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Acute Leukemias

ASXL1 exon 12 mutations are frequent in AML with intermediate risk karyotype and are independently associated with an adverse outcome

Abstract

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.

Introduction

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

Patients

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.

Healthy controls

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.

Molecular analysis

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).

Analyses for mutations of NPM1, FLT3-TKD, NRAS, KRAS, DNMT3A, RUNX1, TET2, WT1, IDH1, IDH2, CEBPA as well as MLL-PTD and FLT3-ITD were described previously.5, 26, 27, 28, 29, 30, 31, 32, 33, 34

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

Statistical analysis

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.

Results

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.

Repeated testings

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.

Figure 1
figure1

Localization of all 128 ASXL1 mutations within the coding region. Each single mutation is indicated as a dot (frameshifts in red, nonsense in blue). Missense mutations (polymorphisms) are indicated below the diagram. ASXN, conserved domain at the N-terminus; ASXM, conserved domain in the middle part; Gly, glycine-rich region; Rb, Rb interacting motif; NR box, domain interacting with RAR and RXR; PHD, plant homedomain finger.

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).

Table 1 Patient characteristics

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.

Table 2 Coincidence of ASXL1mut with other mutations
Figure 2
figure2

Distribution and frequency of ASXL1mut and other molecular mutations in the total cohort of 740 patients. Red indicates a mutation within the respective gene, gray indicates no mutation. White cells indicate that the respective gene mutation was not analyzed for this patient. Patients are depicted vertically.

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.

Table 3 Molecular and cytogenetic analysis of paired diagnostic and relapse samples

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).

Figure 3
figure3

Outcome within the total cohort with survival data (n=481). Kaplan–Meier plot showing inferior (a) overall and (b) event free survival of the ASXL1 mutated cases (red) compared with ASXL1 wild-type cases (gray). Median values for OS and EFS are indicated. (c) OS restricted to patients <60 years (n=223). (d) OS restricted to patients 60 years (n=258). (e) Survival within the normal karyotype subcohort (n=376). Kaplan–Meier plot showing inferior overall and (f) event free survival of the ASXL1 mutated cases (red) compared with ASXL1 wild-type cases (gray). Median values for OS and EFS are indicated.

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).

Figure 4
figure4

Survival according to RUNX1 mutation status. (a) Within the subgroup of RUNX1wt patients (n=408) an inferior OS was observed for ASXL1 mutated cases (red) compared with ASXL1 wild-type cases (gray). (b) Within the subgroup of RUNX1mut (n=73) ASXL1 mutation status (red) resulted only in a non-significantly inferior OS as compared with ASXL1 wild-type status (gray). Median values for OS and EFS are indicated.

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).

Table 4 Cox regression analysis

Discussion

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.

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Acknowledgements

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.

Author contributions

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.

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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. CE, SJ, TA, AF, VG and AK are employed by the MLL Munich Leukemia Laboratory.

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Supplementary Information accompanies the paper on the Leukemia website

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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

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Keywords

  • ASXL1 mutations
  • AML
  • prognosis
  • intermediate karyotype

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