2.1 Free circulating plasma EBV DNA

In 1999, Lo et al. [28] discovered the presence of free circulating plasma EBV DNA in 55 of 57 NPC patients and only 3 of 43 healthy controls. The discovery of disproportionately high amounts of free circulating EBV DNA among NPC patients led to an explosion of new detection techniques on various possible EBV genome targets, such as BamHI-W [28], and EBV-encoded RNA-1 (EBER-1) [2]. The rationale of detecting free circulating EBV DNA in NPC management rests on the fact that NPC cells release EBV DNA during apoptosis [29, 30]. These EBV DNA fragments released by NPC are typically of shorter fragment (less than 181 base-pairs) compared to the classical fragments released during the viral lytic phase, making them highly quantifiable using polymerase chain reaction (PCR) assays [20, 29, 31-34]. Additionally, the detection of free circulating plasma EBV DNA can be applied on cerebrospinal fluid (CSF) and other samples, such as dried blood spots, nasopharyngeal swabs and aspirates, sputum and bronchial washings [34].

The detection of plasma EBV DNA in routine clinical application is limited by differences in interpretation of detection threshold and the timing of specimen collection following treatment [34]. Preiksaitis et al. [35] demonstrated significant inter-laboratory variation in reported EBV DNA levels amongst 28 international laboratories, of which only 47.0% were within acceptable standards. Currently, no US Food and Drug Administration (FDA)-approved assay exists, making harmonization and standardization of various assays challenging [34]. This deficiency in standardization of EBV DNA assays was addressed in the first International Standard for EBV assay released by the World Health Organization in 2011 [36]. In 2014, the Stanford Laboratory established commutability [37, 38] for two EBV PCR assays: the BamHI W assay and the commercial QIAsymphony Rotor-Gene Q (QS-RGQ) assay. The latest 2015 National Cancer Institute Workshop on Harmonization of EBV Testing for Nasopharyngeal Cancer further set sequential goals for test standardization [34]. The first step is to ensure standardization of sample type (plasma or serum) and DNA isolation protocol. Secondly, it recommends the development of a standardized PCR assay that targets both a single-copy EBV gene [e.g., Epstein-Barr virus nuclear antigen 1 (EBNA-1)] and a multi-copy EBV gene (e.g. BamHI-W). This serves to maximize sensitivity of detection (via multi-copy targets) while permitting accurate quantification of viral load (via single-copy targets) [34]. At present, the single-copy approach is preferred for measuring tumor burden across different patients, while the multi-copy approach is preferred when tumor cells are present in low amounts (e.g., NPC screening of asymptomatic patients and post-treatment monitoring for NPC recurrence). Ultimately, clinical validation of these assays across multi-institutions is necessary in order to define test sensitivity and specificity and establish clinically relevant cut-off values in each of these clinical applications.

Presently, most commercial assays use BamHI-W on plasma samples because this target yields the most consistent results across various studies. Additionally, an enhancement of the detection of BamHI-W was made by amplifying the CpG repeats on this DNA fragment, resulting in an even higher sensitivity in EBV detection among NPC patients [39]. Furthermore, Lam et al. [40] found that the positive predictive value (PPV) of detecting plasma EBV in NPC patients was improved from 11.0% to 19.6% when a sequencing-based DNA analysis was used. This technique revealed higher amounts of plasma EBV DNA and generally longer fragment lengths of plasma viral molecules in NPC patients than in non-NPC subjects [40].

2.2 EBV serology markers

Since NPC arises from the nasopharyngeal mucosa, a high IgA response is anticipated [33]. NPC cells produce latency-associated EBV gene products, such as latent membrane protein 1 (LMP1), LMP2, and BamHI-A rightward frame 1 (BARF1), but only EBNA-1 induces strong IgG and IgA antibody responses [33]. Lytic phase EBV proteins, such as the early antigen (EA) and viral capsid antigen (VCA), are produced during the rapid viral replication phase of differentiating NPC cells. Therefore, strong IgA antibody responses against these proteins are often identified among NPC patients. However, these serological markers are not exclusively identified in NPC. IgA against EBV VCA may be raised in repeated infections of EBV or frequent reactivation of latent EBV in B cells, and can revert back to normal titers for unknown reasons without developing NPC [41]. It may also be present in systemic lupus erythematosus, possibly implicating EBV in the pathogenesis of systemic lupus erythematosus; or indicating a difficulty in suppressing the underlying latent EBV infection [42]. This relatively non-specific nature of positive IgA results limits its accuracy in diagnosing NPC [43].

