Maintenance therapy with a combination of azacitidine, danazol, and thalidomide after intensive chemotherapy in patients with acute myeloid leukemia

ABSTRACT

Objective

This study aimed to evaluate the efficacy of azacitidine (AZA) combined with danazol (DNZ) and thalidomide (THD) maintenance therapy after intensive chemotherapy (IC) in patients with acute myeloid leukemia (AML).

Methods

we retrospectively analyzed the clinical data of 11 patients treated with AZA combined with DNZ and THD as maintenance therapy after IC at the Baiyun Hospital were between February 2017 and March 2021. The patients' clinical features, relapse-free survival (RFS), and overall survival (OS) were analyzed.

Results

Eleven cases fulfilled the AML criteria per the 2016 World Health Organization classification. Of the 11 patients, five were females, and six were males, with a median age of 45 years (range, 23-65 years). Ten patients were in the first complete remission (CR1), and one patient was in the second complete remission (CR2). All patients received AZA combined with DNZ and THD maintenance therapy after IC. The median number of AZA cycles received was 7 (6-12). Until June 2022, the median follow-up period was 37 (14-63) months; one patient had a relapse, and three died. RFS at 1 year and 3 years was 100% and 71.1%, respectively, and OS at 3 years was 100%.

Conclusion

AZA combined with DNZ and THD maintenance therapy is effective for patients with AML who are ineligible for allogeneic hematopoietic stem cell transplantation. Further studies with large sample sizes and randomized are needed to verify these findings.

KEYWORDS:

• Azacitidine
• danazol
• thalidomide
• maintenance therapy
• acute myeloid leukemia

Introduction

Although more patients with acute myeloid leukemia (AML) have had better outcomes in recent decades, the relapse rates after intensive chemotherapy (IC) remain high. This is especially true for patients who are ineligible for allogeneic hematopoietic stem cell transplantation (allo-HSCT) for various reasons (financial constraints, lack of an acceptable donor, comorbidities, etc.) [1–3]. The cost of allo-HSCT is one of the most important reasons in our country. Most of the patients with AML cannot affort the cost, some patients even have to borrow the money to pay for chemotherapy. Outcomes in patients with relapse remain poor. Thus, how to prevent relapse in patients with AML is still a major therapeutic challenge.

No maintenance therapy is used as a standard treatment for AML because of its insufficient efficacy [4]. Previous studies have shown that the clinical benefits of maintenance therapies with interleukin-2 (IL-2), low-dose chemotherapy, and gemtuzumab ozogamicin are controversial [5–7]. Recently, several drugs, including hypomethylating agents (azacitidine (AZA) and decitabine) [8,9], immunomodulating agents (lenalidomide) [10], androgen [11], kinase inhibitors (sorafenib, midostaurin, etc.) [12–15], which are maintenance therapies in patients with AML, have shown better event-free survival (EFS). Particularly, oral azacytidine significantly improves the overall survival (OS) and relapse-free survival (RFS) in patients with AML and recommended by NCCN guidelines as maintenance therapy in AML indicating that maintenance therapy is an important aspect of AML treatment [16].

Previous reports had shown that maintenance therapy with AZA, androgen and lenalidomide alone could improve AML patients DFS through different mechanisms [8,10,11]. Let us considered that AZA in combination with danazol (DNZ) and thalidomide (THD) as maintenance therapy may have a synergistic anti-leukemia effect, and DNZ may reduce the adverse effects of AZA and THD, such as cytopenia, fatigue,etc. So we designed maintance therapy including those three medicines.

Here, the clinical data of 11 AML patients treated with AZA combined with DNZ and THD as maintenance therapy were retrospectively analyzed. This study aimed to observe the efficacy of maintenance therapy, along with RFS and OS.

Patients and methods

Patients

Eleven patients diagnosed with AML and treated at the Baiyun Hospital between February 2017 and March 2021 were treated with this maintenance treatment. All patients met the AML criteria of the 2016 World Health Organization classification. All patients signed an informed consent document before treatment and complied with the Declaration of Helsinki and institutional guidelines.

