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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 44  |  Issue : 4  |  Page : 195-203

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


1 Department of Clinical Pathology, Faculty of Medicine, Zagazig University, Zagazig, Egypt
2 Department of Medical Oncology, Faculty of Medicine, Zagazig University, Zagazig, Egypt

Date of Submission20-Jul-2019
Date of Acceptance05-Aug-2019
Date of Web Publication20-Jul-2020

Correspondence Address:
Weaam I Ismail
Clinical Pathology Department, Faculty of Medicine, Zagazig University, Zagazig 44159, Egypt. Tel: 00201223140501;
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ejh.ejh_26_19

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  Abstract 


Background The Wilms’ tumor 1 (WT1) gene is overexpressed in patients with acute leukemias. Many studies have reported the importance of quantitative assessment of WT1 expression as a molecular marker of minimal residual disease monitoring. However, biological significance and the prognostic effect of WT1 overexpression in acute myeloid leukemia (AML) remain elusive.
Subjects and methods A total of 42 patients with newly diagnosed AML were included in the study. Also, 42 individuals matched for age and sex were enrolled as controls. Immunophenotyping, cytogenetic analysis, and quantitative assessment of WT1 gene transcripts were performed using real-time PCR.
Results WT1 overexpression was detected in 73.8% of our patient group. There was a statistically significant decrease in the probability of achieving complete remission with shorter overall survival and event-free survival in the WT1 overexpression group compared with the downexpression group (P=0.035, 0.045, 0.011, respectively). Application of multivariate analysis showed that WT1 overexpression by itself is an independent and negative indicator for predicting overall survival and disease-free survival of AML patients.
Conclusion WT1 overexpression is an independent negative prognostic marker that could be used to evaluate response to induction chemotherapy and prognosis of AML patients.

Keywords: acute myeloid leukemia, real-time PCR, Wilms’ tumor 1 expression


How to cite this article:
Ahmad EI, El-Akad GM, Ismail WI, Al Nagar AA. Study of Wilms’ tumor 1 gene expression in patients with acute myeloid leukemia. Egypt J Haematol 2019;44:195-203

How to cite this URL:
Ahmad EI, El-Akad GM, Ismail WI, Al Nagar AA. Study of Wilms’ tumor 1 gene expression in patients with acute myeloid leukemia. Egypt J Haematol [serial online] 2019 [cited 2020 Aug 12];44:195-203. Available from: http://www.ehj.eg.net/text.asp?2019/44/4/195/290228




  Introduction Top


Acute myeloid leukemia (AML) is a heterogeneous disease characterized by diverse genetic abnormalities and variable morphology, immunophenotypes, and clinical outcomes [1].

The Wilms’ tumor 1 (WT1) gene located at the chromosomal band 11p13 is considered as a tumor suppressor gene that encodes a zinc-finger transcription factor. It was first identified through its involvement in the pathogenesis of Wilms’ tumor [2],[3].

WT1 regulates the genes involved in cellular growth and metabolism. It was documented that it can influence cell survival, proliferation, and differentiation processes. Therefore, it is considered that it also act as an oncogene [4],[5].

The normal expression of WT1 is limited to early hematopoietic progenitor cells which suggests that it plays a critical role in hematopoietic development [4],[6].

However, many studies have demonstrated that WT1 is consistently overexpressed in AML, acute lymphoblastic leukemia, myelodysplastic syndrome, and blastic crisis of chronic myeloid leukemia [7],[8],[9].

The WT1 expression might thus be considered as a universal molecular marker of malignant hematopoiesis, and many studies claimed the usefulness of quantitative assessment of WT1 expression level as a molecular marker for minimal residual disease monitoring [7],[10],[11]. Also, it is suggested that its expression level might have prognostic implications with regard to the remission therapy and the overall survival (OS) of AML patients [12],[13],[14],[15],[16].

The aim of our study is to detect quantitative expression of WT1 gene in AML patients in a trial to assess the clinical relevance of this expression.


