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 Table of Contents  
ORIGINAL ARTICLE
Year : 2012  |  Volume : 37  |  Issue : 4  |  Page : 221-227

The impact of CLLU1 expression as a prognostic marker in chronic lymphocytic leukemia


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

Date of Submission16-Feb-2012
Date of Acceptance30-Mar-2012
Date of Web Publication21-Jun-2014

Correspondence Address:
Maha Atfy
Department of Clinical Pathology, Zagazig University, 20 Atfy Street, Al kawmeya, Zagazig P.44155
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.7123/01.EJH.0000418702.60734.04

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  Abstract 

Background

B-cell chronic lymphocytic leukemia (B-CLL) is a clinically heterogeneous disease; some patients rapidly progress and die within a few years of diagnosis, whereas others have a long life expectancy with minimal or no treatment.

Aim of the study

The aim of this study is to assess the pretreatment levels of CLLU1 in relation to other prognostics markers and to evaluate its impact on the prognosis of CLL patients.

Patients and methods

Seventy-three cases of CLL were characterized for CLLU1 level by a semiquantitative RT-PCR. Univariate analysis and correlations were carried out to test the markers’ ability to identify patients at risk and as prognostic markers in the context of established prognostic factors.

Results

CLLU1 expression was determined in 42/73 (57.5%) of B-CLL patients. There was a highly significant difference in the age and Binet staging in both CLL patients with positive CLLU1 and negative CLLU1 expression (P<0.0001 and P=0.0274, respectively). There was a significant increase in CLLU1 expression in CLL patients with positive CD38 and short lymphocyte doubling time (LDT) (P<0.001 and P<0.0044); this significance was not found with high ZAP-70 and β2 microglobulin (B2M) markers. The relative risk of early death with positive CLLU1 was increased with ZAP-70 and LDT (P=0.0204 and 0.0084) and reduced with CD38 and B2M (P=0.0787 and 0.0750). The poor clinical outcome was associated with positive CLLU1 and ZAP-70 and LDT (P=0.0421 and 0.0084, respectively). B-CLL with positive CLLU1 coexpressed with high B2M and/or CD38 had a slower disease progression (P=0.06 and 0.235, respectively).

Conclusion

CLLU1 expression is an important prognostic marker in B-CLL that could potentially be applied to predict outcome in CLL patients. The CLLU1 may be particularly useful in refining the prognosis of CLL patients.

Keywords: B-cell chronic lymphocytic leukemia, chronic lymphocytic leukemia upregulated gene 1, prognostic marker


How to cite this article:
Atfy M, El-Gohary T. The impact of CLLU1 expression as a prognostic marker in chronic lymphocytic leukemia. Egypt J Haematol 2012;37:221-7

How to cite this URL:
Atfy M, El-Gohary T. The impact of CLLU1 expression as a prognostic marker in chronic lymphocytic leukemia. Egypt J Haematol [serial online] 2012 [cited 2020 Mar 28];37:221-7. Available from: http://www.ehj.eg.net/text.asp?2012/37/4/221/134968


  Introduction Top


Chronic lymphocytic leukemia (CLL) is characterized by the progressive accumulation of monoclonal lymphocytes with a distinctive immunophenotype (i.e. CD5+, CD19+, CD20dim, CD23+, SmIgdim) in peripheral blood, bone marrow, and lymphoid tissues 1,2. Although some patients show an indolent disease and never require treatment, others have a much more aggressive course requiring an intensive treatment shortly or immediately after diagnosis. Identification of these subgroups and an insight into the prognosis for each individual patient will be important to determine individualized treatment strategies 3. Numerous studies have searched for reliable prognostic markers for predicting the progression and outcome of the disease. Clinical staging systems have been identified as important, independent prognostic factors. However, these systems fail to identify individual patients at risk for progression, especially in the early stage 4.

