We aimed at evaluating ASXL1mut in 740 AML with intermediate risk karyotype for frequency, association with other mutations and impact on outcome. Five hundred fifty-three cases had a normal karyotype (NK) and 187 had intermediate risk aberrant cytogenetics. Overall, ASXL1mut were detected in 127/740 patients (17.2%). ASXL1mut were more frequent in males than in females (23.5% vs 9.9%, P<0.001). They were associated with higher age (median: 71.8 vs 61.8, P<0.001), a history of preceding myelodysplastic syndromes, and with a more immature immunophenotype compared with patients with wild-type ASXL1 (ASXL1wt). ASXL1mut were more frequent in patients with aberrant karyotype (58/187; 31.0%), especially in cases with trisomy 8 (39/74; 52.7%), than in those with NK (69/553; 12.5%; P<0.001). ASXL1mut were observed more frequent in RUNX1mut (P<0.001), and less frequent in NPM1mut (P<0.001), FLT3-internal tandem duplication (ITD) (P<0.001), FLT3-TKD (P=0.001) and DNMT3Amut (P<0.001). Patients with ASXL1mut had a shorter overall survival (OS) (P<0.001) and event free survival (P=0.012) compared with ASXL1wt. In multivariable analysis, ASXL1mut was an independent adverse factor for OS (P=0.032, relative risk: 1.70). In conclusion, ASXL1mut belong to the most frequent mutations in intermediate risk group AML. Their strong and independent dismal prognostic impact suggests the inclusion into the diagnostic work-up of AML.
Acute myeloid leukemia (AML) patients can be classified into different prognostic subgroups according to presence or absence of distinct cytogenetic abnormalities. In the past years, various novel molecular genetic markers have been identified enabling further stratification of this heterogeneous disease. Screening for mutations in genes such as FLT3, NPM1, CEBPA, IDH1, IDH2, and RUNX1 allow a better prognostic prediction, in particular in AML with normal karyotype (NK) or intermediate cytogenetic risk profile.1, 2, 3, 4, 5, 6, 7, 8
Recently, another promising candidate gene, ASXL1 (additional sex combs-like 1), has been identified to be mutated in myeloproliferative neoplasms.9 The gene is located in the chromosomal region 20q11 encoding a protein of the polycomb group and trithorax complex family. Mutations of ASXL1 can be found particularly in exon 12 and virtually all are heterozygous.9 Mainly frameshift and stop mutations were found that are predicted to lead to loss of the carboxyterminal plant homeodomain finger on the protein level.10 This motif can be found in nuclear proteins involved in chromatin modifications. Indeed, ASXL1 can interact with retinoic acid receptor and seems to be involved in chromatin remodeling, though the exact function remains unknown thus far.11, 12
Several studies indicate that ASXL1 mutations occur frequently in various myeloid malignancies, including myelodysplastic syndromes (MDS), AML, chronic myeloid leukemia, chronic myelomonocytic leukemia and myeloproliferative neoplasms,9, 13, 14, 15, 16, 17, 18, 19, 19 and published data points to a poor prognostic impact in patients with these mutations.14, 18, 19
In AML, the results regarding frequency and associations with karyotype abnormalities are quite diverse. In different studies, ASXL1 mutations have been detected in about 6 to 30% of AML.15, 16, 17, 20 Furthermore, mutual exclusiveness of NPM1 mutations was described.16 In a previous study, ASXL1 mutations occurred with a similar frequency both in patients with NK (8.9%) and with cytogenetic abnormalities (12.9%).18 Interestingly, there was not only an inverse association observed with NPM1 mutations, but also with FLT3-ITD and WT1 mutations. In addition, an association with RUNX1 mutations was found. Moreover, patients with ASXL1 mutations had a shorter overall survival (OS), but the significance was lost in a multivariable analysis.18 A further study showed that ASXL1 mutations identify a high-risk group of older patients within the ELN ‘favorable’ genetic category.21
To evaluate the impact and frequency of ASXL1 mutations in a large cohort of adult AML not selected for age but only for karyotype, we here analyzed 740 cases with cytogenetically intermediate-risk AML and detected a frequency of 17.2% ASXL1 mutations. There was an association to male sex, higher age, more immature phenotype, aberrant karyotype, a strong positive correlation to RUNX1 mutations, and a negative correlation to NPM1, FLT3-ITD, FLT3-TKD and DNMT3A mutations. For the first time we could show that ASXL1 mutations have strong independent negative impact on survival. In larger subsets than previously reported, we could show that ASXL1 mutations are stable mutations in paired diagnostic/relapse samples and are highly correlated with trisomy 8. In addition, we could show that only frameshift and stop mutations in ASXL1 are somatic mutations.
Patients, controls and methods
All 740 patient samples were referred to our laboratory for first diagnosis of AML between September 2005 and September 2010. AML was diagnosed according to the FAB (French-American-British) and WHO (World Health Organization) classifications.22, 23 Three hundred forty-five patients were female, 395 male and the median age was 66.9 years (range 18.5–100.4 years). Five hundred fifty-three cases had a NK and 187 carried non-recurrent intermediate risk aberrant cytogenetics (according to the refined MRC (United Kingdom Medical Research Council) classification24). Six hundred ninety-seven (94.2%) patients showed de novo AML, whereas 26 (3.5%) patients presented with secondary AML following either MDS or myeloproliferative neoplasms, and 17 (2.3%) showed therapy-related AML.
