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 Table of Contents  
ORIGINAL ARTICLE
Year : 2014  |  Volume : 26  |  Issue : 1  |  Page : 2-7

Correlation of radiotherapy with serum total and lipid-bound sialic acid in OSCC patients


1 Department of Oral Medicine and Radiology, Babu Banarasi Das College of Dental Sciences, Lucknow, Uttar Pradesh, India
2 Department of Oral Medicine and Radiology, NIMS Dental College and Hospital, Jaipur, Rajasthan, India
3 Department of Oral Medicine and Radiology, Chhattisgarh Dental College, Chhattisgarh, India

Date of Submission06-Jun-2014
Date of Acceptance10-Sep-2014
Date of Web Publication26-Sep-2014

Correspondence Address:
Saurabh Srivastava
C-2053/3, Indira Nagar, Lucknow - 226 016, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0972-1363.141823

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   Abstract 

Context: Increased quantities of glycoconjugates such as Total Sialic Acid (TSA) and Lipid-bound Sialic Acid (LSA) have been detected in the plasma and serum of patients with various malignancies, indicating their usefulness in diagnosis or monitoring of the treatment modality. Aims: (1) To estimate and compare the serum TSA and LSA levels in Oral Squamous Cell Carcinoma (OSCC) patients before and after radiotherapy, as also in healthy individuals. (2) To determine the correlation, if any, between Tumor-Node-Metastasis (TNM) staging and levels of TSA and LSA. (3) To determine the use of serum TSA and LSA as biomarkers of OSCC. Settings and Design: The study was designed as a case-control study and was undertaken in a dental college and cancer hospital. Materials and Methods: It was planned to estimate the serum TSA and LSA levels of 20 healthy individuals and of 20 OSCC patients, spectrophotometrically; before starting and one month after completion of radiotherapy. Statistical Analysis: The Statistical Package for Social Sciences (SPSS Version 10.0) was used. Results: The mean serum TSA and LSA levels in OSCC patients decreased significantly after radiotherapy; however, they were still higher than the levels in the controls. In untreated OSCC patients, a statistically significant positive correlation was observed between the TNM stage of the disease and the serum TSA levels; but the same was not found between the TNM stage of the disease and the serum LSA levels. In OSCC, the serum TSA and LSA levels had a positive relationship with the TNM stages. These levels decreased significantly after radiotherapy. Conclusion: Serum TSA and LSA can be utilized as potential diagnostic and prognostic indicators in OSCC.

Keywords: Lipid-bound sialic acid, oncology, oral squamous cell carcinoma, radiotherapy, total sialic acid


How to cite this article:
Srivastava S, Sathawane RS, Mody RN. Correlation of radiotherapy with serum total and lipid-bound sialic acid in OSCC patients . J Indian Acad Oral Med Radiol 2014;26:2-7

How to cite this URL:
Srivastava S, Sathawane RS, Mody RN. Correlation of radiotherapy with serum total and lipid-bound sialic acid in OSCC patients . J Indian Acad Oral Med Radiol [serial online] 2014 [cited 2019 Apr 20];26:2-7. Available from: http://www.jiaomr.in/text.asp?2014/26/1/2/141823


   Introduction Top


Early detection of oral squamous cell carcinoma (OSCC) is the most important for its management. The therapeutic modalities currently offered are based on traditional stage-predicting indices and on the histological grading of the tumor. Studies on malignant cells have revealed alterations in cell surfaces and membranes. There is a significant role of glycoproteins in the malignant transformation, [1] as they are released into circulation through increased turnover, secretion, and/or shedding from malignant cells. [2] The carbohydrate portion contains amino sugars (glucosamine, galactosamine or sialic acid) and hexoses (galactose, mannose) or fucose. [3]

During malignant transformation, one of the most common changes in the glycoconjugates, is the increase in size of the oligosaccharides, resulting in branching sites for incorporation of sialic acid. Sialic acid occupies a position at the terminal or near to terminal, which underlies its vital role in determining the surface characteristics of cells and secreted glycoproteins. Being a non-reducing terminal, sialic acid has gained outstanding importance in cancer research. [1]

