**3. EBV DNA load detection in plasma or nasopharyngeal swab/brushing**

Plasma EBV DNA load detected with polymerase chain reaction (PCR) has been also explored to detect early-stage NPC in asymptomatic patients. Research has demonstrated that EBV DNA could be quantitatively measured in the blood of NPC patients by PCR [32]. A study of 175 patients in New York City found that EBV DNA test had much higher specificity and positive predictive value than IgA test alone [33]. A systematic review reported that the EBV DNA load test had the largest area of 0.932 under the summary receiver operator curve with high sensitivity (73%) and specificity (89%), which suggested that EBV DNA detection in plasma could be an efficient marker in NPC screening [34].

In Hong Kong, a prospective study of 20,174 participants revealed that EBV DNA load in plasma samples was particularly useful in screening for early asymptomatic NPC [35]. The participants were ethnically Chinese men at 40 to 60 years of age. The subjects with initial positive results were detected again after about four weeks, while the subjects with persistent positive EBV DNA in plasma were performed to check by nasal endoscopy and magnetic resonance imaging (MRI). The median duration of follow-up was 22 months (range, 12 to 44 months). This study showed that the sensitivity and specificity of this method in NPC screening were 97.1% and 98.6% respectively.

However, a study reported that EBV DNA load had a little poor sensitivity and specificity for NPC screening among high-risk family members compared with EBV-IgA serology [36].

The patients with early NPC may only release a limited amount of viral DNA to the blood, making it impossible to detect blood circulation. The potential value of plasma EBV DNA detection in screening for early NPC remains controversial. NPC mainly originates from epithelial cells in the nasopharynx fossa or posterior wall of the nasopharynx. EBV genome can be detected in almost all tumor cells of NPC cases [37]. Clonal EBV genome can be continuously detected in invasive cancer and precancerous high-grade dysplasia [38]. It is suggested that direct detection of EBV genome from nasopharyngeal brushing or swab specimens had highly predictive value for screening asymptomatic NPC.

In the 1990s, a prospective study was designed to assess the feasibility of a new method for NPC screening by using of PCR coupled with nasopharyngeal swab [39]. In this study, 55 patients were enrolled. The result showed that this method had a similar sensitivity to serological methods, indicating this new method was a good supplement to NPC screening. Nasopharyngeal swab is a quite simple procedure with little discomfort. SP Hao et al. detected the expression of EBV-derived latent membrane protein 1 (LMP-1) by nasopharyngeal swab, and he found that this strategy could serve as part of a screening program for high-risk populations with a sensitivity of 87.3% and a specificity of 98.4% [40]. Raymond's study has also confirmed the effectiveness of this new method of screening for NPC. This study performed on 578 patients yielded a sensitivity of 98.9% and a specificity of 99.3% with a positive predictive value of 96.9% and a negative predictive value of 99.7% [41]. In a prospective and population-based study, the detection of EBV load in the nasopharynx by nasopharyngeal swab was demonstrated to be a useful tool as a supplement to serological tests [42]. Studies of both Zheng and Zhang also verified the same conclusion [43, 44]. Notably, Nasopharyngeal swab detection of EBV load

alone should not be used as a mean of NPC screening because of its high falsepositive rate [42]. However, nasopharyngeal swab serving as an applicable sampling method for NPC screening is great feasible, but more research will be needed in the future.

#### **4. Novel biomarkers/technology for NPC screening**

As mentioned above, EBV-related test has been widely used for early NPC screening, especially the combination of EBV-antibody VCA-IgA and EBNA1-IgA. With the development of research technologies, other biomarkers also develope for NPC screening. Liu et al. reported that a combination of PCR and MWCNT-Fe3O4 nanocomposites had the higher detection rate and higher sensitivity compared with the traditional ELISA method [45]. MWCNT-Fe3O4 nanocomposites are a combination of multi-walled carbon nanotubes and iron oxide nanoparticles, which can provide a large surface areas for antigen–antibody binding. A nested case–control study including 20 patients with NPC and 88 normal control showed that EBV microRNA BART2-5p had been proved to be a valuable biomarker for NPC screening with a sensitivity of 90.9% [46]. Thirteen genes including DNAAF1, PARPBP, TTC18, GSTA3, RCN1, MUC5AC, POU2AF1, FAM83B, SLC22A16, SPEF2, ERICH3, CCDC81, and IL33 have been associated with NPC detection based on comprehensive bioinformatics analyses [47]. A recent study showed that higher methylation rates of EGFR and ZNF6671 in circulating cell-free DNA (ccfDNA) could predict NPC, which was a potential novel molecular marker for NPC screening [48]. However, all these researches need more evidence and more data to demonstrate their effectiveness in NPC screening.

Many studies have shown that intestinal flora disruption was associated with malignant tumors. C. ramosum bacteria that promotes the secretion of 5-HT was found to be a strong risk factor for NPC. The establishment of a disease prediction model based on C. ramosum might be used for the prediction of disease risk in a high-risk population and early non-invasive screening of NPC [49].

Raman spectroscopy combined with multivariate analysis technology has been reported to analyze the sera of NPC patients and healthy individuals. In NPC samples, the lipid content, phenylalanine, and β-carotene decreased while amide III, tyrosine and tryptophan increased. The changes in these biomolecular concentrations may be applied for NPC diagnosis [50]. A unmodified nanotechnology based on surface-enhanced Raman spectroscopy was used to detect the blood circulating DNA; and the diagnostic sensitivity and specificity for differentiating the NPC patients from the normal control were 83.3% and 82.5% respeactively. Nanotechnology which was sensitive, rapid, and easy-to-use may have the potential to become a better method for NPC detection and screening based on liquid biopsy [51].

#### **5. Cost-effectiveness of NPC screening**

A Markov stimulation model was constructed to evaluate the cost-effectiveness of different screening strategies for serological tests in China. In this study, NPC detection rate, cost, quality-adjust life, and incremental cost-effectiveness ratio were considered. Results showed that strategy (annual screening for EBVseropositive subject, triennial screening for seronegative subjects) was the economical and practical option [52]. In 2019, a Markov cohort model was also reported to use to estimate the screening for NPC with plasma EBV DNA for

#### *Screening of Nasopharyngeal Carcinoma DOI: http://dx.doi.org/10.5772/intechopen.97398*

50-year-old Asian American men in the United States. The study suggested that because of its high false-positive results in high-risk regions and its uncertain clinical value in non-endemic areas this method wasn't the most cost-effective, despite its specificity and sensitivity were high [53]. Therefore more research will be required in the future.

Studies about NPC screening based on EBV-related test have been widely reported, but there are few studies on the association between EBV-antibody screening and NPC mortality. Recently a study about prospective, clusterrandomized, controlled trial in southern China for NPC screening was revealed that the combination of EBV antibody EBNA1-IgA and VCA-IgA could effectively identify the high-risk population and improve diagnosis of NPC in the interim analysis. Although the mortality of the screening group was not significantly reduced, the specific mortality of NPC in the screening participants was significantly reduced [54]. That was the first report which presented a mortality reduction by NPC screening. It is expected to further improve the participation rate in the future, and finally confirm the effectiveness of NPC screening based on EBV detection.