Data on the molecular markers NPM1, FLT3-ITD, MLL-partial tandem duplication (PTD), CEBPA, and RUNX1 was available in all cases. In addition, data on other molecular markers were available for: FLT3-TKD: n=692, IDH1R132: n=598 and IDH2R142+IDH2R172: n=534, WT1: n=587, NRAS: n=475; DNMT3A: n=204, TET2: n=166. Clinical follow-up data were available in 639 patients, but for prognostic analyses only de novo AML with intensively treatment strategies (like standard protocols including ‘7+3’ or combinations of chemotherapeutics, such as TAD (thioguanine, cytarabine and daunorubicin) and HAM (high-dose cytarabine and mitoxantrone) were included (n=481). All patients gave their informed consent for scientific evaluations, for example, molecular 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.
The KORA (Cooperative Health Research in the Region of Augsburg, Germany) participants were selected from the F4 visit (2006–2008), the follow-up survey for the KORA S4 cohort sample, recruited between 1999 and 2001.25 The KORA F4 visit population comprises 3080 male and female residents of the city and region of Augsburg in southern Germany. Altogether 491 individuals from KORA were analyzed.
Isolation of mononuclear cells, DNA extraction and mRNA extraction as well as random primed cDNA synthesis were performed as described previously.26 In 611 cases bone marrow and in 129 cases peripheral blood were used for the molecular analysis.
Screening for ASXL1 mutations in exon 12 was performed at the DNA level by direct Sanger sequencing of six different amplicons using BigDye terminator v1.1 cycle sequencing chemistry (Applied Biosystems, Weiterstadt, Germany). The primers for PCR and sequencing were described previously.13 For PCR Qiagen Master Mix (Qiagen, Hilden, Germany) was used; solely for amplicon 12.4, the GC-rich-Kit was used (Roche Applied Science, Mannheim, Germany).
Cytomorphology, cytogenetics and immunophenotyping
Cytomorphologic assessment was based on May–Grünwald–Giemsa stains, myeloperoxidase reaction and non-specific esterase using alpha-naphtyl-acetate as described before and was performed according to the criteria defined in the French-American-British and the World Health Organization classifications.22, 23, 35 Cytogenetic studies were performed after short-term cultures. Karyotypes, analyzed after G-banding, were described according to the International System for Human Cytogenetic Nomenclature.36 Cytogenetic classification as ‘intermediate’ risk group was performed according to the refined MRC criteria.24 Cytogenetic results were available for all patients in the study. Immunophenotyping was performed in 388 cases as described previously.37, 38
Survival curves were calculated for OS and event free survival (EFS) according to Kaplan–Meier and compared using the two-sided log rank test. OS was the time from diagnosis to death or last follow-up. EFS was defined as the time from diagnosis to treatment failure, relapse, death or last follow-up in complete remission. Relapse was defined according to Cheson et al.39 Cox regression analysis was performed for OS and EFS with different parameters as covariates. 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. Results were considered significant at P<0.05. Parameters that were significant in univariable analyses were included into multivariable analyses. Dichotomous variables were compared between different groups using the χ2-test and continuous variables by Student́s t-test. All reported P-values are two-sided. No adjustments for multiple comparisons were performed. SPSS (version 19.0.0) software (IBM Corporation, Armonk, NY, USA) was used for statistical analysis.
Frequency and characterization of ASXL1 alterations
Overall, 135 ASXL1 alterations were detected in 134/740 patients (18.1%). The majority of these alterations were frameshift mutations caused by deletion or duplication of a nucleotide (n=100; 74.1%). Further, 28 mutations (20.7%) were base exchanges leading to a premature stop of translation. Seven alterations were single-base exchanges leading to missense mutations (5.2%).
Detailed evaluation of molecular variants
To evaluate whether the detected alterations were somatic mutations or even rare constitutional polymorphisms, we did (1) in silico analysis, (2) analysis of remission samples and (3) evaluation of healthy controls. (4) In addition, to assure the validity of the detected muations in homopolymeric regions, we performed repeated testing to exclude sequencing artefatcs.
G646WfsX12 (Gly646TrpfsX12) has repeatedly been discussed not to be a somatic mutation but more likely a polymorphism or even a sequencing artefact due to an 8-bp guanine homopolymere at that site.40 In this study, we excluded a technical problem as all G646WfsX12 cases remained positive and all G646wt samples remained negative upon repeated testing of 20 samples up to 10 times. In addition, this aberration disappears in remission (see below).
In silico analysis
For in silico analysis we used two different algorithms: SIFT (http://sift.jcvi.org),41 and PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/index.shtml).42 All frameshift and nonsense mutations were predicted to confer a damaging character for the protein structure of ASXL1. Results of the prediction analysis were ambigious: whereas the missense mutations were predicted to be damaging in some instances they were predicted to be tolerated in others and thus in part was inconsistent between the different methods (Supplementary Table 1).