  Subjects and methods Top


Subjects

This study was carried out in the Clinical Pathology Department; Scientific and Medical Research Center and Medical Oncology Department, Faculty of Medicine, Zagazig University Hospitals during the period from August 2017 to September 2018. A total of 42 patients with newly diagnosed AML were included in the study after obtaining approvals from the Institutional Review Board of Zagazig University Hospital, Ethics Committee in Faculty of Medicine and from Clinical Pathology Department Committee. Patients were 24 men and 18 women; their mean±SD age was 42±13.53 years and ranged from 18 to 58 years. They were followed up for 1 year. Also, 42 individuals, age and sex matched, were selected as controls. They were 22 men and 20 women, their mean±SD age was 43.2±11.38 years and ranged from 21 to 56 years.

Samples

Peripheral blood (PB) and bone marrow (BM) samples were collected from all patients; samples were collected at the time of presentation, before therapy was initiated. Venous blood samples were aseptically withdrawn from each patient by venipuncture. One ml of the blood sample was delivered into a sterile vacutainer containing EDTA for CBC examination; 3 mLs were delivered into a sterile plain vacutainer tube for liver, kidney function testing, lactate dehydrogenase (LDH) estimation, and serological testing for viral markers and 1.8 ml was delivered into a sterile vacutainer tube containing trisodium citrate for prothrombin time and partial thromboplastin time analysis. We delivered 0.5 ml of the blood sample into a sterile vacutainer containing EDTA for molecular determination of WT1 gene expression by quantitative real-time PCR (RQ-PCR). Two mLs of the BM were aspirated from patients under complete aseptic conditions. BM smears were prepared; 1 ml of BM was delivered into an EDTA vacutainer tube for immunophenotyping by flowcytometry and 1 ml was delivered into a vacutainer containing lithium heparin for cytogenetic analysis.

Treatment plane

Patients were treated by an induction 3+7 regimen consisting of continuous infusion cytarabine (100 mg/m2) daily for 7 consecutive days combined with 3 days of doxorubicin (30 mg/m2). Patients with poor performance status were treated by 2+5 regimen (cytarabine 100 mg/m2 daily for 5 days combined with 2 days of doxorubicin 25 mg/m2) or low dose cytarabine 10 mg/m2/12 h for 14 days. Patients who achieved complete remission (CR) received consolidation therapy which is composed of three to four courses of high-dose cytosine arabinoside (3 g/m2 every 12 h on days 1, 3, and 5; total, 18 g/m2) [17].

Patients’ follow-up

The patients were followed up by CBC and BM aspiration to assess the responsiveness to therapy, CR, and relapse if found. They were followed up for 1 year to evaluate OS, event-free survival (EFS), and disease-free survival (DFS).

Methods

The participants enrolled into the study were subjected to the following: full history taking; clinical examination and abdominal ultrasonography; CBC; liver, kidney function tests; LDH; prothrombin time, partial thromboplastin time, and molecular detection of WT1 gene expression by RQ-PCR. The patients were subjected to BM aspiration and examination followed by cytochemistry and immunophenotyping by flowcytometry using Becton Dickinson (Franklin Lakes, New Jersey, USA) FACScan device using the following markers: CD34, CD13, HLA-DR, CD33, myeloperoxidase, CD14, and CD64. Conventional cytogenetic analysis by G banding technique and karyotyping according to the International System for Human Chromosome Nomenclature was also done for the patient group, which was followed with fluorescence in-situ hybridization studies in order to assess for the presence of conventionally detected cytogenetic abnormalities using specific probes (Vysis; Abbott Laboratories, Lake Bluff, Illinois, USA).

Cytogenetic analysis

Cultivation and harvesting

BM samples were cultured on RPMI-1640 (Sigma-Aldrich, St Louis, Missouri, USA) to which fetal calf serum, l-glutamine, and penicillin/streptomycin were added; the tubes were then incubated at 37°C in a CO2 incubator for 24 and 48 h. Colcemid was added to arrest mitosis followed by hypotonic treatment of the cell pellets and subsequent fixation with methanol–acetic acid solution.