Increased quantities of glycoconjugates like Total Sialic Acid (TSA), Lipid-bound Sialic Acid (LSA), mucoid proteins, and Protein Bound Hexoses (PBH) have been detected in the plasma or serum of patients with different types of malignancies, indicating their usefulness in diagnosis or monitoring therapy. [3]

Monitoring the therapy in cases of OSCC by serum tests is alluring in view of its ease, economic advantage, convenience of repeated sampling, and minimal invasiveness. There is no literature regarding the alterations in serum sialic acid levels in OSCC patients, who have undergone only radiotherapy as a treatment modality, to assess the effectiveness of the therapy during various stages of the disease; hence, this study has been carried out to estimate and compare serum TSA and LSA levels, before and after radiotherapy, in OSCC patients and in healthy individuals and correlate them with the TNM staging.


   Materials and Methods Top


Population included (Inclusion criteria)

The present study included two groups:

  1. Group Ia - Consisting of 20 clinically and histopathologically proven patients of OSCC, planned to be treated only by radiotherapy. (30-35 fractions of 200 cGy given daily five times a week for six-seven weeks),
  2. Group Ib - Consisting of 15 OSCC patients from group Ia, who had completed the full course of radiotherapy, as mentioned (Five patients did not complete radiotherapy; hence were excluded), and
  3. Group II - Consisting of 20 healthy individuals without any history of local or systemic disease, from the same age group as that of the study group.


Exclusion criteria

  1. Patients with a history of major illness, such as, chronic liver disease, diabetes mellitus, inflammatory disease (pneumonia), rheumatoid arthritis, psoriasis, or any other malignancy.
  2. Patients who had already undergone surgery as the primary treatment modality.
  3. Patients who were to undertake concomitant chemotherapy along with radiotherapy.


A detailed case history of all patients was taken, along with the clinical examination. Clinical staging (as per American Joint Committee of Cancer (AJCC)) of all OSCC patients was done and the histopathological cell differentiation was recorded [Table 1]. Approval of the Institutional Ethical Committee as well as a written and informed consent was taken from all patients included in this study.
Table 1: Clinical details of cases

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Sample collection

Five millilitres of fasting blood samples were collected by venipuncture from the cubital vein, maintaining meticulous asepsis, between 9 a.m. and 11 a.m., to avoid any diurnal variations. The samples were centrifuged and the serum was separated and stored at −70 °C till analyzed. [4]

Follow-up sample

Fasting blood samples were again collected one month after completion of radiotherapy, between 9 a.m. and 11 a.m., centrifuged, serum separated, and stored till analysis.

Assays

Estimation of serum total sialic acid

The thiobarbituric acid assay (TBA) [5] was used to determine serum total sialic acid (TSA). Samples of glycoprotein (0.5 ml) were hydrolyzed in an equal volume of 0.05 M-H 2 SO 4 at 80°C for 60 minutes and 0.05 ml of 25 mM-periodic acid was added. Oxidation was terminated by the addition of 0.05 ml of 2% (w/v) sodium arsenite, followed by the addition of 0.1 ml of 6% (w/v) thiobarbituric acid. The color was intensified by the addition of 1.5 ml of dimethyl sulfoxide. The optical density was measured spectrophotometrically at 549 nm and 532 nm against a blank, to overcome any interference from 2-deoxy-D-ribose.

Estimation of serum lipid-associated sialic acid

As per the method suggested by Katopodis and Stock, [6] 150 μl of distilled water was taken in a culture tube and 50 μl of serum was added to it. Three millilitres of cold (4-5°C) chloroform:methanol 2: 1 (v/v) was added to the tube. To this mixture 0.5 ml of cold distilled water was added, the tube was capped, centrifuged for five minutes at room temperature, at 2500 rpm, and then one ml of the upper layer was transferred into a culture tube. Fifty microliters of phosphotungstic acid solution (1 g/ml) was added to it. The tube was centrifuged for five minutes, at 2500 rpm, and the supernatant was removed by suction. One ml of water and one ml of the resorcinol reagent were added, mixed, and placed in boiling water for exactly 15 minutes. Immediately after 15 minutes, the tube was transferred to an ice and water bath, and left for ten minutes. To the ice-cold tube, 2 ml of 85: 15 (v/v) butyl acetate: N-butyl alcohol was added at room temperature and centrifuged for five minutes at 2500 rpm. The extracted color was read colorimetrically, at 580 nm.

