Two-Step Epstein-Barr Virus Immunoglobulin A Enzyme-Linked Immunosorbent Assay System for Serological Screening and Confirmation of Nasopharyngeal Carcinoma

Undifferentiated nasopharyngeal carcinoma (NPC; WHO type III) is 100% associated with Epstein-Barr virus (EBV) infection and the fourth most prevalent cancer in Indonesian males. Therapy failure is high, since most patients come to the hospital at an advanced stage of disease. Screening for early-stage NPC is needed. Here, a simple and economical two-step enzyme-linked immunosorbent assay (ELISA) system is proposed for diagnosing NPC in high-risk populations, employing the peptide-based immunoglobulin A (IgA) EBNA1 plus viral capsid antigen p18 ELISA as an initial screening test and the IgA early antigen (EA) ELISA using a different set of EBV antigens as a confirmation test. A total of 151 NPC patients and 199 regional healthy EBV carriers were used to evaluate the two-step ELISA approach. Routinely, EBV IgG immunoblotting is used as a standard confirmation test. The sensitivity and specificity for diagnosing NPC by the two-step ELISA approach increased from 85.4% to 96.7% and 90.1% to 98%, respectively, with positive predictive values and negative predictive values increasing from 78.7 and 93.9% to 97.3 and 97.5%, respectively, relative to the immunoblotting confirmation system. On discrepant samples, additional testing was done by EBV DNA load quantification in blood. Results showed that 5/11 discrepant NPC samples with an elevated IgA EA ELISA also had elevated an EBV DNA load in the circulation (range, 3,200 to 25,820 copies/ml). Therefore, the IgA EA ELISA is proposed as a confirmation test in first-line NPC serological screening studies. This two-step EBV ELISA system provides a standardized approach for NPC screening and may be used in combination with dried blood sampling in future field studies for identification of early-stage NPC in high-risk regions.Nasopharyngeal carcinoma (NPC) is a common cancer in China and Southeast Asia and closely associated with Epstein-Barr Virus (EBV) (26). In Indonesia, especially in the southern part of central Java, undifferentiated carcinoma (WHO type III) is the most common head and neck cancer and among the five most prevalent cancers overall. Due to unspecific symptoms and the hidden localization of the primary tumor at the early stage, more than 80% of the patients come to the hospital at a late stage (III or IV), when they already have metastasis in the cervical lymph node. Whereas late-stage disease has a poor prognosis and requires combined chemo-radiotherapy, early-stage NPC may reach complete remission by radiotherapy only (17). Therefore, screening for early-stage NPC among the population is important and clinically relevant. For developing countries, such an approach should be economical, employing standardization methods suited for mass screening.Patients with NPC have high-level broad-spectrum anti-EBV antibodies, especially immunoglobulin A (IgA), compared to regional healthy carriers and patients with other head and neck diseases (13, 14). Our group recently demonstrated that the molecular diversity underlying anti-EBV IgG and IgA responses in NPC patients was different, requiring multiple EBV antigens for complete serological coverage (7). Prior studies in China and Taiwan have shown the feasibility of using IgA serology for population screening (2, 15, 27). However, in these studies laborious and poorly standardized cell-based serological techniques were used. Nevertheless, these studies revealed the appearance of serological abnormalities, i.e., positive EBV IgA responses 2 to 3 years prior to onset of NPC (2, 15), which clearly demonstrated the opportunity of using EBV serology for early-stage detection of NPC. This particularly applies for screening in high-risk groups, such as family members of NPC patients and patients with suspicious head and neck symptoms (18, 21).For NPC serodiagnosis, cell-based indirect immunofluorescent assay (IFA) methods are still widely considered the gold standard. IFA involves the separate analysis of antibody responses to viral capsid antigen (VCA), early antigen (EA), and nuclear antigens (EBNA), each comprising multiple proteins and requiring different cell lines for specific analysis (10, 12, 13). However, this method shows considerable variation among laboratories and is time-consuming, subjective, and not suitable for large-scale automatic handling. Enzyme-linked immunosorbent assay (ELISA) techniques are increasingly used and have shown a better sensitivity and specificity compared to IFA and are suitable for large-scale application (4, 10, 11, 16, 20, 21).Recently, we developed an EBV IgA ELISA based on a combination of VCA p18- and EBNA1-derived synthetic peptides which is routinely used as an NPC diagnostic test in our local hospital (Sardjito Hospital, Yogyakarta, Indonesia). This EBV IgA ELISA combines the separate features of IgA VCA and IgA EBNA1, each of which has its value in NPC diagnosis. The combination of these markers in a single assay provided sensitivity and specificity of 85.4% and 90.1%, respectively (8). The presence of NPC-related serological abnormalities can be confirmed by immunoblotting to reveal the spectrum of antibody responses, which has diagnostic value by itself (5, 7, 16, 25). The combined EBV IgA ELISA and immunoblot assay showed increased sensitivity and specificity and positive predictive value (PPV) and negative predictive value (NPV) of more than 95% (7, 8). Because immunoblot studies revealed a diagnostic value of multiple EBV proteins, in particular certain EBV-EA markers, we recently developed a separate IgA EA ELISA using native EA proteins (22). In addition to their role in primary diagnosis, anti-EBV IgA responses, in particular the IgA EA response, also have a distinct role for posttreatment follow-up monitoring, as declining responses correlate with a good prognosis and increasing responses are related to persistence of relapsing tumor (6, 16, 24). The availability of two distinct and biochemically well-defined EBV IgA ELISA systems addressing responses to different EBV antigens for NPC-specific serology may add to further standardization. In this study we evaluate the combination of these two tests for primary diagnosis of NPC in a high-incidence population in Indonesia.