A number of serum markers may reflect disease burden and cellular activity. Different membrane proteins can be released into the plasma, and the levels of these soluble CD molecules can be used as tumor markers in CLL. Major breakthroughs were achieved with the identification of specific cytogenetic aberrations associated with clinical outcome and by the observation that the IgVH mutation status allowed to distinguish between two different prognostic subgroups. The difficulty in carrying out this mutation analysis led to numerous studies on possible surrogate markers and alternative markers of prognosis in CLL, resulting in an increasing number of publications 5.

The Danish group of Buhl et al. 6 used differential display to screen for (new) genes associated with a poor outcome in CLL. They identified a novel gene, CLL upregulated gene 1 (CLLU1), that was uniquely overexpressed in patients with an unfavorable prognosis, defined by unmutated IgVH genes and elevated ZAP-70 expression. Although CLLU1 mapped to chromosome 12q22, overexpression of CLLU1 was independent of the occurrence of trisomy 12. They further elaborated on the prognostic value of CLLU1 and found significant associations with clinical stage, mutation status, ZAP-70 and CD38 expression, adverse chromosomal aberrations, and treatment-free and overall survival. Nevertheless, the data of Buhl et al. 6 indicate that CLLU1 is the first disease-specific gene identified in CLL and that determination of CLLU1 expression levels may become a useful tool for the prediction of risk in CLL.

The aim of this study is to assess the pretreatment levels of CLLU1 in relation to other prognostics markers and to evaluate its impact on the prognosis of CLL patients.


  Patients and methods Top


Patients

This study was carried out on 73 CLL newly diagnosed patients between December 2007 and January 2010. Morphologic diagnosis was made at the Clinical Pathology Department and was confirmed by flow cytometry. The patients were followed up to 30 months or until death. All patients were treated using approved protocols and were followed regularly at the Oncology Department in Zagazig university hospitals.

Patients were classified according to their response to treatment; the response was judged to be good or poor, according to the clinical and laboratory response to standard CLL therapy (CHOP: cyclophosphamide plus doxorubicin plus vincristine plus prednisone). Good responders included patients who achieved partial or complete remission and patients free from CLL-related complications. Poor responders included those with refractory Bone marrow or peripheral blood lymphocytes and those with progressive disease or CLL-related complications.


  Methods Top


All individuals were subjected to full history taking, thorough clinical examination especially for organomegaly and lymphadenopathy, and complete blood count using Sysmex SF 3000 (Kobe, Japan), with examination of Leishman-stained films. Bone marrow aspiration was carried out (for some patients) and liver and kidney function tests were carried out using a Dimension autoanalyzer (Dade Behring, Newark, Germany).

Quantitative determination of β2 microglobulin (B2M) was carried out using a commercial immunoturbidimetric assay kit (Tina-quant β2 microglobulin; Roche) on a Roche/Hitachi Cobas C system (Cobas C701, Roche-Diagnosics, Manheim, Germany).

Immunophenotypic analysis

Immunophenotyping of CLL cases was carried out using the whole blood lysis technique by FACscan flow cytometry (Becton Dickinson, BD, San Diego, USA). Blood samples were stained with the following FITC or PE-labeled panel of monoclonal antibodies: CD19, CD20, CD22, CD23, CD5, CD43, CD79b, and FMC7. Negative isotype-matched controls were used to determine the nonspecific binding. At least 10 000 cells/sample were acquired; an appropriate leukemic cell gate based on both forward and side scatter or CD45 expression and side scatter was selected and analyzed using Cell Quest software (Becton Dickinson). The cut-off point of positivity for leukemic cells was at least 20%.