The TSA and LSA calibration was done by the standard curves [Figure 1] and [Figure 2] obtained for various concentrations of N-acetylneuraminic acid (Sigma, St. Louis, MO, USA).
Figure 1: Standard calibration curve for TSA

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Figure 2: Standard calibration curve for LSA

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To calculate the concentration of TSA and LSA, the following formula was used:




   Statistical Analysis Top


Statistical analysis of the data was done using the Statistical Package for Social Sciences (SPSS Version 10.0). Values were given in mean ± standard deviation. The student's unpaired t-test was used to analyze the variability in measurement of the TSA and LSA levels in groups I and II. The student's paired t-test was used to analyze the variability in measurement of the TSA and LSA levels in groups Ia and Ib. Spearman's rank correlation was used to evaluate the association between alterations in the marker levels and stage-wise disease activity in group Ia and group Ib.


   Results Top


Comparison of mean serum TSA and LSA levels between group Ia (cases) and group II (controls)

The mean serum TSA level in group Ia was 61.667 ± 10.989 mg/dl and in group II it was 28.569 ± 2.322 mg/dl. The mean serum LSA level in group Ia was 39.600 ± 8.083 mg/dl and in group II it was 17.429 ± 1.369 mg/dl. Comparison of the mean serum TSA and LSA levels of group Ia with those in group II, using the student's unpaired t-test, revealed the difference in the means to be statistically significant (P < 0.05).

Comparison of the mean serum TSA and LSA levels between group Ia (before radiotherapy) and group Ib (after radiotherapy)

Five patients did not complete the treatment, and hence, were not considered for the comparison. The mean serum TSA level in group Ia was 59.619 ± 10.947 mg/dl and in group Ib it was 54.521 ± 10.815 mg/dl. The mean serum LSA level in group Ia was 36.648 ± 5.673 mg/dl and in group Ib it was 34.362 ± 5.334 mg/dl. Comparison of the mean serum TSA and LSA levels of group Ia with Ib, using the student's paired t-test revealed the difference in the means to be statistically significant (P < 0.05).

Comparison of mean serum TSA and LSA levels between group Ib (after radiotherapy) and group II (controls)

Mean serum TSA level in group Ib was 54.520 ± 10.815 mg/dl and in group II it was 28.569 ± 2.322 mg/dl. The mean serum LSA level in group Ib was 34.362 ± 5.334 mg/dl and in group II it was 17.429 ± 1.369 mg/dl. Comparison of the mean serum TSA and LSA levels in group Ib with those in group II using the student's unpaired t-test revealed the difference in the means to be statistically significant (P < 0.05).

Comparison of serum TSA and LSA levels with clinical stages of OSCC in group Ia

The difference in the mean serum TSA levels in stages II, III, and IV was statistically significant (P < 0.05). However, the difference in the mean serum LSA levels in stages II, III, and IV was statistically non-significant (P > 0.05).

Correlation of serum TSA and LSA levels with stages of OSCC in group Ia

The Spearman's rank correlation test was used to find the association between the alterations in the marker levels and stage-wise disease activity in group Ia. A positive correlation (r = 0.927) was found between the TNM stage of the disease and the serum level of TSA, and it was statistically significant (P < 0.05). A positive correlation (r = 0.092) between TNM stage of disease and serum level of LSA was also found, but it was statistically nonsignificant (P > 0.05).

Comparison of serum TSA and LSA levels with clinical stages of OSCC in group Ib

The difference in the mean serum TSA levels in stages II, III, and IV was statistically significant (P < 0.05). However, the difference in the mean serum LSA levels in stages II, III, and IV was statistically nonsignificant (P > 0.05).

