
figure3

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=111: sole RUNX1-RUNX1T1: 66.7%, 1 additional mutation: 43.7%, 2 additional mutations: 37.3%, P=0.038), (b) ASXL1 mutation status (EFS in the total cohort, n=111: ASXL1mut 28.6% vs ASXL1wt 56.7%, P=0.021), (c) cytogenetic aberrations (OS in the total cohort, n=111: sole t(8;21)(q22;q22): 62.9%, 1 ACA: 75.3%, 2 ACAs: 79.8%, P=NS), (d) present or absent loss of sex chromosomes (EFS in the total cohort, n=111: 66.9% vs 43.0%, P=0.031) and (e) with or without +8 (EFS in the total cohort, n=111: 26.7% vs 55.9%, P=0.020). (f) De novo AML patients (n=95), with or without additional KIT mutations (D816, exon 8 and exon 11) (OS, KITmut: 68.9% vs KITwt: 80.9%, P=NS), (g) with or without additional KITD816 mutation (OS, KITD816mut: 59.1% vs KITD816wt: 82.0%, P=0.03) and (h) with (mut) or without (wt) additional ASXL1 mutation (EFS, ASXL1mut 20.0% vs ASXL1wt 59.1%, P=0.011).

Next, the analysis was restricted to de novo AML. Like in the total cohort, EFS after 2 years was significantly worse in patients with 1 molecular mutation (43.1% vs 70.1%, P=0.015). Also, separate analysis for KIT, NRAS and ASXL1 mutations were performed. ASXL1 and NRAS mutation status did not impact significantly on the OS. Furthermore, for cumulating KIT mutations (KITD816, exon 8 and exons 9–11), no significant impact on survival was found (68.9% vs 80.9%, P=NS) (Figure 3f). However, when restricting the analysis to KITD816 mutations, OS was significantly worse in the mutated patients (59.1% vs 82.0%, P=0.03; Figure 3g), while mutations in exon 8 or exon 11 had no significant impact on survival (Table 5). There was also a trend to a worse EFS and OSalloTX after 2 years for patients harboring the KITD816 mutation (EFS: 40.9% vs 59.5%, P=0.074; OSalloTX: 64.2% vs 82.3%, P=0.052) (Table 5). However, like for the total cohort, in patients with de novo AML ASXL1 mutations had a significant negative impact on EFS (20.0% vs 59.1%, P=0.011) (Table 5; Figure 3h) and patients with loss of sex chromosomes had a better EFS after 2 years (69.6% vs 43.5%, P=0.030) (Table 5).

Discussion

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 being described in roughly 30% of patients in the literature. We aimed to deepen insights into the cooperating genetic events and their clinical impact by comprehensive investigation of a large 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 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 additional molecular mutations listed in the above second category were similar to previously published cohorts.4, 11, 15 In the murine model, Wang et al.40 had been able to demonstrate that KIT mutations cooperate with RUNX1-RUNX1T1 to induce AML. 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 occur in t(8;21)/RUNX1-RUNX1T1-positive AML. Moreover, ASXL1 mutations mediate an adverse prognostic impact on EFS in t(8;21)/RUNX1-RUNX1T1-positive AML which is probably corresponding to 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 However, in our cohort, this effect was seen only if we restrict the 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, ACAs were prognostically relevant, as loss of sex chromosomes (either 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 sex chromosome loss, Schlenk et al.43 had shown an adverse prognostic impact of loss of Y chromosome in male patients, with t(8;21)/RUNX1-RUNX1T1-positive AML receiving different postremission therapeutic strategies. This discrepancy may slightly varying therapies or different age distributions, as they included only young AML patients.

In accordance with previous studies,44, 45 in our cohort, patients with t-AML showed worse OS as compared to de novo t(8;21)/RUNX1-RUNX1T1-positive AML, but there were no significant differences in the frequencies of ACAs or in the frequencies of the specific additional molecular mutations between both subgroups. KIT mutations were also detected at similar frequencies in de novo AML and t-AML. Therefore, the prognostic difference between de novo AML and t-AML with t(8;21)/RUNX1-RUNX1T1 cannot be explained by the so far revealed underlying genetic features.

