|Year : 2014 | Volume
| Issue : 7 | Page : 179-185
Clinical significance of preoperative serum tumor markers in esophageal squamous cell carcinoma
Hongguang Zhao1, Wenhu Chen2, Jie Wu3, Lifang Wang2, Weimin Mao1
1 Department of Thoracic Surgery, Zhejiang Cancer Hospital; Zhejiang Key Laboratory of Diagnosis and Treatment Technology on Thoracic Oncology (Lung and Esophagus), Zhejiang Cancer Hospital, Hangzhou 310022, China
2 Department of Biochemistry, Institute of Basic Medical Science, Zhejiang Medical College, Hangzhou 310022, China
3 Department of Clinical Laboratory, Zhejiang Cancer Hospital, Hangzhou 310022, China
|Date of Web Publication||29-Nov-2014|
Zhejiang Key Laboratory of Diagnosis and Treatment Technology on Thoracic Oncology (Lung and Esophagus), Zhejiang Cancer Hospital, Hangzhou 310022
Source of Support: This work was supported by Natural Science Foundation
of Zhejiang (LQ12H16001) and General Research Project of Zhejiang Medical
College (2013XZB02), Conflict of Interest: None
Background: Serum tumor markers (TMs) were seldom reported in esophageal carcinoma (ESCC), and the results were still unsatisfactory.
Materials and Methods: We retrospectively analyzed carcinoembryonic antigen, CA125, CA199, CA724 and CA242 in ESCC patients. The preliminary relations between serum TMs and clinicopathological factors or prognosis were analyzed by Fisher's exact test and Kaplan-Meier method firstly. Then, the cut-off values of these serum TMs were delimited according to lymph node metastasis, hematogenic metastasis and 2-year survival or 5-year survival of ESCC by receiver operating characteristic curve. Based on these cut-off values, the relations between the serum TMs and clinicopathological factors or prognosis were analyzed again. Univariate and multivariate analyses of Cox regression proportional hazard model were performed to evaluate the prognostic parameters for survival.
Results: We chose 13.65 U/mL, 9.945 U/mL and 6.25 U/mL as new cut-off values of CA125, CA199 and CA724, respectively, and chose 25.35 U/mL as the cut-off value of CA125 for ESCC hematogenous metastasis. with these optimal cut-off values, CA199, CA125 and CA724 were associated with ESCC hematogenous metastasis, and CA199 and CA125 were associated with ESCC lymph node metastasis, but not associated with other clinicopathological factors. The prognosis was better in patients with CA125 ≤ 13.65 U/mL than those with CA125 > 13.65U/mL. Vascular tumor thrombus, grading, T grade, lymph node metastasis and CA125 were independent prognostic factors.
Conclusion: CA125 could predict lymph node metastasis, hematogenic metastasis and prognosis with the cut-off value 13.65 U/mL.
Keywords: Esophageal squamous cell carcinoma, metastasis, prognostic factor, tumor markers
|How to cite this article:|
Zhao H, Chen W, Wu J, Wang L, Mao W. Clinical significance of preoperative serum tumor markers in esophageal squamous cell carcinoma. J Can Res Ther 2014;10, Suppl S3:179-85
|How to cite this URL:|
Zhao H, Chen W, Wu J, Wang L, Mao W. Clinical significance of preoperative serum tumor markers in esophageal squamous cell carcinoma. J Can Res Ther [serial online] 2014 [cited 2019 Sep 20];10:179-85. Available from: http://www.cancerjournal.net/text.asp?2014/10/7/179/145863
| > Introduction|| |
Tumor markers (TMs) refer to a series of substances, which are produced by tumor cells during their growth and proliferation, or secreted abnormally by the body due to the reaction to the tumor. They express in a low level in healthy people but increase in cancer patients. They can reflect the presence of the tumor. Studies have shown that serum TMs play an important role in cancer diagnosis, treatment and prognosis, monitoring. ,, Due to their convenience and noninvasion, serum TMs are widely applied in clinical practice.
