3.1 Patient Characteristics

Between January 2020 and June 2023, 161 newly diagnosed AML patients received frontline VEN/HMA treatment, among whom 22 patients had AML1/ETO fusion. Baseline characteristics are listed in Table 1. All AML1/ETO-positive AML patients treated with VEN/HMA (Cohort A) were in the ELN 2022 favorable risk group, while 57% of AML1/ETO-negative patients treated with VEN/HMA (Cohort B) were in the ELN 2022 adverse risk group (p < 0.001). Patients in Cohort A had more concomitant KIT mutations (50% vs 3.6%, p < 0.001) and fewer concomitant DNMT3A mutations (0.0% vs 29.5%, p = 0.003) than those in Cohort B. In addition, patients in Cohort A were likely to be younger than those in Cohort B, but the difference was not significant. Other characteristics between the two cohorts were similar.

Table 1 Baseline characteristics of patients

During the same study period, we included 99 patients who had AML1/ETO-positive AML and received frontline IC therapy in Cohort C. Four patients in Cohort C had no information on concomitant gene mutations and were unable to be classified into European ELN 2022 risk groups. Patients in Cohort C were younger (p < 0.001) and had better performance status (p = 0.003) than those in Cohort A. Furthermore, a greater proportion of patients underwent allo-HSCT during follow-up in Cohort C than in Cohort A (38.4% vs 12.6%, p = 0.027). Other characteristics between Cohort A and Cohort C were similar (Table 1).

3.2 Outcomes of Patients Treated with VEN/HMA According to AML1/ETO Fusion Status

When treated with frontline VEN/HMA, patients with AML1/ETO-positive AML had a significantly lower ORR than patients with AML1/ETO-negative AML (40.9% vs 71.2%, p = 0.005), as well as a lower MRD-negative rate (36.4% vs 66.9%, p = 0.006). The 60-day mortality rates were similar in the two cohorts. The ORR of salvage therapy for patients who were primarily resistant to frontline therapy or relapsed after remission was 64.3% in AML1/ETO-positive AML and 44.9% in AML1/ETO-negative AML, with no significant difference (Fig. 1a). Of the 11 patients who were primarily resistant to VEN/HMA, 8 (72.7%) patients responded to the follow-up salvage chemotherapy. The median EFS and OS in AML1/ETO-positive AML patients were 2.7 months and not reached, respectively, which were not significantly different from those in AML1/ETO-negative patients (Fig. 1b, c).

Fig. 1

Outcomes in patients treated with frontline VEN/HMA according to AML1/ETO fusion status. a Response and early death, b EFS, and c OS in all patients. d Response and early death, e EFS, and f OS in patients in the ELN favorable risk group. g Response and early death, h EFS, and i OS in AML1/ETO-positive patients and patients with normal karyotype. AML acute myeloid leukemia, EFS event-free survival, ELN European LeukemiaNet, HMA hypomethylating agent, MRD measurable residual disease, OS overall survival, VEN venetoclax

AML1/ETO-positive AML patients were all in the ELN 2022 favorable risk group, while most AML1/ETO-negative AML patients were in the ELN 2022 adverse risk group, which may lead to bias in survival. Thus, we further analyzed the treatment outcomes of patients in the ELN 2022 favorable risk group, including 22 patients with AML1/ETO-positive AML and 34 patients with AML1/ETO-negative AML. The ORR was 97.1% and the MRD-negative rate was 91.2% in AML1/ETO-negative AML patients with an ELN favorable risk, which were both much higher than those in AML1/ETO-positive AML patients (Fig. 1d). The median EFS in AML1/ETO-positive AML patients with ELN favorable risk was significantly shorter than that in AML1/ETO-negative AML patients (2.7 months vs 14 months, p = 0.003, Fig. 1e). The median OS in AML1/ETO-negative patients with an ELN favorable risk was 20.5 months, with no significant difference compared with that in AML1/ETO-positive patients (Fig. 1f).