Slide preparation and banding

Fixed cells were then dropped onto a frosted, alcohol-cleaned slide. Aging of slides for 24 h in an incubator at 37°C was done for proper banding. Five slides were prepared for each patient and examined under the phase contrast microscope for metaphases. Banding with trypsin solution and counterstaining with Giemsa was done. The slides were examined microscopically by an oil emersion lens. At least 20 metaphases were subjected to analysis [18]. We used an Imstar image analyzer for karyotyping (Paris, France).

Fluorescence in-situ hybridization studies assays

That were performed according to the probe manufacturer’s instructions. Slides were analyzed using an epifluorescence microscope (Olympus, BX63, Olympus Corporation Shinjuku, Tokyo, Japan) and computerized image analysis software (Cytovision Genetics Workstation; Leica Biosystem, Wetzlar, Germany). A minimum of 200 cells per specimen/probe were scored.

Wilms’ tumor 1 gene expression analyses

It included the following steps: RNA extraction from whole blood, RNA quantitation and quality assessment, cDNA synthesis, and RQ-PCR.

RNA extraction, RNA quantification, and cDNA synthesis

All subjects included in the study were subjected to total RNA extraction from EDTA anticoagulated PB using PureLink RNA Mini Kit (Invitrogen, Carlsbad, California, USA) according to manufacturer’s protocol. The concentration of RNA was measured in each sample using Quantus fluorometer (Promega, Madison, Wisconsin, USA). The input amount of total RNA in each sample was adjusted to 50 ng/µl. Single-stranded cDNA was synthesized from purified 10 µl RNA samples using the High-Capacity cDNA Reverse transcription Kit for RNA reverse transcription according to the manufacturer’s protocol (Applied Biosystems, Foster City, California, USA) on a PCR thermocycler (Verti; Applied Biosystems, Foster City, California, USA).

Quantification of Wilms’ tumor 1 expression

The analysis of WT1 gene expressions was constructed using RQ-PCR which was performed using TaqMan real-time PCR methods. A housekeeping gene β-actin was used as an internal control for calibration of possible variations caused by variable efficiencies of RNA extraction and reverse transcription reactions. The operation was performed on the Stratagene Mx3005P platform (Agilent Technologies, Santa Clara, California, USA). The quantification of both WT1 and β-actin genes was performed according to the manufacturer’s instructions. The assay IDs were: TaqMan universal master mix II, no UNG (Applied Biosystems, Foster City, California, USA), WT1 readymade TaqMan gene expression assays (Hs 011030751_m1), and β-actin readymade TaqMan gene expression assays (Hs 01060665_g1). The cycle threshold (Ct) values were obtained for WT1 and then normalized to β-actin. Relative quantitative analysis was performed using healthy controls as the calibrator and then the fold changes were calculated using the method [19].

Response to therapy

CR was characterized by morphologically normal marrow with less than 5% blasts, neutrophil count more than 1.5×109/l, and platelet count more than 100×109/l. Relapse was defined as more than 5% leukemic blasts in the BM aspirate or new extramedullary leukemia.

OS was measured from the protocol on-study date until the date of death regardless of the cause, censoring for those alive at the last follow-up. EFS was defined as the time from diagnosis to treatment failure, disease relapse or death by any causes. DFS was estimated from the time of first CR to relapse or death in CR. Patients who were still alive and disease free were censored at the date of last follow-up.

Statistical analysis

Analysis of data was performed using the SPSS computer program (version 24; SPSS Inc., Chicago, Illinois, USA). χ2 and Mann–Whitney tests were used for statistical analysis. Kaplan–Meier method was used to estimate survival and the difference between groups was analyzed by the log-rank test. Multivariate analysis was done. Odds ratio or hazard ratio (HR) with its 95% confidence interval (CI) was used for risk estimation. P value less than 0.05 was considered to be significant.


