Study design and setting
Case-control study of patients with AML. The patients were recruited from hematology unit, Clinical Pathology Department during the years 2018-2019.
Subjects
The current study was conducted on 65 subjects (35 newly diagnosed AML adult patients (before initiation of chemotherapy) and 30 adult controls). The diagnosis of AML was based on World Health Organization (WHO), 2016 AML diagnostic criteria [11]: blast cells constitutes > 20% of all BM nucleated cells, morphology, especially presence of Auer rods, immunophenotyping (IPT) to detect lineage specific cluster of differentiation (CD) markers and myeloperoxidase. Classification of AML according to the French-American-British (FAB) classification criteria was done [12].
Follow-up of the patients by BM blast % was done at day 28 and afterwards till either the end of the study or last contact with the patient (at least for 1 year from the start of the study). Accordingly, patients were classified as responders (BM blasts % less than 5% at day 28 of starting chemotherapy), non-responders (BM blasts % more than 5% at day 28 of starting chemotherapy), and relapsed (BM blasts % increased more than 5% during follow up) patients.
Thirty non-leukemic adult patients who are candidates for bone marrow aspiration for causes other than hematological malignancies as immune thrombocytopenic purpura and hypersplenism were studied as controls.
Inclusion criteria
All included patients are adults newly diagnosed AML patients.
Exclusion criteria
Cases of transformation of myelodysplastic syndrome (MDS), acute transformation of CML, relapsed or therapy-related AML, and other types of malignant tumors were excluded from this study.
Ethics approval and consent to participate
The approval of the study was taken from the Institutional Ethics Committee (Ethical Committee’s reference number: 138/2018; 27 May 2018). Written informed consent was taken from all patients who were invited to participate in the research.
All patients were subjected to full history taking, thorough clinical examination, complete blood count (CBC) on Sysmex-XN 1000TM using peripheral blood (PB) samples with examination of Leishman-stained PB films, BM aspiration with examination of Leishman-stained BM smears, IPT carried on BM blasts/blast equivalent cells using a standard panel of monoclonal antibodies using Navios flowcytometer (Coulter, Electronics, Hialeah, FL, USA) and cytogenetic analysis carried on BM samples, if feasible, searching for translocation (t)(15;17), t(8;21), t(9;22), inversion (inv)16, and duplication of X chromosome using Laika. HOTAIR expression level on BM mononuclear cells was done on BM samples by qRT-PCR using (Biometra, Germany) for reverse transcription and (Rotor-Gene, Germany) for real time PCR. This was performed on fresh samples taken from both controls and patients at time of diagnosis before initiation of chemotherapy. Another sets of samples were taken from patients at day 28 after treatment and during follow-up for assessment of BM blasts %. No stored samples were used.
Sampling
Three milliliters (mL) of venous blood were aseptically collected from each patient, dispensed in a K2-ethylene diamine tetraacetic acid (K2-EDTA) tube to be used for CBC and Leishman-stained PB smear. BM aspiration was done for all subjects and several BM smears were spread to be examined by Leishman stain. In total, 1.5 mL of BM aspiration was drawn and dispensed into sterile K2-EDTA vacutainer tube to be used for IPT. A total of 0.5 mL BM samples were drawn and dispensed into heparin anticoagulated tubes for cytogenetic analysis. In total, 1.5 mL of BM aspiration was drawn and dispensed into sterile K2-EDTA vacutainer tube to be used for measurement of HOTAIR expression level on BM mononuclear cells by qRT-PCR.
These samples were taken from both controls and patients at time of diagnosis before initiation of chemotherapy. Another sets of BM aspiration samples were taken from patients at day 28 after treatment and during follow-up for BM blasts % assessment.
HOTAIR expression level on BM mononuclear cells measurement by qRT-PCR
Ribonucleic acid (RNA) extraction
RNA extraction was done using QIAamp RNA Blood Mini Kit, Qiagen (04/2010), catalog (cat.) number (no.): (52304) (http://www.qiagen.com). RNA species longer than 200 bases bind to the QIAamp silica-based membrane using a specialized high-salt buffering system. Erythrocytes are lysed and leukocytes are recovered by centrifugation then lysed using highly denaturing conditions that immediately inactivate ribonucleases (RNases), allowing the isolation of intact RNA. Homogenization of the lysate is done by a brief centrifugation through a QIAshredder spin column. Ethanol is added to adjust binding conditions and the sample is applied to the QIAamp spin column. RNA is bound to the silica membrane during a brief centrifugation step. Contaminants are washed away and total RNA is eluted in 30 microliters (uL) or more of RNase-free water for direct use.
Integrated removal of genomic DNA contamination and complementary DNA (cDNA) synthesis
This was done using QuantiTect® Reverse Transcription, Qiagen (1056039 03/2009), cat. no.: (204654, 204754) (http://www.qiagen.com).