Analysis of remission samples
In five cases with a missense mutation, remission material was available. All these five cases were also positive for an NPM1 mutation at diagnosis. For the detection of the NPM1 mutation a highly sensitive real-time PCR assay with a sensitivity of 10−5–10−6 was available.43 Although all cases were negative for the NPM1mut in the respective remission sample, in all cases the missense exchange in ASXL1 was retained with a load of 50%, which is highly suggestive of a constitutional polymorphism. Thus, these cases were assigned to the ASXL1 wild-type group. For the remaining two missense mutations no remission samples were available, but these were clearly assigned as tolerated with in silico analysis. In patients with the p.G646WfsX12 mutation who achieved a complete remission after chemotherapy, this variant disappeared (n=9) or its load diminished in the respective remission sample (n=7). Thus, this variant was proven to be a somatic mutation. An example is depicted in Supplementary Figure 1.
Evaluation of healthy controls from KORA (n=491)
We analyzed 491 age and sex matched individuals from a population-based cohort panel (KORA=Cooperative Health Research in the Region of Augsburg, Germany). In KORA, only one p.G646WfsX12 positive sample (0.2%) with a mutation load of only 10% was identified. This was in contrast to the leukemia samples that all had a mutation load of around 50%. This incidence in the KORA cohort is significantly below the one observed in myeloid malignancies.44 Because of the low mutation load the one positive case was interpreted as presumably having a small pre-malignant clone or an early yet undetected clonal disease. From this data we conclude that the p.G646WfsX12 is a somatic mutation and was regarded as true mutation throughout the paper.
Besides the common silent p.Ser1253Ser (302/491, 61.5%) with 14.5% (71/491) homozygotes and the intronic c.*22 A>G (292/491, 59.5%) with 11.4% homozygotes, 26 different rare missense mutations were detected in 44 individuals of the KORA cohort with frequencies between 0.1 and 0.4% (Supplementary Table 2). Only few of them have been assigned as polymorphisms before (Supplementary Table 2). In addition, 11 further rare variants were described in the literature that were not detected in the KORA cohort (Supplementary Table 3). This data suggest that a number of rare inborn variants exist in ASXL1.
In conclusion, this data suggest that all missense mutations in ASXL1 are inborn polymorphisms and in the following only frameshift and stop mutations in ASXL1 were regarded as somatic mutations.
Frequency and characterization of somatic ASXL1 mutations
After exclusion of the missense alterations, a total of 128 somatic mutations were observed in 127 cases. All had a deleterious effect on the protein structure due to their character as stop or frameshift mutations. In detail, the most frequent mutation was p.Gly646TrpfsX12 (n=69, 53.9%). The p.Gly646TrpfsX12 at the protein level was a result of c.1934dupG (n=65), c.1927_1928insA (n=2) or c.1935dupT (n=2) at the DNA level. The second most frequent mutation was p.Glu635ArgfsX15 (n=18), followed by p.Tyr591X (n=5), p.Arg693X (n=4) and p.Gln733X (n=2). The remaining 30 mutations were non-recurrent consisting of 14 frameshift and 16 nonsense mutations. The majority of the mutations were detected with a mutation/wild-type load of 40–50%. One of the patients had two ASXL1 mutations: a p.Gln829X with a mutation/wild-type load of 10% and a p.Ala1172LeufsX2 with a mutation/wild-type load of 50%. The positions of the mutations within the gene are indicated in Figure 1.
Association with biological characteristics
ASXL1mut were more frequent in males than in females (93/395, 23.5% vs 34/345, 9.9%, P<0.001) and were associated with higher age (mean±s.d. 71.8±9.4 vs 61.8±14.9 years, P<0.001) and lower white blood cell (WBC) counts (mean±s.d. 34.2±49.6 vs 46.8±61.9 × 109/l, P=0.025) (Table 1). There was no association of ASXL1mut to platelet counts or hemoglobin levels. ASXL1mut were detected more frequently in s-AML after MDS/myeloproliferative neoplasms (11/26; 42.3%) compared with de novo AML (114/697; 16.4%) and therapy-related AML (2/17; 11.8%) (P=0.002 for heterogeneity between the three groups). With respect to morphology, ASXL1mut were more frequent in French-American-British M2 (58/240; 24.2%) compared with all other subtypes (65/474; 13.7%; P=0.001) as well as in M5a (8/18, 44.4%) compared with all other subtypes (115/696; 16.5%; P=0.006), but less frequent in M1 (19/221, 8.6%) compared with all other subtypes (104/493, 21.1%; P<0.001).
In 388 cases, immunophenotyping data were available. Cases with ASXL1mut (n=66) had a stronger expression of CD13 (% positive cells, mean±s.d., 51±26 vs 43±25%, P=0.025), CD34 (44±28% vs 29±29%, P<0.001), CD133 (27±24% vs 20±25%, P=0.047) and HLA-DR (40±24% vs 33±24%, P=0.034) as well as a weaker expression of CD33 (68±25% vs 75±23%, P=0.014) and thus had a more immature immunophenotype as compared with ASXL1wt.