Although serum TMs were reported in many tumors, such as pancreatic, ovarian, gastrointestinal, breast and lung cancer, there were few reports in esophageal carcinoma (ESCC). Even in those reports, the results were still unsatisfactory. First, the main pathological type of ESCC is squamous cell carcinoma, especially in china, while that of other gastrointestinal tumors is adenocarcinoma. Different pathological type may cause different expression of serum TMs, which is why serum TMs are higher in gastrointestinal tumors, but lower in ESCC. Second, because of the lower serum TMs expression, the common cut-off values of these serum TMs are of little significance to ESCC. The normal value criteria of serum TMs drew up according to the difference between cancer patients and healthy people while it is more meaningful to compare the difference between cancer patients. Therefore, the cut-off value of serum TMs should be delimited anew in ESCC.
In this research, some serum TMs were retrospectively analyzed in ESCC patients. The preliminary relations between serum TMs and clinicopathological factors or prognosis were analyzed firstly. Then, the cut-off values of these serum TMs were delimited according to lymph node metastasis, blood metastasis and 2-year survival or 5-year survival of ESCC. Based on these cut-off values, the relations between the serum TMs and clinicopathological factors or prognosis were analyzed again. Finally, the role of serum TMs in ESCC was evaluated.
| > Materials and methods|| |
Totally 314 cases of ESCC (243 cases were treated by surgery, 38 cases with neck or celiac lymphatic metastasis and 33 cases with hematogenous metastasis were treated by nonsurgical therapy) in Zhejiang Cancer Hospital (Hangzhou, China) from March 2008 to July 2009 were enrolled. Their clinicopathological data were collected.
Serum tumor markers measurement
After signing informed consent forms, each subject donated 3 mL of blood to be used for detecting serum TMs. Blood samples were collected with venipuncture before surgery. Serum was separated by centrifuge and then stored at −20°C until detection. The repeated thawing and freezing of samples was avoided.
Serum concentrations of carcinoembryonic antigen (CEA), CA125, CA199 were measured by i4000 light-emitting apparatus (Abbott Laboratories Ltd., US) with chemiluminescence. Serum levels of CA724 was tested by cobas e601 light-emitting apparatus (F. Hoffmann-La Roche Ltd., US) with an electrochemical luminescence. Serum CA242 concentrations was determined by automatic luminous instrument maglumi (New industries biomedical engineering Ltd., Shenzhen, China) with chemiluminescence. Reagents are all provided by above company.
The cut-off concentrations of CEA, CA125, CA199, CA724, CA242 were 20 U/mL, 35 U/mL, 37 U/mL, 6.9 U/mL, 20 U/mL, respectively, according to the manufacturer's instructions.
| > Follow-up|| |
All surgical patients were follow-up regularly. Computed tomography and endoscopy were carried out at regular intervals (every 3-6 months). The means of follow-up and data collection included regular outpatient follow-up, telephone or mailings follow-up. Overall survival was defined as the interval from the date of operation to the date of death or last follow-up. The follow-up period was 60 months.
Statistical analyses were carried out using SPSS 16.0. (SPSS, Chicago, IL) Fisher's exact test and Kaplan-Meier method were used to analyze the preliminary relations between TMs and clinicopathological factors or prognosis.
Receiver operating characteristic (ROC) curve was employed to delimited new cut-off values of these TMs according to lymph node metastasis, hematogenous metastasis, 2-year survival or 5-year survival, respectively. Based on these new cut-off values, the relations between serum TMs and clinicopathological factors were analyzed again using Chi-square test, and the relations between serum TMs and prognosis were analyzed again by Kaplan-Meier method. Differences between the Kaplan-Meier curves were evaluated with Log-rank test. Univariate and multivariate analyses of Cox regression proportional hazard model were performed to evaluate the prognostic parameters for survival.
Differences were considered as statically significant with P < 0.05.
| > Results|| |
0 Preliminary relations between serum tumor markers and clinicopathological factors
The positive rates of CEA, CA125, CA199, CA724, CA242 were 9.9% (31/314), 4.5% (14/314), 7.0% (22/314), 15.6% (49/314), 2.5% (8/314), respectively. CEA, CA125, CA199, CA724, except CA242, were related to hematogenous metastasis [Table 1]. Other clinicopathological factors were not associated with above serum TMs.
