
==== Front
Discov Oncol
Discov Oncol
Discover Oncology
2730-6011
Springer US New York

39287751
1348
10.1007/s12672-024-01348-8
Research
Clinical efficacy and immune response of BCL-2 inhibitors combined with hypomethylating agents in the treatment of acute myeloid leukemia
Peng Xiaohuan 12
Yu Jianing 1
Tang Futian 3
Li Yanhong 2
Bai Jun 2
Li Lijuan doctorjuan@sina.com

12
Zhang Liansheng doctorzhanglsh@sina.com

12
1 https://ror.org/01mkqqe32 grid.32566.34 0000 0000 8571 0482 Department of Hematology, The Second Hospital & Clinical Medical School, Lanzhou University, Lanzhou, China
2 https://ror.org/01mkqqe32 grid.32566.34 0000 0000 8571 0482 Key Laboratory of the Hematology of Gansu Province, The Second Hospital & Clinical Medical School, Lanzhou University, Lanzhou, China
3 https://ror.org/01mkqqe32 grid.32566.34 0000 0000 8571 0482 Key Laboratory of the Digestive Tumor of Gansu Province, The Second Hospital & Clinical Medical School, Lanzhou University, Lanzhou, China
17 9 2024
17 9 2024
12 2024
15 45128 7 2024
13 9 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Objective

Acute myeloid leukemia (AML) is a malignant clonal proliferative disease with a high mortality rate. The combination therapy of BCL-2 inhibitor Venetoclax (VEN) and hypomethylating agents (HMAs) has significant anti-leukemia activity.

Methods

We analyzed the efficacy, safety and immune response characteristics of AML patients who were unfit for high-dose chemotherapy and accepted the medication of VEN + HMAs.

Results

After VEN + HMAs treatment, 31 newly diagnosed AML patients had the morphologic leukemia-free state rate (MLFS%) of 80.6% (25/31), complete response rate (CR%) of 54.8% (17/31), the minimal residual disease negative rate (MRD-%) of 51.6% (16/31), and the median progression-free survival (PFS) of 14 months. After treatment, the proportion of bone marrow primitive cells, the MRD level, white blood cell (WBC) count, fibrinogen (FIB) level and the proportion of B cells were significantly decreased. The red blood cell (RBC) count, hemoglobin (HGB) level, platelet count (PLT) count, activated partial thromboplastin time (APTT), the proportion of total T cells, CD8 + T cells and the IFN-γ level were significantly increased. After VEN + HMAs treatment, 12 relapsed AML patients had a MLFS% of 50% (6/12), CR% of 33.3% (4/12), MRD-% of 25% (3/12), and a median PFS of 7 months. After treatment, the proportion of bone marrow primitive cells and MRD level were slightly decreased, the proportions of CD8 + T cells and NK cells were significantly increased, the proportion of B cells and IL-10 level were significantly decreased. 12 AML patients who receive microtransplantation (MST) treatment using VEN + HMAs as a pretreatment regimen had a PFS of 20.5 months, which was much greater than VEN + HMAs group alone. Hematological recovery was better in the MST group with significantly increased RBC count, HGB level and PLT count. The most common adverse events were myelosuppression, agranulocytosis, infection and cardiovascular toxicity. No fatal adverse events were reported.

Conclusion

The combination of BCL-2 inhibitors and HMAs had good efficacy and safety in AML patients who were unfit for high-dose chemotherapy, which may improve the immune microenvironment and enhance anti-leukemia immune response.

Keywords

Acute myeloid leukemia
Clinical efficacy
Immune response
BCL-2 inhibitors
Hypomethylating agents
Fundamental Research Funds for the Central Universities of Lanzhou Universitylzujbky-2022-sp08 Tang Futian Medical Research Improvement Project of Lanzhou Universitylzuyxcx-2022-154 Tang Futian Medical Innovation and Development Project of Lanzhou Universitylzuyxcx-2022-141 Tang Futian Clinical Medical Research Center of Hematological Diseases in Gansu Province21JR7RA435 Li Yanhong Natural Science Foundation of Gansu Province21JR11RA104 Li Yanhong National Natural Science Foundation of China82360029 Zhang Liansheng issue-copyright-statement© Springer Science+Business Media, LLC 2024
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pmcBackground

Acute myeloid leukemia (AML) is a malignant clonal proliferative disease with a high mortality rate and poor prognosis. Approximately 70% of AML patients aged 65 years and older die within the first year of diagnosis [1]. For elderly AML patients, low-intensity treatment such as hypomethylating agents (HMAs) or low-dose cytarabine (Ara-C) have good tolerability but poor treatment responsiveness [2, 3]. In recent years, various targeted drugs have emerged including the highly selective BCL-2 inhibitors Venetoclax (VEN), and VEN + HMAs treatment has significant anti-leukemia activity. A clinical study published by Maiti A et al. revealed that VEN + HMAs treatment provided better results than intensified chemotherapy in newly diagnosed elderly AML patients, having a significantly greater complete response rate (CR%), a lower relapsed rate and a longer overall survival [4]. The Food and Drug Administration (FDA) has approved VEN in combination with HMAs or low-dose Ara-C for older adults with newly diagnosed AML patients who cannot tolerate intensified therapy [5].

