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
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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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