Association with karyotype
The total cohort was comprised of 553 cases with NK and 187 cases with intermediate risk karyotype aberrations comprising the following recurrent aberrations: trisomy 8 (n=74/187 39.6%); loss of chromosome Y (n=13/109; 11.9%), trisomy 13 (n=10/187; 5.3%) and trisomy 21 (n=10/187; 4.9%). ASXL1mut were more frequent in patients with aberrant karyotype than in those with NK (P<0.001, Table 1). Particularly, a strong correlation to trisomy 8 was observed as 39 of these 74 cases (52.7%) had an ASXL1 mutation compared with only 19 of 113 (16.8%) in other aberrant karyotypes (P<0.001).
Association with other molecular mutations
Generally, ASXL1mut were observed together with all other molecular mutations. There was a strong correlation with RUNX1mut (P<0.001) and a trend to increased frequency in IDH2mut cases (P=0.079). A negative correlation was found for NPM1mut (P<0.001), FLT3-ITD (P<0.001), FLT3-TKD (P=0.001), DNMT3Amut (P<0.001) and a negative trend for WT1mut (P=0.068). No significant associations were observed for CEBPA, RUNX1, IDH1R132, NRAS and TET2 mutations. A detailed description of the mutation coincidences is given in Table 2 as well as in Figure 2.
Stability during follow-up
Paired samples of diagnosis and relapse time points were available in 16 cases with an ASXL1 mutation at diagnosis. At diagnosis, nine of these cases had a NK, six had trisomy 8 and one had trisomy 11. In addition, in 15/16 patients one or two additional molecular mutations were detected (in 5 and 10 cases, respectively). At relapse, all ASXL1 mutations and all other molecular mutations (with the exception of one BCOR mutation) were retained. Thus, ASXL1 is a stable mutation. However, this pattern does not allow any conclusions on the hierarchy of all these mutations.
In contrast, in 5 of 14 cases (with available cytogenetics at relapse) additional chromosomal aberrations were detected at relapse, which were not present at diagnosis (Table 3). We would like to outline that within this cohort of 16 relapsed AML 11 cases also had a RUNX1mut, one a CEBPAmut, and two an NPM1mut. Only two cases did not reveal any of these three mutations and one of these two even relapsed with a t(8;21)(q22;q22)/RUNX1-RUNX1T1. In addition to ASXL1mut, this particular case also was IDH2R140 mutated at both time points. Thus, this represents a very unusual case with ASXL1 and IDH2R140 mutations at diagnosis and additional t(8;21)(q22;q22) at relapse 17 months later. The RUNX1-RUNX1T1 was backtracked with highly sensitive real-time PCR and nested PCR but was not present at diagnosis.
In addition, in four cases with s-AML paired samples from the MDS phase were analyzed. All four cases were ASXL1 mutated already at the MDS phase of the disease. One case was in addition CEBPAmut and IDH2R140mut at both time points, one gained an IDH2R140mut and a trisomy 8 at the time point of diagnosis of AML. The third case was RUNX1mut at both time points and gained an FLT3-ITD at the time point of transformation to AML, while in the fourth case, the ASXL1 mutation was the sole mutation detected at both time points.
Prognostic relevance of ASXL1 mutations
Only patients with de novo AML who received intensive treatment (n=481) were included into the prognostic analyses. Patients with ASXL1mut had shorter OS (11.0 vs 62.2 months in ASXL1wt, P<0.001) and EFS (median: 9.1 vs 16.3 months in ASXL1wt, P=0.012) (Figures 3a and b).
In a next step, patients were subdivided according to age ⩾60 years (ASXL1wt: n=217, ASXL1mut: n=47) and <60 years (ASXL1wt: n=213; ASXL1mut: n=10). In the younger as well as in the older cohort, OS was shorter in the ASXL1mut compared with the ASXL1wt subset (median: 11.5 vs 36.3 months, P=0.040 in the older and not reached for both in the younger (median 2 years survival: 60% vs 78%, P=0.049)) (Figures 3c and d).
Furtheron, also within the cohort of patients with NK (n=376) patients harboring an ASXL1mut had shorter OS (median: 9.8 vs 62.2 months in ASXL1wt, P<0.001) and EFS (median: 7.5 vs 17.7 months in ASXL1wt, P=0.001) (Figures 3e and f). In contrast, in the cohort with aberrant intermediate karyotypes, the difference of OS between ASXL1mut (n=24) and ASXL1wt (n=81) (median: 20.0 vs 36.7 months) was not significant.
As we observed a high coincidence of ASXL1 with RUNX1 mutations, which were previously shown to have a negative impact on prognosis, we also investigated the prognostic impact of ASXL1mut according to RUNX1 mutational status. The prognostically adverse effect of ASXL1mut was seen within the RUNX1wt cohort (n=408; median OS: 10.1 vs 62.2 months, P=0.001) (Figure 4a), but there was only a trend toward an adverse effect in the RUNX1mut subgroup (n=73; median OS: 15.3 vs 24.9 months, NS) (Figure 4b).