Correlation of serum TSA and LSA levels with stages of OSCC in group Ib

The Spearman's rank correlation test was used to find the association between alterations in the marker levels and stage-wise disease activity in group Ib. A positive correlation (r = 1) between the TNM stage of disease and serum level of TSA was found, and it was statistically significant (P < 0.05). A negative correlation (r = −0.5) between the TNM stage of disease and serum level of LSA was found to be statistically nonsignificant (P > 0.05).


   Discussion Top


It has been reported that the carbohydrate compositions of cell-surface glycoproteins and glycolipids change in malignant cells, which alters the normal biochemical circuit of the cells. These altered glycoproteins and glycolipids are released into the circulation through increased turnover, secretion, and/or shedding. They are of considerable interest for their potential diagnostic and prognostic value. This carbohydrate moiety may influence differentiation, growth, and cell-to-cell interactions, and thus, may play a crucial role in malignant transformation. [1],[2],[7],[8],[9] The increase of plasma TSA concentration in patients with malignant diseases could be explained by an increased output of protein from the liver as a nonspecific secondary reaction, and by an intensified output of tumor cells with a high content of sialic acid. [8]

It has also been found that the estimation of more than one form of sialic acid is more suitable for diagnosis, monitoring of the disease extent, and anticancer therapy, than of a single form. [1],[2],[7],[8],[10],[11] The earlier studies have not compared the alterations of serum TSA and LSA levels in patients with OSCC treated only by radiotherapy. Hence, the present study has been carried out to evaluate and compare the levels of serum TSA and LSA, before and after radiotherapy, in OSCC patients.

Among the colorimetric methods in use for the quantification of sialic acid (N-Acetyl Neuraminic Acid (NANA)), the thiobarbituric acid test is the most sensitive due to its highest molar extinction coefficient. [5] Determination of LSA has been done by a rapid assay developed by Katopodis and Stock. Its sensitivity in the detection of cancer ranged from 77 to 97%. [6]

The present study comprised of 17 males and three females. This variation in sex distribution could be due to the increased prevalence of habits like tobacco chewing, smoking, and so on, in the males than the females. [12] Age-distribution in the present study ranged from 35 to 70 years, with a mean age of 52.65 years. In other studies by Rajpura et al., in 2005, and Raval et al., in 2003, the age ranged from 14-80 years. This variation could be due to the stringent exclusion criteria, increased frequency and duration of habits in the older population than in the younger ones, or a smaller sample size.

One month after radiotherapy, clinically, none of the patients showed the complete disappearance of the lesion, but there was reduction in the size of the lesion along with relief from symptoms, such as pain and burning sensation. This could be due to the fact that all the cases enrolled in the present study were ulcerative lesions of varying size.

In the present study, group Ia had a mean serum TSA level of 61.667 ± 10.989 mg/dl, which was comparable with the findings of previous studies on untreated OSCC patients by various investigators such as Rao et al., in 1998, (82.1 ± 21.8 mg/dl), Rajpura et al., in 2005, (63.70 ± 19.40 mg/dl), Baxi et al., in 1991, (54.78 mg/dl), and Raval et al., in 2003, (63.20 mg/dl).

The alteration of serum TSA levels between the various stages [Table 2] in group Ia was found to be statistically significant (P < 0.001). There was a positive correlation (r = 0.927) of the serum TSA levels with the TNM stages, which was statistically significant (P < 0.001) [Table 3]. Rao et al., in 1998, also found good correlation (r = 0.83; P < 0.001) between the serum TSA and LSA levels with the TNM stage, in untreated cancer patients. Rajpura et al., in 2005, found the alterations in the serum TSA levels and the TNM stages to be statistically significant (P = 0.0001). The alterations in the serum TSA levels between the various histopathological variants in group Ia were found to be statistically nonsignificant (P = 0.987), which was in accordance with the study done by Rajpura et al. in 2005 (P = 0.265).
Table 2: Comparison of serum TSA and LSA levels with the clinical stages of OSCC in group Ia

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Table 3: Spearman's rank correlation between serum TSA and LSA levels with stages of OSCC in group Ia

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Group II had a mean serum TSA level of 28.569 ± 2.322 mg/dl and the difference in TSA levels compared with group Ia were statistically significant (P < 0.001). Similar findings (30.25 ± 2.49 mg/dl, P < 0.001) were reported by Rajpura et al., in 2005.