  Results Top


Wilms’ tumor 1 expression levels and clinical characteristics at diagnosis

The median expression value of the WT1 transcript in AML patients at diagnosis was 6.5 (range: 0.14–32), which was significantly greater than the expression found in healthy controls (median: 0.39; range: 0.03–1.41) (P<0.001; [Table 1]).
Table 1 Level of Wilms’ tumor 1 gene expression among the studied acute myeloid leukemia patients and control group

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We applied the receiver operating characteristic curve analyses for the definition of the minimum expression level above which the sample should be considered as WT1 overexpression. We used the value of 2.23 as a cutoff with an area under curve of 0.66 (95% CI: 0.47–0.84), sensitivity of 87.5%, and specificity of 32.1% ([Table 2] and [Figure 1]).
Table 2 Level of WT1 gene expression among the studied AML patients

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Figure 1 ROC curve for the level of WT1 gene expression among the studied AML patients.

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Using this cutoff value, our patients were divided into two groups: those with WT1 overexpression and those with WT1 downexpression. At diagnosis, 31 of the 42 (73.8%) patients had WT1 overexpression while 11 (26.2%) patients had WT1 downexpression. All subjects of the control group were included in the downexpression group ([Table 3]).
Table 3 Comparison between the control and patient groups as regards Wilms’ tumor 1 gene expression

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Comparing the level of WT1 expression with the clinical characteristics of the patients, we found that there is no statistically significant differences between overexpression and downexpression groups as regards age, sex, total leukocytes count, hemoglobin (Hb) level, platelets count, or LDH (P>0.05). The medians of BM and PB blast percent were statistically higher in the WT1 gene overexpression group than the WT1 gene downexpression group (P=0.009 and 0.019, respectively; [Table 4]).
Table 4 Comparison between the Wilms’ tumor 1 overexpression and downexpression groups regarding demographic and laboratory data

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WT1 expression demonstrated significant heterogeneity among the French–American–British subtypes. M0, M1, and M5 classes showed no significant difference between WT1 overexpression and downexpression groups; M2 class was statistically higher in the WT1 downexpression group compared with the overexpression group (P=0.035), while M4 was statistically higher in the WT1 gene overexpression group than in the downexpression group (P=0.009; [Table 5]).
Table 5 Comparison between Wilms’ tumor 1 overexpression and downexpression groups regarding French–American–British classification and cytogenetic analysis data

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We did not find any statistical difference between the WT1 overexpression group and the downexpression group as regards the karyotyping analysis results as being normal or abnormal (P=0.657). As regards cytogenetic risk categorization of AML patients as favorable, intermediate or poor risk, there was no statistical difference between the WT1 overexpression group and the downexpression group as regards the three risk groups (P=0.831; [Table 5]).

As regards immunophenotypic markers and their association with WT1 expression, there was no statistical difference between the WT1 overexpression group and the downexpression group as regards any of the surface markers used in the panel of diagnosis (P>0.05; [Table 6]).
Table 6 Association between Wilms’ tumor 1 expression and markers of immunophenotyping expression among the studied acute myeloid leukemia patients

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Prognostic significance of Wilms’ tumor 1 overexpression at diagnosis

We evaluated the potential role of WT1 overexpression in predicting the treatment outcome of AML patients. It was noted that the CR rates were statistically lower in the overexpression group than that in the downexpression group. Of the 31 patients with WT1 overexpression, 17 (54.8%) achieved CR while, of the 11 patients with WT1 downexpression, 10 (90.9%) reached CR and the difference between the two groups was significant (P=0.035). We found that patients with WT1 overexpression had a higher risk of relapse than patients with WT1 downexpression (23.5 vs. 0%; P=0.019; [Table 7]).
Table 7 Comparison between Wilms’ tumor 1 overexpression and downexpression groups regarding response to induction therapy, relapse risk, and survival rates