AML patients have various T cell dysfunctions such as exhaustion and senescence, and AML blasts can directly alter the viability, expansion and senescence of CD8 + T-cell [6]. During the treatment process, chemotherapy or molecular targeted therapy may further affect the activity, proliferation and phenotypic characteristics of immune cells [7]. Recent studies have confirmed that HMAs can regulate T-cell function and T-cell-mediated anti-tumor immune responses [8]. HMAs can enhance the immunogenicity of tumor cells, making them be more easily recognized and killed by immune cells [9]. Low-dose Decitabine (DAC) can enhance anti-tumor response by promoting T-cell proliferation, and IFN-γ + T cells may be a potential prognostic biomarker [10].

BCL-2 inhibitors have achieved good effects in various hematological tumors, but little is known about how these compounds affect immune cells. Research has shown that VEN can enhance T-cell-mediated anti-tumor activity by increasing ROS production [11]. A team from the University of Chicago also reported that VEN imparts distinct cell death sensitivity and adaptivity patterns in T cells [12]. The above studies indicate that short-term or long-term BCL-2 inhibitors treatment can reshape the immune system and regulate the anti-tumor immune response.

However, it is not clear whether VEN + HMAs have direct impact on the count, function, and anti-tumor response of immune cells. Our study analyzed the clinical data and detected changes in lymphocyte subsets and common cytokines in peripheral blood of 55 AML patients who accepted VEN + HMAs treatment to evaluate the efficacy and safety of VEN + HMAs, as well as their impact on the immune microenvironment.

Methods

Patients

This study screened 55 AML patients who received VEN + HMAs treatment at Lanzhou University Second Hospital. The diagnostic criteria for AML refers to the 5th edition of the WHO Calssification of Haematolymphoid Tumors. A necessary condition is that the primitive cell count of peripheral blood or bone marrow is greater than or equal to 20%. The inclusion criteria included (1) a clear diagnosis of non-M3 AML; (2) AML patients who were unsuitability or unwillingness to receive intensive treatment; (3) AML patients who were suitability and willing to receive VEN + HMAs treatment. The exclusion criteria included (1) combined with important organ dysfunction; (2) individuals with immunodeficiency or human immunodeficiency virus positivity; (3) merged with other malignant tumors; (4) individuals who are allergic to the VEN or HMAs; (5) individuals with incomplete clinical information..AML patients were divided into three groups including newly diagnosed AML patient group, relapsed AML patient after previous treatment (excluding the VEN + HMAs regimen) group, and microtransplantation (MST) treatment group using VEN + HMAs as a pretreatment regimen. Our research was in accordance with the ethical standards formulated in the Helsinki Declaration and was approved by the institutional review board of Lanzhou University Second Hospital. Informed consent was obtained from all the participants involved in the study.

Treatment

The patient received a dose escalation of VEN in the first treatment course. VEN was administered 100 mg in day 1, 200 mg in day 2 and 400 mg in day 3 continued to day 28. Starting from the second course, VEN was administered 400 mg daily (d1-d28). The usage and dosage of HMAs refer to the recommendations of the Chinese AML guidelines (2021 version). Azacitidine (AZA) (75 mg/m2/d on days1-7) or DAC (20 mg/m2/d on days1-5) was administered. The specific usage and dosage are shown in Fig. 1A–D. Before the new treatment course, morphology of bone marrow cells, minimal residual disease (MRD) level, routine blood tests and coagulation tests were tested to evaluate the efficacy.Fig. 1 The specific usage and dosage of VEN + HMAs regimen in newly-diagnosed and relapse groups: VEN was administered 100 mg in day 1, 200 mg in day 2 and 400 mg in day 3 continued to day 28 and AZA (75 mg/m2/d on days1-7, ih) or DAC (20 mg/m2/d on days1-5, ivgtt) was administered in first course (A). VEN (100 mg/d on days1-28,oral) was administered and AZA (75 mg/m2/d on days1-7, ih) or DAC (20 mg/m2/d on days1-5, ivgtt) was administered in subsequent courses (B). The specific usage and dosage of VEN + HMAs regimen in MST group: VEN was administered 100 mg in day 1, 200 mg in day 2 and 400 mg in day 3 continued to day 28, AZA (75 mg/m2/d on days1-7, ih) or DAC (20 mg/m2/d on days1-3, ivgtt) was administered and G-PBMC(MNC 2.5X108/Kg or CD3 + T cells on day 7, ivgtt) was administered in first course (C). VEN (100 mg/d on days1-28,oral) was administered, AZA (75 mg/m2/d on days1-7, ih) or DAC (20 mg/m2/d on days1-3, ivgtt) was administered and G-PBMC(MNC 2.5X108/Kg or CD3 + T cells on day 7, ivgtt) was administered in subsequent courses (D). ih hypodermic injection, ivgtt: intravenously guttae, G-PBSC: peripheral blood stem cells mobilized by granulocyte colony-stimulating factor, MNC: mononuclear cell

Main outcome measures

We have collected the following data from the Electronic Medical Record System: the all results of bone marrow examination including bone marrow cell morphology, immunophenotypes, MRD level, cytogenetic analysis and molecular biological detection; the results of peripheral blood examination including routine blood tests and coagulation tests. We have conducted the following laboratory tests including lymphocyte subset and twelve common cytokines in peripheral blood. The lymphocyte subset were defined as the proportions of total T lymphocytes (CD3 + CD19-), CD4 + T cells (CD3 + CD4 +), CD8 + T cells (CD3 + CD8 +), natural killer (NK) cells (CD16 + CD56 +), B lymphocytes (CD3-CD19 +) in the lymphocyte population. Regulatory T cells were defined as the proportion of CD4 + CD25 + FOXP3 + T cell in CD4 + T cell population. The twelve cytokines included IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, TNF-α, IFN-γ, IFN-α, and IL-17A. At last, we collected the baseline clinical characteristics of patients including sex, age, FAB classification and World Health Organization (WHO) risk classification. All patients were followed up to monitor their treatment response and safety.