In the present study, group Ia had a mean serum LSA level of 39.6 ± 8.083 mg/dl, which was similar to the findings of previous studies on untreated OSCC patients by various investigators, such as, Rao et al., in 1998, (25.2 ± 10.4 mg/dl), Rajpura et al., in 2005, (32.81 ± 5.10 mg/dl), and Baxi et al., in1991, (37.88 mg/dl), using the same method as developed by Katopodis and Stock for LSA estimation. Other investigators like Raval et al., in 2003, found the serum LSA level in untreated OSCC cases to be 12.06 mg/dl. The possible reason for the difference in the serum LSA level could be the use of a different technique for its estimation, as given by Winzler.

The alteration of the serum LSA levels between the various stages [Table 2] in group Ia were found to be statistically non-significant (P = 0.407). However, there was a positive correlation (r = 0.378) of the serum LSA levels with the TNM stages, which was also statistically non-significant (P = 0.092) in the present study [Table 3]. Rao et al., in1998, (P < 0.01), and Rajpura et al., in 2005, (P = 0.039), found a statistically significant difference in the serum levels of LSA with the TNM stages. This difference in findings could be attributed to the variation in sample size, in various stages among the studies.

The alteration of serum LSA levels between the various histopathological variants in group Ia was found to be statistically non-significant (P = 0.443), which was in accordance with the study by Rajpura et al., in 2005 (P = 0.857).

Group II (controls) had a mean serum LSA level of 17.429 ± 1.369 mg/dl and the difference of LSA level compared with group Ia was statistically significant (P < 0.001), similar to that reported by Rao et al., in 1998, (19.5 ± 4.7 mg/dl), Rajpura et al., in 2005, (16.32 ± 2.97 mg/dl), and Baxi et al., in 1991, (18.64 mg/dl) (P < 0.001).

In the present study, group Ib had a mean serum TSA level of 54.521 ± 10.815 mg/dl, which was less than that in group Ia (59.619 ± 10.947 mg/dl) and this alteration in serum TSA level was statistically significant (P < 0.001). The mean serum LSA level in the present study in group Ib was 34.362 ± 5.334 mg/dl, which was less than that in group Ia (36.648 ± 5.673 mg/dl), and this alteration in the serum LSA level was also found to be statistically significant (P < 0.001).

Alterations in the serum TSA levels between groups Ia and Ib were statistically significant in stage II (P = 0.011) and stage III (P = 0.025), but were statistically non-significant in stage IV (P = 0.177). As the number of patients [Table 4] in stage IV was very less (n = 2), the statistical results were undesirable. There was no variation between the means of the serum LSA levels in group Ia and group Ib, the possible reason for this could be a further reduction of a small sample size, by segregation of the sample into stages, thus increasing the chances of diminished results.
Table 4: Comparison of serum TSA and LSA levels with clinical stages of OSCC in group Ib

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Group Ib had a mean serum TSA level of 54.521 ± 10.815 mg/dl, which was greater than group II (28.569 ± 2.322 mg/dl) and this alteration in the serum TSA level was statistically significant (P < 0.001). There was a positive correlation (r = 1) of the serum TSA levels with the TNM stages, which was statistically significant (P < 0.001) [Table 5]. The mean serum LSA level in the present study in group Ib was 34.362 ± 5.334 mg/dl, which was greater than in group II (17.429 ± 1.369 mg/dl) and this alteration in serum LSA level was statistically significant (P < 0.001). However, there was a negative correlation (r = −0.5) of the serum LSA levels with the TNM stages, which was also statistically non-significant (P = 0.666) in the present study [Table 5].
Table 5: Spearman's rank correlation between serum TSA and LSA levels with stages of OSCC in group Ib

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As there was a paucity of studies relating to the serum TSA and LSA levels in OSCC patients before and after radiotherapy, no comparable data could be found. Raval et al., in 2003, evaluated the efficacy of TSA and LSA levels in the treatment monitoring of OSCC cases after anti-cancer therapy. When comparing the present study with the above-mentioned one, similar results were achieved for the serum TSA level, which had declined from the pretreatment level, but did not reach the control level, both as a group and stage-wise. This study differed in terms of the response of the serum LSA level, which declined from the pretreatment level significantly, but did not reach the control level of the group, whereas, the stage-wise alteration was non-significant. The possible reason could be the further segregation of the small sample into stages.


