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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 46  |  Issue : 4  |  Page : 255-263

Study of Wilms tumor 1 gene expression in patients with acute leukemia


1 MSC of Clinical Pathology, Faculty of Medicine, Tanta University, Egypt
2 Professor of Clinical Pathology, Faculty of Medicine, Tanta University, Egypt
3 Lecturer of Clinical Pathology, Faculty of Medicine, Tanta University, Egypt

Date of Submission26-Apr-2017
Date of Acceptance28-Jul-2018
Date of Web Publication02-Aug-2019

Correspondence Address:
Riham Moustafa Ahmed Abd-Elkodous
MSC of Clinical Pathology, Faculty of Medicine, Tanta University
Egypt
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DOI: 10.4103/tmj.tmj_44_17

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  Abstract 


Background Leukemias are a group of disorders characterized by the accumulation of malignant white blood cells in the bone marrow and blood. They are classified into two types: acute and chronic leukemia, which are further subdivided into lymphoid or myeloid categories according to the cell origin.
The Wilms tumor 1 (WT1) gene product is a regulatory molecule of cell growth and differentiation. Wild-type WT1 gene is overexpressed in hematological cancers [acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia].
Aim The aim of this study was to study the WT1 gene expression in patients with acute leukemia.
Patients and methods This study was carried out on 30 patients with acute leukemia. They were selected from patients admitted to the Tanta University Hospital.
All individuals were subjected to laboratory investigations. Complete blood picture, bone marrow aspiration examination, cytochemical staining, and immunophenotyping and specific investigations by study of WT1 gene expression by real-time PCR were done.
Results Positive WT 1 gene expression was detected in 70% of studied patients and negative WT 1 expression in 30% of studied patients. Moreover, positive WT 1 gene expression was detected in 84.6% of patients with AML. However, positive WT 1 gene expression was detected in 42.9% of patients with ALL.
Conclusion The WT1 gene expression was increased in patients with acute leukemia. Moreover, the level of WT1 gene expression was increased in AML than ALL. The level of WT1 gene expression is decreased in patients with acute leukemia after chemotherapy and in patients during remission than its level before chemotherapy and in relapsed patients after chemotherapy.
WT1 gene positive expression may be a good prognostic factor in patients with acute leukemia.

Keywords: acute lymphoblastic leukemia, acute myeloid leukemia, Wilms tumor 1 gene


How to cite this article:
Abd-Elkodous RA, Abo-Elenein AM, Hamam SA. Study of Wilms tumor 1 gene expression in patients with acute leukemia. Tanta Med J 2018;46:255-63

How to cite this URL:
Abd-Elkodous RA, Abo-Elenein AM, Hamam SA. Study of Wilms tumor 1 gene expression in patients with acute leukemia. Tanta Med J [serial online] 2018 [cited 2020 Jun 6];46:255-63. Available from: http://www.tdj.eg.net/text.asp?2018/46/4/255/263919




  Introduction Top


Leukemias are a group of disorders characterized by the accumulation of malignant white blood cells in the bone marrow and blood. They are classified into two types: acute and chronic leukemia, which are further subdivided into lymphoid or myeloid categories according to the cell origin. Myeloid and lymphoid leukemias differ from one another regarding clinical presentation, course, and response to therapy [1].

The incidence rates of acute leukemia are greater in developed countries and in industrialized cities. Studies revealed increased risk for western European Jews [2]. Leukemia cells accumulate in the bone marrow cavity, ultimately replacing most of the normal hematopoietic cells, thus resulting in the signs and symptoms of the disease. These include most prominently, bone marrow failure and its consequences of anemia, hemorrhage, and infection. Leukemia cells circulate into the blood, tissues, and organs throughout the body, with patterns characteristic of the particular type of leukemia [3].

The Wilms tumor 1 (WT1) gene product is a regulatory molecule of cell growth and differentiation. In intrauterine life, WT1 is expressed primarily in the urogenital system. In adult life, WT1 gene expression is found in the urogenital system, central nervous system (CNS), bone marrow, and lymph nodes [4].

The WT1 gene located at chromosome 11p13 has 10 exons, which through two splicing events generates four variants with different functions [5]. The N-terminus domain is involved in RNA and protein interactions. The C-terminus domain consists of four cysteine, two histidine, and two zinc fingers, which bind to target DNA sequences and regulate many growth and differentiating factors (bcl-2, c-myc, IGF-II, IGF-I receptor, CSF-1, PAX-1, and RAR-α) [6].