Treatment: induction and consolidation treatment

The induction and consolidation therapies for newly diagnosed patients with AML were based on the Chinese guidelines for diagnosing and treating adult patients with AML (not acute promyelocytic leukemia) [17]. Induction therapy involved daunorubicin 60 mg/m2/day intravenously on days 1–3 and cytarabine (Ara-c) 100 mg/m2/day on days 1–7. Acceptance reinduction therapy was administered if the patient did not achieve complete remission (CR). Three post-remission cycles of consolidation chemotherapy with high-dose Ara-c (3 g/m2/q12h d1–3) and two cycles of a reduced dose of chemotherapy (DA/HA/EA) were administered for five days.

Maintenance therapy with AZA combined with DNZ and THD

After the completion of consolidation therapy, patients remained in CR and were treated with 6–8 cycles of AZA combined with DNZ and THD maintenance therapy: AZA 100 mg/day (approximately 50–75 mg/m2/day) d1–7 by subcutaneous injection, DNZ 200 mg b.i.d., and THD 100 mg/day p.o., followed by AZA every four weeks. After completing the 6–8 cycles, patients continued the DNZ and THD maintenance therapy for at least three years unless relapse or intolerance occurred(Figure 1).

Figure 1. Treatment schema of maintenance therapy.

Disease evaluation included bone marrow examinations every three months for the first two years and then every six months until a CR duration of more than five years.

Criteria for response and clinical follow-up

A patient was considered to have achieved CR after meeting all the following conditions: absence of leukemic blasts in blood and <5% blasts in the bone marrow; absolute neutrophil count (ANC) > 1 × 10⁹/L and platelet count (PLTC) > 100 × 10⁹/L; lack of evidence of extramedullary disease. Minimal residual disease (MRD) negative refers to the failure to detect residual leukemia cells in bone marrow specimens by flow cytometric analysis (FCM) and real-time quantitative polymerase chain reaction (RT-qPCR). Relapse was the presence of bone marrow blast cells (>5%) or extramedullary disease. OS was calculated from the time of diagnosis to death or the last follow-up. RFS was calculated from the first remission to the first relapse (during maintenance therapy) or the last follow-up.

Statistical analysis

RFS and OS rates were extracted based on Kaplan–Meier estimates. The results were analyzed using Statistical Package for Social Sciences version 20.

Results

Patient characteristics

Eleven patients treated with AZA combined with DNZ and THD maintenance therapy between February 2017 and March 2021. Table 1 shows the baseline patient and disease characteristics. Of the 11 patients, five were females, and six were males, with a median age of 45 years (range, 23–65 years) at the time of diagnosis. The median white blood cell count was 37.55 × 10⁹/L (range: 1.04–132.96). Before starting maintenance therapy, 10 patients were in the first complete remission (CR1), and one patient was in the second complete remission (CR2). Patients were classified as favorable (4/11), intermediate (5/11), and adverse (2/11) according to the European Leukemia Net genetic risk stratification(2017).

Table 1. Characteristics of the 11 patients with AML.

No	Sex	Age (y)	WBC (10^9/L)	Karyotype	Fusion gene	Gene Mutation	ELN risk group (2017)	Disease status	Consolidation numbers	MRD status prior to maintenance therapy
1	F	42	53.01	46, XX [20]	Negative	Negative	Intermediate	CR1	5	Negative
2	F	45	13.73	46, XX [20]	Negative	NPM1	Favorable	CR1	5	Negative
3	M	50	75.19	46, XY [16]	Negative	NPM1, CEBPA	Favorable	CR1	5	Negative
4	M	59	38.5	46, XY [20]	Negative	NPM1, NRAS, DNMT3A, TET2, RUNX1(NGS)	Adverse	CR1	5	Negative
5	M	55	1.04	46, XY [13]	Negative	IDH2, DNMT3A, BCOR (NGS)	Intermediate	CR1	5	Negative
6	F	50	3.41	46, XX [20]	Negative	Negative	Intermediate	CR1	5	Negative
7	M	65	2.37	46, XY [18]	Negative	DNMT3A, TET2	Intermediate	CR1	5	Negative
8	F	34	55.37	46, XX [20]	Negative	FLT3/ITD(AR = 0.89), NPM1	Intermediate	CR1	5	Negative
9	M	23	132.96	46, XY, t (8;21) (q22; q22) [10]	RUNX1-RUNX1T1	C-KIT(D816)	Favorable	CR2	0	Undetected
10	M	26	37.55	46, XY [20]	Negative	FLT3/ITD (AR = 1.02)	Adverse	CR1	5	Negative
11	F	25	5.37	46, XX, t (8;21) (q22; q22) [20]	RUNX1-RUNX1T1	C-KIT(D816)	Favorable	CR1	0	Negative