Flow cytometry

We collected fasting peripheral blood 2 mL from AML patients and used flow cytometry to test lymphocyte subsets.We used three 12 × 75 mm flow tubes named as A, B and C, pipetted 20 μL of CD45/CD3/CD4/CD8(BD Bioscience, USA, 662,965) multitest antibodies into the bottom of tube A, pipetted 20 μL of CD45/CD3/CD16 + CD56/CD19 (BD Bioscience, USA, 662,965) multitest antibodies into the bottom of tube B and pipetted CD4/CD25/FOXP3 (BD Bioscience, USA, 560,131) multitest antibodies into the bottom of tube C. Then we added 100 μL blood specimen (EDTA-K2 anticoagulated) separately into the bottom of tubes A,B and C, shaked gently, incubated at room temperature in the dark for 15 min. Next, we added 450 μL 1X hemolysin (BD Bioscience, USA, 349,202) to tubes A, B and C, mixed gently by shaking, and then incubated at room temperature in the dark for 15 min. Finally, we centrifuged all tube at a speed of 1500r/min for 5 min, then discarded the supernatant, added 300uL phosphate buffered saline (PBS, ThermoFisher Scientific, China, 70,011,044) into the bottom of three tubes, mixed gently by shaking and tested by FACS Canto flow cytometer (BD Bioscience, USA).

Cytometric bead array

We collected fasting peripheral blood 2 mL(EDTA-K2 anticoagulated) from the AML patients, and obtained the upper layer of serum through centrifugation at a speed of 4000r/min for 10 min. Firstly, We prepared standard samples with different concentrations and microsphere suspension (Hangzhou Sage Biotechnology Co, Hangzhou, China, P010100403) according to the instructions of Human 12 Cytokines Detection Kit. Next, we vortexed microsphere suspension to ensure complete mixing, and incubated at room temperature in the dark for 15 min. We transfered 25 μL of microsphere suspension and 25 μL samples to each sample tubes, including standard samples tubes and tests sample tubes. Then we added 25 μL of fluorescence detection reagent (Hangzhou Sage Biotechnology Co, Hangzhou, China, P010100403) to all experimental tubes, mixed gently by shaking and incubated them for 2.5 h at room temperature in the dark. After 2.5 h, we added 1 mL PBS to each tube, centrifuged at a speed of 200 g/min for 5 min and discarded the supernatant after centrifugation. At last, we added 300 μL PBS into each tube, mixed thoroughly and tested by FACS Canto flow cytometer (BD Bioscience, USA).

Response criteria and evaluation

The follow-up methods included hospitalization examination, outpatient follow-up and telephone follow-up. The efficacy evaluation criteria refer to the 2022 ELN AML guideline including morphologic leukemia-free state (MLFS), CR and MRD negative (MRD-). The primary efficacy endpoint was progression-free survival (PFS). Safety assessments were conducted during each treatment course.

Statistical analysis

The GraphPad Prism 9.0 and SPSS 26.0 software were used to analyze and plot date. We analyzed baseline data by using t test and χ2 test. Paired sample t-test was used for comparisons the changes before and after treatment in the same group, independent sample t-test was used for comparisons between two groups, and analysis of variance (ANOVA) was used for comparisons among three or more groups. We compared the survival differences between different groups by plotting Kaplan–Meier curves.

Results

Patient characteristics

There were 29 females and 26 males in this study, with a median age of 59 (28–82). As we all know that the young patients could benefit more from intensive chemotherapy than VEN + HMAs. The 3 young patients in this study were these who had accepted intensive chemotherapy but multiple relapses, and automatically request to VEN + HMAs treatment. The other 3 young patients in this study were these who used VEN + HMAs treatment as a pretreatment regimen of MST treatment. The newly diagnosed group included 16 females and 15 males, with a median age of 62 (47–82). These patients don't consider intensive chemotherapy because of older age and poor physical fitness. The relapse group included 5 females and 7 males, with a median age of 56 (28–70). The MST group included 8 females and 4 males, with a median age of 51 (31–60). The patients’ characteristics are summarized in Table 1.Table 1 Patient characteristics

Characteristics	Newly diagnosed AML group	Relapse AML
group	MST AML
group	P	
Median age(range)	62(41,82)	56(28,70)	51(31,60)	0.007	
Sex: Male/n (%)	15(48.4%)	7(58.3%)	4(33.3%)	0.463	
FAB classification				0.420	
M0	1(3.2%)	0	0		
M1	1(3.2%)	0	0		
M2	11(35.5%)	4(33.3%)	2(16.7%)		
M4	4(12.9%)	2(16.7%)	5(41.7%)		
M5	7(22.6%)	3(25.0%)	5(41.7%)		
Other	7(22.6%)	3(25.0%)	0		
Prognosis stratification				0.106	
Good, n (%)	1(3.2%)	0	1(8.3%)		
Moderate, n (%)	10(32.3%)	3(25.0%)	8(66.7%)		
Poor, n (%)	20(64.5%)	9(75.0%)	3(25.0%)		
Gene mutation, n (%)	22(71.0%)	11(91.7%)	11(91.7%)	0.163	
Complex karyotype, n (%)	5(16.1%)	1(8.3%)	11(8.3%)	0.691	

