Background
Nasopharyngeal carcinoma (NPC) is highly prevalent in Southern China and South-East Asia. The incidence of NPC in Indonesia is 6.2 cases/100,000 population per year representing the fourth most common cancer [
1]. Based on size of the Indonesian population it is estimated that 13,000 new cases of NPC occur annualy. Early detection is needed to improve patient survival since the majority of cases are currently diagnosed at late stage.
Given the close link between NPC and Epstein-Barr virus (EBV) infection, detection of characteristic antibodies against EBV and elevated viral load has been proposed as useful screening tool [
2‐
4]. Recently, we started a screening protocol in the Yogyakarta region recruiting high risk patients with chronic problems in the head and neck area and testing them using EBV-based assays. Although this study is still ongoing, current observations indicate that neither method provides an adequate stand-alone marker for detecting NPC as a primary screening test due to their relative low positive predictive value (PPV) (Hutajulu et al., unpublished data) confirming other reports [
5,
6]. The low PPV might be due to the large number of subjects presenting with elevated EBV antibody levels and viral load that showed no clinical mass. Indeed, antibody levels against EBV were shown to be elevated for as long as ten years before tumor presentation [
4]. As a consequence, recruitment of subjects by EBV-based markers alone would require long term monitoring. CT-scan examination and nasopharyngeal biopsy are needed for clinical confirmation of tumor presence. This would imply that a huge number of people would need detailed examination resulting in unacceptably high costs. It is therefore essential to define additional NPC progression markers for better selection of high risk patients.
Exploration of altered cellular genes involved in NPC development may provide a complementary test for risk assessment in EBV infected individuals. Promoter DNA methylation is widely considered to be an important epigenetic mechanism in carcinogenesis. In NPC, gene silencing by promoter methylation has been shown for multiple tumor suppressor genes (TSGs). Each TSG potentially contributes to the multistep oncogenesis including alterations of apoptosis, cell cycle and mitotic checkpoint regulation, intracellular adhesion, cytoskeleton organization, and Wnt-signalling pathway [
7‐
9]. Therefore, in the present study we determined the frequency of promoter DNA methylation of multiple genes in Indonesian NPC cases. These TSGs were proven to be frequently methylated in NPC from other ethnic groups [
10‐
16]. To analyse their potential value for detection of early-stage NPC, we compared the methylation pattern of NPC patients, healthy EBV carriers and a high risk population. The latter group consisted of patients with chronic head and neck complaints showing elevated EBV markers. Furthermore, we analysed an additional TSG, the myelin and lymphocyte-associated protein (MAL), which has been proven methylated in head and neck cancer [
17] but has not been tested in NPC. More importantly, MAL gene is frequently methylated in cervical cancer indicating its role in virus-related epithelial malignancy [
18].
Discussion
Methylation of promoter DNA is considered an important epigenetic event in NPC carcinogenesis [
7‐
9] and has been proven promising for diagnosis of multiple type of tumors [
18,
19]. Since the profile of TSG promoter methylation may vary according to tumor type [
20], this study defined epigenetic variability in NPC. Aberrant methylation of five TSGs was identified as independent marker for early detection of NPC with added value to EBV IgA serology and DNA load. For a developing country like Indonesia such a test should be economical with simple but well standardized technology suited for screening in a large population. Therefore qualitative MSP is proposed, being less labor-intensive and inexpensive compared to other assays such as Q-MSP or bisulfite sequencing.
We selected a set of markers that have been evaluated in various NPC populations. Since methylation changes have been reported to occur early in carcinogenesis [
7], epigenetic markers are potentially relevant as early indicators of subclinical presence of NPC. Our present study demonstrated that the promoter methylation frequency of individual TSGs is variable in NPC, confirming previous publications [
10‐
16]. The methylation frequency of single genes varied from 29.2% (RASSF2A gene) to 79.2% (DAPK1 gene). This frequency is comparable to the one presented in other studies except for WIF1 [
12] and RASSF2A [
14] where the frequency in the Indonesian samples was much lower (61.2% and 29.2%, respectively).
For particular genes (DAPK1, CADM1, CDH13 and DLC1), the methylation frequency is not only high in NPC cases, but also in the high risk group and normal EBV carriers, thus limiting their diagnostic value. The high frequency of methylation has not been reported before in other healthy control populations, mostly showing a frequency below 10% [
15,
16,
21,
22]. However, the occurrence of methylation in normal tissue was confirmed in PBMCs and LCLs obtained from healthy individuals. In CADM1, the high frequency of methylation detected by MSP was explained by frequent but low level methylation in both normal and NPC brushing using Q-MSP. This suggested that CADM1 is not a good marker for the early detection of NPC. Similarly DAPK1, CDH13 and DLC1 could be considered as non-specific markers.