There was a trend toward a positive correlation between KIT and mutations with a function for the RAS pathway (FLT3-ITD, NRAS and JAK2), which is suggestive of leukemogeneic cooperation of the respective gene mutations within t(8;21)/RUNX1-RUNX1T1-positive AML, whereas KIT mutations were under-represented in patients with an additional ASXL1 mutation.

With regard to changes of mutation patterns between diagnosis and relapse, the t(8;21)/RUNX1-RUNX1T1 showed 100% stability. However, the additional molecular mutations behaved highly dynamic at relapse, as 66.7% of analyzed patients gained or lost molecular mutations. Most frequent gains were represented by KIT mutations, and most frequent losses were KIT and ASXL1 mutations. Gains or, less frequently, losses of chromosomal alterations were also observed in 33.3% of cases.

However, the importance of cytogenetic alterations as prognostic factors for clinical outcome in AML patients led to comparison of different postinduction treatment strategies. When comparing chemotherapy and allogeneic hematopoietic stem cell transplantation within distinct cytogenetic AML subentities, early hematopoietic stem cell transplantation during first complete remission showed no significant benefit in patients with good-risk AML, for example with t(8;21)/RUNX1-RUNX1T1, or normal karyotype.46, 47 This was also confirmed in larger studies with patients with CBF leukemias.48, 49 At present, tyrosine kinase inhibitors in addition to chemotherapy are evaluated in patients with CBF leukemias.16 ASXL1 mutations should be further studied in this AML subtype, aiming to evaluate whether the presence of these adverse marker in addition to a t(8;21)/RUNX1-RUNX1T1 may justify an up-front intensification of therapy.

In conclusion, t(8;21)/RUNX1-RUNX1T1-positive AML shows high frequencies of additional cytogenetic and molecular lesions. Loss of sex chromosomes is prognostically favorable, whereas an additional +8 is adverse. On the molecular level, mutations with an activating function for the RAS pathway, KIT and ASXL1 mutations are most frequent. KITD816 and ASXL1 mutations had adverse prognostical impact. Our data underscore that screening for the respective mutations should be included in all patients at diagnosis of AML with t(8;21)/RUNX1-RUNX1T1 to improve risk stratification and probably might further personalize future therapies.

References

  1. 1

    Grimwade D, Hills RK, Moorman AV, Walker H, Chatters S, Goldstone AH et al. Refinement of cytogenetic classification in acute myeloid leukemia: determination of prognostic significance of rare recurring chromosomal abnormalities among 5876 younger adult patients treated in the United Kingdom Medical Research Council trials. Blood 2010; 116: 354–365.

    CAS  Article  Google Scholar 

  2. 2

    Mulloy JC, Cammenga J, MacKenzie KL, Berguido FJ, Moore MAS, Nimer SD . The AML1-ETO fusion protein promotes the expansion of human hematopoietic stem cells. Blood 2002; 99: 15–23.

    CAS  Article  Google Scholar 

  3. 3

    Speck NA, Gilliland DG . Core-binding factors in haematopoiesis and leukaemia. Nat Rev Cancer 2002; 2: 502–513.

    CAS  Article  PubMed Central  Google Scholar 

  4. 4

    Kuchenbauer F, Schnittger S, Look T, Gilliland G, Tenen D, Haferlach T et al. Identification of additional cytogenetic and molecular genetic abnormalities in acute myeloid leukaemia with t(8;21)/AML1-ETO. Br J Haematol 2006; 134: 616–619.