Response to treatment

After VEN + HMAs treatment, newly diagnosed patients had a MLFS rate (MLFS%) of 80.6% (25/31), CR% of 54.8% (17/31) and an MRD- rate (MRD-%) of 51.6% (16/31). The proportion of bone marrow primitive cells and the MRD level were significantly decreased (P < 0.0001). The VEN + HMAs treatment effectively reduced the tumor burden in newly diagnosed AML patients (Fig. 2A). After treatment, the white blood cell (WBC) count decreased (P = 0.022), the neutrophil percentage (NE%) increased (P < 0.0001), the monocyte percentage (MO%) decreased (P = 0.001), the red blood cell (RBC) count increased (P = 0.007), the hemoglobin (HGB) level increased (P = 0.008), and the platelet count (PLT) increased (P = 0.005). All of these differences had statistical differences. The VEN + HMAs treatment effectively promoted the recovery of hematopoietic function in newly diagnosed AML patients (Fig. 2B). In addition, this study found that activated partial thromboplastin time (APTT) was significantly increased (P = 0.014), fibrinogen (FIB) level was significantly decreased (P = 0.046), and Dimmer level was slightly decreased (P = 0.163) in newly diagnosed AML patients after VEN + HMAs treatment (Fig. 2C). This finding suggested that VEN + HMAs treatment may affect endogenous coagulation system function. The lower Dimmer levels means that VEN + HMAs treatment may improve the hypercoagulable state of AML patients. The significantly increased APTT and significantly decreased FIB level mean that the risk of bleeding may increase after VEN + HMAs treatment. Therefore, we should monitor changes of APTT and FIB during the treatment process, and if necessary perform APTT mixing study to further clarify the diagnosis. In terms of treatment, clinical physicians should give supplementary coagulation factor therapy to patients such as vitamin K1 injection and fresh frozen plasma infusion according to APTT changes to prevent bleeding events.Fig. 2 The clinical and immune response changes in 31 newly diagnosed AML patients after VEN + HMAs treatment. The proportion of bone marrow primitive cells and the MRD level were significantly decreased (A). The WBC count and MO% were significantly decreased, the NE%, RBC count, HGB level, PLT count were significantly increased (B). The APTT was significantly increased, FIB expression was significantly decreased (C). As for immune indices, the proportions of total T cells and CD8 + T cells were significantly increased, the proportions of CD4 + T cells (P = 0.175) and NK cells (P = 0.058) were slightly increased, the proportions of Treg cells was not change, the proportions of B cells was significantly decreased, IFN-γ expression was significantly increased, TNF-α expression was slightly decreased (P = 0.169) (D). *–p < 0.05; **–p < 0.01; ***–p < 0.001; ****–p < 0.0001

After VEN + HMAs treatment, relapsed patients had a MLFS% of 50% (6/12), CR% of 33.3% (4/12) and an MRD-% of 25% (3/12). The proportions of bone marrow primitive cells (P = 0.17) and MRD level (P = 0.09) decreased (Fig. 3A), but the difference was not statistically significant. There was no significant change of WBC count, RBC count,PLT count and coagulation indicators (Fig. 3B,C). The above results indicated that the VEN + HMAs treatment also had a therapeutic effect on relapsed patients, but the effect was worse compared to newly diagnosed patients.Fig. 3 The clinical and immune response changes in 12 relapsed AML patients after VEN + HMAs treatment. The proportion of bone marrow primitive cells (P = 0.17) and MRD level (P = 0.09) were slightly decreased (A). There were no significant changes in WBC count, NE%, MO%, RBC count, HGB level and PLT count (B). There was no significant change of coagulation indicators such as APTT and FIB level (C). As for immune indices, the proportions of total T cells was slightly increased (P = 0.120), the proportions of CD8 + T cells and NK cells were significantly increased, the proportions of B cells was significantly decreased, the proportions of CD4 + T cells and Treg cells were no significant change, the level of IL-8 was slightly decreased (P = 0.054) and IL-10 was significantly decreased, the level of IFN-γ was also increased but the difference was not statistically significant (D).ns–p > 0.05, *–p < 0.05; **–p < 0.01

