SIMULTANEOUSLY BOTH EXPRESSION OF LMP-1 AND METHYLATION OF E-CADHERIN: MOLECULAR BIOMARKER IN STAGE IV OF NASOPHARYNGEAL CARCINOMA PATIENTS
Lao TD1, Truong PK1, Thieu HH1, Nguyen DH2, Nguyen MT3, Le TAH1,*
*Corresponding Author: Associate Professor Thuy A.H. Le, Department of Pharmaceutical and Medical Biotechnology, Ho Chi Minh City Open University, 35-37 Ho Hao Hon Street, Ho Chi Minh City, Vietnam. Tel.: +84-905-784-471. E-mail: thuy.lha@ou.edu.vn
page: 57

RESULTS

Sample Collection. A total of 93 NPC biopsies and 100 non cancerous samples were collected in local hospital. The characteristics of all 93 NPC samples are summarized in Table 2. The mean age of NPC specimens ranged from 20 to 81 (mean: 53.51 ± 1.43). According to sex distribution, the number of males (accounting for 73.12%) was more than that of females (accounting for 26.88%). Histological type: the number of type 3 (undifferentiated carcinoma) was the highest, accounting for 67.74%. The stage 4 (advanced stage), accounting for 48.39%, was the highest among stages of NPC patients. LMP-1 Expression in Nasopharyngeal Carcinoma and Non Cancerous Samples. The expression of LMP-1 and GAPDH were evaluated by qPCR. The GAPDH gene was used as the internal control for evaluating the expression of LMP-1. As the expression of GAPDH, both NPC samples and control samples were positive. The Ct values of GAPDH were 27.14 ± 1.79 and 27.75 ± 2.14 in case and control groups, respectively. There are no significant differences between the expression of GAPDH in the case and control groups (p >0.05) (Figure 1). According to the expression of LMP-1, 71 of the 93 NPC specimens (accounting for 76.34%) were positive, while none of the controls were positive (Figure 1). A value of p <0.0001 indicated that LMP-1 gene expression was significantly associated with NPC. The relative sensitivity, specificity, negative likelihood ratio, positive predictive value, as well as negative predict value were 76.34% (95% CI = 66.40-84.54), 100.00% (95% CI = 96.37-100.00), 0.24 (95% CI = 0.17-0.34), 100.00%, 81.97% (95% CI = 75.93-86.75), respectively. We found no significant association was found between LMP-1 expression and the clinicopathological characteristics, including patients’ gender (p = 0.06), age (p = 0.19), tumor histological types (p = 0.29) and stage of NPC (p = 0.07) (Table 3). Additionally, the OR value was 638.73 (95% CI = 38.12- 10,703.57, p <0.0001), indicated that the positive association between the expression of LMP-1 and risk of NPC. Methylation of E-Cadherin Status in Nasopharyngeal Carcinoma Samples and Non-Cancerous Samples. In the current case-control study, nested-MSP was applied in evaluation of the methylation status of the E-cadherin gene promoter in Vietnamese samples. The methylation and unmethylation frequencies of the E-cadherin gene promoter in NPC biopsy samples were 73.12% (68 of 93 samples) and 26.88% (25 of 93 samples), respectively. In the case of non-cancerous samples, the methylation and unmethylation frequencies were 30.00% (30 of 100 samples) and 70.00% (70 of 100 samples), respectively. The relative sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, positive predictive value, as well as negative predictive value were 73.12% (95% CI = 62.92-81.79), 70.00% (60.02- 78.76), 2.44 (95% CI = 1.76-3.37), 0.38 (95% CI = 0.27- 0.55), 69.39% (95% CI = 62.12-75.81) and 73.68% (95% CI = 66.17-80.04), respectively. Additionally, the methylation status of the E-cadherin gene promoter in NPC samples was found to be significant higher than in healthy samples, and strongly associated to the cancer of nasopharynx (p <0.0001). The nested-MSP products (stage 2) of methylation and/or un-methylation in the promoter of E-cadherin in samples were observed in electrophoresis with visualized by ethidium bromide staining and are shown in Figure 2. The sequencing of samples methylated in the promoter region of representative sample revealed a conversion of unmethylated cytosine, but not methylated cytosine (Figure 2). By sequencing, comparison between the nonbisulfite- modified and bisulfite-modified, all methylated cytosines were unchanged, which were marked as square symbols. Otherwise, the unmethylated cytosines, which were marked as triangle symbols, were changed into thymine in the bisulfite sequence (Figure 3). In this study, the OR values were computed between the methylation of E-cadherin and NPC. The results show that the OR value was 6.35 (95% CI = 3.39-11.88, p <0.0001). It indicated that there was significant association between the methylation of the E-cadherin gene promoter and risk of NPC. Additionally, only significant association between E-cadherin methylation and patients’ gender was observed (p = 0.05). No significant association was found between E-cadherin methylation and the clinicopathological characteristics, including patients’ age (p = 0.52), tumor histological types (p = 0.16) and stage of NPC (p = 0.14) (Table 3). Association Between E-Cadherin Methylation and LMP-1 Expression. As shown in Table 4, the χ2 test was applied to determine the relationship between the LMP-1 expression and E-cadherin methylation. Based on the positive index (PI = 0: both LMP-1 and methylation of E-cadherin were negative; PI = 0.5: either LMP-1 or E-cadherin was positive; PI = 1.0: both LMP-1 and methylation of E-cadherin were positive) among 93 NPC biopsy samples, 61 cases (accounting for 65.59%) were positive for both expression of LMP-1 and E-cadherin methylation (PI = 1.0). This also meant that among 71 samples that were positive for LMP-1 expression, 61 cases (85.92%) were positive for E-cadherin methylation. The p value (p <0.0001) indicated that the expression of LMP-1 was positively significantly associated with the methylation of E-cadherin. Analysis with clinicopathological characteristics, no significant association was between PI value and the clinicopathological characteristics, including patients’ gender (p = 0.14), age (p = 0.42), and histological type (p = 0.42). Only significant association between PI value and patients’ NPC stage was observed (p = 0.04) (Table 5). The result indicated that there was a significant tendency of LMP-1 expression and methylation of E-cadherin in NPC biopsy samples of the advance stage (Table 5, Figure 4). As shown in Figure 4, among 37 samples that were positive for LMP-1 expression, 35 cases of 37 samples (accounting for 94.60%) were positive for methylation of E-cadherin. Therefore, it could be inferred that the expression of LMP-1 may have an ability to methylate the gene of E-cadherin, and those genes were associated with NPC in advanced stages.



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