WT1 was first described as a tumor-suppressor gene in WT. Inactivation of both alleles of WT1 gene resulted in WT. Its function, however, is more complex and depends on the environment and tissue specificity. Wild-type WT1 gene is overexpressed in hematological cancers [acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia] and other cancers (lung, prostate, CNS, breast cancer, neuroblastoma, ovarian, pancreatic, desmoid tumor, etc.) [7].

WT1 gene is overexpressed in 70–90% of leukemic cells, irrespective of the type of leukemia. WT1 gene expression in peripheral blood may detect minimal residual disease in all types of leukemias [8].


  Patients and methods Top


A prospective randomized study was carried out on 30 patients with acute leukemia. They were 14 males and 16 females, with aging from 3 to 65 years. They were selected from Hematology and Oncology Unit, Internal Medicine Department, Pediatric Department, Tanta University Hospital, and Tanta Cancer Institute. This study was conducted after approval of Research Ethics committee in Faculty of Medicine, Tanta University. An informed consent was obtained from the patients before their enrollment in this study. In addition, 10 apparently healthy controls with matched age and sex were selected to act as a control group.

They were classified into three groups:
  1. Group І: patient group (N=20).
    • Twenty patients of newly diagnosed AML and ALL.
  2. Group ІІ: patient group (N=10).
    • Ten patients of AML and ALL after chemotherapy.
  3. Group ІІI: control group (N=10).


This group included 10 apparently healthy as a control.

Inclusion criteria were patients diagnosed with acute leukemia, and exclusion criteria include any nonhematological malignancies, any other organic diseases, and any other hematological malignancy.

Patients and controls were subjected to the following clinical and laboratory studies:
  1. Detailed history taking, including name, age, sex, residence, bleeding, and ecchymosis.
  2. Clinical examination: general and local examination including examination of the liver, spleen, and lymph nodes.
  3. Laboratory investigations
    1. Routine investigations:
      1. Complete blood picture including hemoglobin concentration, platelets count, and white blood cells count performed by automated cell counter. Peripheral blood films were stained with Leishman stain [9] and examined for detection of blast cells and their percent.
      2. Liver function tests, kidney function tests, and lactate dehydrogenase levels.
    2. Investigations for diagnosis of acute leukemia (for patients only):
      1. Bone marrow aspiration smears: for morphological diagnosis of acute leukemia.
      2. Cytochemical analysis of air-dried peripheral blood and/or bone marrow smears: it was helpful in distinguishing AML from ALL and in subclassifying each type. The cytochemical stains included myeloperoxidase, periodic acid Schiff stain, and nonspecific esterase (α-naphthyl acetate esterase).
      3. Immunophenotyping of leukemic blasts: it was important in distinguishing AML from ALL and in subclassifying each type, using flow cytometer. The panel of monoclonal antibodies included c myeloperoxidase, CD13, CD33, CD117, HLA-DR, CD34, CD14, CD11b, cCD3, CD7, CD10, CD20, and CD19 by flow cytometry, compared with internal negative control cells [10].
    3. Specific investigations:
      1. Detection of WT1 gene expression by real-time PCR.


Sample collection

Six milliliters of peripheral blood was collected under complete aseptic conditions. These were aliquoted as follows: 2 ml on K3 EDTA for complete blood count and morphological studies, and also 4 ml on K3 EDTA for real-time PCR. Cytogenetic studies were collected from data of patients.

Detection of WT1 gene expression by real-time PCR

Isolating RNA from whole blood

RNA extraction

RNA extraction was done using the RNeasy Minikit (Qiagen, USA).

Purified RNA was stored on ice when using the RNA within a few hours of isolation. For long-term storage, purified RNA was stored at −80°C [11].

cDNA synthesis

To reverse transcribe the RNA into cDNA, we used first-strand cDNA synthesis kit. cDNA synthesized with this system could be directly used as a template in PCR or real-time PCR [12].

Expression of Wilms tumor 1 gene in the studied groups determined by real-time PCR

The assay was performed using real-time PCR (Applied Biosystem, USA).

The used primers and probes

WT1 expression was studied using the following forward (f) and reverse (r) primers: (f) 5′-CAGGCTGCAATAAGAGATATTTTAAGCT-3′;

(r) 5′-GAAGTCACACTGGTATGGTTTCTCA-3′; and the hybridization probe: F5′-CTTACAGATGCACAGCAGGAAGCACACTG-3′ T [8].

Each plate should contain a TaqMan gene expression assay for each cDNA sample.

Endogenous control assays

The TaqMan Universal PCR Master Mix may be used for real time or plate read (end point) detection of DNA or cDNA. Analysis was performed using Step One Real-Time PCR System (Applied Biosystem) [12] ([Figure 1] and [Figure 2]).
Figure 1 Example of amplification plot WT1. WT1, Wilms tumor 1.