This study stratified newly diagnosed and relapsed AML patients into two groups based on prognosis. The relatively low-risk group included patients with good and moderate prognoses, while the relatively high-risk group included patients with poor prognoses. We compared the treatment response of relatively low-risk group and relatively high-risk group patients to VEN + HMAs. The results showed that the MLFS% of the relatively low-risk group was 78.6% (11/14), CR% was 57.1% (8/14), MRD-% was 64.3% (9/14). The MLFS% of the relatively high-risk group was 65.5% (19/29), CR% was 44.8% (13/29), MRD-% was 34.5% (10/29). In both the relatively low-risk group and the relatively high-risk group, the VEN + HMAs treatment significantly reduced the proportion of bone marrow primitive cells and the MRD level (Fig. 4A). After treatment, a significant increase in NE%, PLT count and a significant decrease in MO% were observed in both two groups. A significant increase of RBC count and HGB level were observed in the relatively low-risk group. These results suggested that the VEN + HMAs treatment can promote the recovery of hematopoietic function in both the relatively low-risk and high-risk groups, but relatively low-risk group was better (Fig. 4B). After treatment, APTT was significantly prolonged in both two groups, and FIB level was significantly decreased in relatively low-risk group (Fig. 4C). The proportion of primitive cells in the relatively high-risk group was higher than relatively low-risk group after treatment (Fig. 4A), and the RBC, HGB, and PLT counts in the relatively high-risk group were less than relatively low-risk group (Fig. 4B), the difference was not statistically significant. The above studies suggested that the VEN + HMAs treatment has therapeutic effect on both two groups, and relatively low-risk patients were better.Fig. 4 The clinical and immune response changes of AML patients with different risk stratifications after VEN + HMAs treatment. The proportion of bone marrow primitive cells and MRD level were significantly decreased in both relatively low-risk group and the relatively high-risk group after VEN + HMAs treatment, but there was no significant difference between relatively low-risk group and the relatively high-risk group (A). The WBC counts were decreased especially the relatively high-risk group (P = 0.0623), NE% and PLT count were significantly increased and MO% was significantly decreased in both two groups, and RBC count and HGB level were significantly increased in relatively low-risk group (B). APTT was significantly prolonged in both two groups, and FIB level was significantly decreased in relatively low-risk group (C). As for immune indices, there were no significant changes in T cells, CD4 + T cells and CD8 + T cells in the relatively low-risk group. The proportion of NK cells was significantly increased while the proportion of B cells was significantly decreased in the relatively low-risk group. The proportion of T cells, CD8 + T cells were significantly increased and the proportion of NK cells was slightly increased (P = 0.0834), the proportion of B cells was significantly decreased in the relatively high-risk group. In terms of cytokines, IL-8 level was significantly decreased and IL-10 level was slightly decreased (P = 0.0878) in relatively high-risk groups, IFN-γ level was increased while TNF-α level was decreased in both two groups, but the difference was not statistically significant. In addition, compared to the relatively low-risk group, the relatively high-risk group had lower levels of IL-8 and IL-10 after treatment, and the difference had statistically significant. No significant changes were observed in other indicators (D). ns–p > 0.05, *–p < 0.05; **–p < 0.01; ***–p < 0.001. L: relatively low-risk group, H: relatively high-risk group

The AML patients in MST group showed no significant changes in the proportion of bone marrow primitive cells and MRD level after VEN + HMAs treatment, as for they were in a CR state before treatment. The patient's hematopoietic function further recovered after treatment, especially erythroids. The WBC count and PLT count increased to various degrees, and RBC count and HGB level were significantly increased (P = 0.042 for RBC and P = 0.031 for HGB) (Fig. 5). Moreover, APTT was significantly prolonged (P = 0.046) (Fig. 5). The above results all suggested that VEN + HMAs as a pretreatment regimen for MST can further improve the hematopoietic function of AML patients. However, clinical doctors may need to monitor changes in coagulation function.Fig. 5 The clinical and immune response changes of AML patients who accepted MST treatment with VEN + HMAs as a pretreatment regimen. The RBC count, HGB level and APTT were significantly increased, and there was no significant change in immune indices after a single course of VEN + HMAs treatment. ns–p > 0.05, *–p < 0.05

This study also analyzed the differences between the MST group and the non-MST group of AML patients after VEN + HMAs treatment. The MST group had a lower proportion of bone marrow primitive cells (P = 0.169) and MRD level (P = 0.0518) after treatment (Fig. 6A). The WBC count (P = 0.007), NE% (P = 0.033), RBC count (P < 0.0001), HGB level (P < 0.0001) and PLT count (P = 0.002) were significantly increased in MST group (Fig. 6B). Combination treatment of MST and VEN + HMAs can further promote the recovery of hematopoietic function. For coagulation function, we found that the Dimmer level in MST group was significantly lower (P = 0.035) compared to non-MST group (Fig. 6C), indicating that MST treatment may can reduce the risk of thrombosis.Fig. 6 Clinical and immune response differences between the MST group and non-MST group after VEN + HMAs treatment. Compared to non-MST group, the MST group had a lower proportion of bone marrow primitive cells (P = 0.169) and MRD levels (P = 0.0518) (A). Compared to non-MST group, the MST group had a higher WBC count, NE%, RBC count, HGB level and PLT count, the difference had statistically significant (B). Compared to non-MST group, the MST group had a lower Dimmer level, the difference had statistically significant (C). There were no significant differences in immune indices between two groups (D). ns–p > 0.05, *–p < 0.05; **–p < 0.01; ****–p < 0.001