|Table 1: The relations between serum tumor markers and hematogenous metastasis based on the normal cut-off values |
Click here to view
New cut-off values
A series of cut-off values of these TMs were identified according to lymph node metastasis, hematogenous metastasis, 2-year survival or 5-year survival with ROC curve and area under the curve. CA125 and CA199 were significant in lymph node metastasis, and the cut-off values were 13.75 U/mL and 9.945 U/mL, respectively [Figure 1] and [Table 2]. CA125, CA199 and CA724 were significant in hematogenous metastasis, and the cut-off values are 25.35 U/mL, 10.1 U/mL and 6.25 U/mL, respectively [Figure 2] and [Table 3]. CA125 was significant in 2-year survival and 5-year survival, and the cut-off values were 13.65 U/mL and 13.25 U/mL, respectively [Figure 3] and [Figure 4], [Table 4] and [Table 5]. We incorporate the similar results from the same TMs, and chose 13.65 U/mL, 9.945 U/mL and 6.25 U/mL as new cut-off values of CA125, CA199 and CA724, respectively. We chose 25.35U/mL as the cut-off value of CA125 for ESCC hematogenous metastasis.
|Figure 1: The receiver operating characteristic curve for lymph node metastasis prediction were plotted to verify the optimum cut-off point for tumor markers|
Click here to view
|Figure 2: The receiver operating characteristic curve for hematogenous metastasis prediction were plotted to verify the optimum cut-off point for tumor markers|
Click here to view
|Figure 3: The receiver operating characteristic curve for 2-year survival prediction were plotted to verify the optimum cut-off point for tumor markers|
Click here to view
|Figure 4: The receiver operating characteristic curve for 5-year survival prediction were plotted to verify the optimum cut-off point for tumor markers|
Click here to view
Relations between serum tumor markers and clinicopathological factors with new cut-off values
Based on these new cut-off values, the relations between these serum TMs and clinicopathological factor were analyzed [Table 6]. CA199, CA125 and CA724 were associated with ESCC hematogenous metastasis, and CA199 and CA125 were associated with ESCC lymph node metastasis, but not associated with other clinicopathological factors.
|Table 6: Relations between serum tumor markers and clinicopathological factors according to new cut-off values |
Click here to view
A total of 243 cases were treated by surgery. 34 cases were lost after follow-up a period, and treated as Censoring data. The loss ratio of follow-up was 14.00%. The 5-year overall survival was 41.15% by the Kaplan-Meier curves and Log-rank test. The prognosis was better in patients with CA125 ≤ 13.65 U/mL than those with CA125 > 13.65 U/mL [Figure 5]. Tumor length, vascular tumor thrombus, grading, TNM stage, T grade, Lymph node metastasis and CA125 were related with prognosis by univariate analyses [Table 7]. However, multivariate analyses revealed that vascular tumor thrombus, grading, T grade, Lymph node metastasis and CA125 were independent prognostic factors [Table 8].
|Figure 5: CA125 and prognosis of esophageal carcinoma patients with the optimum cut-off value. Patients with CA125 ≤ 13.65 U/mL had significantly better prognosis than patients with CA125 > 13.65 U/mL|
Click here to view
| > Discussion|| |
Carcinoembryonic antigen, which is a special product from cancer of mesodermal origin, is one of the first known TMs. CEA is an important TM in colorectal cancer.  Although the elevation of serum CEA was originally thought to be specific for the development of malignant tumors of the gastrointestinal tract, high levels of CEA have also been detected in extra gastrointestinal malignancies in the lung, breast. 
Carcinoembryonic antigen, determined alone or in combination with others, is still one of the most applied TMs. Its main clinical applications are prognosis, early diagnosis of recurrence and follow-up of patients with carcinomas. CEA is a useful TM for esophageal adenocarcinoma.  However, CEA showed significant difference between ESCC and esophageal adenocarcinoma.  The positive rate of CEA in ESCC ranges from 11.4% to 39%, , and sensitivity ranges from 4.8% to 30% , by other reports. Preoperative CEA did not correlate with clinicopathological factors, , and was not a prognostic factor. , However, some reports revealed that increased CEA levels were associated with distant metastases. , In our study, the positive rate of CEA was 9.9%. CEA was related to hematogenic metastasis, but was not associated with other clinicopathological factors, and was not a prognostic factor, which was similar to other reports. Several reports have suggested that CEA is associated with adhesion of malignant tumors, , which would explicate the relations between high CEA and hematogenic metastasis.
Carcinomic antigen (carbohydrate antigen) is identified from tumor cell lines or strains using monoclonal antibody technology. CA125, CA199, CA724, CA242 are the most common carcinomic antigens.
