Clinical Research for Azacitidine Combined With Low-dose Dasatinib in Maintenance Therapy of Acute Myeloid Leukemia

Study Overview
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Brief Summary
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This project is a prospective, single-center study to evaluate the efficacy, safety and related mechanisms of azacitidine combined with low-dose dasatinib in maintenance therapy in patients with intermediate and high-risk acute myeloid leukemia(AML). The patients were randomly divided into azacitidine group and azacitidine combined with low-dose dasatinib group. The overall survival and disease-free survival were taken as the main end points, and the mortality and recurrence rate were taken as the secondary end points, meanwhile, the incidence of adverse events were evaluated. At the same time, the mRNA expressions of DNA methyltransferase (DNMT1, DNMT3a, DNMT3b), tumor suppressor genes (TP53, P15, P16, P21, CDH1, DOK6, SHP1, PTPN11) and differentiation genes (pu.1, C/EBP α, C/EBP β) were detected. Pyrophosphate sequencing was used to detect the methylation level of the promoter region of these tumor suppressor genes. Western Blot was used to detect apoptosis proteins (caspase3, caspase8) and phosphorylated proteins (pSTAT3, pSTAT5, pAKT). The proportion of apoptotic population of bone marrow cells was determined by flow cytometry. Therefore, the data in this study will reflect the efficacy and safety of azacitidine or azacitidine combined with low-dose dasatinib in real-world maintenance therapy in patients with medium and high-risk AML.

Detailed Description
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In addition to studying the overall survival, disease-free survival and recurrence rates, mortality and incidence of adverse events of patients treated with azacitidine or azacitidine combined with low-dose dasatinib, we will also study its related mechanisms. One of the pathogenesis of AML is that abnormal DNA methylation makes the cell cycle out of control and carcinogenesis by inhibiting the expression of tumor suppressor genes. In addition, the abnormal activation of tyrosine kinase signal pathway also promotes the development of leukemia. Azacitidine, the hypomethylating agents, can not only inhibit the DNA methyltransferase family, but also activate tumor suppressor genes to inhibit a variety of tyrosine kinase signaling pathways, including JAK-STAT. NaShen et al have directly demonstrated that tyrosine kinase inhibitors (TKIs) can not only inhibit the abnormal activation of tyrosine kinase pathway, but also reduce DNA methylation. This study found that the combination of the second generation TKIs and hypomethylating agents can reduce has a synergistic effect on promoting apoptosis and reducing DNA methylation. In addition, TKIs often produces drug resistance due to long exposure time, and the main mechanisms of drug resistance is due to DNA methylation and abnormal reactivation of tyrosine kinase signal pathway. The combination of TKI and azacitidine reduces DNA methylation and inhibits the reactivation of abnormal tyrosine kinase signal pathway, which is helpful to improve TKI drug resistance. Based on the above theory, we assume that patients treated with azacitidine and dasatinib may have more obvious demethylation effect, increased expression of tumor suppressive genes, more obvious apoptosis, and inhibition of phosphorylated protein expression.So we did the lab tests of these mechanisms.We innovatively used azacitidine and TKIs in the treatment of patients with AML maintenance, in order to reduce drug toxicity, enhance drug efficacy, improve patient prognosis and reduce the financial burden of patients.

Official Title
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Clinical Research for Azacitidine Combined With Low-dose Dasatinib in Maintenance Therapy of Acute Myeloid Leukemia

Conditions
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Acute Myeloid Leukemia

Intervention / Treatment
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* Drug: Azacitidine
* Drug: Dasatinib


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Patients with intermediate and high-risk AML who are diagnosed according to the 2016 WHO guidelines, aged ≥18 years; Detect minimal residual disease(-) after induction therapy and consolidation therapy; Eastern Cooperative Oncology Group (ECOG) performance status score 0-2; The heart, pulmonary, liver and kidneys have sufficient organ functions: Cardiac color doppler ultrasound shows cardiac ejection fraction> 50%, heart function classification NYHA III/IV, no heart block or arrhythmia; Patients without severe restrictive/obstructive pulmonary disease; Liver function: total bilirubin (TBIL) < 2 times the upper limit of normal, alanine aminotransferase (ALT) and/or aspartate aminotransferase (AST) <2.5 times the upper limit of normal; Renal function: serum creatinine (Cr) < 1.5 times the upper limit of normal. The patient and family members agree and sign an informed consent form. Exclusion Criteria: Patients with malignant tumors of other organs; HCV positive; or HIV positive; or one of the following HBV test results: HBsAg positive; HBsAg negative, HBcAb positive and HBV DNA titer positive; Pregnant and lactating women, and patients who have family planning during the enrollment period; Patients considered to be unsuitable for enrollment by the investigator.