Immune function changes

T cells especially CD8 + T cells are the main immune cells in killing tumor cells. After VEN + HMAs treatment, the proportions of T cells (P = 0.026), CD4 + T cells (P = 0.175), and CD8 + T cells (P = 0.041) tended to increase in newly diagnosed AML patients (Fig. 2D), indicating that VEN + HMAs treatment may enhance the anti-leukemia immune effect mediated by T cells in vivo. NK cells also play a key role in the anti-tumor immune responses. After VEN + HMAs treatment, the proportion of NK cells in newly diagnosed AML patients was also increased (P = 0.058) (Fig. 2D). Recent studies have shown that B cells may promote tumor progression, possibly through regulatory B (Breg) cells, which are immunosuppressive cells. Breg cells secrete IL-10 and TGF-β to inhibit anti-tumor immunity in the body. This study found that after VEN + HMAs treatment, the proportion of B cells was significantly decreased in newly diagnosed AML patients (P < 0.0001) (Fig. 2D). As for cytokines, this study found that IFN-γ expression was significantly increased (P = 0.043), TNF-α expression was slightly decreased (P = 0.169) after VEN + HMAs treatment (Fig. 2D). Other cytokines changes were various, but the differences were not statistically significant. The above results suggested that VEN + HMAs treatment may improve the immunosuppressive tumor microenvironment of newly diagnosed AML patients and enhance the body's anti-leukemia immune response. Further research such as testing absolute number of immune cells and mechanistic exploration are needed.

After VEN + HMA treatment, the proportions of T cells (P = 0.120) and CD8 + T cells (P = 0.015) tended to increase in relapsed AML patients (Fig. 3D).. The proportion of NK cells was significantly increased (P = 0.003), and the proportion of B cells was significantly decreased (P = 0.046). The level of cytokines such as IL-8 (P = 0.054) and IL-10 (P = 0.015) were decreased after VEN + HMAs treatment (Fig. 3D). The level of IFN-γ also increased, but the difference was not statistically significant (Fig. 3D). Similar results were obtained in relapsed patients and newly diagnosed patients, further confirming that the VEN + HMAs treatment may improve the tumor immune microenvironment in AML patients. For the MST patients, we found that there was no significant change in immune indices after a single course of VEN + HMAs treatment (Fig. 5). We also found that there was no significant change in immune indices between MST group and non-MST group (Fig. 6D).

Immune microenvironment changes were different among AML patients with different risk stratifications after VEN + HMAs treatment. There were no significant changes in T cells, CD4 + T cells and CD8 + T cells in the relatively low-risk group (Fig. 4D). The proportion of NK cells was significantly increased (P = 0.0012), while the proportion of B cells was significantly decreased (P = 0.0022) in the relatively low-risk group (Fig. 4D). The immune function of the relatively high-risk AML patients showed more significant changes after VEN + HMAs treatment. The proportion of T cells (P = 0.0218) and CD8 + T cells (P < 0.001) were significantly increased, the proportion of NK cells slightly increased (P = 0.0834) and the proportion of B cells (P < 0.0001) was significantly decreased (Fig. 4D). As for cytokines, we observed that IFN-γ level increased while TNF-α level decreased in both the relatively low-risk and high-risk groups, but the difference was not statistically significant (Fig. 4D). IL-8 level was significantly decreased (P = 0.0351) and IL-10 level was slightly decreased (P = 0.0878) in relatively high-risk groups. After treatment, the levels of IL-8 and IL-10 were significantly lower in the relatively high-risk group than relatively low-risk group. The above results suggested that the VEN + HMAs treatment has a greater impact on the immune microenvironment of relatively high-risk AML patients and may improve the immunosuppressive tumor microenvironment.

Survival

The median PFS of newly diagnosed AML group was 14 months, the median PFS of relapsed AML group was 7 months, and the median PFS of the MST group was 20.5 months (Fig. 7A), the difference had statistically significant. It can serve as an induction treatment for newly diagnosed elderly AML patients, and increasing MST therapy after CR is more effective. This study found that some AML patients may abandon treatment or use other drugs in the midway due to economic factors, which may be one of the reasons for the low PFS. Therefore, this study also assessed the relationship between the number of courses of VEN + HMAs treatment and the final PFS. Patients who received less than or equal to 3 courses had a median PFS of only 9 months, while those who received 6 or more courses had a median PFS of up to 22 months (Fig. 7B), and the difference had statistically significant.Fig. 7 The survival curve comparison of newly diagnosed, relapsed and MST groups. The MST group had a longest PFS, followed by the newly diagnosed group, and the relapsed group had the shortest PFS, and the difference had statistically significant (A). The survival curve comparison of newly diagnosed and relapsed patients with less than or equal to 3 courses and 6 or more courses. Patients who received less than or equal to 3 courses had a shorter PFS, while those who received 6 or more courses had a longer PFS, and the difference had statistically significant (B)

Safety

This study found that all AML patients treated by VEN + HMAs had varying degrees of bone marrow suppression during treatment process, and 12.7% (7/55) of the patients experienced severe neutropenia. However, bone marrow hematopoietic function can gradually recover. The other most common adverse event was 10.9% infection (6/55), including 4 cases of lung infection and 2 cases of perianal infection. No severe septic shock occurred after active anti-infection treatment. Cardiovascular toxicity was another common adverse event that manifests mainly as arrhythmia, with an incidence rate of 7.3% (4/55). It is worth noting that one relapsed patient had hearing toxicity during treatment, leading to treatment interruption.

