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. 2011 May 2:10:48.
doi: 10.1186/1476-4598-10-48.

Epigenetic markers for early detection of nasopharyngeal carcinoma in a high risk population

Susanna H Hutajulu  1 , Sagung R Indrasari, Luh P L Indrawati, Ahmad Harijadi, Sylvia Duin, Sofia M Haryana, Renske D M Steenbergen, Astrid E Greijer, Jaap M Middeldorp
Affiliations

Epigenetic markers for early detection of nasopharyngeal carcinoma in a high risk population

Susanna H Hutajulu et al. Mol Cancer. .

Abstract

Background: Undifferentiated nasopharyngeal carcinoma (NPC) is strongly related to Epstein-Barr virus (EBV) infection, allowing aberrant antibodies against EBV and viral DNA load as screening tools in high risk populations. Methylation analysis in the promoter of tumor suppressor genes (TSGs) may serve as a complementary marker for identifying early cases. This study determined methylation status of multiple TSGs and evaluated whether it may improve early detection.

Methods: Nasopharyngeal brushings were taken from 53 NPC patients, 22 high risk subjects and 25 healthy EBV carriers. Corresponding NPC paraffin tissue was included. DNA was bisulfite-modified preceding analysis by methylation-specific PCR (MSP). Ten TSGs were studied.

Results: NPC paraffin and brushing DNA revealed an 81.8% concordance so that MSP analysis was done using either one of both specimens. NPC samples showed methylation for individual TSGs (DAPK1 79.2%, CDH13 77.4%, DLC1 76.9%, RASSF1A 75.5%, CADM1 69.8%, p16 66.0%, WIF1 61.2%, CHFR 58.5%, RIZ1 56.6% and RASSF2A 29.2%). High risk individuals, having elevated EBV IgA and viral load, showed high frequency of methylation of CDH13, DAPK1, DLC1 and CADM1, but low frequency of methylation of p16 and WIF1 and undetectable methylation of RASSF1A, CHFR, RIZ1 and RASSF2A. Healthy subjects showed similar patterns as high risk individuals. A combination of RASSF1A and p16 gave good discrimination between NPC and non-NPC, but best results were combined analysis of five methylation markers (RASSF1A, p16, WIF1, CHFR and RIZ1) with detection rate of 98%.

Conclusion: Multiple marker MSP is proposed as a complementary test for NPC risk assessment in combination with EBV-based markers.

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Figures

Figure 1
Figure 1
Box plot of seroreactivity of IgA/[EBNA-1+VCA-p18] ELISA by group (normal, high risk and NPC) with cut-off value of 0.354. The seroreactivity in NPC and high risk group was significantly higher than that in normal subjects (Mann-Whitney U test all p < 0.001). NPC = nasopharyngeal carcinoma, CoV = cut-off value.
Figure 2
Figure 2
Boxplot of (log) EBV DNA load in nasopharyngeal brushings by group (normal, high risk and NPC). The cut-off value was set at (log) 2,300 copies/brushing (3.362). There were highly significant variations between median in the three groups (one-way ANOVA, p < 0.001). Using Tukey analysis, the differrences between median in all groups are all significant. NPC = nasopharyngeal carcinoma, CoV = cut-off value.
Figure 3
Figure 3
Representative results of methylation specific PCR in the three groups. NPC isolates showed more frequent methylated genes compared to high risk and normal isolates. L = marker, UMD = unmodied DNA, C- = negative control, C+ = positive control, NP = nasopharyngeal carcinoma, HR = high risk, N = normal, U = unmethylated alele, M = methylated alele.
Figure 4
Figure 4
Scatter diagram of relative quantity of methylation of (A) CADM1 and (B) MAL gene. The Y axis is expressed in log scale. CADM1 (A) showed an insignificant variations between normal brushing and NPC brushing and between NPC brushing and NPC paraffin (one-way ANOVA using Tukey analysis p = 0.654 and 0.069 respectively). Thus Q-MSP confirmed the result of MSP. MAL (B) showed insignificant variations between normal brushing and NPC brushing (p = 0.070). Methylation level in NPC paraffin was comparable to NPC brushing (p = 0.069) but was significantly higher compared to normal brushing (p = 0.002) indicating that MAL methylation can be a promising marker for NPC detection. HR = high risk, NPC = nasopharyngeal carcinoma.
Figure 5
Figure 5
Scatter diagram of (A) ELISA seroreactivity on 53 NPC sera, (B) log EBV DNA load on 50 NPC brushings and (C) age of 53 NPC cases versus number of methylated tumor suppressor genes. The correlation with methylation rate is significant but weak for EBV DNA load (Pearson correlation r = 0.322, p = 0.023) and insignificant for ELISA seroreactivity (r = -1.88 p = 0.117) and age (r = 0.270, p = 0.05). The latter becomes significant when excluding a young patient from a multiplex family (symbolized with star) (p = 0.007).
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