CA125, which is a repeating peptide epitope of the mucin MUC16, , is a member of the mucin family glycoproteins. CA125 has been used as a biomarker for ovarian cancer detection and management.  It may also be elevated in pancreatic cancer, and CA125 test using in the management of patients with this cancer was also evaluated by previous studies.  The positive rate of CA125 in ESCC was 16% in other report,  and 4.5% in ours' report. There was no report about the correlations between CA125 and clinicopathological factors or prognosis. To the best of our knowledge, this may be the first report the relations between CA125 and clinicopathological factors or prognosis. We discovered CA125 was associated with hematogenic metastasis and lymph node metastasis and was a independent risk factor when the cut-off value was 13.65 U/mL. CA125 is a repeating peptide epitope of the mucin MUC16,  which promotes cancer cell proliferation and inhibits anticancer immune responses.  Maybe, that is why CA125 was associated with hematogenic metastasis, lymph node metastasis and prognosis.
CA199, which was extracted from colon cancer cell lines in 1979, is an isolated Lewis antigen of the MUC1 protein. It was originally defined by a monoclonal antibody produced by a hybridoma prepared from the spleen cells of a mouse that had been immunized with the human colorectal carcinoma cell line, SW1116.  CA199 was applied in the diagnosis of pancreatic cancer, colon cancer, gastric cancer, and other gastrointestinal tumors as a specificity TM and often in combination use with CEA. The positive rate of CA199 in ESCC was 4.4%, and sensitivity ranges from 6.3% to 23%. The seropositivity of CA199 before treatment had no association with resectability, most clinicopathologic parameters of tumor progression or patient survival. In our study, the positive rate of CA199 was 7.0%. However, we discovered CA199 was associated with hematogenic metastasis and lymph node metastasis when the cut-off value was 9.945 U/mL. CA199 is a ligand of E-selectin that may play a role in the adhesion of cancer cells to endothelial cells, resulting in metastasis.  To our knowledge, this may be the first report that CA199 was associated with hematogenic metastasis and lymph node metastasis.
CA242, first isolated in 1985, was obtained by immunization of mice with the human adenocarcinoma cell line COLO 205 and fusion with the Sp 2/0 mouse myeloma cell line. It is a cancer-associated glycoconjugate expressed in mucin and found predominantly in the sera of pancreatic cancer patients.  Hence it was used as a TM for pancreatic and colon cancers ,, and also an important serum TM for the diagnosis of pancreatic cancer.
CA724, first described in 1981, is a high molecular weight mucin-like glycoprotein. It was elevated in a variety of cancers, including gastrointestinal, ovarian, endometrium, and lung cancers. ,,,, CA724 has been identified as a type of Science Translational Medicine specific for gastric cancer  and been suggested to be the most sensitive and specific biomarker for gastric cancer.  The positive rate of CA724 in ESCC was 42%. The sensitivity of CA 724 was 16-18%, which includes not only ESCC but also esophageal adenocarcinoma, and higher concentrations were found in esophageal adenocarcinoma. CA 724 was not associated with different local, lymphonodular, or metastatic tumor stages. The positive rate of CA724 was 15.6% in our study. CA724 was related to hematogenic metastasis, but was not associated with other clinicopathological factors, and was not a prognostic factor.
| > Conclusion|| |
CA125 was nan independent risk factor for the prognosis of ESCC. CEA, CA125, CA199 and CA724 were all associated with hematogenic metastasis, and CA125, CA199 were associated with lymph node metastasis in ESCC. The cut-off values for CA125, CA199 and CA724 should be 13.65 U/mL, 9.945 U/mL and 6.25 U/mL, respectively.
| > Acknowledgment|| |
This work was supported by Natural Science Foundation of Zhejiang (LQ12H16001) and General Research Project of Zhejiang Medical College (2013XZB02). Contributions made by each of authors toward the manuscript were listed as the followings: Design, definition of intellectual content, literature search, manuscript preparation (Hongguang Zhao); data acquisition, data analysis, statistical analysis (Wenhu Chen); clinical studies, experimental studies (Jie Wu), manuscript editing and review (Lifang Wang); concept (Weimin Mao).
| > References|| |
Ni XG, Bai XF, Mao YL, Shao YF, Wu JX, Shan Y, et al.