The following parameters were tested in univariable Cox regression analyses for impact on OS and EFS: sex, age, WBC count, platelet count, hemoglobin level, cytogenetics (normal vs aberrant karyotype), and mutational status of ASXL1, NPM1, FLT3-ITD, MLL-PTD, CEBPA, RUNX1, FLT3-TKD, IDH1, IDH2, WT1, NRAS, DNMT3A and TET2. A significant negative impact on OS was shown for higher age (P<0.001, relative risk (RR) per decade: 1.50), higher WBC count (P<0.001, RR per 10 × 109/l: 1.07), ASXL1 mutations (P<0.001; RR: 2.23), FLT3-ITD (P=0.002, RR: 1.69), MLL-PTD (P=0.006, RR: 2.06), and RUNX1 mutations (P=0.010, RR: 1.64). A favorable impact was found for biallelic CEBPA mutations (P=0.009, hazard ratio: 0.55). No impact was found for the other parameters.
A significant negative impact on EFS was found for higher age (P<0.001, RR per decade: 1.03), higher WBC count (P<0.001, RR per 10 × 109/l: 1.06), ASXL1 mutations (P=0.013; RR: 1.59), FLT3-ITD (P=0.021, RR: 1.38), MLL-PTD (P=0.010, RR: 1.79) and RUNX1 mutations (P=0.030, RR: 1.42). No impact was found for the other parameters.
In multivariable analysis, ASXL1mut revealed an independent prognostic impact on OS (P=0.028, RR: 1.73) besides age (P<0.001, RR per decade: 1.51), WBC count (P<0.001, RR per 10 × 109/l: 1.06) and FLT3-ITD status (P=0.049, RR: 1.40). In multivariable analysis for EFS, ASXL1mut revealed no independent impact and only age (P<0.001, RR per decade: 1.33), WBC count (P<0.001, RR per 10 × 109/l: 1.05) and FLT3-ITD (P=0.046, RR: 1.39) were associated with outcome (Table 4).
Mutations in ASXL1 have been reported in various myeloid malignancies but have not been intensively studied in AML. Still, the incidence, associations with other molecular markers and associations with biologic characteristics were reported variably, mainly because of selected cohorts or different ethnical backgrounds of the analyzed cohorts.16, 17, 18 We concentrated on adult AML with intermediate risk karyotype independent of age. In addition, as mutations have been shown to cluster in exon 12 and were detected very rarely outside this region,45 we sequenced only exon 12, which actually comprises ∼50% of the whole coding region of the gene. We show that ASXL1 mutations occurred in 17.2% and therewith belong to the most common molecular markers mutated in the cytogenetic intermediate risk group AML. They are associated with distinct clinical and biological features like male sex, higher age, immature immunophenotype, concomitant RUNX1 and IDH2 mutations, aberrant intermediate risk karyotype, especially trisomy 8, and with adverse prognosis.
Similar findings have been reported in a Taiwanese population.18 However, in this study, there was no independent prognostic effect of ASXL1mut and this was discussed to be due to the high coincidence with RUNX1mut, which is also a highly adverse prognostic marker in AML.7, 8 We confirmed a high correlation of ASXL1 mutations and RUNX1 mutations. In a multivariable analysis on OS, taking age, WBC count, ASXL1, FLT3-ITD, MLL-PTD and RUNX1 status into account, however, we found ASXL1 mutations to be an independent adverse prognostic factor for OS (P=0.032).
In a previous study, instability of ASXL1mut was reported as two of six patients lost ASXL1mut at relapse or even in primary refractory disease.18 In our cohort, 16 combined diagnosis/relapse samples were available and all these cases retained the same ASXL1 mutation at relapse. Almost all of these cases had two additional mutations of different classes: (1) RUNX1mut (11/16), NPM1mut (2/16), CEBPAmut (1/16). (2) IDH1R132 (2/16), IDH2R140 (5/16), FLT3-ITD (1/16), NRAS (2/16), BCOR (1/3). With the exception of the BCOR mutation, all mutations were stable at relapse and thus a hierarchical pattern of mutation could not be identified. In contrast, in four s-AML cases that were backtracked to the MDS phase the ASXL1mut were already present at the MDS stage of the disease and additional aberrations (FLT3-ITD, IDH2R140 or trisomy 8) were gained in AML transformation suggesting that ASXL1 mutations are an early event in transformation.
A previous study has shown that ASXL1 mutations are five time as frequent in patients older than 60 years and are associated with high risk in the ‘favorable’ cytogenetic category according to ELN criteria.21 We confirmed the considerably higher frequency in older patients. In addition, we showed that ASXL1 mutations have a negative impact on outcome in the AML with intermediate risk karoytpe and also in the subset with NK. In addition, ASXL1 mutations were also correlated with adverse outcome in AML <60 years.
