The Addition of Hypomethylating Agents to Low-Intensity Induction Chemotherapy Does Not Improve Outcomes in Elderly Acute Myeloid Leukemia Patients: A Single-Center Retrospective Study

Background and Objectives: This study aimed to evaluate whether the addition of hypomethylating agents (HMA) to low-intensity chemotherapy can enhance the clinical efficacy of induction treatment for elderly acute myeloid leukemia (AML) patients who are unsuitable for standard induction therapy. Materials and Methods: This study retrospectively analyzed 117 patients over 60 years old who were initially diagnosed with AML and received low-intensity induction treatment in the Department of Hematology in Anhui provincial hospital from January 2015 to December 2020. Twenty-three patients were excluded, and the remaining 94 patients were divided into two groups according to the selection of induction regimens. Results: Forty-four patients received HMA combined with low-intensity chemotherapy, and the other 50 patients received only low-intensity induction chemotherapy. Forty-three patients (45.7%) obtained complete remission (CR) after the initial induction treatment. The CR rate in the HMA plus low-intensity chemotherapy group was 34.1% (15/44), and in the single low-intensity chemotherapy group was 56.0% (28/50) (p = 0.04). The 30 days cumulative early death rates were 9.1% (95% CI: 3.5–22.4%) in the HMA plus low-intensity chemotherapy group and 6.0% (95% CI: 2.0–17.5%) in the single low-intensity chemotherapy group, respectively (p = 0.59), and the one-year cumulative relapse rates were 21.1% (95% Cl: 9.8–41.9%) and 33.3% (95% Cl: 20.3–51.5%), respectively (p = 0.80). The one-year overall survival (OS) rates for patients in the HMA plus low-intensity chemotherapy group and the single low-intensity chemotherapy group were 37.3% (95% Cl: 23.1–51.5%) and 55.4% (95% Cl: 40.5–67.9%), respectively (p = 0.098), and the one-year event-free survival (EFS) rates were 8.5% (95% Cl: 2.2–20.6%) and 20.6% (95% Cl: 9.1–35.3%), respectively (p = 0.058). Conclusions: This study showed that the addition of HMA to low-intensity induction chemotherapy does not improve prognosis in elderly AML patients who are unsuitable for standard induction chemotherapy.


Introduction
Acute myeloid leukemia (AML) is a common hematological malignancy in adults. The median age of patients is 68 years old, and more than half of the patients are over 60 years old when first diagnosed [1]. The traditional "3 + 7" regimen of cytarabine and anthracycline is the first-line induction therapy for AML patients, and the five-years survival rate is 30-35% for patients younger than 60 years old, and for the elderly patients, 2 of 9 the five-years survival rate is only 12.5%, and the early mortality rate is 20-50% [2,3]. The toxicity of chemotherapy may outweigh the benefit for the patients who are older, in poor performance, or not suitable for standard-intensity chemotherapy, and the median survival time was about 6-8 weeks when only receiving the supportive treatment [4,5]. Therefore, it is appropriate for clinicians to select low-intensity treatment for elderly patients who are not suitable for standard-intensity induction.
With the development of epigenetic research, hypomethylating agents (HMA) including decitabine (DEC) and azacytidine (AZA) which can mainly reactivate the silenced tumor suppressor gene due to aberrant DNA methylation, are considered to be the most effective regimens for the treatment of myelodysplastic syndrome (MDS) and AML [6,7]. However, most patients would be relapsed because the single-regimen HMA therapy cannot remove all the malignant clones [8]. Therefore, new, and more effective low-intensity induction schemes need to be further explored. Recently, it has been reported that the complete remission (CR) rate is 67% and the survival time has been prolonged when patients received HMA and venetoclax, a Bcl-2 inhibitor, which has become the new hope for patients who cannot tolerate standard-intensity induction [9]. However, for patients with poor economic conditions who cannot use venetoclax or in areas where venetoclax cannot be supplied, low-intensity chemotherapy is still an important treatment option for elderly AML patients.
In this retrospective study, we compared the CR rate, minimal residual disease (MRD) negative rate, overall survival (OS) and event-free survival (EFS) rates between lowintensity chemotherapy and HMA combinations in newly diagnosed elderly AML patients who are unsuitable for standard induction therapy, in order to evaluate whether the addition of HMA to low-intensity induction chemotherapy could improve the prognosis of these patients.