The clinical value of serum CEA, CA19-9, and CA242 in the diagnosis and prognosis of pancreatic cancer. Eur J Surg Oncol 2005;31:164-9.
Nakai Y, Isayama H, Sasaki T, Takahara N, Hamada T, Uchino R, et al.
A retrospective analysis of early CA19-9 change in salvage chemotherapy for refractory pancreatic cancer. Cancer Chemother Pharmacol 2013;72:1291-7.
Ambrogi V, Mineo TC, For the Multidisciplinary Tor Vergata University Study Group for Malignant Pleural Mesothelioma. Clinical and biologic prognostic factors in malignant pleural mesothelioma. Thorac Cancer 2012;3:289-302.
Fakih MG, Padmanabhan A. CEA monitoring in colorectal cancer. What you should know. Oncology (Williston Park) 2006;20:579-87.
Laurence DJ, Stevens U, Bettelheim R, Darcy D, Leese C, Turberville C, et al.
Role of plasma carcinoembryonic antigen in diagnosis of gastrointestinal, mammary, and bronchial carcinoma. Br Med J 1972;3:605-9.
Clark GW, Ireland AP, Hagen JA, Collard JM, Peters JH, DeMeester TR. Carcinoembryonic antigen measurements in the management of esophageal cancer: An indicator of subclinical recurrence. Am J Surg 1995;170:597-600.
Brockmann JG, St Nottberg H, Glodny B, Heinecke A, Senninger NJ. CYFRA 21-1 serum analysis in patients with esophageal cancer. Clin Cancer Res 2000;6:4249-52.
Shimada H, Takeda A, Arima M, Okazumi S, Matsubara H, Nabeya Y, et al.
Serum p53 antibody is a useful tumor marker in superficial esophageal squamous cell carcinoma. Cancer 2000;89:1677-83.
Munck-Wikland E, Kuylenstierna R, Wahren B, Lindholm J, Haglund S. Tumor markers carcinoembryonic antigen, CA 50, and CA 19-9 and squamous cell carcinoma of the esophagus. Pretreatment screening. Cancer 1988;62:2281-6.
Yamamoto K, Oka M, Hayashi H, Tangoku A, Gondo T, Suzuki T. CYFRA 21-1 is a useful marker for esophageal squamous cell carcinoma. Cancer 1997;79:1647-55.
Mealy K, Feely J, Reid I, McSweeney J, Walsh T, Hennessy TP. Tumour marker detection in oesophageal carcinoma. Eur J Surg Oncol 1996;22:505-7.
Shimada Y, Watanabe G, Kawamura J, Soma T, Okabe M, Ito T, et al.
Clinical significance of osteopontin in esophageal squamous cell carcinoma: Comparison with common tumor markers. Oncology 2005;68:285-92.
Nakashima S, Natsugoe S, Matsumoto M, Miyazono F, Nakajo A, Uchikura K, et al.
Clinical significance of circulating tumor cells in blood by molecular detection and tumor markers in esophageal cancer. Surgery 2003;133:162-9.
Shimada H, Nabeya Y, Okazumi S, Matsubara H, Shiratori T, Gunji Y, et al.
Prediction of survival with squamous cell carcinoma antigen in patients with resectable esophageal squamous cell carcinoma. Surgery 2003;133:486-94.
Kosugi S, Nishimaki T, Kanda T, Nakagawa S, Ohashi M, Hatakeyama K. Clinical significance of serum carcinoembryonic antigen, carbohydrate antigen 19-9, and squamous cell carcinoma antigen levels in esophageal cancer patients. World J Surg 2004;28:680-5.
Munck-Wikland E, Kuylenstierna R, Lindholm J, Wahren B. Carcinoembryonic antigen, CA 19-9 and CA 50 in monitoring human squamous cell carcinoma of the esophagus. Anticancer Res 1990;10:703-8.
Benchimol S, Fuks A, Jothy S, Beauchemin N, Shirota K, Stanners CP. Carcinoembryonic antigen, a human tumor marker, functions as an intercellular adhesion molecule. Cell 1989;57:327-34.
Pignatelli M, Durbin H, Bodmer WF. Carcinoembryonic antigen functions as an accessory adhesion molecule mediating colon epithelial cell-collagen interactions. Proc Natl Acad Sci U S A 1990;87:1541-5.
