|Ahead of print publication
Ezrin is a prognostic biomarker in patients with clear cell metastatic renal cell carcinoma receiving sunitinib
Bulent Cetin1, Ipek Isik Gonul2, Ozge Gumusay3, Baris Afsar4, Irem Bilgetekin5, Ahmet Ozet6, Aytug Uner6
1 Department of Internal Medicine, Division of Medical Oncology, Faculty of Medicine, Suleyman Demirel University, Isparta, Turkey
2 Department of Pathology, Gazi University Faculty of Medicine, Ankara, Turkey
3 Department of Internal Medicine, Division of Medical Oncology, Faculty of Medicine, Gaziosmanpasa University, Tokat, Tokat, Turkey
4 Department of Internal Medicine, Division of Nephrology, Faculty of Medicine, Suleyman Demirel University, Isparta, Turkey
5 Department of Internal Medicine, Division of Medical Oncology, Dr. A. Y. Ankara Oncology Training and Research Hospital, Ankara, Turkey
6 Department of Internal Medicine, Division of Medical Oncology, Gazi University Faculty of Medicine, Ankara, Turkey
Department of Internal Medicine, Division of Medical Oncology, Faculty of Medicine, Suleyman Demirel Univesity, Isparta 32260
Source of Support: None, Conflict of Interest: None
Objective: Sunitinib is a novel oral multitargeted tyrosine kinase inhibitor with antitumor and antiangiogenic activities. This study evaluates ezrin expression in sunitinib-treated metastatic clear cell renal cell carcinoma (ccRCC) patients and elucidates its role as a possible marker for survival.
Materials and Methods: The expression of ezrin was measured by immunohistochemistry in 80 patients with ccRCC treated by first-line sunitinib between January 2007 and June 2012. Kaplan–Meier curves and log-rank tests were used for analysis of progression-free survival and overall survival (OS), and a multivariate Cox proportional hazard model was employed to identify factors with an independent effect on the survival.
Results: In multivariate analysis, liver metastasis (P = 0.018; hazard ratio [HR]: 3.707 (1.257–10.931) and overexpression of ezrin (P = 0.006; HR: 2.993 (1.373–6.523 95% confidence interval) were remained significant factors influencing OS. Overexpression of ezrin in the patients who had progressed in the first 3 months was higher than in the patients who had progressed after 3 months (P = 0.003). The median OS was longer in patients with low levels of ezrin expression (27 months) compared to patients overexpressing ezrin (12 months) (P = 0.001).
Conclusion: This is the first study in the literature showing that ezrin status is related with prognosis in patients with metastatic ccRCC.
Keywords: Clear cell renal cell carcinoma, ezrin, sunitinib
|How to cite this URL:|
Cetin B, Gonul II, Gumusay O, Afsar B, Bilgetekin I, Ozet A, Uner A. Ezrin is a prognostic biomarker in patients with clear cell metastatic renal cell carcinoma receiving sunitinib. J Can Res Ther [Epub ahead of print] [cited 2020 Oct 28]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=264695
| > Introduction|| |
Renal cell carcinoma (RCC) occurs in 64,000 new patients each year in the United States and results in about 14,000 deaths. Clear cell RCC (ccRCC) is the most common histologic subtype, representing approximately 70% of all sporadic RCCs. ccRCC is highly metastatic. Antiangiogenesis drugs have become the new standard of care in the first-line setting for metastatic ccRCC. The primary targeted approaches in this first-line setting include pazopanib, sunitinib, cabozantinib, and bevacizumab plus interferon-alfa (IFN-α). Ezrin might play an important role in the development of cancer. Ezrin regulates cell–cell and cell–matrix adhesions by interacting with adhesion molecules E-cadherin and beta-catenin and has an important role in the regulation of adhesive and invasive behaviors of cancer cells and also in the tumor progression and metastasis. Better understanding of the molecular biology of RCC will lead to the identification of many molecular pathways which can be targeted for the treatment of patients with advanced stage/metastatic RCC.