    CAS  Article  Google Scholar 

  5. 5

    Yuan Y, Zhou L, Miyamoto T, Iwasaki H, Harakawa N, Hetherington CJ et al. AML1-ETO expression is directly involved in the development of acute myeloid leukemia in the presence of additional mutations. Proc Natl Acad Sci USA 2001; 98: 10398–10403.

    CAS  Article  PubMed Central  Google Scholar 

  6. 6

    Higuchi M, O'Brien D, Kumaravelu P, Lenny N, Yeoh EJ, Downing JR . Expression of a conditional AML1-ETO oncogene bypasses embryonic lethality and establishes a murine model of human t(8;21) acute myeloid leukemia. Cancer Cell 2002; 1: 63–74.

    CAS  Article  Google Scholar 

  7. 7

    Schnittger S, Schoch C, Dugas M, Kern W, Staib P, Wuchter C et al. Analysis of FLT3 length mutations in 1003 patients with acute myeloid leukemia: correlation to cytogenetics, FAB subtype, and prognosis in the AMLCG study and usefulness as a marker for the detection of minimal residual disease. Blood 2002; 100: 59–66.

    CAS  Article  Google Scholar 

  8. 8

    Schessl C, Rawat VP, Cusan M, Deshpande A, Kohl TM, Rosten PM et al. The AML1-ETO fusion gene and the FLT3 length mutation collaborate in inducing acute leukemia in mice. J Clin Invest 2005; 115: 2159–2168.

    CAS  Article  PubMed Central  Google Scholar 

  9. 9

    Beghini A, Peterlongo P, Ripamonti CB, Larizza L, Cairoli R, Morra E et al. C-kit mutations in core binding factor leukemias. Blood 2000; 95: 726–727.

    CAS  Google Scholar 

  10. 10

    Wang YY, Zhou GB, Yin T, Chen B, Shi JY, Liang WX et al. AML1-ETO and C-KIT mutation/overexpression in t(8;21) leukemia: implication in stepwise leukemogenesis and response to Gleevec. Proc Natl Acad Sci USA 2005; 102: 1104–1109.

    CAS  Article  Google Scholar 

  11. 11

    Schnittger S, Kohl TM, Haferlach T, Kern W, Hiddemann W, Spiekermann K et al. KIT-D816 mutations in AML1-ETO-positive AML are associated with impaired event-free and overall survival. Blood 2006; 107: 1791–1799.

    CAS  Article  Google Scholar 

  12. 12

    Paschka P, Du J, Schlenk RF, Gaidzik VI, Bullinger L, Corbacioglu A et al. Secondary genetic lesions in acute myeloid leukemia with inv(16) or t(16;16): a study of the German–Austrian AML Study Group (AMLSG). Blood 2013; 121: 170–177.

    CAS  Article  Google Scholar 

  13. 13

    Bowen DT, Frew ME, Hills R, Gale RE, Wheatley K, Groves MJ et al. RAS mutation in acute myeloid leukemia is associated with distinct cytogenetic subgroups but does not influence outcome in patients younger than 60 years. Blood 2005; 106: 2113–2119.

    CAS  Article  PubMed Central  Google Scholar 

  14. 14

    Kiyoi H, Naoe T, Nakano Y, Yokota S, Minami S, Miyawaki S et al. Prognostic implication of FLT3 and N-RAS gene mutations in acute myeloid leukemia. Blood 1999; 93: 3074–3080.

    CAS  PubMed  Google Scholar 

  15. 15

    Boissel N, Leroy H, Brethon B, Philippe N, de Botton S, Auvrignon A et al. Incidence and prognostic impact of c-Kit, FLT3, and Ras gene mutations in core binding factor acute myeloid leukemia (CBF-AML). Leukemia 2006; 20: 965–970.

    CAS  Article  PubMed Central  Google Scholar 

  16. 16

    Schlenk R Dasatinib (Sprycel™) in patients with newly diagnosed core binding factor (CBF) acute myeloid leukemia (AML). ClinicalTrials gov 2013; NCT00850382.