2.3 Other emerging EBV detection markers

Other potential liquid biopsy markers include circulating tumor cells (CTCs, which may comprise of micro-metastatic tumor cells that precede distant metastasis [44]), miRNAs (small 21-23 nucleotide non-coding RNAs [45] that are encoded during cellular replication [46]), and DNA hypermethylation (hypermethylation of potential tumor suppressor genes [47] that occurs early in pre-malignant cells [32, 48]). There is emerging evidence on the utility of these detection methods in NPC management. An overview of these existing and emerging detection modalities of various EBV detection markers is summarized in Table 1.

CTCs and CTC clusters or microemboli are a rare and highly heterogenous group of cells in the peripheral blood, that exhibit different molecular features and play different roles in cancer progression [49, 50]. There are only a handful of studies on CTCs in NPC detection, and these studies differ in the method of CTC isolation, making the application of CTCs in the detection of NPC controversial. He et al. [51] reported that CTC enumeration was correlated with EBV VCA-IgA and EBV DNA load in all NPC patients with or without distant metastasis, but Vo et al. [39] concluded that EBV DNA levels showed no correlation with CTC counts. Furthermore, You et al. [52] observed that CTCs had superior specificity (86.0% vs. 41.0%) and inferior sensitivity (42.3% vs. 81.3%) when compared with plasma EBV DNA load in diagnosing distant metastasis. It is worth noting that the first two studies used size-based isolation systems to isolate CTCs, namely Isolation by Size of Epithelial Tumour Cells (ISET) [51] and Microsieve [39], whereas the latter study used CellSearch technology [52]. Size-based isolation attempts to separate leukocytes from CTCs, as leukocytes are smaller, but some overlap in the size distribution of the two entities impedes accurate separation [53]. CellSearch is currently the only CTC isolation system approved by the US FDA [54]. This platform uses a marker-based CTC enrichment technique with magnetic nanoparticles which capture antibodies that are tagged onto the epithelial cell adhesion molecules (EpCAM) expressed on CTCs. Similarly, different studies use different definitions to classify CTCs. While cells isolated by ISET method were considered CTC-positive if they were Cytokeratin (CK)-positive or P63-positive, Microsieve classified CK-positive and CD45-negative cells as canonical CTCs, and CellSearch classified CK-positive, EpCAM-positive, 4′,6-diamidino-2-phenylindole (DAPI)-positive, and CD45-negative cells as CTCs. These differences in the methods used to isolate and classify CTCs could be the primary reason behind the different conclusions reached in different studies. As CTCs are highly heterogenous, marker-based CTC enrichment methods might miss certain sub-populations of CTCs [55]. Hence, it is important to standardize the methods and definitions used to classify CTCs in future studies.

There are numerous reports on the role of miRNAs in the pathogenesis of NPC [56]. However, the mechanism by which deregulation of these epigenetic regulators affects cell signaling pathways in NPC is not well understood. Of the two families of EBV-encoded miRNAthe BamHI fragment H rightward open reading frame 1 (BHRF1) and the BamHI-A rightward transcripts (BART), the former has low expression in NPC cell lines, while the latter has been closely linked to the tumorigenesis of NPC [57]. A handful of studies have evaluated the usefulness of circulating BART miRNAs as potential biomarkers for detection of NPC [22, 46], as will be discussed later in this paper.

Hypermethylation of the CpG island of promoter region results in the silence of tumor suppressor genes, and this feature is a characteristic abnormality seen in EBV-associated cancers [58-60]. Using a viral-wide genome approach, Lam et al. [61] found distinct EBV DNA methylation profiles among different EBV-associated diseases; and by analyzing methylated DNA sequences allowed differentiation between NPC and non-NPC subjects. Aberrant methylation of tumor suppressor genes such as cadherin 4 (CDH4), cyclin-dependent kinase inhibitor 2A (CDKN2A/p16), and RAS association domain family protein 1A (RASSF1A) are early events in NPC tumorigenesis [62-64]. This hypermethylation is mediated by EBV-encoded LMP1 and LMP2A proteins that up-regulates the expression of DNA methyltransferases (DNMTs), namely, DNMT1, DNMT3A, and DNMT3B [65]. The presence of early and widespread methylation changes, frequently occurring in several chromosomal locations such as 3p21.3, 9p21, and 6p21.3, led to the evaluation of these liquid biomarkers for early detection of NPC. In the first study evaluating the detection rate of hypermethylation, Chang et al. [47] compared detection rates of hypermethylated p15, p16, RASSF1A, E-cadherin, and death-associated protein kinase (DAPK) in tumor samples, nasopharyngeal swabs, mouth and throat rinsing fluid, plasma, and buffy coat. However, hypermethylation was found in less than 20% of peripheral blood samples as compared to 17%-63% of nasopharyngeal swabs and 17%-50% of mouth and throat rinsing fluids. Later on, Wong et al. [66] detected hypermethylation in at least one of the 5 genes (CDH1, DAPK1, CDKN2B, RASSF1, and CDKN2A) in 71% of plasma samples from NPC patients before treatment, with a specificity of 91%. Subsequently, the use of hypermethylation panels was heavily explored, and this will be discussed in the next section on sensitivity and specificity section.