We also compared the 22 AML1/ETO-positive patients with the normal karyotype subgroup of the 139 AML1/ETO-negative patients receiving VEN/HMA. The ORR was 73.8% and the MRD-negative rate was 68.2% in AML1/ETO-negative AML patients with normal karyotype, which were much higher than those in AML1/ETO-positive AML patients (Fig. 1g). No significant differences were found in EFS and OS between the two groups (Fig 1h, i).

3.3 Outcomes of Patients with AML1/ETO-Positive AML According to Frontline Treatment Strategies

We analyzed the response and survival of AML1/ETO-positive patients treated with frontline VEN/HMA (n = 22) or frontline IC (n = 99). The ORR and MRD-negative rate in AML1/ETO-positive AML patients treated with frontline IC were 80.8% and 78.8%, respectively, which were much higher than those in AML1/ETO-positive patients treated with frontline VEN/HMA (p < 0.001; Fig. 2a). The 60-day mortality and ORR of salvage therapy were similar in the two cohorts (Fig. 2a). The median EFS in patients treated with VEN/HMA was significantly shorter than that in patients treated with IC (2.7 months vs 14.9 months, p = 0.003; Fig. 2b). The median OS in both cohorts was not reached, with no significant difference (p = 0.070, Fig. 2c).

Fig. 2

Outcomes in patients with AML1/ETO-positive AML according to frontline treatment strategies. a Response and early death, b EFS and c OS in all patients. d Response and early death, e EFS, and f OS in patients after propensity score matching for age, Eastern Cooperative Oncology Group performance status and follow-up allogeneic hematopoietic stem cell transplantation. AML acute myeloid leukemia, EFS event-free survival, HMA hypomethylating agent, IC intensive chemotherapy, MRD measurable residual disease, OS overall survival, VEN venetoclax

However, there were significant differences in the baseline characteristics, such as age, performance status, and follow-up allo-HSCT, between patients treated with VEN/HMA and patients treated with IC. Thus, we analyzed the outcomes of patients after propensity matching for age, ECOG PS and follow-up HSCT. After PSM, 40 patients, including 20 patients with frontline VEN/HM treatment, were matched by a 1:1 matching ratio, and all the baseline characteristics were similar between the two matched cohorts (Table 2). The response and EFS in the propensity-matched VEN/HMA cohort were significantly inferior to those in the propensity-matched IC cohort (ORR 40.0% vs 90.0%, p = 0.001; MRD-negative rate 35.5% vs 85.0%, p = 0.001; median EFS 2.4 months vs 14.0 months, p = 0.029; Fig. 2d and e). No significant OS differences were found between the two propensity-matched cohorts (Fig. 2f).

Table 2 Baseline characteristics of patients with AML1/ETO-positive AML after propensity score matching3.4 Subgroup Analysis Stratified by KIT Mutation Status

KIT mutations have been reported to be associated with a poor prognosis in core binding factor (CBF)-AML patients receiving IC [15,16,17,18]. We therefore performed analyses stratified by KIT mutation status. We first analyzed the impact of KIT mutations on the outcomes of patients with AML1/ETO-positive AML receiving VEN/HMA. The ORR and EFS in KIT-mutated patients with AML1/ETO-positive AML receiving VEN/HMA were much inferior to those in KIT wild-type patients (ORR 0.0% vs 81.8%, p = 0.001; EFS 1.2 months vs not reached, p < 0.001, Table 3, Fig. 3a). OS between the two cohorts was not significantly different (Fig. 3b).