Tumor metastasis begins with breakdown of epithelial integrity, followed by malignant cells invading into the surrounding stroma and lymphovascular space. Cell adhesion molecules and actin cytoskeleton play an important role in tumor metastasis. Ezrin, a membrane-cytoskeletal linking protein, which is associated with aggressive tumor behavior by involving all stages of tumor metastasis, including cell adhesion, survival, motility, and signal transduction.,, Previous studies showed that ezrin is strongly expressed in a variety of invasive cancers, and its overexpression is associated with poor prognosis. Yu et al. found that the expression of miR-96 was negatively correlated with the metastatic ability of RCC, and its downregulation could prevent metastasis via downregulation of ezrin expression.
Our study has demonstrated that the high ezrin expression is significantly associated with poor survival in metastatic ccRCC patients. No published study has assessed the prognostic effect of ezrin expression first-line therapy with sunitinib in ccRCC.
| > Materials and Methods|| |
Data were obtained in 90 consecutive patients with metastatic ccRCC who were treated with tyrosine kinase inhibitors (TKI) in the Department of Oncology at the University of Gazi between January 2007 and June 2012. These patients were treated with the antiangiogenic TKI sunitinib (Sutent ®) as a first-line treatment. Sunitinib was administered 50 mg once daily on 28 consecutive days of a given 6-week cycle. The relationship between immunohistochemical staining of ezrin response to treatment with TKIs was investigated in patients with metastatic RCC. Patients with the intermediate-risk group according to the Memorial Sloan Kettering Cancer Center included in the study.
Ten patients were excluded from the analysis (unavailable tissue for the accurate reevaluation [n = 4], patients with brain metastases [n = 4], incomplete records [n = 1], and administration of therapy outside the study institution [n = 1]). For each case, clinical, biological, and radiological features were obtained from referring physicians and medical records. The response to treatment was assessed using response evaluation criteria in solid tumors; the first evaluation was assessed after two cycles of sunitinib (3 months). For the purposes of this study, progression-free survival (PFS) was defined as time from the start of treatment with sunitinib to clinical and/or radiological evidence of disease progression, and overall survival (OS) was defined as the time from the date of metastatic RCC diagnosis to the last follow-up visit date or death of the patient.
Data analysis was conducted using Statistical Package for the Social Sciences 17.0 software package (IBM Corporation, Armonk, NY, USA). Descriptive analyses were used for demographic data and Kaplan–Meier test for survival analysis. OS and PFS were estimated using Kaplan–Meier method, and a log-rank test was used for comparison of the study groups for survival. Variables were found to be significant if the two-sided P value was <0.05 on univariate testing. We also employed the Cox proportional hazards model for multivariable analysis. The variables that reached statistical significance (P < 0.05) in this model were then deemed to be independent predictors of the treatment outcome concerning the OS.
H and E-stained tumor sections of the patients were reexamined by a uropathologist, and immunohistochemical staining for ezrin was performed on the 4-μ sections from corresponding paraffin blocks using the standard streptavidin biotin-peroxidase procedure. Sections underwent heat-induced epitope retrieval in 0.01 M sodium citrate buffer at pH 6.0 for 20 min. As primary antibodies, Ezrin antibody (Ezrin Ab-1, Thermo Scientific, Clone 3C12 mouse polyclonal antibody, MS-661-P1, at 1/200 dilution) was used. For each staining session, cc RCC for lung parenchyma for ezrin was used as positive controls. The pathology slides were reviewed by an expert urologic pathologist who had no knowledge of patients' treatment and outcomes. Tumors were scored positive for ezrin if tumor cells showed definite nuclear and/or membranous staining and negative if tumor nuclei and cell membrane had no immunoreactivity. Staining intensity (weak, 1 point; moderate, 2 points; strong, 3 points) and percentage groups of positive tumor cells (≤25%, 1 point; 26%–50%, 2 points; 51%–75%, 3 points; >75%, 4 points) were multiplied to achieve a score between 1 and 12. Subsequently, patients were divided into two groups (underexpressing or overexpressing) based on their median values for each stain [Figure 1]. Cutoff levels for the scoring were presented as follows: scores of >6 were classified as a high expression and scores of ≤6 were classified as low expression.
