Clinical features of ASXL1 + AML patients
ASXL1 mutations were found in 8.7% (91 of 1047) of the patients in the whole cohort. The median age of the patients was 50 (33–58) years, with 20 cases older than 60 years and 49 cases being male, as indicated in Table 1. The median white blood cell (WBC) count was 7.5 (2.4–33.3) ×109/L, with 18 cases (19.78%) having a value of ≥ 50 ×109/L. Bone marrow blast percentage of more than 80% was seen in 15 cases (16%). According to the 2017 ELN risk criteria, 27 cases (20%) were favorable-risk AML (including 4 cases of APL), 1 case (1%) was intermediate-risk AML, and 63 cases (69%) were adverse-risk AML. Allo-HSCT was applied in 12 patients (13%). Three cases died within 30 days after induction therapy, and 50 cases (63%) died at the end of the follow-up.
Table 1
Clinical characteristics of ASXL1 + AML
Characteristics
|
Median (Interquartile range) or N (%)
|
Gender male(n[%])
|
49(54%)
|
Age (years)
|
50(33–58)
|
Age ≥ 60 years(n[%])
|
20 (22%)
|
Type (APL vs. non-APL)
|
4(4%)
|
ASXL1 type(n[%])
|
|
G652S
|
38(41.76%)
|
G642fs
|
11(12.09%)
|
H630fs
|
8 (8.79%)
|
ASXL1 VAF(%)
|
49.17 (22.9-57.11)
|
ASXL1 VAF (≥ 49.17%)
|
46(51%)
|
WBC counts (× 109/L)
|
7.5 (2.4–33.3)
|
WBC counts (≥ 50 × 109/L)
|
18(19.78%)
|
HGB counts (g/L)
|
79(66–93)
|
HGB counts (≥ 110 g/L)
|
13 (14%)
|
PLT counts ( × 109/L)
|
48 (20–93)
|
PLT counts (≥ 100 vs × 109/L)
|
19 (21%)
|
Bone marrow blasts (%)
|
51 (26–72)
|
Bone marrow blasts (≥ 80%)
|
15(16%)
|
AML1-ETO
|
17 (19%)
|
CBFβ-MYH11
|
3(3%)
|
Risk group
|
|
Favorable
|
27 (30%)
|
Intermediate
|
1 (1%)
|
Adverse
|
63(69%)
|
APL, acute promyelocytic leukemia; VAF, variant allele frequency; WBC, white blood cell; HGB, hemoglobin; PLT, platelet. |
The molecular mutations of ASXL1 were detected in 30 different nucleotide sites, all of which were located in exon 12, including G652S (41.76%),G642fs (12.09%)༌H630fs (8.79%)༌S1231F and R693X (5.49%)༌N986S (4.40%)༌T1139K (3.33%)༌G643fs and Y591X (2.20%). The distribution of all nucleotide sites was shown in Additional file 1. Most of the patients carried a single-point mutation, 7 (7.69%) patients carried two-point mutations, and one patient carried three-point mutations (G642fs, G643fs and G645fs). The median VAF value of ASXL1 mutation was 49.17% (1.02%-79.28%).
Companion gene mutations and fusion genes in ASXL1 + AML patients
One or more co-mutation of genes was observed in 83 patients (86.46%) of ASXL1 + AML (Fig. 1). TET2 had the highest mutation frequency (48.35%), followed by U2AF1 (16.48%), CEBPA (15.38%), NRAS (14.29%), FLT3-ITD (13.19%), DNMT3A (10.99%), IDH2 (8.79%), RUNX1 (7.69%), KIT (6.59%), SRSF2 (5.49%). Other mutant genes (including FLT3-TKD, ETV6, IDH1, CBL, SETBP1, NPM1, TP53, EZH2, SF3B1, JAK) are found in fewer than 5% of ASXL1 + AML patients; PHF6 and ZRSR2 mutations are not seen in ASXL1 + AML patients.
The fusion genes were screened in 83 of 91 ASXL1 + AML cases. There were 31 cases (37.35%) with fusion gene mutations, including AML1-ETO in 17 cases (20.48%), PML-RARα in 4 cases (4.82%), BCR-ABL, MLL-AF9 and CBFβ-MYH11 in 3 cases (3.61%), MLL-ELL in 1 case (1.20%). The remaining 52 cases (62.65%) were with negative fusion genes.