Discussion

AML is a malignant clonal proliferative disease with a high mortality rate and poor prognosis. The prognosis of AML patients had significantly improved because the rapid development of molecular-targeted agents. But, the prognosis of elderly AML patients is very poor still [13]. Researchers found that some AML patients had a high BCL-2 expression level, which is associated with treatment resistance and poor overall survival [14]. BCL-2 inhibitors may be the promising treatment agents which can improve the prognosis of AML patients. Venetoclax (VEN) is the first BCL-2 inhibitor approved by the FDA for the treatment of AML. However, the therapeutic effect of VEN monotherapy is poor [15]. Konopleva M found that the overall response rate of VEN monotherapy was only 19% in AML patients [16].

Vitro studies have shown that VEN can increase the anti-leukemia effect of HMAs, and HMAs can reduce the level of MCL-1 and delay the emergence of VEN resistance [17]. Subsequent clinical trials have also confirmed that VEN combined with HMAs had a significant anti-leukemia activity in AML patients. A phase III clinical study showed that the CR + CRi% was 66.4% and the median PFS was 14.7 months of VEN + HMAs treatment among 283 newly diagnosis AML patients who were not suitable for intensified therapy [5]. The FDA had approved the combination therapy of VEN and HMAs for newly diagnosed elderly AML patients or AML patients who were unfit for high-dose chemotherapy [18]. This study achieved similar or even better clinical responses. In this study, newly diagnosed AML patients had a MLFS% of 80.6%, a CR% of 54.8% and an MRD-% of 51.6% after VEN + HMAs treatment. VEN + HMAs can reduce the tumor burden and effectively promote hematological recovery in newly diagnosed AML patients. After VEN + HMAs treatment, the MLFS% of the relapsed patients in this study reached 50%, CR% reached 33.3%, and MRD-% reached 25%. VEN + HMAs treatment has therapeutic effect on some relapsed AML patients, but its efficacy is limited compared to newly diagnosed patients. In our study, the median PFS of newly diagnosed patients was 14 months and the median PFS of relapsed patients was 7 months. The similar conclusions were obtained in the relevant literature. One study showed that the CR + CRi% rate of VEN + HMAs treatment for relapsed AML patients was approximately 40%, with a median OS of approximately 6 months [19]. This study found that some AML patients may stop or change VEN + HMAs treatment during the treatment process, which is often related to the expensive drug costs. The results found that patients with less than or equal to 3 treatment courses had a PFS of only 9 months, while patients with 6 or more treatment courses had a PFS of 22 months. If AML patients can tolerate VEN + HMAs treatment, they should persist in taking medication for long time, which can significantly prolong survival.

MST refers to a new transplantation method for hematological malignancy [20]. In 2011, BLOOD first published a MST clinical trial for the treatment of AML. The 2-year leukemia-free survival (LFS) rates of the MST group was 39% and chemotherapy group was 10% [21]. Duke University reported that the CR% of AML patients after MST treatment was 63% [22]. For é s R et al. reported the CR% of four AML patients after MST treatment was 75% [23]. Our research team completed MST treatment for 56 AML patients in the past six years, with a median OS time of 25.5 (3–103) months and a median PFS time of 18 (2–102) months. The efficacy and safety were greater than chemotherapy alone. Before 2021, the pretreatment regimen for MST mainly was high-dose Ara-C. With the emergence of BCL-2 inhibitors, many research groups have optimized the pretreatment regimen for MST, and the VEN + HMAs regimen has become the main approach. This study found that the 1-year CR rate of AML patients using VEN + HMAs as the pretreatment regimen for MST was 83.3%, with a median PFS of 20.5 months. Our research findings suggested that VEN + HMAs combined with MST can significantly improve both short-term and long-term outcomes of AML patients. The specific mechanism still needs further research.

Regarding safety, previous studies have shown that the common adverse events of the VEN + HMAs treatment include hematological toxicity, infection, cardiovascular toxicity, etc. [24]. Hematological toxicity is an inevitable adverse event during the treatment of AML patients, and the VEN + HMAs treatment also has varying degrees of hematological toxicity. The results of this study showed that the level of granulocytes and platelets can recover to relatively safe level in approximately 3 weeks. Infection was the other common adverse event. Sandy et al. reported that the infection rate after VEN treatment was 33.6% and pulmonary infections were the most common infection [25]. Our research had reached similar conclusions. 4 cases had lung infection and 2 cases had perianal infection in our study. Johnson et al. reported that the VEN + HMAs combination treatment had a greater incidence of cardiovascular disease in AML patients than HMA monotherapy. This may be due to the high blood potassium and uric acid level caused by VEN, which further increases the risk of atrial fibrillation and other heart diseases [26]. This study reached similar conclusions. The 4 patients had cardiovascular toxicity, but no fatal cardiovascular accidents occurred. Research has shown that the 30-day mortality rate of VEN + HMAs induction therapy is approximately less than 3% [27]. No fatal adverse events were observed in this study.

AML patients often had coagulation disorders such as bleeding, thrombosis, which seriously affect their prognosis. This study found that newly diagnosed AML patients showed a significant increase in APTT, a significant decrease in FIB expression and a mild decrease in Dimmer level after VEN + HMAs treatment. These results suggested that the VEN + HMAs treatment may affect the endogenous coagulation system. Through extensive literature review, no relevant reports have been found about whether VEN + HMAs treatment impact coagulation function in AML patients. Further researches are needed to investigate the effects and underlying mechanisms.