|Figure 1: Immunohistochemical staining of ezrin (a) Tumor cells displaying diffuse and prominent ezrin expression in a membranous pattern (b) Tumor cells showing rare membranous ezrin expression|
Click here to view
| > Results|| |
This study enrolled a total of 80 patients diagnosed with metastatic ccRCC who had taken first-line sunitinib treatment during follow-up visits. The mean age at diagnosis was 57 years (34–80). A total of 49 patients (61.25%) had an Eastern Cooperative Oncology Group performance status of 0. In all ccRCC tissue samples, positive staining signals of ezrin were detected in 18 (45%) with low immunoreactivity and in 22 (55%) with high immunoreactivity, respectively. The mean follow-up period was 23 months (range: 1–53 months) from sunitinib introduction, and 74 patients (92.5%) died. The median OS was 21.2 months (95% confidence interval [CI], 18–33). The median PFS was 6 months (95% CI, 3–9). Demographic, clinical, and pathological characteristics of patients are shown in [Table 1]. We compared baseline clinical characteristics and known prognostic factors between patients with high and low ezrin expressions [Table 2]. Liver metastases, age, bone metastases, lung metastases, high ezrin expression, and a number of metastatic organ sites 2 were associated with PFS in the univariate analysis. The two variables that were significantly predictive of PFS with sunitinib were liver metastases (P = 0.019; hazard ratio [HR]: 3.488 [1.224–9.939]) and high ezrin expression (P = 0.045; HR: 2.080 [1.006–4.300]) [Table 3] and [Figure 2]a. Ezrin overexpression, liver metastasis, bone metastasis, and a number of metastatic organ sites ≥2 were statistically significant factors (P < 0.05) for OS in the univariate analysis. Factors that reached a P value below 0.05 in univariate analysis entered into the multivariate model. In multivariate analysis, liver metastasis (P = 0.018; HR: 3.707 [1.257–10.931]) and overexpression of ezrin (P = 0.006; HR: 2.993 [1.373%–6.523 95% CI]) were remained significant factors influencing OS [Table 3] and [Figure 2]b. Overexpression of ezrin in the patients who had progressed in the first 3 months was higher than in the patients who had progressed after 3 months [Figure 3]. The difference was statistically significant (P = 0.003).
|Table 1: Demographic, clinical, and pathological characteristics of patients|
Click here to view
|Table 2: Distribution of clinicopathologic prognostic factors stratified by high- and low-ezrin expression|
Click here to view
|Table 3: Estimation of parameters of the Cox regression model for progression-free survival and death|
Click here to view
|Figure 2: Progression-free survival and overall survival according to ezrin expression. (a) Progression-free survival for the low ezrin expression group and high ezrin expression group. (b) Overall survival for low and high ezrin cc(RCC) patients|
Click here to view
|Figure 3: The ezrin expression of the patients who experience progression at 3 months was compared with ezrin expression of patients who did not progress at 3 months|
Click here to view
| > Discussion|| |
RCC accounts for approximately 3% of adult malignancy and more than 90% of renal cancers. Despite improvements in RCC treatment, the prognosis remains poor for metastatic RCC. Thus, it is important to identify biomarkers associated with the invasive and metastatic potential of RCC, which may provide the new targets for treatment.
Ezrin, cytoskeletal protein, is involved in a number of signaling pathways that is crucial to cancer progression. In this study, we sought to conduct an analysis to estimate the prognostic importance of ezrin level for OS and PFS among patients with metastatic ccRCC, aiming to gain insights into whether ezrin could provide useful guidance in the biological understanding and treatment of metastatic RCC. In recent years, promising results have been obtained in the treatment of metastatic RCC with the introduction of targeted agents owing to molecular advances. Antiangiogenic vascular endothelial growth factor (VEGF)-targeted therapies have been approved as a first-line treatment in metastatic ccRCC. Sunitinib malate is an oral multikinase inhibitor that blocks VEGF receptor -1, 2, and 3, platelet-derived growth factor receptor -B, the Fms-like tyrosine kinase 3, and the c-KIT receptor tyrosine kinase. Two Phase 2 trials in patients with cytokine-refractory disease reported a response rate of 41% and PFS of 8.2 months. These studies led to the Food and Drug Administration approval of sunitinib for the second-line therapy in 2006., A randomized Phase 3 trial compared sunitinib with IFN-α as first-line therapy demonstrated a 47% objective response rate, and a median PFS and OS of 11 months and 26.4 months, respectively, in the sunitinib arm compared with a 12% objective response rate, 5-month median PFS, and 21.8-month median OS in the IFN arm.