ASXL1 and NPM1 mutations have been suggested to be mutually exclusive.16 Different routes of leukemogenesis rather than two alternate hits on the same route were discussed. We confirmed a negative correlation of ASXL1 and NPM1, however, in our cohort in 8 ASXL1mut cases, we also detected NPM1 mutations. This leads to the more likely hypothesis that certain routes of gene mutations are more prevalent than others but do not exclude each other. This is supported by one of our cases demonstrating both ASXL1mut and RUNX1mut at diagnosis and at relapse who additionally gained a t(8;21)/RUNX1/RUNX1T1 at relapse. In this line, during the past years it has become clear that the concept of a two-hit event in leukemia with a classical ‘type 1’ (proliferation) and ‘type 2’ (differentiation) mutation46 cannot fully explain all recent findings on molecular mutations. More and more mutations have been shown to be important for the development of leukemia including alterations in genes relevant for genomic stability like TP53,47 metabolic enzymes (IDH1, IDH2, ND4)5, 48, 49 or proteins with effects on epigenetic modification (TET2, EZH2, DNMT3A)50, 51.52 This leads to a high probability of multiple mutations from different pathways are randomly combined and thereby underlie the pathogenesis of AML.
All ASXL1 mutations detected in this study were heterozygous, which is consistent with the hypothesis of a dominant negative effect of truncated ASXL1 proteins. All mutations were either (1) frameshift mutations caused by deletion or duplication of one nucleotide or (2) base exchanges leading to stop mutations. The most common mutation was p.Gly646TrpfsX12, which accounted for 53.9% of all deleterious mutations, followed by p.Glu635ArgfsX15 in 14.2%. Despite previous suggestions that these mutations40 may be germline or even technical artifacts, we could clearly show that these are true somatic mutations. This conclusion was based on analysis of a large healthy control cohort, analysis of remission samples and repeated testings. In constrast, we could show that missense mutation in ASXL1 are highly likely to be always rare inborn polymorphisms.
As has been suggested before17 the majority of mutations (59%) in our cohort are localized to one particular region within exon 12, around the Gly-rich domain spanning amino acids 642–685 (Figure 1). Three mutations that lead to a truncated protein are located upstream of the C-terminal nuclear receptor box (amino acid 1107–1112), which is predicted to interact with the retinoic acid receptor. The predicted truncated protein would lack its plant homeodomain, thus compromising the function of the associated chromatin modifiers.9 Although the function of ASXL1 is not completely understood, the presented defects suggest an important role in pathogenesis of AML.
In conclusion, our findings indicate that ASXL1 mutations are one of the most commonly occurring molecular mutations in intermediate risk AML and they have to be considered to significantly contribute to leukemogenesis. There is a strong association of ASXL1 mutations with male sex, MDS prephase, higher age, immature immunophenotype and mutations in RUNX1. Given their strong and independent dismal prognostic impact, ASXL1 mutations should be included in the diagnostic work-up of patients with cytogenetically intermediate-risk AML.
Falini B, Bolli N, Liso A, Martelli MP, Mannucci R, Pileri S et al. Altered nucleophosmin transport in acute myeloid leukaemia with mutated NPM1: molecular basis and clinical implications. Leukemia 2009; 23: 1731–1743.
Dohner H, Estey EH, Amadori S, Appelbaum FR, Buchner T, Burnett AK et al. Diagnosis and management of acute myeloid leukemia in adults: recommendations from an international expert panel, on behalf of the European LeukemiaNet. Blood 2010; 115: 453–474.
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. Lyon: International Agency for Research on Cancer (IARC); 2008.
Preudhomme C, Sagot C, Boissel N, Cayuela JM, Tigaud I, De Botton S et al. Favorable prognostic significance of CEBPA mutations in patients with de novo acute myeloid leukemia: a study from the Acute Leukemia French Association (ALFA). Blood 2002; 100: 2717–2723.
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.
Boissel N, Nibourel O, Renneville A, Gardin C, Reman O, Contentin N et al. Prognostic impact of isocitrate dehydrogenase enzyme isoforms 1 and 2 mutations in acute myeloid leukemia: a study by the Acute Leukemia French Association group. J Clin Oncol 2010; 28: 3717–3723.
Tang JL, Hou HA, Chen CY, Liu CY, Chou WC, Tseng MH et al. AML1/RUNX1 mutations in 470 adult patients with de novo acute myeloid leukemia: prognostic implication and interaction with other gene alterations. Blood 2009; 114: 5352–5361.
Schnittger S, Dicker F, Kern W, Wendland N, Sundermann J, Alpermann T et al. RUNX1 mutations are frequent in de novo AML with noncomplex karyotype and confer an unfavorable prognosis. Blood 2011; 117: 2348–2357.
Carbuccia N, Murati A, Trouplin V, Brecqueville M, Adelaide J, Rey J et al. Mutations of ASXL1 gene in myeloproliferative neoplasms. Leukemia 2009; 23: 2183–2186.
Vainchenker W, Delhommeau F, Constantinescu SN, Bernard OA . New mutations and pathogenesis of myeloproliferative neoplasms. Blood 2011; 118: 1723–1735.
Cho YS, Kim EJ, Park UH, Sin HS, Um SJ . Additional sex comb-like 1 (ASXL1), in cooperation with SRC-1, acts as a ligand-dependent coactivator for retinoic acid receptor. J Biol Chem 2006; 281: 17588–17598.