Patients
This study retrospectively analyzed 117 patients over 60 years old who were initially diagnosed with AML and received low-intensity induction treatment in the Department of Hematology in Anhui provincial hospital from January 2015 to December 2020. All patients underwent bone marrow aspiration and were diagnosed according to morphology, immunology, cytogenetics, and molecular biology (MICM). Among the patients studied, we excluded 23 patients who received only single HMA induction therapy and divided the remaining 94 patients into two groups according to the selection of induction regimens. Forty-four patients received HMA combined with low-intensity chemotherapy, and the other 50 patients received only low-intensity induction chemotherapy. We collected patient data from the hospital's electronic medical record system. The characteristics of the patients in both groups at their first visit are shown in Table 1. The protocol was approved by the Ethics Committee of Anhui Provincial Hospital (approval number: 2022-RE-144) and carried out in accordance with the Declaration of Helsinki.
Patients who achieved CR/CRi entered consolidation treatment phase and were treated with sequential low-dose chemotherapy regimens (such as low dose-IA or DA, CAG, HAG, HMA + low-dose cytarabine, HMA +CAG, HMA + HAG, etc.).

Definitions and Statistical Analysis
Early death was defined as death from any cause from the start of diagnosis to the first 30 days. The overall response rate (ORR) included CR, complete remission with incomplete hematologic recovery (CRi) and partial remission (PR). Overall survival (OS) was defined from initial diagnosis to death from any cause or the time until follow-up. Event-free survival (EFS) was defined from initial diagnosis to relapse or death. Cumulative incidence of relapse (CIR) was defined from CR to disease relapse. MRD was examined by analysis of bone marrow fluid using an eight-color flow cytometer, which was identified by leukemiaassociated immunophenotype, and MRD levels < 0.01% were considered negative. The continuous variables were compared between the two groups using the Mann-Whitney U test, and categorical variables were compared using the chi-square test or Fisher's exact test. The probability of OS and EFS is calculated according to the Kaplan Meier curve. The cumulative incidence rate function method was used to estimate the probability of early death and relapse. The statistical analysis was carried out using R statistical software (R statistical calculation basis). Differences with p < 0.05 were considered significant.

Clinical Characteristics
The median age was 70 years (range 60-83) in the whole cohort. The median age of patients in HMA plus low-intensity chemotherapy group was 69 years old (range 60-83), and there were 8 (18.2%) patients over 75 years old. The median age of patients in single low-intensity chemotherapy group was 70 years old (range 60-83), and 7 (14.0%) patients were over 75 years old. According to the available cytogenetic and molecular biological data, risk stratification was carried out according to the 2017 European leukemia (ELN) guidelines [10]. In HMA plus low-intensity chemotherapy group, 35 (79.5%) patients belonged to the favorable-or intermediate-risk group, and 9 (20.5%) patients belonged to the adverse-risk group. In the single low-intensity chemotherapy group, 42 (84.0%) patients belonged to the favorable-or intermediate-risk group, and 8 (16.0%) patients belonged to the adverse-risk group.

Early Death
During the induction treatment, the incidence of serious infection (pulmonary fungal infection, severe pneumonia, sepsis, etc.) in the HMA plus low-intensity chemotherapy group and the single low-intensity chemotherapy group were 54.5% vs. 52.0%, respectively (p = 0.838). Within 30 days after the first diagnosis, seven patients (7.4%) died: four patients (9.1%) in the HMA plus low-intensity chemotherapy group, including two patients with cerebral hemorrhage and two patients with severe infection and multiple organ failure. Three patients (6.0%) died in the single low-intensity chemotherapy group, including two patients with cerebral hemorrhage and one patient with severe infection. The 30 days cumulative early death rates were 9.1% (95% CI: 3.5-22.4%) in the HMA plus low-intensity chemotherapy group and 6.0% (95% CI: 2.0-17.5%) in the single low-intensity chemotherapy group, respectively (p = 0.59) (Figure 2a).