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Figure 2 Example of amplification plot GAPDH.

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Statistical analysis

Statistical presentation and analysis of the present study was conducted, using the mean, SD, and χ2 test by statistical package for the social science, version 20 (Faculty of Medicine, Tanta University).


  Results Top


Regarding clinical findings, there were no significant differences between both studied groups as shown in [Table 1].
Table 1 Statistical comparison between group I and group II regarding clinical findings

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Moreover, there was a statistically significant difference between the three studied groups regarding hemoglobin concentration (g/dl), platelet (×103/cmm), and WBCs (×103/cmm), and also blast % in bone marrow, as shown in [Table 2].
Table 2 Statistical comparison between the three studied groups regarding hemoglobin concentration (g/dl), platelet (×103/cmm), and WBCs (×103/cmm)

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There was a statistically significant difference between group I and group II regarding blast % in bone marrow, as shown in [Table 3].
Table 3 Statistical comparison between group I and group II regarding blast % in bone marrow

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[Figure 3] and [Figure 4] show significant difference between both studied groups regarding WT1 gene expression.
Figure 3 Statistical comparison between group I and group II regarding WT1 gene expression. WT1, Wilms tumor 1.

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Figure 4 Statistical comparison between the studied groups regarding CT value for WT1 gene expression. CT, cycle threshold; WT1, Wilms tumor 1.

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There was a statistically significant difference among the three studied groups regarding cycle threshold (CT) value of WT1 gene expression, and between group I and group II and between group I and group III. However there is no statistically significant difference between group II and group III.

There was a significant difference between both AML and ALL regarding WT1 gene expression, as P value was 0.049, as shown in [Table 4].
Table 4 Wilms tumor 1 gene expression in both acute myeloid leukemia and acute lymphoblastic leukemia in group I

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[Figure 5] shows a statistically significant difference between cases of remission and relapse after induction chemotherapy regarding CT value for WT1 gene expression.
Figure 5 Statistical comparison between cases of remission and relapse after induction chemotherapy regarding CT value for WT1 gene expression. CT, cycle threshold; WT1, Wilms tumor 1.

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There were statistically significant strong positive correlations between WT1 gene expression with blast % in bone marrow and CD34 in patients with acute leukemia before chemotherapy, as shown in [Figure 6]a and b, and statistically significant negative correlation between WT1 gene expression with blast % in bone marrow in patients with acute leukemia after chemotherapy, as shown in [Figure 6]c.
Figure 6 Significant strong positive correlation between WT1 gene expression and blast bone marrow % (a) and CD34 (b) in bone marrow in patients with acute leukemia before chemotherapy and significant negative correlation between WT1 gene expression and blast bone marrow % in patients with acute leukemia after chemotherapy (c). WT1, Wilms tumor 1.

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  Discussion Top


Leukemias are a group of disorders characterized by the accumulation of malignant white blood cells in the bone marrow and blood. They are classified into two types: acute and chronic leukemia, which are further subdivided into lymphoid or myeloid categories according to the cell origin. Myeloid and lymphoid leukemias differ from one another regarding clinical presentation, course, and response to therapy [1].

The WT1 gene was originally defined as a tumor-suppressor gene, but we proposed, on the basis of the accumulating evidence, that the WT1 gene plays an oncogenic function in leukemogenesis and tumorigenesis. Inactivation of both alleles of WT1 gene resulted in WT. Its function, however, is more complex and depends on the environment and tissue specificity. Wild-type WT1 gene is overexpressed in hematological cancers (AML, ALL, and chronic lymphocytic leukemia) and other cancers (lung, prostate, CNS, breast cancer, neuroblastoma, ovarian, pancreatic, desmoid tumor, etc.) [8].

WT1 gene is overexpressed in 70–90% of leukemic cells, irrespective of the type of leukemia. WT1 gene expression in peripheral blood may detect minimal residual disease in all types of leukemias [8].

To determine the expression levels of the target (WT1) in unknown samples, the CT was determined. Its increased level means lower WT1 gene expression and vice versa. The value of negative control was 39.82, and the value of positive control was 22.93 [13].

In this study, positive WT 1 gene expression was detected in 70% of studied patients and negative WT 1 expression in 30% of studied patients. These findings were in agreement with Adrienne et al. [8], who found that WT1 gene is overexpressed in 70–90% of leukemic cells.

Moreover, in the present study, positive WT 1 gene expression was detected in 84.6% of patients with AML. However, positive WT 1 gene expression was detected in only 42.9% of patients with ALL. The results showed that increased WT1 expression in AML was more than ALL. The same results were obtained by Østergaard et al. [14], who found that WT1 was overexpressed in ∼70–90% of patients with AML, and Asgarian Omran et al. [15], who found that WT1 was overexpressed in 51.6% of patients with ALL.