4.1 General population screening in a Chinese community

Historically, two large-scale serological screening studies were performed to detect NPC in a general Southern Chinese cohort in a community setting (Table 3). Zeng et al. [15] utilized both IgA EA and VCA serological screening among 12,932 adults. The NPC detection rate was 100.5/100,000 persons, which was almost double that of the indigenous detection rate of NPC. However, in a larger study by Deng et al. [16] in 1995, a lower detection rate of 31.4/100,000 persons was identified using both IgA EA and VCA serological screening of 318,912 Chinese adults aged 30-70 years. For screened adults aged 45-49 years, the detection rate was 68.9/100,000 persons [16].

Presently, serological detection of anti-EBV antibodies utilizes ELISA, which is less tedious and time-consuming and has lower intra-observer variability [18] than traditional, indirect immunofluorescence (IF) techniques [80]. Additionally, serological detection tends to use a combination of IgA VCA and IgA EBNA-1 [81], which has higher sensitivity and specificity than combining IgA VCA with IgA EA [18].

In this regard, two large-scale studies using ELISA and the IgA EBNA-1 and IgA VCA combination have been performed. Yu et al. [18] studied a population of 16,712 persons between 2009 and 2015 and obtained an increased detection rate of 149.59/100,000 persons. When both IgA EBNA-1 and IgA VCA were seropositive, the sensitivity was 48.9%, and the specificity was 99.5%; when either of them was positive, the sensitivity was 99.5%, but the specificity dropped to 90.1% [18]. Liu et al. [17] in 2013 screened 28,688 Cantonese using the combination of IgA EBNA-1 and IgA VCA serology, which yielded a similar detection rate of 142.9/100,000 persons.

Interestingly, Yu et al. [18] boosted NPC detection rates through implementing a longer follow-up protocol. By stratifying the population into various risk levels based on serological titers and follow-up duration, they eventually detected 47 NPC patients (281.24/100,000). Notably however, the compliance rate by the second year of follow-up fell to approximately half the initial number (43.7%-53.9%). Hence, longer-term follow-up protocols may be able to boost NPC detection rates, but compliance and additional expenses may limit the widespread adoption of this method.

Furthermore, despite these advancements, the PPV of serological tests remained low (Yu et al. [18]: 2.3-2.6%) compared with that of EBV DNA (Chan et al. [20]: 11%). This implies that more test-positive individuals may be subject to costly confirmatory tests, such as endoscopy and magnetic resonance imaging. Even though serological test retain the logistical and technical advantages in being a cheap, high-throughput, and rapid test [81], the overall costs may still be higher than that of plasma EBV DNA detection due to the low PPV. Suggested solutions usually involve the combination of serology with alternative methods. For example, Jiang et al. [82] proposed that miRNA BART2-5p detection has the potential to improve the PPV of mass serological screening, by distinguishing between pre-clinical NPC cases and matched serological high-risk controls (higher EBV VCA IgA and EBNA-1 IgA values) with a sensitivity of 90.9% and a specificity of 54.5%.

In the recent decade, EBV DNA assays targeting BamHI-W gene fragments have yielded much higher detection rates than earlier studies utilizing serology. Zeng et al. [15] in 1982 yielded NPC detection rate of 100.5/100,000 persons using IgA EA, followed by Deng et al. [16] in 1995 yielding detection rate of 17.2/100,000 persons using IgA EA and VCA in combination. However, when plasma EBV targeting the BAM-HIW fragment was used to screen NPC, tChan et al. [20] in 2017 showed a higher detection rate of 168.5/100,000 persons. Similarly, a direct comparison study by Chan et al. [19] in 2013 on 1318 individuals aged 40-60 years yielded an NPC detection rate of 227.6/100,000 using plasma EBV DNA detection, but only 75.9/100,000 when IgA VCA detection was used as the detection method.