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The Kaplan–Meier survival analysis was used to calculate the OS, EFS, and DFS. The potential difference between the overexpression and down expression groups was analyzed by the log-rank test. Patients with WT1 overexpression had a statistically shorter OS (1 year OS: mean 7.7 vs. 11 months with percent probability of OS 54.8 vs. 90.9%, log-rank=4.02, P=0.045) and shorter EFS (1 year EFS: mean 7.3 vs. 11 months with percent probability of EFS 41.9 vs. 90.9%, log-rank=6.41, P=0.011) than those with WT1 downexpression but nonstatistical difference in DFS was found between the two groups (1 year DFS: 11.05 vs. 12 months with percent probability of DFS 76.5 vs. 100%, log-rank=2.59, P=0.107) ([Table 7] and [Figure 2]). On the basis of these results, we suggest that WT1 overexpression could play an important negative role in predicting OS and EFS of the AML patients.
Figure 2 Kaplan–Meier curve shows probability of (a) overall survival, (b) event-free survival, and (c) disease-free survival for the Wilms’ tumor 1 (WT1) overexpression and WT1 downexpression groups.

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Multivariate analysis of Wilms’ tumor 1 overexpression and its role in prognostic outcome of acute myeloid leukemia

In this study, we were interested in further testing if WT1 overexpression is an independent prognostic factor in predicting response to induction therapy, OS or DFS of AML patients. Multivariate modeling including WT1 expression, hepatomegaly, splenomegaly, Hb level, total leukocytes count, PB, and BM blast percentage was designed. Multivariate analysis was carried out using the logistic regression model (odds ratio) for response to induction therapy. It showed that Hb level is an independent prognostic factor (P=0.04); odds ratio was 1.996 (95% CI: 1.034–3.856); WT1 overexpression was an independent factor but with borderline significance (P=0.05) which affects the response to induction therapy. Multivariate analysis using Cox regression model (HR) for survival analysis was done. WT1 overexpression was the only independent prognostic factor which significantly affects the OS in the AML group (<0.001) with HR 1.190 (95% CI: 1.081–1.309). Also, WT1 overexpression was the only independent prognostic factor which significantly affects the DFS in the AML group (P=0.003) with HR 1.088 (95% CI: 1.028–1.151) ([Table 8]).
Table 8 Multivariate analysis (Cox regression) for clinical and laboratory variables of overall survival and disease-free survival in the patient group

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  Discussion Top


The study of WT1 involvement in malignancy had shown its potential role as an oncogene. The prime evidence supporting this is the overexpression of wild-type WT1 in a variety of human cancers of both hematological and nonhematological origin. The most studied of these is leukemia where there is a great evidence demonstrating WT1’s biological and clinical importance in cell survival, differentiation, and proliferation [5]. WT1 has been described to be both overexpressed and mutated in AML patients, and this overexpression has been reported to play a prognostic role. However, the precise mechanism through which WT1 may play a role in leukemogenesis has remained elusive [20].

In this study, the incidence of WT1 overexpression was73.8% in AML patients, which corresponds with other reports [12],[21],[22] but it was higher than that detected by other researches [23],[24]. This great discrepancy between different studies could be attributed to the use of different reference genes in determining the level of WT1 gene expression, different cutoff values for normal WT1 expression recommended in the different studies, and to other molecular aberrations that downregulates WT1 expression in the process of leukemia cell proliferation [23].

No statistical difference was observed between the WT1 overexpression and downexpression groups as regards age and sex, which comes in accordance with other studies [21],[23],[24],[25]. There was no significant difference observed between overexpression and downexpression groups as regards Hb level and platelet count at the time of diagnosis which was in agreement with the study of Assem et al. [21].

A significant association was observed between higher BM and PB blasts percent and WT1 overexpression (P=0.009 and 0.019, respectively) which came in congruence with Lane et al. [26]. Contradictory results were reported by Assem et al. [21] and Ibrahim et al. [24] who did not find a significant difference between BM and PB blast percent and WT1 expression level. The high WT1 expression level was hypothesized to originate from leukemic cells and it is precisely those leukemic cells that are the cause of treatment resistance [8]. WT1 overexpression is one of the the main features of leukemic stem cells and that leukemic stem cells are responsible for the occurrence of therapy resistance [27].