Ezrin is synthesized by the glomerular and tubular epithelial cells of the kidney. Ezrin is an important member of ezrin/radixin/moesin family and is derived from epithelilal or nonepithelial cells in various cancer types. In human, the ezrin gene located on chromosome 6q25.2–q26, composed of 585 aminoacids with certain specificity. Ezrin protein can reduce expression of E-cadherin on the cell surface, thereby reducing the adhesion between the cells, resulting in the migration to a distant organ. Ezrin is released in an inactive form and subsequently transformed into active form with treonin and tirozin phosphorylation. Its main function is to bind transmembrane proteins to actin cytoskeleton., In addition, it regulates many cellular processes including formation of microvilli, maintenance of cellular structure, cell–cell adhesion, cell motility, and invasion. The biological pathways associated with ezrin include protein kinase C, Rho-kinase, NF-kB, and PI3-kinase/Akt.
There is increasing evidence that the increase in ezrin level is associated with tumor progression and invasion. High ezrin is associated with a poor prognosis in a variety of solid tumors. However, the results about the prognostic value of ezrin expression in metastatic renal cancer patients remain unknown. Some studies reported that upregulated ezrin was a negative prognostic factor for survival for cancer patients such as digestive system cancer,,,,,,,,,, osteosarcoma,,,,,,,, squamous-cell carcinoma of the head and neck,,,,, gynecologic cancer,,, and hepatobiliary cancer. In the systematic review and meta-analysis published by Li et al., the relation between ezrin expression and prognosis was investigated in cancer patients, and it was reported that overexpression of ezrin is associated with poor survival in cancer patients.
In the present study, we investigated the effect of ezrin on prognosis in metastatic cRCC. We found that ezrin high expression was also associated with poor survival in patients with metastatic RCC treated with sunitinib. To our best knowledge, there are no studies which investigated the prognostic significance of ezrin in metastatic ccRCC patients receiving TKIs. Ezrin, most likely, has important effects on tumor biology, given the important role of the matrix in regulating cellular differentiation and tumor invasiveness and metastasis. This study of ezrin in ccRCC suggests new windows into the future targets for disease treatment. It is important to acknowledge the limitations of our study. First, bias in patient selection and data collection which is typical for a retrospective study and particularly differences in characteristics of patients selected for histopathological examination were shortcomings of the study. The sample size is limited and partially explained by our rigorous inclusion criteria such as homogeneous histology ccRCC and sunitinib as the first-line treatment.
| > Conclusion|| |
New molecular models are needed for making decisions on treatment with targeted agents giving consideration to clinical factors as well as molecular characteristics of the tumor, which affect the prognosis. Prospective studies in a larger number of patients are needed to demonstrate such molecular models in metastatic RCC. This study is the first to demonstrate the independent prognostic significance of ezrin expressions on OS histopathologically in addition to clinical factors.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| > References|| |
Siegel R, Ma J, Zou Z, Jemal A. Cancer statistics, 2014. CA Cancer J Clin 2014;64:9-29.
Hiscox S, Jiang WG. Ezrin regulates cell-cell and cell-matrix adhesion, a possible role with E-cadherin/beta-catenin. J Cell Sci 1999;112(Pt 18):3081-90.
Hunter KW. Ezrin, a key component in tumor metastasis. Trends Mol Med 2004;10:201-4.
McClatchey AI. Merlin and ERM proteins: Unappreciated roles in cancer development? Nat Rev Cancer 2003;3:877-83.
Yu N, Fu S, Liu Y, Xu Z, Liu Y, Hao J, et al.
MiR-96 suppresses renal cell carcinoma invasion via downregulation of ezrin expression. J Exp Clin Cancer Res 2015;34:107.
Mendel DB, Laird AD, Xin X, Louie SG, Christensen JG, Li G, et al
. In vivo
antitumor activity of SU11248, a novel tyrosine kinase inhibitor targeting vascular endothelial growth factor and platelet-derived growth factor receptors: Determination of a pharmacokinetic/pharmacodynamic relationship. Clin Cancer Res 2003;9:327-37.
Motzer RJ, Rini BI, Bukowski RM, Curti BD, George DJ, Hudes GR, et al.