A largescale EBV DNA study by Chan et al. [20] in 2017 screened 20,178 adults aged 40-62 years. By using qPCR to target the BamHI-W fragment of EBV DNA, the results surpassed earlier serological studies, with an NPC detection rate of 168.5/100,000 and PPV of 11.0%, and with high sensitivity (97.1%) and specificity (98.6%). Furthermore, the majority of patients (70.6%, 24 out of 34) had early-stage disease (stage I or II), contrary to an unscreened cohort in the same period (19.3%, 149 of 773) [20, 33]. Earlier detection was associated with longer survival [15, 16, 19, 20], such that a 97% 3-year progression-free survival (PFS) rate was achieved in the landmark study [20]. Therefore, at present, EBV DNA detection targeting the Bam HI-W fragment has demonstrated a superior detection rate and PPV as compared to serological methods of EBV detection.

4.2 NPC screening in high-risk cohorts (first-degree relatives of NPC patients)

There is paucity of data using EBV detection methods to screen for NPC among high-risk cohorts. When IgA VCA and IgA EA serology were used to screen for NPC among 929 first-degree relatives of index NPC patients, the detection rates were 433/100,000 and 499/100,000 person-years among males and females, respectively [83]. This detection rate was more than double compared to the serological detection rates of NPC among the general population: 142.9/100,000 by Liu et al. [17] in 2012 and 149.6/100,000 by Yu et al. [18] in 2018. In 2010, Ng et al. [84] used IgA VCA to screen for NPC among 1,199 asymptomatic first-degree relatives of index NPC patients and identified 15 patients with NPC (detection rate: 1,251/100,000 persons). The majority of these patients had early-stage disease (Stage I: 41.2%, Stage II: 17.6%). The utility of other EBV detection methods, especially using plasma EBV DNA assay warrants further exploration to screen for NPC among high-risk individuals.

5.1 Pre-treatment plasma EBV DNA and other EBV status detection techniques in prognostication

Lin et al. [85] in 2004 found that with a pre-treatment plasma EBV DNA cut-off value of 1500 copies/mL, 83.4% of those with detectable EBV DNA before treatment survived at least 2 years, as compared to 100% of those with undetectable EBV DNA (P < 0.001). Prayongrat et al. [86] in 2017 found that with a pre-treatment plasma EBV DNA cut-off value of 600 copies/mL, 67% of those with detectable EBV DNA before treatment survived at least 5 years, as compared to 82.6% of those with undetectable plasma EBV DNA (P < 0.05).

Across all studies using plasma EBV DNA assay, those with detectable plasma DNA before treatment had 2- to 5-year overall survival (OS) rates lower than those with undetectable EBV DNA [85-89] (Table 4). Sengar et al. [68] found the mean OS to be 26.7 months shorter in patients with detectable pre-treatment EBV DNA levels than in those with undetectable pre-treatment EBV DNA levels (42.4 months vs 69.1 months). Unfortunately, cut-off values varied across these studies, and an optimal cut-off value is still being determined [90].

Additionally, pre-treatment plasma EBV DNA may be incorporated into existing staging systems because it is highly predictive of survival. Leung et al. [91] found that stage II NPC patients could be further stratified into high-risk (with a survival rate similar to stage I patients: about 90%) and low-risk groups (with a survival rate similar to stage III patients: approximately 63%) according to the pre-treatment plasma EBV DNA level, although the cut-off value was unreported. Kitpanit et al. [92] found that integration of pre-treatment plasma EBV DNA levels (with a cut-off value of 2300 copies/mL) into the 8^th edition of the American Joint Committee on Cancer (AJCC) TNM staging system allows better survival prediction than using the TNM staging system alone (Harrell's C-concordance index increased from 0.690 to 0.760 for OS and from 0.650 to 0.744 for PFS). This improved staging system using pre-treatment plasma EBV DNA may possibly allow a more accurate selection of high-risk patients who might benefit from treatment intensification [92].

On the other hand, IgA EBV serology has not been shown to be consistent in prognosticating NPC patients [88, 89]. Twu et al. [88] showed that while the presence of plasma EBV DNA before treatment was associated with survival, pre-treatment IgA and IgG VCA titers did not demonstrate any association with survival or risk of cancer relapse. In a separate NPC cohort, Ling et al. [89] found that those with undetectable IgA EA/VCA before treatment had higher 5-year survival rate than those with detectable values. In this study, there was no end-to-end comparison with plasma EBV DNA in prognosticating this cohort of NPC patients.

CTCs have been explored to be possibly linked to survival [111], although a head-to-head comparison by Vo et al. [39] showed that pre-treatment EBV DNA was better associated to survival than pre-treatment CTC.

