Leukemia is caused by the unrestrained proliferation of hematopoietic stem cells and was reported to account for 3.8% of cancer deaths as of 2020 . Acute lymphoblastic leukemia (ALL) is a clonal malignancy of the lymphoid line of blood cells. It is the most common childhood cancer and the most common cause of cancer death in individuals younger than 20 years in the United States . Based on the involved cell line, the World Health Organization divides ALL into B-ALL and T-ALL, each of which can be detected by immunophenotyping. B-ALL is the most prevalent type among both children (88%) and adults (75%) . Despite numerous studies on the etiology of ALL, its causative agents have not been precisely identified. However, heredity, infections, and environmental factors such as radiation exposure play an essential role in ALL pathogenesis [2, 3]. Numerous studies have shown angiogenesis in leukemia by reporting increased levels of various angiogenic factors in patients and have suggested that anti-angiogenic drugs can act as a treatment agent for leukemia [4, 5]. The balance between angiogenic and anti-angiogenic factors secreted by vascular endothelial cells regulates angiogenesis, thus determining leukemia progression. Different types of angiogenic factors and their role in angiogenesis have been previously described; these factors include VEGF, FGF, angiopoietin, TGF, PIGF, HGF, HIF, and TNF [4, 6, 7].
The angiopoietin family (Ang1, Ang2, and Ang4) and its receptors (Tie1 and Tie2) are some of the most significant and well-known angiogenic factors . Ang1 and Ang2 play antithetical roles in the process of angiogenesis, and
The primary receptor of angiopoietins is Tie2, which upon binding with ligands, is activated by autophosphorylation and activates its downstream pathways [16, 17]. A soluble type of the Tie2 protein regulates angiopoietin activity by binding to one angiopoietin type (most commonly, Ang1) to increase the activity of the other angiopoietins . Tie1 is another angiopoietin receptor. No ligand is known for this receptor, and its function is induced through the formation of heterodimers with Tie2. Tie1 binds to Tie2 at the cell surface and prevents angiopoietins from binding to Tie2 . The Ang-Tie system plays a key role in the angiogenesis, proliferation, and maturation of vascular endothelial cells and is a useful therapeutic target in solid tumors and hematologic malignancies . However, few studies have been performed to determine expression level changes and the function of the angiopoietin family in ALL [20, 21].
In this study, we aimed to quantify the expression levels of the genes in the Ang-Tie system (
Forty bone marrow (BM) samples from newly diagnosed ALL patients (aged 0–19 yr) and 15 normal BM samples (from individuals aged 0–19 yr) were obtained from the bone marrow transplantation section of Taleghani Hospital in Tehran from March to October 2020. Pre-B-ALL and early pre-B-ALL cases, which are the most common types of ALL, were included in this study. Normal BM specimens were selected from individuals who were referred to our hospital because of high peripheral blood (PB) WBC count, but their BM specimen examination and flow cytometric tests did not confirm any malignancy or other diseases influencing the BM. The patients signed a consent form, and questionnaires were filled out for demographic data collection. The study protocol was approved by the ethical committee of Tabriz University of Medical Sciences (code: IR.TBZMED.REC.1397.1040).
The nucleotide sequences of designed primers for the target and normalizer genes are shown in Table 1. Total RNA was extracted and purified using the Qiagen RNeasy Mini Kit (Cat. No: ID: 74104; Qiagen, Germany) according to the manufacturer's protocol. Quality and concentration assessments of the RNA samples were carried out using a Thermo Scientific NanoDrop One (Thermo Fisher Scientific Inc., Waltham, MA, USA). The Thermo Scientific SuperScript IV Reverse Transcriptase kit was used for cDNA synthesis. We chose the
Real-time PCR efficiencies and the cycle threshold (CT) were used in the ^^CT formula [Δct (treated sample)–Δct (untreated sample)] to calculate the relative expression levels of the
In this study, 40 samples from newly diagnosed ALL patients and 15 samples from control individuals were collected. Demographic characteristics and data obtained from laboratory diagnoses of patients were collected using diagnostic tests or questionnaires filled out by the patients. Demographic and laboratory information of patients are shown in Table 2.
Statistical analyses were performed using the
We performed a correlation analysis between the expression levels of Ang-Tie system genes and ALL subtypes, patient age, blast count, sex, and PB WBC count. Table 3 shows that Ang-Tie system gene expression levels did not show a significant statistical associations across either patient demographic or clinical data.
ROC curves were plotted to evaluate the diagnostic roles of
ALL is caused by excessive proliferation and lack of differentiation in lymphoid blast cells, which eventually overwhelm the BM and PB. It is the most common cause of cancer-related deaths in individuals younger than 20 years in the United States .
Angiogenesis and its causative factors play critical roles in different types of cancers. In solid tumors, hypoxic conditions follow tumorigenesis, and the balance between pro-angiogenic and anti-angiogenic factors tends to tilt towards pro-angiogenic factors, which cause angiogenesis and tumor spread [4, 22]. Angiogenesis is also responsible for the spread of leukemia, and several studies have been performed to discern the effects of angiogenic factors in various forms of leukemia [4, 5]. The angiopoietin family and their receptors are among the most fundamental angiogenic factors [9, 10]. In the present study, we investigated the expression of genes belonging to this family, as well as of their receptors, in patients with ALL.
Our results indicated that Ang1 was expressed at lower levels in ALL patients than in control samples. The expression level of this gene was not significantly different across demographic or clinical characteristics (Table 3). Ang1 competes with Ang2 for binding to the Tie2 receptor, with Ang1 having a higher affinity. The expression of Ang1 is likely decreased in patients with ALL, leading to Ang2 activation. Examinations of
Another gene in the angiopoietin family is
The least studied member of the Ang-Tie system is Ang4. The precise function of Ang4 in angiogenesis has not yet been defined, due to the operational similarities of Ang4 with Ang1 . However, the expression of
In a study by Kivivuori
We performed ROC curve analysis to evaluate Ang-Tie system genes as diagnostic biomarkers in ALL samples. The combination of AUC values, sensitivity, and specificity of the Ang-Tie system genes can be considered as diagnostic biomarkers for patients with ALL. However, since we included only two types of ALL (pre-B and early pre-B-ALL), further studies with a large sample size that includes all types of ALL are needed to improve the sensitivity and specificity criteria of diagnostic markers. We recommend following up on the condition of patients after treatment to measure the effects of these proteins on prognosis, treatment, and survival. Furthermore, we believe it is crucial to investigate
In brief, we observed a significant decrease in
We thank all the individuals and their family members for their participation and support in this study.
*This study was supported by a grant from Tabriz University of Medical Sciences.
No potential conflicts of interest relevant to this article were reported.