However, this was not in agreement with Ibrahim et al. [16] who found that the WT1 transcript was overexpressed in 34% of patients with AML at diagnosis and found that WT1 was expressed in only 14% of newly diagnosed patients with ALL, as the WT1 gene has been shown to be overexpressed in immature leukemia cells, especially those with myeloid characteristics [17].

The results of the present work showed statistically significant increase of WT1 gene expression in group I before chemotherapy and cases of relapse after chemotherapy in group II when compared with control group III (P=0.0001) and this was in agreement with that of Rosenfeld et al. [6] and Asgarian Omran et al. [15] who found that high levels of WT1 expression detected both in bone marrow and peripheral blood samples have been demonstrated to be of adverse prognostic significance.

CT value of WT1 gene expression in patients with ALL after induction chemotherapy was significantly less than their levels in the patients before chemotherapy. It ranged between 21.5 and 39.8; however, their value before chemotherapy ranged between 21 and 39, with P value 0.018. This was in agreement with Rosenfeld et al. [6], Asgarian Omran et al. [15], and Adrienne et al. [8] who found that the WT1 expression decreased to very low levels or even disappeared during remission and reached high levels several months before clinical relapse, which qualified WT1 gene as a sensitive tool in leukemia monitoring.

Regarding immunophenotyping, there was a statistically significant difference between group I and group II with respect to CD34 (P=0.004), CD33 (P=0.032), HLA-DR (P=0.001), and CD64 (P=0.020), with no expression of CD14 in the studied groups, but there was no statistically significant difference between group I and group II regarding CD19, CD10, and TdT. This indicates that WT1 gene was expressed more in the immature leukemia cells. This was confirmed by Ishikawa et al. [17] who found that the WT1 gene has been shown to be overexpressed in immature leukemia cells, especially those with myeloid characteristics.There was a significant positive correlation regarding cells that express CD34 (r=0.663 and P=0.001), and this was in agreement with Hosen et al. [18] and Ishikawa et al. [17] who suggest that WT1 is expressed in human CD34+ bone marrow cells, but not in CD34− bone marrow cells or in peripheral blood mononuclear cells, which is owing to expression of WT1 in human cells of hematopoietic origin.

They suggested a role for WT1 in control of proliferation and/or differentiation of hematopoietic cells and characterization of its expression pattern, which indicates that WT1 is expressed in primitive immature cells.

On the contrary, Adrienne et al. [8] found that WT1 gene is expressed in low levels in CD34+ primitive cells, which was explained by induced controlled self-renewal of cells.

Regarding blasts in the bone marrow, there was a significant positive correlation between WT1 gene expression and blasts count in bone marrow (r=0.771 and P=0.001), and this was in agreement with that of Ibrahim et al. [16] and Gallo et al. [19] who reported that blasts seen in nearly all patient either circulating in the peripheral blood or in bone marrow, as the WT1 gene has been shown to be overexpressed in immature leukemia cells.

This study proved that detection of WT1 gene is important as leukemia-associated molecular marker that may be used for the diagnosis and for monitoring clinical progress in ALL.


  Conclusion and recommendations Top


The WT1 gene expression is increased in patients with acute leukemia. The level of WT1 gene expression was increased in AML than ALL. The level of WT1 gene expression is decreased in patients with acute leukemia after chemotherapy than its level before it. The level of WT1 gene expression is decreased in patients with acute leukemia during remission than its level in relapsed patients after chemotherapy. There was no significant correlation between WT1 gene expression and the clinical findings in patients with acute leukemia. There was a significant positive correlation between WT1 gene expression and blast number in bone marrow in patients with acute leukemia.

WT1 gene positive expression may be a good prognostic factor in patients with acute leukemia. The recommendations are wide scale of study of WT1 gene expression in patients with acute leukemia. The study of WT1 gene expression in patients with acute leukemia before and after chemotherapy with observation of its level at complete remission and relapse to detect if its level decreased at complete remission or not. Early detection of WT1 gene expression in newly diagnosed patients with acute leukemia to predict their prognosis and response to therapy. WT1 protein expression could be detected in various leukemia cell lines as a prognostic marker. WT1 can be considered as possible target for immunotherapy in acute leukemia.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Østergaard M, Olesen LH, Hasle H, Kjeldsen E, Hokland P. WT1 gene expression: an excellent tool for monitoring minimal residual disease in 70% of acute myeloid leukaemia patients − results from a single-centre study. Br J Haematol 2004; 125:590–600.  Back to cited text no. 14
    
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
 
 
    Tables

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