Risk factors on the prognosis of ASXL1 + AML
In order to understand the prognostic impacts of clinical features and molecular profiles on the outcomes of ASXL1 + AML patients, we analyzed the risk factors on OS and EFS including gender (female vs. male), age (≥ 60 vs. < 60 years), ASXL1 nucleotide sites, ASXL1 VAF (≥ 49.17% vs. < 49.17%), WBC counts (≥ 50 vs. < 50 × 109/L), HGB (≥ 110 vs. < 110 g/L), PLT counts (≥ 100 vs. < 100 × 109/L), bone marrow blasts (≥ 80% vs. < 80%), peripheral blood blasts (≥ 20% vs. < 20%), allo-HSCT (yes vs. no), risk stratification (adverse vs. inter/favorable -risk), AML1-ETO fusion gene (positive vs. negative), CBFβ-MYH11 fusion gene (positive vs. negative), and the mutation status of other common AML co-mutation genes. The median follow-up time was 12.93 (0.37–53.53) months. Table 2 and Additional file 2A revealed that older patients (age ≥ 60 years) had a shorter OS (P = 0.034). Higher WBC counts (≥ 50 × 109/L) were associated with a shorter OS (P = 0.035, Additional file 2C) and EFS (P = 0.006, Additional file 2D). Cases who accepted allo-HSCT had a longer OS (P = 0.024, Additional file 2E) and a better EFS (P = 0.013, Additional file 2F). The adverse risk group had a lower OS (P = 0.005) and EFS (P = 0.004). AML1-ETO coexistence was related to a prolonged OS (P = 0.010, Additional file 3A) and EFS (P = 0.013, Additional file 3B). FLT3-ITD co-mutation was related to a shorter OS (P < 0.001, Additional file 3C) and EFS (P < 0.001, Additional file 3D). However, neither the ASXL1 mutation sites nor the ASXL1 VAF had impacts on EFS or OS.
Table 2
Comparison of EFS and OS between different clinical and molecular characteristic groups in ASXL1 + AML.
Variables
|
OS
|
|
EFS
|
|
χ2
|
P-value
|
χ2
|
P-value
|
Sex (female vs. male)
|
0.69
|
0.406
|
0.719
|
0.395
|
Age (≥ 60 vs. <60 years)
|
4.513
|
0.034
|
2.96
|
0.085
|
ASXL1 type(n[%])
|
|
|
|
|
G652S
|
0.911
|
0.34
|
1.528
|
0.216
|
G642fs
|
1.243
|
0.265
|
1.737
|
0.188
|
H630fs
|
0.592
|
0.442
|
0.214
|
0.643
|
ASXL1 VAF (≥ 49.17% vs.<49.17%)
|
0.005
|
0.944
|
0.344
|
0.557
|
WBC counts (≥ 50 vs. <50 × 109/L)
|
4.471
|
0.035
|
7.564
|
0.006
|
HGB counts (≥ 110 vs. <110 g/L)
|
0.131
|
0.717
|
0.085
|
0.77
|
PLT counts (≥ 100 vs. <100 × 109/L)
|
1.216
|
0.27
|
2.674
|
0.102
|
bone marrow blasts (≥ 80% vs.<80%)
|
0.611
|
0.434
|
0.364
|
0.546
|
peripheral blasts (≥ 20% vs. <20%)
|
1.242
|
0.537
|
1.939
|
0.379
|
risk group (high-risk vs. low/inter)
|
7.719
|
0.005
|
8.231
|
0.004
|
allo-HSCT (yes vs.no)
|
5.066
|
0.024
|
6.105
|
0.013
|
AML1-ETO (positive vs. negative)
|
6.583
|
0.01
|
6.229
|
0.013
|
CBFβ-MYH11 (positive vs. negative)
|
0
|
0.993
|
0.018
|
0.894
|
TET2 (mutated vs. wild type)
|
0.738
|
0.39
|
1.206
|
0.272
|
FLT3-ITD (positive vs. negative)
|
14.081
|
0
|
11.395
|
0.001
|
U2AF1 (mutated vs. wild type)
|
3.056
|
0.08
|
2.273
|
0.132
|
CEBPA (mutated vs. wild type)
|
0.954
|
0.329
|
0.476
|
0.49
|
NRAS (mutated vs. wild type)
|
1.338
|
0.247
|
0.986
|
0.321
|
DNMT3A (mutated vs. wild type)
|
1.824
|
0.177
|
1.205
|
0.272
|
IDH2 (mutated vs. wild type)
|
3.21
|
0.073
|
2.199
|
0.138
|
RUNX1 (mutated vs. wild type)
|
0.608
|
0.436
|
0.391
|
0.532
|
KIT (mutated vs. wild type)
|
1.557
|
0.212
|
1.949
|
0.163
|
SRSF2(mutated vs. wild type)
|
0.323
|
0.570
|
0.537
|
0.464
|
VAF, variant allele frequency; WBC, white blood cell; HGB, hemoglobin; PLT, platelet; allo-HSCT, allogenic hematopoietic stem cell transplantation. |
The factors with P < 0.05 in univariate analyses were included in the multivariate analysis. FLT3-ITD co-mutation had an independent predictive impact on poor OS (Table 3). Allo-HSCT was an independent protective factor for the OS and EFS of ASXL1 + AML patients (Table 3).