O'Brien TJ, Beard JB, Underwood LJ, Dennis RA, Santin AD, York L. The CA 125 gene: An extracellular superstructure dominated by repeat sequences. Tumour Biol 2001;22:348-66.
Yin BW, Lloyd KO. Molecular cloning of the CA125 ovarian cancer antigen: Identification as a new mucin, MUC16. J Biol Chem 2001;276:27371-5.
Armstrong A, Otvos B, Singh S, Debernardo R. Evaluation of the cost of CA-125 measurement, physical exam, and imaging in the diagnosis of recurrent ovarian cancer. Gynecol Oncol 2013;131:503-7.
Haglund C. Tumour marker antigen CA125 in pancreatic cancer: A comparison with CA19-9 and CEA. Br J Cancer 1986;54:897-901.
Xiang X, Feng M, Felder M, Connor JP, Man YG, Patankar MS, et al.
HN125: A novel immunoadhesin targeting MUC16 with potential for cancer therapy. J Cancer 2011;2:280-91.
Schultes BC, Whiteside TL. Monitoring of immune responses to CA125 with an IFN-gamma ELISPOT assay. J Immunol Methods 2003;279:1-15.
Koprowski H, Steplewski Z, Mitchell K, Herlyn M, Herlyn D, Fuhrer P. Colorectal carcinoma antigens detected by hybridoma antibodies. Somatic Cell Genet 1979;5:957-71.
Tsumatori G, Ozeki Y, Takagi K, Ogata T, Tanaka S. Relation between the serum E-selectin level and the survival rate of patients with resected non-small cell lung cancers. Jpn J Cancer Res 1999;90:301-7.
Lindholm L, Johansson C, Jaiisson EL, Hallberg C, Nilsson O. An immunoradiometric assay (IRMA) for the CA 50 antigen. In: Holmgren J, editor. Tumour Marker Antigen. Lund: Student Literature; 1985. p. 123-33.
Nilsson O, Johansson C, Glimelius B, Persson B, Nørgaard-Pedersen B, Andrén-Sandberg A, et al.
Sensitivity and specificity of CA242 in gastro-intestinal cancer. A comparison with CEA, CA50 and CA 19-9. Br J Cancer 1992;65:215-21.
Costache MI, Iordache S, Karstensen JG, Saftoiu A, Vilmann P. Endoscopic ultrasound-guided fine needle aspiration: From the past to the future. Endosc Ultrasound 2013;2:77-85.
Yang XQ, Chen C, Peng CW, Liu SP, Li Y. Carbohydrate antigen 242 highly consists with carbohydrate antigen 19-9 in diagnosis and prognosis of colorectal cancer: Study on 185 cases. Med Oncol 2012;29:1030-6.
Guadagni F, Roselli M, Amato T, Cosimelli M, Perri P, Casale V, et al.
CA 72-4 measurement of tumor-associated glycoprotein 72 (TAG-72) as a serum marker in the management of gastric carcinoma. Cancer Res 1992;52:1222-7.
Kornek GV, Depisch D, Rosen HR, Temsch EM, Scheithauer W. Comparative analysis of CA72-4, CA195 and carcinoembryonic antigen in patients with gastrointestinal malignancies. J Cancer Res Clin Oncol 1992;118:318-20.
Udagawa Y, Aoki D, Ito K, Uejima T, Uemura M, Nozawa S. Clinical characteristics of a newly developed ovarian tumour marker, galactosyltransferase associated with tumour (GAT). Eur J Cancer 1998;34:489-95.
Hareyama H, Sakuragi N, Makinoda S, Fujimoto S. Serum and tissue measurements of CA72-4 in patients with endometrial carcinoma. J Clin Pathol 1996;49:967-70.
Cramer DW, Bast RC Jr, Berg CD, Diamandis EP, Godwin AK, Hartge P, et al.
Ovarian cancer biomarker performance in prostate, lung, colorectal, and ovarian cancer screening trial specimens. Cancer Prev Res (Phila) 2011;4:365-74.
Chen XZ, Zhang WK, Yang K, Wang LL, Liu J, Wang L, et al.
Correlation between serum CA724 and gastric cancer: Multiple analyses based on Chinese population. Mol Biol Rep 2012;39:9031-9.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8]