A panel of methylation markers consisting of CHFR, RIZ1, p16, WIF1 and RASSF1A is proposed as a complementary test for early NPC detection. These five TSGs showed high methylation in NPC cases and not more than 4% in the regional healthy EBV positive controls providing good differentiation between cancer cases and normal controls. Detection of aberrant methylation in at least one of this panel showed a rate of 98% in NPC group compared to individual markers. In particular, detection of aberrant methylation of p16 and RASSF1A may provide more simple testing with specificity and sensitivity above 90%.
Pilot testing of MAL methylation using Q-MSP revealed this gene as an additional discriminator between NPC and normal controls. As explored previously in cervical [
18] the MAL gene may play a specific role in viral-epithelial malignancies. Since the MAL gene has not been analysed in NPC before, this report is the first to describe it as a promising marker for further investigation.
Comparing paraffin tissue and brushing samples of NPC cases revealed a good detection of methylation for both sample types, supporting the results of Sun
et al. [
16]. Nasopharyngeal brushing is proposed for sampling in population screening studies considering it is convenient and simple. When determined only on brushing samples, methylation of any marker of the selected genes was detected in 39 out of 41 NPC cases (95.1%). This is comparable to the assessment using either paraffin or brushing which detected methylation in 98% of NPC. Moreover, this minimal invasive procedure yielded high PPV (>90%) compared to biopsy and reproducible high yields of cellular DNA (10
6-10
7 genome copies/brush). Nasopharyngeal brushing is also an excellent sampling method for monitoring EBV DNA load and EBV mRNA expression, each having good diagnostic value [
23].
Our present study did not demonstrate a significant difference between methylation frequency in early and late stage NPC cases (data not shown). To evaluate the capacity of epigenetic markers to detect preclinical disease, a longitudinal study is currently in progress in the local high risk population. Compared to the baseline observation, this cohort revealed an increased frequency of methylation of p16 and RIZ1. The presence of p16 methylation supports previous reports showing p16 gene silencing in precancerous lesions of NPC. However our data do not confirm RASSF1A methylation in premalignant tissues [
7]. This indicates that epigenetic changes leading to malignancy in the Indonesian population may differ from other populations. Which gene silencing event initiates NPC development and which contributes to further progression of the multistep carcinogenic process remains to be investigated.
The correlation found between methylation status and viral DNA load in Indonesian NPC cases indicates a link between epigenetic events and EBV infection. The appropriate level of DNA load in our NPC samples may directly reflect a (pre)malignant process. This is in agreement with a previous data on matched NPC tumor and adjacent tissues. The nearer to the NPC tissue, the higher the levels of gene promoter methylation and EBV DNA load found [
11]. More recent studies implied the role of EBV genes in epigenetic silencing either through activation of DNA methyltransferases [
24] or interaction with transcriptional repression [
25].
Factors known to contribute to methylation alterations include age, diet and lifestyle [
26,
27]. Regarding the factor of age, our result on Indonesian NPC showed a tendency for methylation frequency to increase with age. This reflects accumulative epigenetic events under the influence of environmental exposures that may increase with time. Considering the factor of diet, our observation in the local population demonstrates that diet might also predispose one towards cancer development. Formaldehyde, boric acid, Rhodamine B, and yellow Metanyl are among many reported chemicals found in the food of local markets in Indonesia [
28]. Persistent exposure to environmental toxins is indicated as an initial step of epigenetic alteration preceding cancer development [
29‐
31]. This might explain the high frequency of methylation of certain genes observed in the Indonesian healthy population. The potential relationship between carcinogenic exposure and promoter methylation status is subject to further study. Analysis of the methylation status of selected genes is proposed as additive test for NPC risk assessment using EBV-based assays in primary screening. The epigenetic markers described here provide complementary information in a subgroup of high risk individuals with aberrant EBV IgA seroreactivity and elevated viral load. In a case-finding approach, screening subjects presenting with symptoms suspicious of NPC, this approach may identify those subjects at highest risk of NPC development.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
JMM conceived the study. SHH carried out the molecular work, statistical analysis, data interpretation and wrote the initial draft of the manuscript. SRI and LPLI participated in clinical sampling. H participated in sample selection and pathological expertise. SD carried out quantitative methylation-specific PCR and participated in data interpretation. SMH, RDS and AEG supervised the molecular work, participated in data interpretation and edited the manuscript. JMM and SHH had the primary responsibility for interpreting the data and editing the final manuscript. All authors provided comments of various drafts, participated in direction setting discussions and reviews and have read and approved the final version.