The aim, design, and setting of the study
This study aims to determine the TLR9 expression in AML patients and its relation to the prognosis of the disease.
This is a prospective study conducted in the Hematology Department of the hospital from Aug, 2016 to Aug, 2018.
Participants and description of materials
Participants were 40 newly diagnosed AML patients managed in the hospital in addition to 20 sex and age matched normal volunteers as control.
The AML patients with mixed lineage expression, autoimmune diseases, coexisting secondary malignancy, hepatitis C viral infection, and bilharziasis were excluded. In patients presented with infection, the flow cytometry test for TLR9 was done after infection control by antimicrobial therapy for 7 to 10 days.
AML was diagnosed based on the finding of ≥ 20% blasts in the bone marrow, and the flow cytometry characterized the leukemic blasts using acute panel of monoclonal antibodies labeled by fluorescein isothiocyanate (FITC) (green) or phycoerythrin (PE) (red).
Cytogenetic studies were done by fluorescence in situ hybridization (FISH) analysis, and the patients were classified as favorable, intermediate, and poor risk groups based on the cytogenetic findings [11]. Favorable cytogenetic risk is defined by the presence of t(8;21)(q22;q22) and/or detection of an AML1-ETO translocation product, independent from additional cytogenetic findings. Or the presence of an inv(16)(p13p22) or a t(16;16)(p13;p22) and/or detection of a CBFβ-MYH11 translocation product, independent from additional cytogenetic findings. Poor cytogenetic risk is defined by the presence of del(5q) or monosomy 5, del(7q) or monosomy 7, inv(3)(q21q26.2) or t(3;3)(q21;q26.2), 11q23 aberrations, or complex-aberrant karyotype comprising of three or more aberrations not involving low-risk aberrations. Intermediate cytogenetic risk is defined by the presence of all other aberrations not included in low or high risk [11].
The assay of TLR9 expression
The assay of TLR9 expression was performed on peripheral blood samples of AML cases before the start of treatment as well as the healthy controls. TLR9 is an intracytoplasmic antigen, detected after permeabilization using Tween 20 and paraformaldehyde for gentle fixation of cells. The monoclonal antibodies (MoAbs) of Abcam, Anti-TLR-9 antibody [5G5] (FITC) ab58864, the United Kingdom was used in analysis of washed cells by flow cytometer.
Immunofluorescence on the viable leukemic cells in suspension was analyzed using Becton Dickinson, FACS caliber flow cytometer equipped with the Cell Quest software [USA]. Gating was done around the leukemic blasts on the forward versus side scatter plots in the initial diagnostic specimens, while in the control specimens gating was done around the mononuclear cells on the forward versus side scatter plots. Representative histograms for gating and expression in one of the cases and one of the controls as follows (Fig. 1, 2).
Clinical outcomes and survival analysis
Clinical outcomes were based on the response to treatment. The response to treatment was defined according to the 2017 European Leukemia Network (ELN) recommendations [12], with evidence of persistent leukemia by blood and/or bone marrow examination.
Complete remission (CR) was defined by bone marrow blasts < 5% durable for at least 28 days, absence of blasts with Auer rods, absence of extramedullary disease, absolute neutrophil count > 1.0 × 109/L, platelet count > 100 × 109/L, and independence of red cell transfusions. Partial remission (PR) was defined by all hematologic criteria of CR, decreased bone marrow blast percentage from 5 to 25% and decreased pretreatment bone marrow blast percentage by at least 50%. Refractory disease (RD) was defined as the failure to achieve CR or PR, only includes patients surviving ≥ 7 days following completion of initial treatment, with evidence of persistent leukemia by blood and/or bone marrow examination.
The follow-up period was 24 months, from Aug, 2016 to Aug, 2018. Survival analysis was described as the overall survival time.
This study was ethically approved by the ethical committee of the hospital. Patients and their relatives were informed about the objective of the study and their written consents were taken prior to inclusion to this study.
Statistical analysis
Data processing was performed by the SPSS v.24 which revealed non-parametric distributed data a part of age and hemoglobin level of the studied groups. For these, parametric t tests were used. For other parameters, non-parametric tests were conducted (Mann-Whitney U test for two medians and Kruskal-Wallis test for more than 2 medians). Qualitative variables were tested by the chi-square test. The ROC curve for TLR9 was conducted to estimate the cutoff value with the highest sensitivity. The Kaplan Meier method was used to describe the median overall survival time in regard to the cutoff value of TLR9 and tested by the log-rank test. All tests were conducted with the 95% confidence interval and p values of ≤ 0.05 were considered statistically significant.