All patients were treated with DA induction therapy: 10 patients achieved remission after one course of treatment, whereas one patient achieved remission after salvage therapy. Ten patients completed all consolidation therapy courses, including three courses of high-dose Ara-c and two courses of a low-dose 5-day regimen. One patient diagnosed with AML with t(8;21) refused to continue chemotherapy because of renal failure after remission and was directly switched to maintenance therapy. Ten patients completed all consolidation chemotherapy cycles, nine of whom began maintenance therapy. One patient with AML with t(8;21) relapsed after stopping chemotherapy for two months and achieved CR2 by FLAG reinduction therapy, refused continuous chemotherapy and transplantation, and directly switched to maintenance therapy. Only one patient was not evaluated for MRD, and all others were MRD negative before starting maintenance therapy.

All patients received a median of seven (6–12) courses of AZA combined with DNZ and THD continuous therapy. Treatment was well tolerated and accepted by the patients. All patients received continuous DNZ and THD maintenance therapy after finishing AZA. Common adverse events included mild neutropenia, thrombocytopenia, and elevated aminotransferase(Table 2). One patient had a significant increase in urea nitrogen and creatinine because the patient was diagnosed with renal failure after remission before maintenance therapy. No patient discontinued the maintenance therapy due to adverse events. The patient in CR2, after reinduction therapy, received 12 courses of AZA. AZA was given every two months after 10 cycles.

Table 2. Adverse events of patients during maintenance treatment.

AE	Grade 1	Grade 2	Grade 3	Grade 4
Haematological adverse events
Neutropenia	7(63.6%)	2(18.2%)	0	0
Anaemia	4(36.4%)	1(9.0%)	0	0
Thrombocytopenia	2(18.2%)	1(9.0%)	0	0
Non-haematological adverse events			0	0
Nausea	6(54.5%)	2(18.2%)	0	0
Vomiting	2(18.2%)	0	0	0
Fatigue	6(54.5%)	1(9.0)	0	0
ALT elevation	7(63.6%)	1(9.0)	0	0
AST elevation	6(54.5%)	0	0	0
Hyperbilirunaemia	4(36.4%)	0	0	0
Creatinine increase	0	0	0	1(9.0%)
BUN increase	0	0	0	1(9.0%)
Cardiac disorder	0	0	0	0

Outcomes

The median follow-up period was 37 (14–63) months until June 2022. The expected RFS at 1 and 3 years was 100% and 71.1%, respectively, and OS at 3 years was 100% (Figure 2(a and b)). Three of the 11 patients died, and eight were alive with EFS. Of the three patients who died, one relapsed at 36 months and died of disease progression at 46 months. The blood test of the second patient was abnormal at 30 months, and a bone marrow puncture was performed, confirming the diagnosis of mixed phenotypic acute leukemia (T/My, NOS), and the patient died at 43 months. The third patient died of a traffic accident at 37 months while in CR1. Notably, two cases of AML with t(8; 21) (one patient relapsed after consolidation chemotherapy, and another patient developed renal failure after DA induction therapy) remained in CR by the end of the follow-up period. The CR duration was 24 and 13 months, respectively. The latest bone marrow MRD test showed AML/ETO 0%. Figure 2(c) shows the Swimmer plot of the 11 patients.

Figure 2. Response and long-term outcomes of the entire cohort.

Note: (a) Relapse-free survival of the 11 patients. (b) Overall survival of the 11 patients. (c) Swimmer plot of the 11 patients.