  17. 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 (FAB) co-operative group. Br J Haematol 1976; 33: 451–458.

    CAS  Article  Google Scholar 

  18. 18

    Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA, Gralnick HR et al. 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.

    CAS  Article  Google Scholar 

  19. 19

    Buchner T, Berdel WE, Schoch C, Haferlach T, Serve HL, Kienast J et al. Double induction containing either two courses or one course of high-dose cytarabine plus mitoxantrone and postremission therapy by either autologous stem-cell transplantation or by prolonged maintenance for acute myeloid leukemia. J Clin Oncol 2006; 24: 2480–2489.

    Article  Google Scholar 

  20. 20

    Büchner 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—combined prospective analysis by the German AML Intergroup. J Clin Oncol 2012; 30: 3604–3610.

    Article  Google Scholar 

  21. 21

    Arber DA, Brunning RD, Le Beau MM, Falini B, Vardiman J, Porwit A et al. 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 Research on Cancer (IARC): Lyon, France, 2008; pp 110–123.

    Google Scholar 

  22. 22

    Schoch C, Schnittger S, Bursch S, Gerstner D, Hochhaus A, Berger U et al. Comparison of chromosome banding analysis, interphase- and hypermetaphase-FISH, qualitative and quantitative PCR for diagnosis and for follow-up in chronic myeloid leukemia: a study on 350 cases. Leukemia 2002; 16: 53–59.

    CAS  Article  Google Scholar 

  23. 23

    Haferlach C, Rieder H, Lillington DM, Dastugue N, Hagemeijer A, Harbott J et al. Proposals for standardized protocols for cytogenetic analyses of acute leukemias, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorders, and myelodysplastic syndromes. Genes Chromosomes Cancer 2007; 46: 494–499.

    CAS  Article  Google Scholar 

  24. 24

    Shaffer LG, Tommerup N . ISCN 2013: An International System for Human Cytogenetic Nomenclature. Karger: Basel, Switzerland and New York, NY, USA, 2013.

    Google Scholar 

  25. 25

    Kern W, Voskova D, Schoch C, Hiddemann W, Schnittger S, Haferlach T . Determination of relapse risk based on assessment of minimal residual disease during complete remission by multiparameter flow cytometry in unselected patients with acute myeloid leukemia. Blood 2004; 104: 3078–3085.

    CAS  Article  Google Scholar 

  26. 26

    Kern W, Bacher U, Haferlach C, Schnittger S, Haferlach T . The role of multiparameter flow cytometry for disease monitoring in AML. Best Pract Res Clin Haematol 2010; 23: 379–390.

    CAS  Article  PubMed Central  Google Scholar 

  27. 27

    Schnittger S, Weisser M, Schoch C, Hiddemann W, Haferlach T, Kern W . New score predicting for prognosis in PML-RARA+, AML1-ETO+, or CBFBMYH11+ acute myeloid leukemia based on quantification of fusion transcripts. Blood 2003; 102: 2746–2755.

    CAS  Article  Google Scholar 

  28. 28

    Schnittger S, Eder C, Jeromin S, Alpermann T, Fasan A, Grossmann V et al. ASXL1 exon 12 mutations are frequent in AML with intermediate risk karyotype and are independently associated with an adverse outcome. Leukemia 2013; 27: 82–91.

    CAS  Article  PubMed Central  Google Scholar 

  29. 29

    Bacher U, Haferlach C, Kern W, Haferlach T, Schnittger S . Prognostic relevance of FLT3-TKD mutations in AML: the combination matters—an analysis of 3082 patients. Blood 2008; 111: 2527–2537.

    CAS  Article  Google Scholar 

  30. 30

    Kohl TM, Schnittger S, Ellwart JW, Hiddemann W, Spiekermann K . KIT exon 8 mutations associated with core-binding factor (CBF)-acute myeloid leukemia (AML) cause hyperactivation of the receptor in response to stem cell factor. Blood 2005; 105: 3319–3321.