Although good treatment results for AML patients have been obtained with the use of VEN + HMAs treatment, little is known about how these compounds affect immune cells. In theory, targeted therapy can impair the number and function of lymphocytes, thereby hindering normal anti-tumor immune responses. Research has found that VEN can enhance the ability of T cells to recognize and kill AML cells by promoting the generation of reactive oxygen species [11]. An increase in PD-1 + T cells and CD8 + T effector memory cells were observed in the tumor-bearing mice after VEN treatment [28]. Studies have shown that HMAs may promote the expression of immune activating molecules and tumor antigens, increase recognition ability of T cell for tumor cells, and thus enhance anti-tumor effects. AZA can affect T-cell polarization, significantly reduce the Treg cells count and regulate the killing activity of cytotoxic T cells to tumor cells. [29–31]. It has also been proven that DAC can enhance anti-tumor immunity by reducing Treg count [32]. Luo Yao H et al. reported that DAC can promote CD8 + T-cell tumor infiltration and inhibit tumor growth, which may be related to the up-regulation of perforin and granzyme B expression [33]. However, there are few reports about how the combination therapy of VEN and HMAs affect tumor immune microenvironment..

T cells are the important component in the immune system. This study found that the proportions of T cells, CD4 + T cells and CD8 + T cells in AML patients were increased after VEN + HMAs treatment. CD8 + T cells are the most significant It confirmed that VEN + HMAs can directly enhance anti-leukemia function of T cells. NK cells are other important immune cells and also play a key role in the anti-tumor immune responses. Researchers have reported that VEN can enhance the function and anti-leukemic ability of NK cells and that low-dose VEN has the least impact on NK cells proliferation and viability [34]. There is no report about whether HMAs affects NK cell function. Our study revealed that the proportion of NK cells in AML patients significantly increased after VEN + HMAs treatment, indicating that the VEN + HMAs treatment can enhance NK cell-mediated anti-leukemia effects. The specific mechanism still needs further research. Recent studies have shown that B cells may promote tumor progression, possibly through Breg cells which secrete IL-10 and TGF-β to inhibit anti-tumor immunity. A preliminary study revealed that the proportion of Breg cells in the bone marrow and peripheral blood of newly diagnosed AML patients significantly increases, which is associated with poor survival [35]. Our study found that the proportion of B cells significantly decreased after VEN + HMAs treatment. But we did not further detect the changes in B cell subsets, especially Breg cells. We will conduct relevant research in the future.

TNF-α is a cytokine that regulates inflammation and immune cell function [36]. Studies have shown that the high TNF-α level of AML patients after chemotherapy may be related to infection, leading to poor prognosis [37]. This study found that the expression level of TNF-α decreased in newly diagnosed AML patients after VEN + HMAs treatment. IFN-γ is an important immune cytokine which can activate and enhance immune ability. Studies have shown that low-dose HMAs can promote IFN-γ secretion to enhance anti-tumor immunity [38]. VEN can promote the secretion of IFN-γ of NK cells to enhance anti-leukemia effect [34]. However, there is one study found that VEN + HMAs treatment may inhibit the secretion of IFN-γ by CD8 + T cells [39]. Our study found that IFN-γ level increased in newly diagnosed AML patients after VEN + HMAs treatment, which confirmed that VEN + HMAs treatment enhances anti-tumor immune responses. We observed more significant cytokine changes such as significantly reduced level of IL-8 and IL-10 in relapsed AML patients after VEN + HMAs treatment. In the tumor microenvironment, IL-8 is an important immunosuppressive factor and plays a role in promoting the occurrence and development of tumors. IL-10 is a multifunctional cytokine with a strong anti-inflammatory properties and a negative regulatory effect on the anti-tumor immune response. The above results suggested that the VEN + HMAs treatment may improve the immunosuppressive tumor microenvironment and enhance the anti-leukemia immune response of AML patients. Further research and mechanistic exploration are needed.

Conclusion

AML is a malignant clonal proliferative disease with a high mortality rate. VEN + HMAs treatment has good efficacy and safety in AML patients, especially in newly diagnosed AML patients. It may improve the immune microenvironment, enhance anti-leukemia immune response and prolong survival time of AML patients. VEN + HMAs combined with MST treatment may have a better outcome. Further researches with large samples, long-term follow-up, and mechanistic exploration are needed to verify our conclusion.

Acknowledgements

Informed consent was obtained from all the participants involved in the study. The authors thank to all patients for permitting us to use clinical data for presentation.

Author contributions

XH. P., LS. Z. and LJ. L. involved in manuscript writing and revision; YJ. N. assisted with statistical analysis; FT. T. assisted with data interpretation and manuscript editing; YH. L. and J. B.involved in scientific discussion and manuscript preparation. All authors reviewed the manuscript.

Funding

This work has been supported by the National Natural Science Foundation of China (82360029), Fundamental Research Funds for the Central Universities of Lanzhou University (lzujbky-2022-sp08), Medical Research Improvement Project of Lanzhou University (lzuyxcx-2022–154), Medical Innovation and Development Project of Lanzhou University (lzuyxcx-2022–141), Clinical Medical Research Center of Hematological Diseases in Gansu Province (21JR7RA435), Natural Science Foundation of Gansu Province (21JR11RA104).

Data availability

The data that support the findings of this study are available on request from the authors.

Declarations

Competing interests

The authors have no relevant financial or non-financial interests to disclose.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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