Table 3 Overall response rates for patientsFig. 3

a EFS and b OS in AML1/ETO-positive patients treated with frontline VEN/HMA according to KIT mutation status. AML acute myeloid leukemia, EFS event-free survival, HMA hypomethylating agent, OS overall survival, VEN venetoclax

We then conducted subgroup analysis by KIT mutation status. In KIT wild-type patients treated with frontline VEN/HMA, no significant differences in ORR and EFS were found between patients with AML1/ETO-positive AML and AML1/ETO-negative AML (ORR 81.8% vs 70.8%, p = 0.708; median EFS not reached vs 7.1 months, p = 0.190; Table 3, Fig. 4a). In KIT-mutated patients treated with frontline VEN/HMA, the ORR and EFS for patients with AML1/ETO-positive AML were significantly worse than those for AML1/ETO-negative AML (ORR 0.0% vs 60.0%, p = 0.018; median EFS 1.2 months vs 10.0 months, p = 0.011; Table 3, Fig. 4c). In AML1/ETO-positive AML without KIT mutations, no significant differences in ORR and EFS were found between patients treated with VEN/HMA and IC (Table 3, Fig. 4e). In AML1/ETO-positive AML with concomitant KIT mutations, the ORR and EFS for patients treated with VEN/HMA were significantly worse than those for patients treated with IC (ORR 0.0% vs 78.7%, p < 0.001; median EFS 1.2 months vs 12.1 months, p < 0.001; Table 3, Fig. 4g). The OS between cohorts was not significantly different (Fig. 4b, d, f, h).

Fig. 4

Outcomes in patients stratified by KIT mutation. a EFS and b OS in patients treated with frontline VEN/HMA in the KIT wild-type group according to AML1/ETO fusion status. c EFS and d OS in patients treated with frontline VEN/HMA in the KIT-mutated group according to AML1/ETO fusion status. e EFS and f OS in patients with AML1/ETO-positive AML in the KIT wild-type group according to frontline treatment strategies. g EFS and h OS in patients with AML1/ETO-positive AML in the KIT-mutated group according to frontline treatment strategies. AML acute myeloid leukemia, EFS event-free survival, HMA hypomethylating agent, IC intensive chemotherapy, OS overall survival, VEN venetoclax

3.5 Different Gene Expressing Analysis, Pathway Analysis and SNV Analysis of Patients with AML in Datasets

VEN/HMA did not show a satisfactory efficacy for patients with AML1/ETO-positive AML. We analyzed open-source RNA-seq datasets and SNV datasets to explore the reason for poor results in these patients. The differential expressing genes analysis showed that AML1/ETO-positive patients expressed significantly lower BCL2 than AML1/ETO-negative patients (Fig. 5a), which indicated low dependency on BCL2 of AML1/ETO-positive patients. Meanwhile, AML1/ETO-positive patients expressed higher CD34/CD117 and lower CD33/CD11b, which indicated less differentiation (Fig. 5a). Gene set enrichment analysis shows the down-regulation of the mitochondrion morphogenesis pathway in the BEATAML data set (Fig. 5b).

Fig. 5

Different gene expressing analysis, pathway analysis, and SNV analysis of AML patients in datasets. a BCL2 expression between AML patients with and without AML1/ETO fusion. Expression was in logarithm. p-Value was calculated by DESeq2. b Pathway enrichment result. X axis value presented the quantitative results of pathway enrichment. Down-regulated pathways were at the left side of zero. c Gene mutations and d mutation abundance of AML patients with and without AML1/ETO fusion. AML acute myeloid leukemia, GSEA gene set enrichment analysis, SNV simple nucleotide variation

Concomitant gene mutations in patients with and without AML1/ETO fusion are shown in Fig. 5c. Unfortunately, the differences in gene mutations between AML1/ETO-positive patients and AML1/ETO-negative patients were not significant, which may be the result of a small sample size of AML1/ETO-positive patients. We paid special attention to the mutations in genes involved in DNA methylation such as DNMT3A and TET2. The analysis showed that although lacking statistical significance, relatively fewer patients with AML1/ETO fusion had DNMT3A mutations compared with patients without AML1/ETO fusion (8.3% vs 17.9%, p = 0.287). The probabilities of TET2 mutations were similar in AML1/ETO-positive patients and AML1/ETO-negative patients (16.7% vs 12.3%, p = 0.527, Fig. 5d).