Using nasopharyngeal carcinoma as a model, we found that it was feasible to use analysis of circulating DNA to screen for cancers in asymptomatic persons. Of the 20,174 participants who underwent screening, only 309 (1.5% of all participants and 27.8% of those who initially tested positive) had persistently detectable EBV DNA in plasma at baseline and at follow-up. Among these 309 participants, nasopharyngeal carcinoma was confirmed in 34 (11.0%). Low positive predictive values are typical for cancer screening studies performed in asymptomatic populations. For example, a recent Korean study that screened 45,855 asymptomatic participants for hepatocellular carcinoma with the use of alpha-fetoprotein analysis showed a positive predictive value of only 1.66%.30 The positive predictive value of 11% in this study is superior to the typical 3% value of existing blood-based tumor markers in a population-screening context.31 Furthermore, nasal endoscopic examination is safe, quick, and inexpensive as compared with tests to confirm most other solid tumors.

The screening for nasopharyngeal carcinoma identified participants with early-stage disease. Remarkably, 16 participants (47%) had stage I disease. This proportion is substantially higher than the typical proportion of 5 to 7% in historical cohorts.21,22 Between 2006 and 2010, of 2671 consecutive participants with nasopharyngeal carcinoma who received treatment at the Sun Yat-sen University Cancer Center, the largest treatment center for nasopharyngeal carcinoma in China, only 120 participants (4.5%) had stage I disease.21 In patients with stage I disease, lesions that are localized in the nasopharynx can be effectively treated by means of intensity-modulated radiotherapy alone so that the side effects associated with more extensive radiotherapy and chemotherapy can be avoided.32

The 3-year rate of progression-free survival among the participants in whom nasopharyngeal carcinoma was identified by screening was superior to that among those in the historical cohort (97% vs. 70%; hazard ratio, 0.10).20 In this study, the effect of length-time bias (which can occur when the lengths of intervals are analyzed by means of random selection or with respect to relatively nonprogressive cancers) is likely to be small because nasopharyngeal carcinoma is an aggressive cancer with frequent early progression and metastases.33,34 Even if a patient presents with carcinoma in situ at the time of diagnosis, the disease often progresses to invasive nasopharyngeal carcinoma in 40 to 48 months. The potential confounding effect of lead-time bias (in this case, the interval between early diagnosis with screening and later diagnosis with standard techniques) could be addressed only in randomized, controlled trials. However, because curative treatment for early-stage nasopharyngeal carcinoma is available, the increase in progression-free survival is unlikely to be driven by an earlier diagnosis only, but more likely by the timely administration of effective treatments.

In this cohort of 20,174 participants, on the basis of an annual incidence of 35 cases per 100,000 persons in the target age group, nasopharyngeal carcinoma would be expected to develop in approximately 7 participants in a year.22 The 34 cases identified is approximately the number of cases expected to be encountered in 5 years. One possible explanation is that participants who would originally present with more advanced nasopharyngeal carcinoma over the next few years had been identified by our screening program at earlier stages. This hypothesis is supported by the lower-than-expected number of cases diagnosed during the follow-up period and the presentation of advanced-stage nasopharyngeal carcinoma in 1 screen-positive participant who had declined to undergo further assessment.

Among the 20,140 participants in whom nasopharyngeal carcinoma was not identified by screening, 19,626 (97.4%) were interviewed by telephone 1 year after screening, and nasopharyngeal carcinoma was reported to have developed in only 1 participant within the first year. This number is much lower than expected from the annual incidence. One of the 9 participants who declined further workup presented with an advanced nasopharyngeal carcinoma 32 months after enrollment. It is likely that the cancer had already been present in this participant at the time of screening. Had he not declined further assessment, the tumor might have been diagnosed much earlier and a better treatment outcome might have been expected.

In this study, screen-positive participants were assessed with the use of both nasal endoscopy and MRI. This arrangement can maximize the power for detecting nasopharyngeal carcinoma so as to provide the best ascertainment of the performance of screening to detect EBV DNA in plasma. In 3 participants, the tumors were not revealed on the endoscopic examination but were detected on MRI. These results are compatible with those in previous studies showing that MRI is more sensitive than nasal endoscopy for detecting small nasopharyngeal carcinomas in symptomatic patients.28 If MRI screening is not readily available, assessment of test-positive participants with nasal endoscopy alone is a reasonable alternative, since this test could detect 91% (31 of 34) of the cancer cases.

The costs for each EBV DNA analysis, endoscopic examination, and MRI were $30, $80, and $1,000 (U.S. dollars), respectively. On the basis of the results of this study, to detect 1 case, 593 participants would need to be screened at a cost of $28,600. Considering the potential decrease in mortality and morbidity, as well as treatment-cost savings associated with the shift in stage distribution, screening for nasopharyngeal carcinoma appears to be a feasible practice in regions with a high incidence of this disease.

Although the current study focused only on men between the ages of 40 and 62 years, this screening protocol should also be applicable to women and persons in other age groups, in whom EBV DNA in plasma is also detected.18,25 However, a lower positive predictive value would be expected owing to the lower incidence of nasopharyngeal carcinoma among women and persons in other age groups.

With a median follow-up of 22 months, the current study shows that the chance of the development of a nasopharyngeal carcinoma within 2 years after a negative screening is low. However, the most appropriate time interval for the screening can be addressed by a longer-term follow-up and rescreening of the cohort.

This study has shown the potential of analysis of circulating DNA to screen for early nasopharyngeal cancer. Even small tumors could release sufficient amounts of DNA into the circulation to allow sensitive detection. Since the half-life of EBV DNA clearance in plasma is only 2 hours,35 to maintain an equilibrium level of EBV DNA in plasma at 20 EBV genomes per milliliter, the lower detection limit of our assay, 1 million EBV genomes need to be released from the tumor cells into the plasma each day. On the basis of the assumption that each tumor cell carries 50 genomes of EBV,36 this is equivalent to a turnover of 200,000 cancer cells per day. This turnover of nasopharyngeal carcinoma cells is remarkable, given that 47% of the participants with disease that was identified through screening had stage I disease. Screening for cancers that are not associated with viral infections would be more challenging technically because detection of genetic and epigenetic markers other than viral DNA sequences would be needed. In each of the 50 genomes of EBV in nasopharyngeal carcinoma cells, there are approximately 10 repeats of the BamHI-W region (our PCR target). Thus, we estimate that 500 molecular markers would be required to achieve a similar level of performance in detecting cancer-derived DNA as shown in this study.

In conclusion, we found that analysis of EBV DNA in plasma was useful for screening an at-risk population for nasopharyngeal carcinoma. The men with nasopharyngeal carcinoma who were identified by screening had significantly earlier stage distribution and superior progression-free survival than an unscreened population in a historical cohort.