Table 3
Multivariate analysis for OS and EFS in ASXL1 + AML.
Variables
|
OS
|
|
EFS
|
|
HR (95%CI)
|
P-value
|
HR (95%CI)
|
P-value
|
Age ≥ 60 years
|
1.382(0.720–2.653)
|
0.331
|
|
|
Risk group
|
1.734(0.655–4.585)
|
0.268
|
2.219(0.847–5.813)
|
0.105
|
Allo-HSCT
|
0.204(0.061–0.085)
|
0.01
|
0.184(0.056–0.605)
|
0.005
|
WBC counts (≥ 50 × 109/L)
|
1.194(0.566–2.517)
|
0.641
|
1.826(0.862–3.867)
|
0.116
|
FLT3-ITD
|
2.894(1.260–6.647)
|
0.012
|
1.848(0.810–4.215)
|
0.144
|
AML1-ETO
|
0.611(0.176–2.123)
|
0.438
|
0.760(0.231–2.507)
|
0.653
|
OS, overall survival; EFS, event-free survival; HR, hazard ratio; CI, confidence interval; allo-HSCT, allogenic hematopoietic stem cell transplantation; WBC, white blood cell. |
Then, we assessed the prognosis effect of the aforementioned factors in the adverse-risk group. The survival study revealed that decreased HGB levels (༜110g/L), FLT3-ITD mutations, and RUNX1 mutations had a negative influence on the OS of ASXL1 + AML patients (P = 0.045, P = 0.047, and P = 0.027, respectively; Additional file 3E-F). These variables had no impact on EFS. Allo-HSCT recipients had a longer OS and EFS (P = 0.024 and P = 0.013, respectively). HGB levels < 110g/L and the FLT3-ITD mutations were found to have an independent predictive influence on poor OS in the multivariate analysis.
Increased number of risk factors may shorten the OS and EFS of ASXL1 + AML patients.
The aforementioned factors that had adverse impact on OS and EFS are defined as high risk factors, including age ≥ 60 years, WBC count ≥ 50 × 109/L, FLT3-ITD mutations, RUNX1 mutations, and the absence of AML1-ETO fusion gene. ASXL1 mutations without any risk factor were referred to as single-hit ASXL1 + AML. ASXL1 mutations with one risk factor was referred to as double-hit ASXL1 + AML. ASXL1 mutations with two or more risk factors were referred to as triple-hit ASXL1 + AML. The combination of these risk factors had a negative influence on the prognosis of ASXL1 + AML (Fig. 2). The median OS was not attained in single-hit ASXL1 + AML, 29.53 months in double-hit ASXL1 + AML, and 6.67 months in triple-hit ASXL1 + AML (P = 0.003, Fig. 2A). The median EFS in single-hit ASXL1 + AML was not attained in single-hit ASXL1 + AML, 29.53 months in double-hit ASXL1 + AML, and 5.47 months in triple-hit ASXL1 + AML (P = 0.003, Fig. 2B).
Allo-HSCT improved the survival of double/triple-hit ASXL1 + AML patients
In our study, 12 patients received allo-HSCT as the consolidation management. Eleven of them carried one or more risk factors in addition to ASXL1 mutations. As shown in Fig. 3, allo-HSCT significantly improved the OS (median 29.53 months vs. 11.33 months, P = 0.008, Fig. 3A) and EFS (median 29.53 months vs. 8.53 months, P = 0.007, Fig. 3B) in double or triple-hit ASXL1 + AML patients.