الفهرس 
Acute Lymphoblastic LeukemiaOnly 14 pages are availabe for public view
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Abstract
Background: Recent advances in the molecular characterization of B-ALL genetic profiling offer opportunities to identify novel genetic aberrations, providing insight into the pathogenesis of high risk ALL associated with treatment failure. It has been reported that overexpression of the CRLF2 gene is associated with poor outcome in pediatric precursor B- cell acute lymphoblastic leukemia (B-ALL).
Objectives: This study analyzed CRLF2 gene expression in precursor B-ALL pediatric patients and studied its correlation with various clinicopathological characteristics and standard prognostic factors.
Results: CRLF2gene was expressed in all studied patients and this expression was significantly higher in patients than controls with P-value <0.001, moreover, 21out of 40 patients (52.5%) showed high gene expression.
There was statistically significant positive correlation between CRLF2 gene expression and WBCs count. In our patients, 28 (70%) were of low risk subset, of which 13 had CRLF2 overexpression. There was no statistically significant correlation found between CRLF2 gene expression and other standard prognostic factors of the studied patients.
Conclusion: CRLF2 expression at diagnosis was significantly correlated with WBC count in pediatric B-ALL patients. CRLF2 expression detection at diagnosis might be a good supplement to evaluate B-ALL and needs further studies on larger scale of patients
Keywords: Acute Lymphoblastic Leukemia, CRLF2
INTRODUCTION
B‐cell acute lymphoblastic leukemia (B‐ALL) is a major childhood leukemia with a first peak distribution occurring around 5 years of age, but it also represents around 20% of adult leukemia cases, with a second peak distribution around 50 years of age [1]
ALL cases are broadly classified as B-ALL or T-ALL based on immunophenotyping, with B-ALL comprising approximately 85% of cases, although this percentage can differ depending on age at diagnosis, race, or ethnicity [2]
B-ALL is characterized by the acquisition of recurrent and clonal genetic aberrations. Although significant progress has been made in treating B‐ALL by risk‐adapted approach, approximately 20% of patients with childhood B‐ALL fail to respond to current therapeutic regimens, and those who relapse often show a dismal outcome [3][4]
It appears that chromosomal alterations and mutations that are associated with the disease may be inherited from pregnancy or develop through infancy and childhood, and these alterations and mutations then interact with certain environmental exposures which can lead to at least some types of ALL [5].
Recent advances in the molecular characterization of B‐ALL genetic profiling offer opportunities to identify novel genetic aberrations in essential lymphoid development and signal transduction pathways, providing insight into the pathogenesis of high risk ALL associated with treatment failure [6].
Among the novel lesions that were identified in pediatric cohort, a deregulated expression of the cytokine receptor-like factor 2 (CRLF2) gene. The CRLF2 gene encodes a subunit of the thymic stromal lymphopoietin (TSLP) receptor that heterodimerizes with the interleukin‐7 receptor (IL7R) to form the type I cytokine receptor. The type I cytokine receptor is a functional receptor for TSLP that is an important physiologic signal for early B‐cell development. Upon ligand binding, the type I cytokine receptor interacts with JAK proteins which in turn phosphorylate certain targets of STAT5 to promote transcription of proliferation and anti‐apoptotic factors [7].
Importantly, overexpression of CRLF2 is associated with a particularly poor prognosis, with these patients demonstrating significantly worse relapse-free survival relative to patients without CRLF2 overexpression. While the molecular basis for this clinical observation is currently unknown, this suggests that these patients may have intrinsic resistance to conventional chemotherapy [8].
We aimed to evaluate CRLF2 gene expression in pediatric B-ALL patients and to investigate its correlation with various clinicopathological characteristics and standard prognostic factors.
PATIENTS AND METHODS:
This case Control study was conducted on 40 newly diagnosed precursor B-ALL children recruited from pediatric hematology clinic, Ain Shams University hospital during the period from January 2021 until January 2022. Their ages ranged from 2-12 years (Median 5.5 years). Twenty two patients were males and 18 were females with male to female ratio of 1.2:1. An informed consent was obtained from patient’s guardians before participation in the study. Twenty age and sex matched non-leukemic subjects were enrolled as controls
The procedures applied in this study were approved by the Ethical Committee of Human Experimentation of Ain Shams University, and are in accordance with the Helsinki Declaration of 1975.
Patients were diagnosed on the basis of: i) complete history taking and through clinical examination; ii) laboratory investigations including complete blood count (CBC) using Sysmex XN 1000 (Sysmex corporation, Japan), examination of Leishman stained peripheral blood (PB) films, bone marrow (BM) aspiration and examination, cytochemical studies using myeloperoxidase stain together with flow cytometric immunophenotyping using EPICS XL Coulter flowcytometer (Coulter Electronics, USA).
ALL patients receiving treatment, in addition to patients with malignant disorders other than ALL were excluded from the study.
CRLF2 gene expression analysis:
CRLF2 gene expression was analysed by real time quantitative reverse transcription Polymerase chain reaction (qRT-PCR) using (Biometra, Germany) for reverse transcription and (Rotor-Gene, Germany) for real time PCR.
Data analysis and interpretation was done by viewing the amplification blots and setting threshold values. The cycle threshold (Ct) was determined for CRLF2 and GAPDH housekeeping gene in each experimental sample which is the intersection between an amplification curve and a threshold line.
Results were reported in relative quantification and normalized to the endogenous reference gene through the following steps:
1. ΔCT = CT target gene – CT house keeping (patient group)
2. The average (mean) ΔCT was calculated for samples of the control
3. (ΔΔCT) = ΔCT of the patient – mean of ΔCT of control samples
4. The relative expression level for CRLF2 in each sample was calculated by the comparative cycle threshold (2-ΔΔCT)
Sample collection:
1. 2 mL of PB were collected on dipotassium ethylene diaminetetraacetic acid (K2-EDTA) solution (1.2 mg/dL) and were used for CBC and preparation of Leishman-stained PB smears.
2. Adequate amount of BM aspirate was used for BM examination, 1.5 ml of BM was collected and subdivided into two EDTA tubes, and one was used for IPT and other tube for PCR.
Statistical analysis
Data were collected, revised, coded and entered to the Statistical Package for Social Science (IBM SPSS) version 23.
The confidence interval was set to 95% and the margin of error accepted was set to 5%. So, the p-value was considered significant as the following:
P-value > 0.05: Non significant (NS)
P-value < 0.05: Significant (S)
P-value < 0.01: Highly significant (HS).