   Conclusion Top


Serum TSA and LSA levels increase in OSCC. The serum TSA and LSA levels increase with the stage of the disease (OSCC). The levels of serum TSA and LSA after radiotherapy are decreased, but are still higher than the levels in the control group. Hence, serum TSA and LSA can be utilized as potential prognostic indicators for OSCC. To strengthen our conclusions, further studies need to be carried out on a larger population and for a longer duration, for serial measurements of these indicators after radiotherapy.

 
   References Top

1.Rajpura KB, Patel PS, Chawda JG, Shah RM. Clinical significance of total and Lipid-bound sialic acid levels in oral pre-cancerous conditions and oral cancer. J Oral Pathol Med 2005;34:263-7.  Back to cited text no. 1
    
2.Rao VR, Krishnamoorthy L, Kumaraswamy SV, Ramaswamy G. Circulating levels in serum of total sialic acid, lipid-associated sialic acid, and fucose in precancerous lesion and cancer of the oral cavity. Cancer Detect Prev 1998;22:237-40.  Back to cited text no. 2
    
3.Baxi BR, Patel PS, Adhvaryu SG, Dayal PK. Usefulness of serum glycoconjugates in precancerous and cancerous diseases of the oral cavity. Cancer 1991;67:135-40.  Back to cited text no. 3
    
4.Plucinsky MC, Riley WM, Prorok JJ, Alhadeff JA. Total and lipid-associated serum sialic acid levels in cancer patients with different primary sites and differing degrees of metastatic involvement. Cancer 1986;58:2680-5.  Back to cited text no. 4
[PUBMED]    
5.Skoza L, Mohos S. Stable thiobarbituric acid chromophore with dimethyl sulphoxide. Application to sialic acid assay in analytical de-O-acetylation. Biochem J 1976;159:457-62.  Back to cited text no. 5
[PUBMED]    
6.Katopodis N, Hirshaut Y, Geller NL, Stock CC. Lipid-associated sialic acid test for the detection of human cancer. Cancer Res 1982;42:5270-5.  Back to cited text no. 6
[PUBMED]    
7.Bhuvarahamurthy V, Balasubramanian N, Vijayakumar S, Govindasamy S. Effect of radiation on serum glycoproteins and glycosidases in patients with cervical carcinoma. Int J Gynaecol Obstet 1995;48:49-54.  Back to cited text no. 7
    
8.Painbeni T, Gamelin E, Cailleux A, Le Bouil A, Boisdron-Celle M, Daver A, et al. Plasma sialic acid as a marker of the effect of the treatment on metastatic colorectal cancer. Eur J Cancer 1997;33:2216-20.  Back to cited text no. 8
    
9.Riley WM, Tautu C, Verazin G, Gregory J, Josiah S, Prorok JJ, et al. Evaluation of sialic acid concentrations in serum for the diagnosis and staging of breast cancer. Clin Chem 1990;36:161-2.  Back to cited text no. 9
    
10.Raval GN, Parekh LJ, Patel DD, Jha FP, Sainger RN, Patel PS. Clinical usefulness of alterations in sialic acid, sialyltransferase and sialoproteins in breast cancer. Indian J Clin Biochem 2004;19:60-71.  Back to cited text no. 10
    
11.Raval GN, Patel DD, Parekh LJ, Patel JB, Shah MH, Patel PS. Evaluation of serum sialic acid, sialyltransferase and sialoproteins in oral cavity cancer. Oral Dis 2003;9:119-28.   Back to cited text no. 11
    
12.Malati T. Tumor markers: An overview. Indian J Clin Biochem 2007;22:17-31.  Back to cited text no. 12
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    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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