Sunitinib in patients with metastatic renal cell carcinoma. JAMA 2006;295:2516-24.
Motzer RJ, Hutson TE, Tomczak P, Michaelson MD, Bukowski RM, Rixe O, et al.
Sunitinib versus interferon alfa in metastatic renal-cell carcinoma. N Engl J Med 2007;356:115-24.
Vaheri A, Carpén O, Heiska L, Helander TS, Jääskeläinen J, Majander-Nordenswan P, et al.
The ezrin protein family: Membrane-cytoskeleton interactions and disease associations. Curr Opin Cell Biol 1997;9:659-66.
Turunen O, Wahlström T, Vaheri A. Ezrin has a COOH-terminal actin-binding site that is conserved in the ezrin protein family. J Cell Biol 1994;126:1445-53.
Tsukita S, Oishi K, Sato N, Sagara J, Kawai A, Tsukita S, et al.
ERM family members as molecular linkers between the cell surface glycoprotein CD44 and actin-based cytoskeletons. J Cell Biol 1994;126:391-401.
Heiska L, Kantor C, Parr T, Critchley DR, Vilja P, Gahmberg CG, et al.
Binding of the cytoplasmic domain of intercellular adhesion molecule-2 (ICAM-2) to alpha-actinin. J Biol Chem 1996;271:26214-9.
Chuan YC, Iglesias-Gato D, Fernandez-Perez L, Cedazo-Minguez A, Pang ST, Norstedt G, et al.
Ezrin mediates c-myc actions in prostate cancer cell invasion. Oncogene 2010;29:1531-42.
Brambilla D, Fais S. The Janus-faced role of ezrin in “linking” cells to either normal or metastatic phenotype. Int J Cancer 2009;125:2239-45.
Yu Y, Khan J, Khanna C, Helman L, Meltzer PS, Merlino G, et al.
Expression profiling identifies the cytoskeletal organizer ezrin and the developmental homeoprotein six-1 as key metastatic regulators. Nat Med 2004;10:175-81.
Yeh TS, Tseng JH, Liu NJ, Chen TC, Jan YY, Chen MF, et al.
Significance of cellular distribution of ezrin in pancreatic cystic neoplasms and ductal adenocarcinoma. Arch Surg 2005;140:1184-90.
Gao SY, Li EM, Cui L, Lu XF, Meng LY, Yuan HM, et al.
Sp1 and AP-1 regulate expression of the human gene VIL2 in esophageal carcinoma cells. J Biol Chem 2009;284:7995-8004.
Lam EK, Wang X, Shin VY, Zhang S, Morrison H, Sun J, et al.
A microRNA contribution to aberrant Ras activation in gastric cancer. Am J Transl Res 2011;3:209-18.
Wang YY, Chen WL, Huang ZQ, Yang ZH, Zhang B, Wang JG, et al.
Expression of the membrane-cytoskeletal linker ezrin in salivary gland adenoid cystic carcinoma. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2011;112:96-104.
Patara M, Santos EM, Coudry Rde A, Soares FA, Ferreira FO, Rossi BM, et al.
Ezrin expression as a prognostic marker in colorectal adenocarcinoma. Pathol Oncol Res 2011;17:827-33.
Li L, Wang YY, Zhao ZS, Ma J. Ezrin is associated with gastric cancer progression and prognosis. Pathol Oncol Res 2011;17:909-15.
Xie JJ, Xu LY, Wu ZY, Zhao Q, Xu XE, Wu JY, et al.
Prognostic implication of ezrin expression in esophageal squamous cell carcinoma. J Surg Oncol 2011;104:538-43.
Lin LJ, Chen LT. Association between ezrin protein expression and the prognosis of colorectal adenocarcinoma. Mol Med Rep 2013;8:61-6.
Arumugam P, Partelli S, Coleman SJ, Cataldo I, Beghelli S, Bassi C, et al.
Ezrin expression is an independent prognostic factor in gastro-intestinal cancers. J Gastrointest Surg 2013;17:2082-91.
Piao J, Liu S, Xu Y, Wang C, Lin Z, Qin Y, et al.
Ezrin protein overexpression predicts the poor prognosis of pancreatic ductal adenocarcinomas. Exp Mol Pathol 2015;98:1-6.
Salas S, Bartoli C, Deville JL, Gaudart J, Fina F, Calisti A, et al.