Discussion

Relapse is still a major problem for patients with AML, especially for those who cannot undergo allo-HSCT. Thus, an effective and safe post-remission maintenance therapy is needed to improve RFS and OS for patients with AML remission after induction and consolidation.

Although clinicians have been using various maintenance therapies after IC or HSCT to improve RFS and OS for many years, maintenance therapies are not a part of the definite treatment schedule for AML. Multiple recent studies evaluating maintenance therapies after IC or HSCT using different agents, such as lenalidomide, androgens, HMAs (AZA, oral azacitidine, and decitabine), kinase inhibitors (sorafenib, midostaurin, and gilteritinib), and BCL-2 inhibitors, have shown encouraging results with improved RFS and OS.

In this study, we evaluated the efficacy and safety of AZA combined with DNZ and THD as maintenance therapy after IC for patients with AML who cannot undergo allo-HSCT. This combination has never been reported. We combined the three drugs for the following reasons: (i) AZA was used because previous studies have shown its anti-leukemia effect, and a recent study has shown that AZA maintenance therapy after IC improves disease-free survival in elderly patients with AML [8]. (ii) DNZ was used due to Pigneux reported that maintenance therapy with norethandrolone improved survival in elderly patients with AML [11], on the other hand DNZ may reduce adverse events such as hemocytopenia, fatigue caused by AZA and THD so that patients would not interrupt treatment. (iii) THD’s precise mechanism of action in hematological tumors remains unclear. Previous studies have shown that THD has the following effects: anti-cell proliferation, induction of apoptosis, blocking tumor-stroma interactions, and changes in immune responses. We are now conducting pre-clinical work to investigate the synergistic effect or potential synergistic mechanism of these three drugs.

This study demonstrated that AZA combined with DNZ and THD could be safely administered to patients after IC. All our patients received a median of seven courses of AZA (6–12) combined with DNZ and THD continuous therapy and continued DNZ and THD oral maintenance therapy. The major side effects included mild neutropenia, thrombocytopenia, and elevated aminotransferase. None of the patients interrupted treatment because of adverse events.

By the end of the follow-up period, three patients had died. However, only one case died of disease relapse and progression. Of the remaining two patients, one was diagnosed with mixed phenotypic acute leukemia (T/My, NOS) and died, while the other died in a road accident. RFS at 1 and 3 years was 100% and 71.1%, respectively; OS at 3 years was 100%. The reasons for this result are as follows: (1) our maintenance treatment; (2) most of the patients entering maintenance therapy belonging to the favorable (4/11) or intermediate (5/11) risk group and being MRD negative (10/11). Due to most of our patients did not proceed the NGS test, the ELN risk of some patients may not precise. Two patients were diagnosed with AML with t(8;21). One relapsed, and the other received only one induction therapy because of renal failure but remained in CR until the end of the follow-up period. The CR duration was 24 and 13 months, respectively, which implies that AZA combined with DNZ and THD is an effective treatment. Although the number of patients was small, AZA combined with DNZ and THD maintenance therapy showed efficacy and safety. It provides an excellent treatment for patients who cannot undergo allo-HSCT.

In conclusion, this is the first study to report the efficacy and safety of AZA combined with DNZ and THD as maintenance therapy in patients with AML. Based on the results of this study, we are conducting a randomized trial in order to verify these findings.

Author's contributions

Fang Zheng and Qianqian Li collected the data and wrote the manuscript. Sisi Yang, Zhen Zhou, Qingfan Zeng and Fang Zheng were involved in patient management and clinical data collection. Kaiqi Liu and Fangzheng designed therapeutic regimen. Kaiqi Liu revised the manuscript and provided valuable advice.

Availability of data and materials

The datasets used during the current study are available from the corresponding author on reasonable request.

Consent for publication

All authors have read and approved the manuscript for publication.

Ethics approval and consent to participate

All studies involving human participants were in accordance with the ethical standards of the institutional and national research committee and with the Helsinki Declaration 2013. Written informed consent was obtained from the patients or patients’ parents/legal guardians.

Acknowledgements

Not applicable.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

The author(s) reported there is no funding associated with the work featured in this article.