    CAS  Article  Google Scholar 

  31. 31

    Schnittger S, Kern W, Tschulik C, Weiss T, Dicker F, Falini B et al. Minimal residual disease levels assessed by NPM1 mutation-specific RQ-PCR provide important prognostic information in AML. Blood 2009; 114: 2220–2231.

    CAS  Article  Google Scholar 

  32. 32

    Schnittger S, Schoch C, Kern W, Mecucci C, Tschulik C, Martelli MF et al. Nucleophosmin gene mutations are predictors of favorable prognosis in acute myelogenous leukemia with a normal karyotype. Blood 2005; 106: 3733–3739.

    CAS  Article  PubMed Central  Google Scholar 

  33. 33

    Weisser M, Kern W, Schoch C, Hiddemann W, Haferlach T, Schnittger S . Risk assessment by monitoring expression levels of partial tandem duplications in the MLL gene in acute myeloid leukemia during therapy. Haematologica 2005; 90: 881–889.

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34

    Schnittger S, Haferlach C, Ulke M, Alpermann T, Kern W, Haferlach T . IDH1 mutations are detected in 6.6% of 1414 AML patients and are associated with intermediate risk karyotype and unfavorable prognosis in adults younger than 60 years and unmutated NPM1 status. Blood 2010; 116: 5486–5496.

    CAS  Article  PubMed Central  Google Scholar 

  35. 35

    Bacher U, Haferlach T, Schoch C, Kern W, Schnittger S . Implications of NRAS mutations in AML: a study of 2502 patients. Blood 2006; 107: 3847–3853.

    CAS  Article  PubMed Central  Google Scholar 

  36. 36

    Schnittger S, Bacher U, Alpermann T, Reiter A, Ulke M, Dicker F et al. Use of CBL exon 8 and 9 mutations in diagnosis of myeloproliferative neoplasms and myelodysplastic/myeloproliferative disorders: an analysis of 636 cases. Haematologica 2012; 97: 1890–1894.

    CAS  Article  PubMed Central  Google Scholar 

  37. 37

    Schnittger S, Bacher U, Kern W, Schroder M, Haferlach T, Schoch C . Report on two novel nucleotide exchanges in the JAK2 pseudokinase domain: D620E and E627E. Leukemia 2006; 20: 2195–2197.

    CAS  Article  Google Scholar 

  38. 38

    Cheson BD, Bennett JM, Kopecky KJ, Buchner T, Willman CL, Estey EH et al. Revised recommendations of the international working group for diagnosis, standardization of response criteria, treatment outcomes, and reporting standards for therapeutic trials in acute myeloid leukemia. J Clin Oncol 2003; 21: 4642–4649.

    Article  Google Scholar 

  39. 39

    Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H et al. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues 4th edn. International Agency for Research on Cancer (IARC): Lyon, France, 2008.

    Google Scholar 

  40. 40

    Wang YY, Zhao LJ, Wu CF, Liu P, Shi L, Liang Y et al. C-KIT mutation cooperates with full-length AML1-ETO to induce acute myeloid leukemia in mice. Proc Natl Acad Sci USA 2011; 108: 2450–2455.

    CAS  Article  Google Scholar 

  41. 41

    Paschka P, Marcucci G, Ruppert AS, Mrozek K, Chen H, Kittles RA et al. Adverse prognostic significance of KIT mutations in adult acute myeloid leukemia with inv(16) and t(8;21): a Cancer and Leukemia Group B Study. J Clin Oncol 2006; 24: 3904–3911.

    CAS  Article  Google Scholar 

  42. 42

    Kim HJ, Ahn HK, Jung CW, Moon JH, Park CH, Lee KO et al. KIT D816 mutation associates with adverse outcomes in core binding factor acute myeloid leukemia, especially in the subgroup with RUNX1/RUNX1T1 rearrangement. Ann Hematol 2013; 92: 163–171.