Flow cytometric detection of ZAP-70 and CD38

Cytoplasmic ZAP-70 expression was determined by flow cytometry on fresh peripheral blood samples. Cells were fixed and permeabilized using the Fix and Perm kit (Caltag, Caltag-Medsystems, Buckingham, UK) according to the manufacturer’s instructions. ZAP-70 PE monoclonal antibody (clone 1F7.2; Caltag) was added to 1×106 cells, and the mixture was incubated for 15 min at room temperature; the cells were washed twice in PBS. CD5-FITC (BD Biosciences, BD, San Diego, USA) and CD19-PerCp (BD Biosciences) were used for staining samples. Lymphocyte cells were gated further to select CD5+CD19− cells (T cells), which were used as an internal positive control, and CD5+CD19+ cells (CLL cells). Isotype controls were run with each sample to distinguish positive cells from negative cells. Data acquisition and analysis were carried out using a FACSCalibur flow cytometer (BD Biosciences) and Cell Quest software (BD Biosciences). After appropriate lymphocyte gating, a marker that included greater than 97% of the T cells (positive control) was used to define the percentage of CLL cells that expressed ZAP-70 with the same intensity of fluorescence as the T cells. The cut-off point for ZAP-70 positivity in CLL cells was greater than 20%. CD38 PE (clone HB-7; BD Biosciences) was a surface marker; its cut-off point for positivity in CLL cells was greater than 30%.

Semiquantitative RT-PCR analysis for CLLU1

Bone marrow or peripheral blood samples of patients with CLL were obtained at the time of routine clinical procurement. Total RNA was extracted from bone marrow or peripheral blood mononuclear cells (PBMNs) using spin-column technology (Qiagen, Hilden, Germany). First-strand cDNA was synthesized from 1 mg of RNA with a commercially available kit (Gibco, Nanjing, Jiangsu, China) using oligo (dT) primers according to the manufacturer’s instructions. The use of TaqMan One-Step RT-PCR Master Mix Reagents (Applied Biosystems, Invitrogen, California, USA) has been described previously 7. A volume of 5 µl of cDNA was added to a final PCR reaction mixture of 50 µl, composed of 1 µl of Taq polymerase, 5 ml of recommended buffer (10× Taq PCR buffer), 1 µl dNTP Mix (10 mmol/l), 2 µl of 20 µmol/l CDS-specific primers (forward primer: 5′-ATGTTCAACAAATGCTCCTTTCA-3′, reverse primer: 5′-TACCAACACACGCGCAACAG-3′) or 2 µl of 20 µmol/l GAPDH endogenous control primers (forward primer: 5′-GAAGGTGAAGGTCGGAGTC-3′, reverse primer: 5′-GAAGATGGTGATGGGATTTC-3′), and 36 µl of H2O. Cycle conditions were 1 cycle for 2 min at 94°C, 35 cycles for 40 s at 94°C, 1 min at 55°C, 1 min at 72°C, and then 1 cycle for 10 min at 72°C. PCR products were electrophoresed on 1.5% agarose gels and visualized by ethidium bromide staining. Then, the amounts of PCR products were evaluated according to the relative intensity of CLLU1 and GPDH bands using the computed densitometry assay of the Bandscan software (Silk Scientific, USA).

Nineteen normal healthy individuals were included in a normal group to determine the cut-off level of CLLU1; we estimated the PBMNs of the healthy individuals and found that these cells had very low to negligible expression of CLLU1. CLLU1 expression levels were measured as a fold upregulation continuum ranging from 1.5 to 10-fold compared with that of normal PBMNs, with a median of three-fold upregulation.

Statistical analysis

LDT, defined as the period of time required for lymphocytes to double in number from the amount found at the time of diagnosis, was obtained in each patient by linear regression 8.

All calculations were carried out using the software package SPSS version 10 (SPSS Inc., version 10, Chicago, IL, USA) and MedCalc software (Medcalc Software, Marakerke, Belgium). Differences between positive and negative CLLU1 groups were analyzed using the Mann–Whitney rank sum test for independent groups. The χ2-and Student t-tests were used for statistical comparisons between patients’ parametric data. In addition, correlations between continuous variables were assessed by the Spearman rank correlation coefficient (r). Survival curves were estimated using the Kaplan–Meier method. Overall survival was the primary outcome of the studies and was calculated from the date of first diagnosis to death from any cause. Univariate Cox regression analysis was carried out to determine the predictive effect of each prognostic factor. P-values less than 0.05 were considered significant.