Ezrin and alpha-smooth muscle actin are immunohistochemical prognostic markers in conventional osteosarcomas. Virchows Arch 2007;451:999-1007.
Kim MS, Song WS, Cho WH, Lee SY, Jeon DG. Ezrin expression predicts survival in stage IIB osteosarcomas. Clin Orthop Relat Res 2007;459:229-36.
Kim C, Shin E, Hong S, Chon HJ, Kim HR, Ahn JR, et al.
Clinical value of ezrin expression in primary osteosarcoma. Cancer Res Treat 2009;41:138-44.
Khanna C, Wan X, Bose S, Cassaday R, Olomu O, Mendoza A, et al.
The membrane-cytoskeleton linker ezrin is necessary for osteosarcoma metastasis. Nat Med 2004;10:182-6.
Ferrari S, Zanella L, Alberghini M, Palmerini E, Staals E, Bacchini P, et al.
Prognostic significance of immunohistochemical expression of ezrin in non-metastatic high-grade osteosarcoma. Pediatr Blood Cancer 2008;50:752-6.
Boldrini E, Peres SV, Morini S, de Camargo B. Immunoexpression of ezrin and CD44 in patients with osteosarcoma. J Pediatr Hematol Oncol 2010;32:e213-7.
Wang YF, Shen JN, Xie XB, Wang J, Huang G. Expression change of ezrin as a prognostic factor in primary osteosarcoma. Med Oncol 2011;28 Suppl 1:S636-43.
Mu Y, Zhang H, Che L, Li K. Clinical significance of microRNA-183/Ezrin axis in judging the prognosis of patients with osteosarcoma. Med Oncol 2014;31:821.
Madan R, Brandwein-Gensler M, Schlecht NF, Elias K, Gorbovitsky E, Belbin TJ, et al.
Differential tissue and subcellular expressionof ERM proteins in normal and malignant tissues: Cytoplasmic ezrin expression has prognostic signficance for head and neck squamous cell carcinoma. Head Neck 2006;28:1018-27.
Gao W, Zhang C, Feng Y, Chen G, Wen S, Huangfu H, et al.
Fascin-1, ezrin and paxillin contribute to the malignant progression and are predictors of clinical prognosis in laryngeal squamous cell carcinoma. PLoS One 2012;7:e50710.
Wang X, Liu M, Zhao CY. Expression of ezrin and moesin related to invasion, metastasis and prognosis of laryngeal squamous cell carcinoma. Genet Mol Res 2014;13:8002-13.
Schlecht NF, Brandwein-Gensler M, Smith RV, Kawachi N, Broughel D, Lin J, et al.
Cytoplasmic ezrin and moesin correlate with poor survival in head and neck squamous cell carcinoma. Head Neck Pathol 2012;6:232-43.
Wang Y, Lin Z, Sun L, Fan S, Huang Z, Zhang D, et al.
Akt/Ezrin Tyr353/NF-κB pathway regulates EGF-induced EMT and metastasis in tongue squamous cell carcinoma. Br J Cancer 2014;110:695-705.
Köbel M, Langhammer T, Hüttelmaier S, Schmitt WD, Kriese K, Dittmer J, et al.
Ezrin expression is related to poor prognosis in FIGO stage I endometrioid carcinomas. Mod Pathol 2006;19:581-7.
Köbel M, Gradhand E, Zeng K, Schmitt WD, Kriese K, Lantzsch T, et al.
Ezrin promotes ovarian carcinoma cell invasion and its retained expression predicts poor prognosis in ovarian carcinoma. Int J Gynecol Pathol 2006;25:121-30.
Ma L, Liu YP, Zhang XH, Geng CZ, Li ZH. Relationship of RhoA signaling activity with ezrin expression and its significance in the prognosis for breast cancer patients. Chin Med J (Engl) 2013;126:242-7.
Kang YK, Hong SW, Lee H, Kim WH. Prognostic implications of ezrin expression in human hepatocellular carcinoma. Mol Carcinog 2010;49:798-804.
Li J, Wei K, Yu H, Jin D, Wang G, Yu B, et al.
Prognostic value of ezrin in various cancers: A systematic review and updated meta-analysis. Sci Rep 2015;5:17903.
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]