It comprises 2 main steps: elimination of genomic DNA and reverse transcription (RT) [13] (http://www.qiagen.com).
Elimination of genomic DNA
The purified RNA sample is briefly incubated in genomic DNA Wipeout Buffer at 42 °C for 2 min to effectively remove contaminating genomic DNA, the RNA sample is then used directly in reverse transcription.
Reverse transcription
The entire reaction takes place at 42 °C and is then inactivated at 95 °C. Quantiscript reverse transcriptase has a high affinity for RNA and is optimized for efficient and sensitive cDNA synthesis from 10 picograms (pg) to 1 microgram (ug) of RNA. This high RNA affinity, in combination with quantiscript RT buffer, enables high cDNA yields. RT primer mix ensures cDNA synthesis from all regions of RNA transcripts, even from 5′ regions. This allows high yields of cDNA template for real-time PCR analysis regardless of where the target region is located on the transcript.
qRT-PCR
HOTAIR TaqManTM Gene Expression Assay, ThermoFisher, cat. no.: (4448892) (Fig. 1) (https://www.thermofisher.com/taqman-gene-expression/) with primer sequence (Forward 5′-GCA GTA GAA AAA TAG ACA TAG GAGA-3′, Reverse 5′-AAT GAT AGG GAC ACA TCG GGG AAC T-3) (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4774541/) was used.
TaqManTM Gene Expression Master Mix, ThermoFisher (Part No: 4371135 Rev. C, 07/2010) (https://www.thermofisher.com), was used for qRT-PCR which contains AmpliTaq Gold®, DNA Polymerase, UP (Ultra Pure), Uracil-DNA Glycosylase (UDG), deoxyribonucleotide triphosphates (dNTPs) with deoxyuridine triphosphate (dUTP), ROX™ Passive Reference and Buffer components optimized for sensitivity, precision, specificity, and duplexing. The PCR reaction exploits the 5′ nuclease activity of AmpliTaq® Gold DNA Polymerase, UP (Ultra Pure) to cleave a TaqMan® probe during PCR. The TaqMan probe contains a reporter dye at the 5′ end of the probe and a quencher dye at the 3′ end of the probe. Cleavage of the probe separates the reporter dye and the quencher dye, resulting in increased fluorescence of the reporter. Accumulation of PCR products is detected directly by monitoring the increase in fluorescence of the reporter dye. The nuclease activity is fork-like and structure dependent. When the probe is intact, the proximity of the reporter dye to the quencher dye results in suppression of the reporter fluorescence primarily by Förster-type energy transfer, the probe specifically anneals to the target. The 5′ to 3′ nucleolytic activity of the AmpliTaq Gold, UP enzyme cleaves the probe between the reporter and the quencher only if the probe hybridizes to the target. The probe fragments are then displaced from the target, and polymerization of the strand continues. The 3′ end of the probe is blocked to prevent extension of the probe during PCR. This process occurs in every cycle, and it does not interfere with the exponential accumulation of product. Increased fluorescence signal is detected only if the target sequence is complementary to the probe and if it is amplified during PCR.
Housekeeping gene GABDH was used as an endogenous control to normalize the amount of total mRNA in each sample of HOTAIR between different samples. HOTAIR expression was measured as fold of control using the equation of ΔRn = (Rn+)−(Rn−), (where Rn+ = emission Intensity of Reporter PCR with template Emission Intensity of Passive Reference and Rn− = Emission Intensity of Reporter PCR without template or early cycles of a real-time Emission Intensity of Passive Reference reaction). Then the results is calculated as 2−∆∆CT (where CT= cycle threshold).
Statistical analysis
Descriptive and analytical statistical procedures were conducted. Data entry and statistical analysis of collected data was performed using Statistical Package for Social Science (SPSS version 23.0). Comparison between two groups regarding qualitative data was performed using chi-square test (X2). The comparison between two independent groups regarding quantitative data with parametric distribution was done by using independent t test (t) while comparison between two independent groups regarding quantitative data with non-parametric distribution was done by using Mann-Whitney test. Parameters correlations (correlation coefficient “r”) were performed by using Spearman’s correlation. Kruskal-Wallis test and post HOC analysis were performed for comparison among groups regarding quantitative non-parametric data. Receiver operating characteristic (ROC) as a graphical plot was done to determine the best cut-off value for HOTAIR as a diagnostic marker for AML. Kaplan-Meier analysis with log-rank test was used to assess the relation of HOTAIR with relapse free survival (event free survival (EFS)). Regarding power of significance, the probability level (p value) was considered significant (S) if p value was < 0.05, non-significant (NS) if p value was ≥ 0.05 and highly significant (HS) if p value was <0.01.