  Results Top


Seventy-three patients participated in this study, 48 (65.8%) men and 25 (34.2%) women. The CLL patients were grouped according to the positivity of the CLLU1 into a CLLU1-positive group, 42 (57.5%) patients, and a CLLU1-negative group, 31 (42.5%) patients. The biological and clinical data of the patients are presented in [Table 1] and [Table 2].
Table 1: Biological and clinical characteristics of the study cohort

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Table 2: Associations between some prognostic markers and positive CLLU1 expression in CLL patients

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CLLU1 expression in CLL

There were 48 men and 25 women; 30 of 48 CLL male patients had a positive expression of CLLU1 and only 12 of 25 female patients had a positive expression of CLLU1, with no significant difference (χ2=0.88; P<0.3482). There was a highly significant difference (U=1122; P<0.0001) between the age of CLL patients with a positive expression of CLLU1 (median: 56; range: 46–78) and the age of CLL patients with a negative expression of CLLU1 (median: 70, range: 51–79). There were statistically significant differences between the number of CLL patients with a positive expression of CLLU1 and the number of patients with a negative expression of CLLU1 in terms of organomegaly (χ2=4.31; P=0.0378) and Binet staging (χ2=4.86; P=0.0274), with an increase in the CLL patients with positive CLLU1 in stage B/C [Table 1].

Association between CLLU1 expression and some prognostic markers

The probability of relations between CLLU1 expression and CD38, Zap-70, B2M, and LDT was assessed in our study. Twenty-five of 28 patients were defined as positive for both CD38 and CLLU1, with highly significant differences between those patients who had positive CLLU1 with CD38+ and CLLU1− CD38 CLL cases (χ2=16.69; P<0.001). However, CLL cases with positive ZAP-70 showed no statistically significant differences in terms of the expression of CLLU1 (χ2=0.16; P<0.6891). Also, no statistically significant differences were found between CLL patients with greater than 2.2 mg/ml β2M with positive CLLU1 and those patients with greater than 2.2 mg/ml β2M, but with negative CLLU1 (χ2=0.03; P<0.8624).

Data for the LDT were available for 45 patients; only 24 of these patients had a positive expression of CLLU1. Ten patients had LDT less than 6 months had a positive expression of CLLU1. There were highly significant differences between the number of patients expressing positive CLLU1 and negative CLLU1 (χ2=10.81; P<0.0044; [Table 2].

The level of CLLU1 expression was studied in all poor risk prognostic groups. The CD38 level was significantly highly expressed in CLLU1-positive cases, median and range 35.5 (10–93) in comparison with CLLU1-negative cases, median and range 15 (4–39) (U=202; P<0.0001). An evaluation of the levels of CD38 and CLLU1 showed a highly significant correlation (r=0.41319; P<0.0065). The median and range of ZAP-70 was 17.5 (10–72) in positive CLLU1 patients and 15 (1–40) in negative CLLU1 patients , with highly significant difference (U=399; P<0.0025). No significant correlation was found between the levels of ZAP-70 and CLLU1 in these patients (r=0.10512; P<0.5079; [Figure 1] and [Table 3].
Figure 1: Levels of different prognostic markers in terms of positive and negative CLLU1 expression. CD38, ZAP-70, and LDT showed highly significant differences between their levels in association with positive and negative CLLU expressions, but B2M showed no significant difference. B2M, β2-microglobulin; LDT, lymphocyte doubling time.

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Table 3: Correlation between the expression of CLLU1 and other prognostic markers

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There was no statistically significant difference in the B2M level in CLL patients with a positive expression of CLLU1, median and range 2.6 (0.4–8.1), in comparison with CLLU1-negative cases 2.4 (0.9–6.9) (U=680; P<0.3745). This relation showed no significant correlation (r=0.2443; P<0.1921). The median and range of LDT was 13 (2.8–19) in positive CLLU1 patients and 6.35 (2.5–12) in negative CLLU1 patients, with a highly significant difference (U=262; P<0.0001). A comparison of the expression levels of the LDT and CLLU1 showed a highly significant correlation (r=0.4942; P<0.0001; [Table 3] and [Figure 1].