Discussion
At present, there is no literature report on whether the addition of HMA on the basis of low-intensity chemotherapy can improve the poor prognosis of elderly AML. It is unclear whether HMA can enhance the clinical efficacy of low-intensity chemotherapy for elderly AML patients, including raising the CR rate, reducing the relapse rate and prolonging the survival.
In most clinical circumstances, monotherapy with HMA (azacitidine or decitabine) or low-dose cytarabine was recommended for older adults with AML not suitable for intensive antileukemic therapy. Until now, there are only two clinical studies have proved that combination treatment is effective in elderly AML. A multicenter phase II clinical trial [11] showed that for AML patients who were not eligible for intensive chemotherapy (n =

Discussion
At present, there is no literature report on whether the addition of HMA on the basis of low-intensity chemotherapy can improve the poor prognosis of elderly AML. It is unclear whether HMA can enhance the clinical efficacy of low-intensity chemotherapy for elderly AML patients, including raising the CR rate, reducing the relapse rate and prolonging the survival.
In most clinical circumstances, monotherapy with HMA (azacitidine or decitabine) or low-dose cytarabine was recommended for older adults with AML not suitable for intensive antileukemic therapy. Until now, there are only two clinical studies have proved that combination treatment is effective in elderly AML. A multicenter phase II clinical trial [11] showed that for AML patients who were not eligible for intensive chemotherapy (n = 116), the use of low-dose cytarabine combined with glasdegib prolonged survival OS to 8.3 months in the combination group and only 4.3 months in the group with low-dose cytarabine alone, (p = 0.0002), with manageable adverse effects. Dinardo et al. [9] proved that venetoclax combined with HMA was well tolerated in elderly AML, with an increased overall response rate (ORR) of 67%. However, this study shows that the addition of HMA to low-intensity chemotherapy did not improve the prognosis of elderly AML. There were no significant differences in the one-year cumulative relapse rate and the one-year OS rate in the combined HMA group compared to the single low-intensity chemotherapy group (p = 0.802, 0.098). Especially in the patients with favorable-or intermediate-risk, the use of the single low-intensity chemotherapy regimen resulted in a significantly higher CR rate than in the combined HMA group (62.0% vs. 28.6%) (p = 0.006) and a significantly higher one-year OS rate (63.7% vs. 31.4%) (p = 0.016). Although the combination of HMA in this study did not increase the incidence of severe infection events, it has been reported [12] that the incidence of agranulocytosis with fever (infection) increased significantly (42% vs. 19%) when venetoclax was combined with AZA. Another phase III multicenter randomized open trial [13] showed that comparing low-intensity cytarabine with DEC, the risk of severe agranulocytosis (24% and 15%) and infection (21% and 15%) was significantly increased in the DEC group.
Poor cytogenetic characteristics and secondary AML in elderly patients are the main causes of poor prognosis and affect the long-term survival [14]. Although this study shows that the addition of HMA to the initial induction cannot improve the survival of elderly AML, in certain clinical situations, such as treatment of patients with complex karyotype, previous MDS history, TP53 mutation, HMA-based chemotherapy might be favored, because patients with adverse biology are not likely to respond to induction with low-intensity chemotherapy only, such as low-dose cytarabine. Xing-Nong et al. [15] conducted a clinical trial to investigate the effectiveness of the sequential combination of DEC followed by low-dose chemotherapy in high-risk myeloid neoplasms and found that epigenetic priming with DEC had an increased anti-leukemia effect. This study also supports the additional use of HMA to low-intensity chemotherapy in patients with adverse-risk patients; although the statistical difference is not significant, the combined HMA treatment group has a higher CR rate (55.6% vs. 25.0%), a longer one-year OS rate (47.6% vs. 12.5%) and a lower one-year cumulative relapse rate (33.3% vs. 68.8%).

Conclusions
In conclusion, this study showed that the addition of HMA to low-intensity induction chemotherapy did not significantly improve the outcome in elderly AML patients who are unsuitable for standard induction chemotherapy. In particular, patients in the favorableor intermediate-risk groups are more likely to achieve CR and prolong OS using single low-intensity chemotherapy. However, this is a retrospective clinical study with a limited number of patients in each group; and the regimens in low-intensity chemotherapy were heterogeneous which includes low-dose IA, DA, CAG and HAG, etc. A large number of patients with unified low-intensity chemotherapy regimens should be enrolled in a prospective study in the future.
Author Contributions: D.L. collected and analyzed data and wrote the original paper. X.W. collected and analyzed data. J.T., L.Z., E.C., Z.Z., L.X., X.Z., G.S. participated in patient care and contributed to data interpretation. C.Z. designed the research, performed the research, analyzed and interpreted data, critically reviewed the manuscript. All authors have read and agreed to the published version of the manuscript.