We demonstrated an association between M2 subtype and WT1 downexpression (P=0.035), while the M4 subtype was associated with WT1 overexpression (P=0.009). Polák et al. [28] found that the highest expression detected was in M4 and M1 cases and the least expression was observed in M5 and M2 cases while Marjanovic et al. [25] observed that the highest WT1 overexpression was observed in M2 and M4 cases but it was statistically nonsignificant. On the other hand, other studies found no significant difference as regards WT1 expression level and French–American–British subtypes [15],[21],[23],[24].

Concerning the association between WT1 expression and markers of immunophenotyping expression among the studied AML patients, it was found that there was no statistically significant difference between WT1 overexpression and downexpression groups as regards any of CD34, CD13, HLA-DR, CD33, myeloperoxidase, CD14, or CD64 expression (P>0.05). In different studies, investigators could not find any association between CD34 positivity and WT1 expression [21],[25].

Evaluation of WT1 expression in association with karyotype as being normal or abnormal showed nonsignificant results. As regards cytogenetic risk stratification, there was no significant difference among the three cytogenetic risk groups in relation to WT1 expression level which came in accordance with other studies [22],[24],[29]. Contradictory results were noted with both Ayatollahi et al. [22] who found that the highest WT1 expression was observed in normal karyotype followed by inversion 16 and least in t(8;21) and Lane et al. [26] who observed that WT1 levels correlated with the cytogenetic risk group with a higher level found in the favorable risk group.

We examined the possible role of WT1 gene overexpression in AML patient’s responses to induction chemotherapy, and in predicting the therapy outcome. We noted that CR rate was significantly reduced among the overexpression group than that in the downexpression group (P=0.035) which was also noted in other studies [21],[23],[25]. The negative impact which WT1 overexpression had in our patients on CR induction was not applied to other studies which claimed that the WT1 expression level has no influence on patient response to induction chemotherapy [15],[24]. Any therapeutic effect on CR induction is most certainly contributed to multifactorial factors such as patient’s age, physical status, peripheral total white blood counts, and many molecular factors. WT1 gene overexpression is at least one of the molecular factors that could play a negative prognostic role in predicting CR [23].

With follow-up, we found that the relapse risk is significantly higher in the overexpression group than in the downexpression group (P=0.019). In agreement with our results, Barragan et al. [15] observed that WT1 expression level can predict the risk of relapse in AML patients and that WT1 could be used as a tool of initial assessment to establish more defined risk groups.

OS was shortened in patients with WT1 overexpression compared with patients with downexpression (P=0.045, log-rank=4.028). Decreased OS has been reported also in numerous studies of AML patients with WT1 overexpression [12],[21],[23]. In contrast, other studies claimed that WT1 overexpression did not affect the OS [24],[25]. We observed that EFS was also shortened in patients with WT1 overexpression compared with patients with WT1 downexpression (P=0.011, log-rank=6.41) which was in congruence with Lane et al. [26] who observed that elevated WT1 levels were significantly associated with impaired EFS .

The reason for the association of high WT1 expression with a worse long-term outcome remains speculative. There are downstream effectors of WT1 genes, many of those downstream effectors are involved in cellular growth or survival [23]. Using antisense oligonucleotides has shown that WT1 is required not only for proliferation but also for inhibiting apoptosis in tumor cell cultures [30].

We did not find a significant difference between overexpression and downexpression groups according to DFS (P=0.107, log-rank=2.59) which corresponds to other studies [24],[25]. On the other hand, Lyu et al. [23] demonstrated a statistically significantly shorter DFS in the WT1 overexpression group. Controversial data about the prognostic significance of WT1 overexpression are reported which can be attributed to the limited number of patients and the diversity of methods used in the different studies [12],[13],[14],[29],[31],[32].


  Conclusion Top


Our results suggest that WT1 overexpression at diagnosis is considered as an independent negative prognostic biomarker that could potentially be used to evaluate the response to induction chemotherapy and prognosis of AML patients.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8]



 

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Abstract
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