Lee SW, Cho YS, Na JM, Park UH, Kang M, Kim EJ et al. ASXL1 represses retinoic acid receptor-mediated transcription through associating with HP1 and LSD1. J Biol Chem 2010; 285: 18–29.
Gelsi-Boyer V, Trouplin V, Adelaide J, Bonansea J, Cervera N, Carbuccia N et al. Mutations of polycomb-associated gene ASXL1 in myelodysplastic syndromes and chronic myelomonocytic leukaemia. Br J Haematol 2009; 145: 788–800.
Gelsi-Boyer V, Trouplin V, Roquain J, Adelaide J, Carbuccia N, Esterni B et al. ASXL1 mutation is associated with poor prognosis and acute transformation in chronic myelomonocytic leukaemia. Br J Haematol 2010; 151: 365–375.
Abdel-Wahab O, Manshouri T, Patel J, Harris K, Yao J, Hedvat C et al. Genetic analysis of transforming events that convert chronic myeloproliferative neoplasms to leukemias. Cancer Res 2010; 70: 447–452.
Carbuccia N, Trouplin V, Gelsi-Boyer V, Murati A, Rocquain J, Adelaide J et al. Mutual exclusion of ASXL1 and NPM1 mutations in a series of acute myeloid leukemias. Leukemia 2010; 24: 469–473.
Boultwood J, Perry J, Pellagatti A, Fernandez-Mercado M, Fernandez-Santamaria C, Calasanz MJ et al. Frequent mutation of the polycomb-associated gene ASXL1 in the myelodysplastic syndromes and in acute myeloid leukemia. Leukemia 2010; 24: 1062–1065.
Chou WC, Huang HH, Hou HA, Chen CY, Tang JL, Yao M et al. Distinct clinical and biological features of de novo acute myeloid leukemia with additional sex comb-like 1 (ASXL1) mutations. Blood 2010; 116: 4086–4094.
Thol F, Friesen I, Damm F, Yun H, Weissinger EM, Krauter J et al. Prognostic significance of ASXL1 mutations in patients with myelodysplastic syndromes. J Clin Oncol 2011; 29: 2499–2506.
Rocquain J, Carbuccia N, Trouplin V, Raynaud S, Murati A, Nezri M et al. Combined mutations of ASXL1, CBL, FLT3, IDH1, IDH2, JAK2, KRAS, NPM1, NRAS, RUNX1, TET2 and WT1 genes in myelodysplastic syndromes and acute myeloid leukemias. BMC Cancer 2010; 10: 401.
Metzeler KH, Becker H, Maharry K, Radmacher MD, Kohlschmidt J, Mrozek K et al. ASXL1 mutations identify a high-risk subgroup of older patients with primary cytogenetically normal AML within the ELN Favorable genetic category. Blood 2011; 118: 6920–6929.
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.
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.. Lyon: International Agency for Research on Cancer (IARC), 2008; pp 110–123.
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.
Illig T, Gieger C, Zhai G, Romisch-Margl W, Wang-Sattler R, Prehn C et al. A genome-wide perspective of genetic variation in human metabolism. Nat Genet 2010; 42: 137–141.
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.
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.
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.
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.
Dicker F, Haferlach C, Kern W, Haferlach T, Schnittger S . Trisomy 13 is strongly associated with AML1/RUNX1 mutations and increased FLT3 expression in acute myeloid leukemia. Blood 2007; 110: 1308–1316.
Kohlmann A, Grossmann V, Klein HU, Schindela S, Weiss T, Kazak B et al. Next-generation sequencing technology reveals a characteristic pattern of molecular mutations in 72.8% of chronic myelomonocytic leukemia by detecting frequent alterations in TET2, CBL, RAS, and RUNX1. J Clin Oncol 2010; 28: 3858–3865.
Grossmann V, Schnittger S, Schindela S, Klein HU, Eder C, Dugas M et al. Strategy for robust detection of insertions, deletions, and point mutations in CEBPA, a GC-rich content gene, using 454 next-generation deep-sequencing technology. J Mol Diagn 2011; 13: 129–136.
Schnittger S, Kinkelin U, Schoch C, Heinecke A, Haase D, Haferlach T et al. Screening for MLL tandem duplication in 387 unselected patients with AML identify a prognostically unfavorable subset of AML. Leukemia 2000; 14: 796–804.
Grossmann V, Kohlmann A, Zenger M, Schindela S, Eder C, Weissmann S et al. A deep-sequencing study of chronic myeloid leukemia patients in blast crisis (BC-CML) detects mutations in 76.9% of cases. Leukemia 2011; 25: 557–560.
Haferlach T, Kern W, Schoch C, Hiddemann W, Sauerland MC . Morphologic dysplasia in acute myeloid leukemia: importance of granulocytic dysplasia. J Clin Oncol 2003; 21: 3004–3005.
ISCN (1995). Guidelines for cancer cytogenetics, Supplement to: an International System for Human Cytogenetic Nomenclature Mitelman F, Karger S ed. 1995.
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.
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.
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.