    CAS  Article  Google Scholar 

  43. 43

    Schlenk RF, Benner A, Krauter J, Buchner T, Sauerland C, Ehninger G et al. Individual patient data-based meta-analysis of patients aged 16 to 60 years with core binding factor acute myeloid leukemia: a survey of the German Acute Myeloid Leukemia Intergroup. J Clin Oncol 2004; 22: 3741–3750.

    CAS  Article  Google Scholar 

  44. 44

    Gustafson SA, Lin P, Chen SS, Chen L, Abruzzo LV, Luthra R et al. Therapy-related acute myeloid leukemia with t(8;21) (q22;q22) shares many features with de novo acute myeloid leukemia with t(8;21)(q22;q22) but does not have a favorable outcome. Am J Clin Pathol 2009; 131: 647–655.

    CAS  Article  Google Scholar 

  45. 45

    Kayser S, Dohner K, Krauter J, Kohne CH, Horst HA, Held G et al. The impact of therapy-related acute myeloid leukemia (AML) on outcome in 2853 adult patients with newly diagnosed AML. Blood 2011; 117: 2137–2145.

    CAS  Article  PubMed Central  Google Scholar 

  46. 46

    Burnett AK, Wheatley K, Goldstone AH, Stevens RF, Hann IM, Rees JH et al. The value of allogeneic bone marrow transplant in patients with acute myeloid leukaemia at differing risk of relapse: results of the UK MRC AML 10 trial. Br J Haematol 2002; 118: 385–400.

    Article  Google Scholar 

  47. 47

    Suciu S, Mandelli F, de Witte T, Zittoun R, Gallo E, Labar B et al. Allogeneic compared with autologous stem cell transplantation in the treatment of patients younger than 46 years with acute myeloid leukemia (AML) in first complete remission (CR1): an intention-to-treat analysis of the EORTC/GIMEMAAML-10 trial. Blood 2003; 102: 1232–1240.

    CAS  Article  Google Scholar 

  48. 48

    Nguyen S, Leblanc T, Fenaux P, Witz F, Blaise D, Pigneux A et al. A white blood cell index as the main prognostic factor in t(8;21) acute myeloid leukemia (AML): a survey of 161 cases from the French AML Intergroup. Blood 2002; 99: 3517–3523.

    CAS  Article  Google Scholar 

  49. 49

    Delaunay J, Vey N, Leblanc T, Fenaux P, Rigal-Huguet F, Witz F et al. Prognosis of inv(16)/t(16;16) acute myeloid leukemia (AML): a survey of 110 cases from the French AML Intergroup. Blood 2003; 102: 462–469.

    CAS  Article  Google Scholar 

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Correspondence to S Schnittger.

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SS, WK, CH and TH are part owners of the MLL Munich Leukemia Laboratory GmbH. MTK, CE, TA, UB and NN are employed by the MLL Munich Leukemia Laboratory GmbH.

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Author contributions

SS and MTK were the principal investigators of this study, analyzed the data and wrote the manuscript. CE did analysis of molecular mutations. CH was responsible for chromosome banding analysis. WK was responsible for immunophenotyping and was involved in the statistical analysis. TH was responsible for cytomorphologic analysis, and MTK and UB contributed to cytomorphologic analysis. TA collected and analyzed clinical data. UB contributed writing of the manuscript. NN contributed to statistics and graphics. All authors read and contributed to the final version of the manuscript.

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Krauth, MT., Eder, C., Alpermann, T. et al. High number of additional genetic lesions in acute myeloid leukemia with t(8;21)/RUNX1-RUNX1T1: frequency and impact on clinical outcome. Leukemia 28, 1449–1458 (2014). https://doi.org/10.1038/leu.2014.4

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Keywords

  • acute myeloid leukemia
  • t(8;21)/RUNX1-RUNX1T1
  • secondary mutations
  • KIT mutation
  • ASXL1 mutation
  • prognosis

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