CLLU1 as a prognostic marker of poor clinical outcome

Positive CLLU1 prognostic significance was not altered in patients with positive CD38 and B2M in comparison with negative CLLU1 [hazard ratio (HR)=0.5638; 95% confidence interval (CI), 0.26–1.19; P=0.0787 and HR=0.5656; 95% CI, 0.2841–1.1258; P=0.0750, respectively]. There was an increase in the relative risk of early death in the coexpression of positive CLLU1 and a high level of ZAP-70 and LDT less than 6 months (HR=2.0608; 95% CI, 0.9845–4.3140; P=0.0204 and HR=2.6813; 95% CI, 1.001–7.182; P=0.0084, respectively; [Table 4].
Table 4: Relative risk of early death associated with positive CLLU1 expression in CLL patients

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Disease progression (as regard to TTFT) was more expressed in B-CLL patients with positive CLLU1 and High Zap70. CLL cases with positive CLLU1 and LDT less than 6 months had a poor clinical outcome (P=0.0084). B-CLL with positive CLLU1, with high B2M and/or CD38 greater than 30%, had no significant differences in terms of disease progression when compared with negative CLLU1 (P=0.06 and 0.235, respectively; [Figure 2].
Figure 2: Curves of treatment-free survival (TTFT) according to the positivity of CLLU1 (a) ZAP-70, (b) B2M, (c) CD38 percent, and (d) LDT. B2M, β2-microglobulin; LDT, lymphocyte doubling time; TTFT, time from diagnosis to first treatment.

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  Discussion Top


B-CLL prognosis is indicated by a number of clinical and biological factors that have been used to divide B-CLL patients into subgroups with different prognoses and treatment requirements 9,10. Clinical staging (Rai or Binet systems), peripheral LDT, marrow histology, B2M, number of cleaved lymphocytes, and karyotyping are all factors that affect B-CLL prognosis 11–14. The mutational status of the immunoglobulin heavy-chain variable-region (IgVH), and ZAP-70 and CD38 expression have emerged as the most useful tools in the identification of patients with a more aggressive course and in monitoring therapy 15. Understanding of the pathophysiology and biology of B-CLL can be improved by identification of novel markers with prognostic significance.

Recently, a unique overexpression has been reported in CLL of a novel CLL-specific gene CLL, upregulated gene 1 (CLLU1) 6. Although CLLU1 mapped to chromosome 12q22, overexpression of CLLU1 was independent of the occurrence of trisomy 12 16. The fact that the novel gene CLLU1 is disease specific, albeit the function is unknown, might imply that CLLU1 expression has some prognostic information not provided by other biologic markers in CLL 16.

In the current study, CLLU1 was found to be expressed in 57.7% of CLL patients; this result was nearly comparable with that of the study of Chen et al. 17, who reported that CLLU1 was expressed in 50% of patients with CLL. It was also found that the predictive power of CLLU1 was restricted to patients who had been diagnosed with CLL before the age of 70 years. A highly significant difference was found between the age of CLL patients with positive CLLU1 and those with negative CLLU1. This result was explained by Josefsson et al. 16, who suggested that CLLU1 is expressed from a very early CLL onset, but may exert its clinical effect over a long time. In younger patients, CLLU1 expression was able to improve all other prognostic subgroups, except the group of patients with unmutated IgVH gene 16.

CD38 functions in the regulation of cell activation, proliferation, and adhesion. It is found on hematopoietic cells of various lineages, including B cells 18. Around the time of the discovery of the prognostic significance of mutation properties, CD38 was implicated as a possible alternative marker correlating with mutation 19. It is clear that the presence of CD38 on its own is indicative of a poorer prognosis. Deaglio et al. 20 have presented the hypothesis that CD38 functions specifically in CLL to promote both the continued proliferation and the prolonged survival of the leukemic cell population. This hypothesis of the signaling role played by CD38 in CLL is strengthened by the cellular location of CD38 in a variety of cell types that are close to the receptor primarily responsible for signaling in each respective cell type.

In this study, 28 of 73 CLL patients analyzed for CD38 showed the expression of the marker. Twenty-five of 28 CLL patients had positive CLLU1. The prognostic role of CD38 in CLL is now established. However, it is tempting to consider that higher CD38 levels reflect an increase in the proliferative capacity of the CLL clone and more progressive disease. The change in CD38 expression could also serve as a useful tool to monitor disease activity 3.