Abdel-Wahab O, Kilpivaara O, Patel J, Busque L, Levine RL . The most commonly reported variant in ASXL1 (c.1934dupG;p.Gly646TrpfsX12) is not a somatic alteration. Leukemia 2010; 24: 1656–1657.
Kumar P, Henikoff S, Ng PC . Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm. Nat Protoc 2009; 4: 1073–1081.
Adzhubei IA, Schmidt S, Peshkin L, Ramensky VE, Gerasimova A, Bork P et al. A method and server for predicting damaging missense mutations. Nat Methods 2010; 7: 248–249.
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.
Gelsi-Boyer V, Brecqueville M, Devillier R, Murati A, Mozziconacci MJ, Birnbaum D . Mutations in ASXL1 are associated with poor prognosis across the spectrum of malignant myeloid diseases. J Hematol Oncol 2012; 5: 12.
Abdel-Wahab O, Pardanani A, Patel J, Wadleigh M, Lasho T, Heguy A et al. Concomitant analysis of EZH2 and ASXL1 mutations in myelofibrosis, chronic myelomonocytic leukemia and blast-phase myeloproliferative neoplasms. Leukemia 2011; 25: 1200–1202.
Gilliland DG, Jordan CT, Felix CA . The molecular basis of leukemia. Hematology Am Soc Hematol Educ Program 2004, 80–97.
Link DC, Schuettpelz LG, Shen D, Wang J, Walter MJ, Kulkarni S et al. Identification of a novel TP53 cancer susceptibility mutation through whole-genome sequencing of a patient with therapy-related AML. JAMA 2011; 305: 1568–1576.
IDH1 Cazzola M. . and IDH2 mutations in myeloid neoplasms--novel paradigms and clinical implications. Haematologica 2010; 95: 1623–1627.
Damm F, Bunke T, Thol F, Markus B, Wagner K, Gohring G et al. Prognostic implications and molecular associations of NADH dehydrogenase subunit 4 (ND4) mutations in acute myeloid leukemia. Leukemia 2012; 26: 289–295.
Delhommeau F, Dupont S, Della V,V, James C, Trannoy S, Masse A et al. Mutation in TET2 in myeloid cancers. N Engl J Med 2009; 360: 2289–2301.
Ernst T, Chase AJ, Score J, Hidalgo-Curtis CE, Bryant C, Jones AV et al. Inactivating mutations of the histone methyltransferase gene EZH2 in myeloid disorders. Nat Genet 2010; 42: 722–726.
Ley TJ, Ding L, Walter MJ, McLellan MD, Lamprecht T, Larson DE et al. DNMT3A mutations in acute myeloid leukemia. N Engl J Med 2010; 363: 2424–2433.
We thank all coworkers in our laboratory for their excellent technical assistance. We also thank Hubert Serve, Johann Wolfgang Goethe-University, Frankfurt; Dietrich Braumann, Asklepios Klinik Altona, Hamburg; Hermann-Josef Pielken, St Johannes Hospital, Dortmund; Clemens-Martin Wendtner, Klinikum Schwabing, Munich; Tanja Hesse, Klinikum Lippe, Lemgo; Hans-Jörg Weh, Franziskus Hospital, Bielefeld; Jürgen Wehmeyer, Gemeinschaftspraxis für Hämatologie und Onkologie, Münster; Heinz-Gert Höffkes, Klinikum Fulda; Michael Flasshove, Krankenhaus Düren, Düren; Michael Rummel, Justus Liebig University, Gießen; Christian Peschel, Klinikum Rechts der Isar der Technischen Universität München, Munich; Andreas Neubauer, Philipps University, Marburg and all other physicians for referring samples to our center.
SS was the principal investigator of this study, analyzed the data and wrote the paper. CE and AF did sequence analysis of ASXL1. AK and VG performed next-generation sequencing. SJ contributed to writing of the paper. KAK, CS, PS, RP, and NS provided patient samples and clinical data. 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. TA collected and analyzed clinical data. All authors read and contributed to the final version of the paper.
SS, WK, CH, and TH are part owners of the MLL Munich Leukemia Laboratory. CE, SJ, TA, AF, VG and AK are employed by the MLL Munich Leukemia Laboratory.
Supplementary Information accompanies the paper on the Leukemia website
About this article
Cite this article
Schnittger, S., Eder, C., Jeromin, S. et al. ASXL1 exon 12 mutations are frequent in AML with intermediate risk karyotype and are independently associated with an adverse outcome. Leukemia 27, 82–91 (2013). https://doi.org/10.1038/leu.2012.262
- ASXL1 mutations
- intermediate karyotype
Cold Spring Harbor Perspectives in Medicine (2021)
Allogeneic hematopoietic stem cell transplantation could improve the survival of acute myeloid leukemia patients with ASXL1 mutations
Clinical significance of FLT3-ITD/CEBPA mutations and minimal residual disease in cytogenetically normal acute myeloid leukemia after hematopoietic stem cell transplantation
Journal of Cancer Research and Clinical Oncology (2021)
Mutant ASXL1 induces age-related expansion of phenotypic hematopoietic stem cells through activation of Akt/mTOR pathway
Nature Communications (2021)