**2. EBV related pathogenesis of NPC**

The pathogenesis of NPC includes multi-stepped process that leads to the development of NPC **(Fig. 1.).** EBV infection alone cannot drive normal cells towards carcinoma development. It is thought that loss of heterozygosity (LOH on chromosome 3p and 9p, which are the location of some tumor suppressor genes), possibly as a result of inherited traits (Chinese ethnicity) as well as exposure to dietary factors (salted fish) and other environmental cofactors (Formaldehyde), is an early stage event in the pathogenesis of this disease. EBV is infected within these low-grade pre-invasive lesions, subsequent to further genetic and epigenetic alterations.

EBV was first suspected to be linked with NPC on the basis of the serological observations by Old and colleagues (Old et al., 1966) in 1966. This link was formally demonstrated later by in situ hybridization of the viral DNA in the nuclei of epithelial cells (zur Hausen et al., 1970). The full length EBV genome is contained in all malignant epithelial cells, but not in most infiltrating lymphocytes. The association with EBV is constant, regardless of the

Epstein-Barr Virus Serology in the Detection and Screening of Nasopharyngeal Carcinoma 85

EBV infection to NPC formation(Choi et al., 2011; Ji et al., 2007; Ng et al., 2010; Ng et al., 2005), the long period of pre-clinical detectable phase (PCDP) offer the opportunity for

Serological studies have shown that the clinical onset of NPC is preceded by the appearance of a high titer of various EBV antibodies such as viral capsid antigens (VCA) IgA(Li et al., 2010; Ng et al., 2010; O et al., 2007), anti-EBV DNase(Chien et al., 2001) and combined EBV EA(early antigen) + EBNA-1 (Nuclear antigen 1) IgA test(Chang et al., 2008)( **Table 1.**).

> VCA IgA (ELISA)

VCA IgA (ELISA and IF)

(ELISA)

VCA IgA (ELISA and IF)

assay)

Table 1. Reported plasma EBV antibody in NPC diagnosis and risk assessment of NPC

For NPC serodiagnosis, cell-based indirect immunofluorescent assay (IF) methods are widely considered the gold standard (Paramita et al., 2009). IF involves the separate analysis of antibody responses to VCA, EA, and EBNA, and requiring different cell lines for specific analysis. However, this method shows considerable variation among laboratories and is time-consuming, subjective, and not suitable for large-scale automatic handling. Enzymelinked immunosorbent assay (ELISA) techniques are increasingly used recently and have shown a better sensitivity and specificity compared to IFA and which are suitable for largescale application. Stage is one of the most important prognostic predictors of NPC. NPC in its early stages are highly curable with radiotherapy. Screening may change the distribution of stage and prognosis. In a cohort study undertaken in Wuzhou (Guangxi province, China) in the early 1980s (Zeng et al., 1985), total 1136 individuals identified as positive for Ig A

EBNA1 IgA (ELISA)

EA+EBNA1 IgA

VCA IgA(IF) and anti-DNase (enzyme neutralization

Sensitivity 90.6% Specificity 93.5%

Sensitivity 83.3% Specificity 87.0%

Sensitivity 94.2% Specificity 82.6%

Sensitivity 92% Specificity 98%

(7.3–66.9)

Rate ratio VCA IgA 22.0

anti-DNase 3.5 (1.4–8.7)

Rate ratio 4.7 (1.4–16)

**3. Applications of EBV antibodies and EBV DNA as markers of NPC in** 

Study type Author Marker Results of the utility

screening and early diagnosis of NPC.

Case-control study O *et al.* 

**3.1 Plasma EBV protein in the diagnosis of NPC** 

(O et al., 2007)

Ng *et al.* (2010) (Ng et al., 2010)

(Li et al., 2010)

Yu *at al.* (2011) (Yu et al., 2011).

IF: immunofluorescent assay; ELISA: enzyme-linked immunosorbent assay

*et al.* (2001) (Chien et al., 2001)

Chang *et al.*  (2008) (Chang et al.,

2008)

Meta-analysis Li *et al.* (2010)

Cohort study Chien

**population** 

patient's geographical origin and is observed in World Health Organization (WHO) types II and III. However, the association of NPC type I with EBV has long been a matter of controversy. It is now clear that WHO type I tumors are frequently associated with EBV in endemic regions, but not in non-endemic regions, where they often result from tobacco and alcohol abuse (Nicholls et al., 1997). Types II and III may be accompanied by an inflammatory infiltrate of lymphocytes, plasma cells, and eosinophils.

Fig. 1. EBV in the pathogenesis model for nasopharyngeal carcinoma

More than 95% adults in all ethnic groups across the world are healthy carriers of EBV (Ooka et al., 1991). This means that NPC oncogenesis is not simply a consequence of EBV infection. It probably results from a form of viral reactivation in combination with other events, such as cellular genetic lesions due to environmental carcinogens and/or some form of immune defects. EBV-encoded RNA signal (EBER) has been shown, by in-situ hybridization, to be present in nearly all tumor cells, whereas EBV-encoded RNA is absent from the adjacent normal tissue, except perhaps for a few scattered lymphoid cells. Premalignant lesions of the nasopharyngeal epithelium have also been shown to harbor EBV, which suggests that the infection occurs in the early phases of carcinogenesis (Gulley, 2001). Consistent with this hypothesis, is the fact that NPC generally occur several years after EBV primary infection. The expression of EBV latent genes provides growth and survival advantages to these infected cells, ultimately leading to the development of NPC. Further genetic and epigenetic alterations post-NPC development can occur, which may result in a more metastatic disease. Because of it takes years for premalignant lesion after EBV infection to NPC formation(Choi et al., 2011; Ji et al., 2007; Ng et al., 2010; Ng et al., 2005), the long period of pre-clinical detectable phase (PCDP) offer the opportunity for screening and early diagnosis of NPC.