Our result confirmed the report of Chen et al. 17, in which the level of CD38 was significantly higher with positive CLLU1 when compared with negative CLLU1. Also, a significant correlation was found between the two markers. This study confirmed the statement of Buhl et al. 6, who confirmed that CLLU1 was significantly upregulated in the CD38-positive expression CLL group. When studying TTFT, positive CLLU1 and positive CD38 cases proved that they work as separate independent predictive factor.

ZAP-70 encodes for T cell-specific ZAP-70 and had been initially identified in T cells as a protein tyrosine kinase that plays a critical role in T cell receptor signaling 21. Using a cut-off set at 20% of positive cells, ZAP-70 expression was found to have a negative prognostic impact in CLL 22,23. The relevance of ZAP-70 as an independent prognostic factor was indicated by multivariate analysis 24.

In this study, out of 73 CLL patients, only 18 patients had a positive expression of CLLU1 and ZAP-70. The high expression of ZAP-70 was found mainly with positive CLLU1 (P=0.037), yielding a relative risk of (HR=2.0608); these results may indicate the fact that a high ZAP-70 expression decreases the threshold for triggering a signal through the B-cell antigen receptor complex, thereby conferring a growth and survival advantage to the leukemic cell; thus, this prognostic marker may be linked to a biologically relevant phenomenon. Therefore, CLLU1 provided additional prognostic information in ZAP-70-positive CLL patients 16.

β2M is an extracellular protein that is noncovalently associated with the α-chain of the class I major histocompatibility complex, which is detectable in the serum. β2M is associated with adverse prognostic characteristics at presentation and higher values have been found in CLL patients with a shorter survival 4. β2M more than 2.2 mg/ml was found in 38/73 CLL patients; 22 patients had a highly positive expression of CLLU1. The risk of these two markers together was limited. Therefore, a statistical relation between CLLU1 and β2M could not be found, indicating that both parameters are independent prognostic variables for disease progression. β2M has been found to be a strong prognostic marker in predictive survival after chemotherapy or chemoimmunotherapy 4.

LDT in CLL patients showed a statistically positive correlation with the level of CLLU1. The high expression of both CLLU1 and LDT showed the worst clinical outcome; patients with a mixed pattern showed an intermediate outcome for the time to first treatment. These findings were in agreement with those of Rampazzo et al. 25, who found that B-CLL with the highest proliferative index (i.e. LDT<6 months) had the shorter survival.

Buhl et al. 26 confirmed in their study that a high expression of the CLLU1 gene is restricted to CLL, and patients with CLL most likely to require early and repetitive treatment will more often have high CLLU1 expression levels. Also, previous studies have indicated that patients with CLL with a high CLLU1 expression have shorter time intervals from diagnosis to initial therapy and decreased overall survival 6, 9, 26.

Recently, it was also found that CLLU1 monitoring might be a dependable means by which it may be possible to detect the minimal residual of CLL cells after therapy 26.


  Conclusion Top


The high expression of CLLU1 and its contribution with other prognostic markers could provide more information about the prognosis in CLL cases. Consequently, the CLLU1 expression level is an essential prognostic parameter that may identify patients at the time of diagnosis who may require aggressive treatment. The prognostic value of CLLU1 needs to be confirmed in other patient studies, and its function needs to be investigated more in detail and to confirm its role in the evaluation of residual disease in the context of therapy and to develop new therapeutic strategies.[26]

 
  References Top

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26.Buhl AM, James DF, Neuberg D, Jain S, Laura Z, Rassenti LZ, Kipps JT. Analysis of CLLU1 expression levels before and after therapy in patients with chronic lymphocytic leukemia. Eur J Hematol. 2011:1–7  Back to cited text no. 26
    


    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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  In this article
Abstract
Introduction
Patients and methods
Methods
Results
Discussion
Conclusion
Introduction
Patients and methods
Methods
Results
Discussion
Conclusion
References
Article Figures
Article Tables

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