5.2 Post-treatment plasma EBV DNA and other EBV status detection techniques in prognostication

While 80%-99% of NPC patients would achieve complete remission after treatment [94, 95], 30%-40% of patients with locally advanced (stage III-IV) NPC would eventually recur [95]. Lo et al. [96] observed that patients who relapsed within the first year had higher median levels of plasma EBV DNA t than those without relapse (median: 41,756 vs. 5807 copies/mL). Furthermore, post-treatment plasma EBV DNA was found to be a prognostic marker of NPC independent of the tumor stage (P = 0.003) [96]. Using a cut-off of 500 copies/mL, Chan et al. [95] showed that the 1-year PFS and OS rates were higher in patients with undetectable post-treatment EBV DNA levels than in those with detectable levels (PFS: 93% vs. 48%; OS: 97% vs. 76%). Hence,post-treatment plasma EBV DNA levels can be used to identify patients at risk of developing recurrence.

Survival is consistently poorer among NPC patients with detectable post-treatment plasma EBV DNA levels than among those with undetectable plasma EBV DNA levels [85, 88, 93, 95] (Table 5). The difference in OS rate between patients with detectable and undetectable plasma EBV DNA levels appears to be more stark using post-treatment plasma EBV DNA (21.0%-76.1%) [85-88, 93, 95] than using pre-treatment EBV DNA (6.6%-32.8%) [85-89] (Tables 4 and 5). It was hypothesized that patients with persistently elevated plasma EBV DNA levels after definitive intensity-modulated radiotherapy may have minimal residual disease [95] and hence, may benefit from adjuvant chemotherapy [97]. However, a randomized phase III study led by Chan et al. [93] did not show any prolonged recurrence free survival with adjuvant chemotherapy (cisplatin and gemcitabine) following definitive treatment among patients with detectable post-treatment plasma EBV DNA. Nevertheless, post-treatment plasma EBV DNA level was associated with the risks of locoregional failure, distant metastasis, and death. More recently, Hui et al. [25] validated that integrating post-treatment plasma EBV DNA levels with the TNM staging system improved risk stratification of NPC. Additionally, low-risk NPC patients were identified using recursive partition model analysis with the view that they could potentially be spared from adjuvant chemotherapy.

The role of real-time monitoring of EBV DNA levels during chemotherapy was also found to be useful. Wang et al. [27] found that during the first month after chemotherapy, a slower EBV DNA clearance rate, determined by longer half-life (T_1/2) value, was associated with poorer survival rates. More recently, Lv et al. [26] also suggested that trajectory of plasma EBV clearance following induction chemotherapy can be used to modulate intensification or de-intensification of subsequent treatment. They measured and tracked plasma EBV DNA levels after each cycle of induction chemotherapy and clustered these NPC patients into early responders, intermediate responders, late responders, and treatment-resistant subjects. Significant differences in disease-free survival were observed between these groups. On the other hand, unlike EBV DNA, post-treatment IgA and IgG VCA titers demonstrated no association with survival or the risk of cancer relapse [88].

5.3 Prognostic value of plasma EBV DNA and other EBV status detection techniques in cancer staging

Across studies, plasma EBV DNA levels have consistently been shown to be associated with NPC stage [28, 39, 86] (Table 6), suggesting a direct correlation between plasma EBV DNA titers and tumor burden. EBV IgA serology [89] and miRNA titers [21, 46] have also been shown to significantly associate with stage, but conflicting reports exist regarding the prognostic role of CTC [39, 98]. Si et al. [98] demonstrated a significant association with stage for epithelial and mesenchymal CTCs (P < 0.05), but Vo et al. [39] found no significant association with stage for canonical and potential CTCs.

AUTHORS’ CONTRIBUTIONS

Development of research topic: R Tan, SKA Phua, YL Soong, LLE Oon, KS Chan, SS Lucky, J Mong, MH Tan, CM Lim

Data accrual and analysis: R Tan, SKA Phua, YL Soong, LLE Oon, KS Chan, SS Lucky, J Mong MH Tan, CM Lim

Manuscript writing, vetting and final approval: R Tan, SKA Phua, YL Soong, LLE Oon, KS Chan, SS Lucky, J Mong, MH Tan, CM Lim.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

Not applicable.

CONSENT FOR PUBLICATION

Not applicable.

CONFLICT OF INTEREST STATEMENT

Minhan Tan is the CEO of Lucence Diagnostics. The rest of the authors declare no conflict of interest.

FUNDING

Not applicable.