The Expression of mi-RNAs Involved in the Hematopoietic Niche Microenvironment in Newly Diagnosed AML Patients

Introduction

Acute myeloid leukemia (AML) is a genetically heterogeneous disease and the most common type of acute leukemia in adults. Despite the progress made in the last years in AML therapy, induction treatment failure and disease relapse still remain the main challenges in clinical practice. miRNAs are involved in the pathogenesis of AML as well as influence the differentiation and proliferation of normal hematopoietic cells. The expressions of specific miRNAs have been identified as potential biomarkers for predicting prognosis both in solid tumors and hematological malignancies.

Methods

The expression of 12 miRNAs involved in hematopoiesis and the microenvironment of the hematopoietic niche: miR-15a-5p, miR-34a-5p, miR-125b-5p, miR-139-5p, miR-146-5p, miR-181a-5p, miR-199b-5p, miR-204-5p, miR-218-5p, miR-299-5p, miR-424-3p, miR-452-5p was determined in duplicates using qPCR in bone marrow samples of newly diagnosed AML patients. The miRCURY LNA SYBR® Green PCR Kit (Qiagen) was used to carry out qPCR. miRNAs expression was normalized using the mean expression of all miRNAs in a given sample. The normalization was performed using the formula ∆Ct = Ct(reference) − Ct(miRNA of interest). This approach results in higher values for higher miRNA expression. Differential expression analysis was carried out using a t-test. Cox proportional hazard regression analyses of clinical factors and miRNAs for predicting overall survival in AML patients were performed.

Results

To the study group we enrolled 45 (23 women and 22 men) newly diagnosed AML adults with the mean age 45.1 ± 12.8 years. Among them, 13.9% patients (pts) had a low-risk, 44.2% intermediate-risk and 41.9% high-risk AML according to EuropeanLeukemiaNet (ELN) 2017 risk stratification (Döhner et al. 2017). In the study group, 16.7% of pts had NPM1 mutation, whereas 30.2% of pts had FLT3-ITD or FLT3-TKD mutation. The majority of patients (88.9%) received intensive treatment: DA-60 (daunorubicin 60mg/m² + cytarabine 200mg/m²) regimen (50%), followed by DAC (DA-60 + cladribine 5mg/m²) (40%) and DA-90 (daunorubicin 90mg/m² + cytarabine 100mg/m²) (10%). Azacitidine in standard dose was used as the low-intensity regimen.

There were no statistically significant differences in miRNAs expression according to complete remission (CR) achievement. Patients with high-risk AML had higher expression of miR-125b-5p (fold change- FC=2.3, p=0.017) and lower expression of miR-34a-5p (FC=0.3, p=0.029), miR-199b-5p (FC=0.5, p=0.039) and miR-15a-5p (FC=0.6, p=0.043) as compared to low- and intermediate-risk pts. In patients with NPM1 mutation, miR-34a-5p, miR-146a-5p and miR-15a-5p were significantly upregulated, whereas miR-139-5p, miR-424-3p and miR-452-5p were downregulated. FLT-3-mutated patients had significantly lower expression of miR-424-3p (FC=0.2, p=0.019) comparing to FLT-3-unmutated controls. In patients with unfavorable karyotype, upregulation of miR-125b-5p (FC=2.8, p=0.002) and miR-139-5p (FC=3.0, p=0.027) was observed meanwhile lower expression of miR-34a-5p (FC=0.3, p=0.035) in these patients was detected.

The median overall survival (OS) in the study cohort was 12.3 months. In univariate Cox regression analysis, hematopoietic cell transplantation-comorbidity index (HCTCI) score >0 (HR 2.86, 95%CI: 1.15-7.12, p=0.024), white blood cell (WBC) count at diagnosis (HR 1.01, 95%CI: 1.00-1.01, p=0.033), and higher expression (>median) of miR-199b-5p (HR 0.31, 95%CI: 0.12-0.81, p=0.027) (Fig.1) were factors influencing the outcome. In multivariate model, higher expression of miR-199-5p (HR 0.27, 95%CI: 0.08-0.91, p=0.036) retained their significant protective effect in the context of established prognostic factors.

Conclusions

Our preliminary results demonstrate that the expression of miRNAs involved in the microenvironment niche is deregulated in AML patients. The upregulation of miR-199-5p at the diagnosis is associated with longer overall survival of AML patients. Significantly lower expression of miR-199-5p was reported in ELN high-risk group. These data indicate that miR-199-5p may be a potential prognostic factor in AML patients. Larger studies are needed to confirm our observation.

Czemerska:Novartis: Honoraria; Pfizer: Honoraria; Celgene: Honoraria; Sandoz: Honoraria; Abbvie: Honoraria. Krawiec:Celgene/BMS: Honoraria. Pluta:Astellas: Honoraria; Celgen/BMS: Honoraria; Angelini: Honoraria; Novartis: Honoraria; Swixx Biopharma: Honoraria, Research Funding. Wierzbowska:Swixx Biopharma: Honoraria, Research Funding; Novartis: Honoraria; Gilead: Honoraria; Astellas: Honoraria; AbbVie: Honoraria; Celgene/BMS: Honoraria; Janssen: Honoraria; Servier: Honoraria.