Ages Eligible for Study
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Minimum Age: 18 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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No

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Treatment
Allocation: Randomized
Intervention Model: Parallel Assignment
Interventional Model Description: The patients were randomly divided into two groups among intermediate and high-risk AML, namely azacitidine group, azacitidine combined with low-dose dasatinib group, with overall survival and disease-free survival as the main research endpoints , taking mortality and recurrence rates as secondary research endpoints, assessing the incidence of adverse events, and studying its related mechanisms.
Masking: Single


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Experimental: experimental group<br>Patients with intermediate and high risk AML were negative for minimal residual disease after intensive induction and consolidation chemotherapy,the patients were randomly divided into two groups, and one group was given azacitidine(75mg/m2, per day on day 1-7]. Dasatinib 100 mg p.o. qd was administered on days 1-28 of each consolidation cycle. | Drug: Azacitidine<br>* Azacitidine, 75mg/m2,d1-7;Treatment cycles every 28 days<br>Drug: Dasatinib<br>* dasatinib,20mg,po,qd,treatment cycles every 28 days<br>|
| Active Comparator: control group<br>Patients with intermediate and high risk AML were negative for minimal residual disease after intensive induction and consolidation chemotherapy,the patients were randomly divided into two groups, and the other group was given azacitidine(75mg/m2, per day on day 1-7)on days 1-28 of each consolidation cycle. | Drug: Azacitidine<br>* Azacitidine, 75mg/m2,d1-7;Treatment cycles every 28 days<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| overall survival | OS is defined as the time from the date of enrollment until the date of death from any cause. | up to 30 months. | 
| disease-free survival | Event-free survival is defined as the time from enrollment until documented refractory disease, relapse after complete remission(CR) or CR with incomplete recovery of blood counts(CRi), or death from any cause. | up to 30 months. | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| mortality | The proportion of patients from enrollment to death was recorded. | mortality rate at 30 months. | 
| recurrence rate | Record the proportion of patients with recurrence in the study. | recurrence rate at 30 months. | 
| adverse events | Number of participants with treatment-related adverse events as assessed by CTCAE v5.0. | Adverse events were assessed weekly during the first and second cycles, and every two cycles thereafter (each cycle is 28 days), up to 30 months. | 
| apoptotic protein and phosphorylated protein | The relative expression of apoptotic protein(caspase3, caspase8) and phosphorylated protein(pSTAT3, pSTAT5, pAKT) were detected by western blot with bone marrow aspirate. | once before enrollment and once after the completion of the study, up to 30 months. | 
| DNA methyltransferase, tumor suppressor genes and differentiation genes | To detect the mRNA expression of DNA methyltransferase(DNMT1, DNMT3a, DNMT3b), tumor suppressor genes(TP53,P15, P16, P21, CDH1, DOK6, SHP1, PTPN11) and differentiation gene(pu.1, C/EBPα, C/EBPβ) with bone marrow aspirate by Q-PCR. | once before enrollment and once after the completion of the study, up to 30 months. | 
| methylation level in the promoter region of some tumor suppressor genes. | Detection of methylation level in the promoter region of the above tumor suppressor genes by pyrophosphate sequencing using patient bone marrow aspirate. | once before enrollment and once after the completion of the study, up to 30 months. | 
| Percentage of bone marrow cell apoptosis population | The percentage of apoptotic population of bone marrow cells was determined by flow cytometry. | once before enrollment and once after the completion of the study, up to 30 months. | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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AML,Maintenance Therapy,Azacitidine,Dasatinib