For the present evaluation, 2,444 unaffected individuals from the NPC Multiplex Family Study in Taiwan contributed a median follow-up time of 6.5 years (range: 0.36–11 years). A total of 15,519 person-years were accrued, within which 14 incident NPC cases were diagnosed. Thirteen NPC cases were identified via linkage to the Taiwan cancer registry; 1 additional case was identified through clinical examination in our follow-up effort. shows the time between recruitment and development of NPC, sex, age at NPC diagnoses, TNM staging, and WHO type for each of the 14 incident NPC cases. As shown, cases with earlier stage diagnoses had overall shorter follow-up period and were younger than later stage diagnoses (stages 1–2: median follow-up = 3 and median age at diagnoses = 47 years vs. stages 3–4: median follow-up = 6 and median age at diagnoses = 59 years). In addition, there was no distinct pattern in EBV seropositivity and staging (data not shown). The rate of NPC observed in our study was 90 per 100,000 person-years, nearly 11 times higher than general population rates for comparably aged individuals in Taiwan (8.4 per 100,000; ). When we excluded subjects with NPC diagnosed within 1 year of study recruitment (n = 2), the incidence was 77 per 100,000 person-years.
Characteristics of 14 subjects with nasopharyngeal carcinoma diagnosed after recruitment
Rate ratio of NPC for specific EBV biomarker profiles in high-risk families
The RRs of NPC are presented in , stratified by each of the 4 EBV seromarkers evaluated and combinations of markers. Standard cutoff points reported in the literature (based on studies of sporadic NPC) were used to define positivity for each of the markers. We observed that anti-EBV EBNA1 IgA was the marker with the strongest association with NPC; individuals positive for anti-EBV EBNA1 IgA had nearly 5 times the rate of NPC (265 per 100,000) compared with individuals who tested negative [56 per 100,000; RR = 4.7; 95% CI: 1.4–16). Rates for the other 3 markers were also elevated [anti-EBV VCA IgA (immunofluorescent) = 110.6/100,000; RR = 1.6 (0.34–6.3); anti-EBV VCA IgA (ELISA) = 175/100,000; RR = 2.0 (0.046–13); anti-EBV DNase = 168.2/100,000; RR = 2.2 (0.39–8.5)], although none reached significant levels. Positivity to any biomarker versus none yielded a RR of 2.3 (95% CI: 0.71–8.9). When risk stratification was examined by number of markers positive, a dose response was observed, with individuals who tested positive for a single marker having an RR = 1.7 (95% CI: 0.39–7.4) and those who tested positive for 2+ markers having an RR = 4.4 (95% CI: 0.87–20) compared with individuals negative for all markers (Ptrend = 0.01). Individuals negative for all measured EBV markers had NPC rates (58.3/100,000) that were nearly 7 times higher than general population rates (8.4/100,000). Exclusion of the 2 incident NPC cases that were diagnosed within a year of study enrollment did not materially alter our findings (data not shown).
We calculated RRs stratified by time of NPC diagnosis relative to study enrollment. There were 10 NPC cases diagnosed within the first 5 years and 4 cases after more than 5 years since study enrollment. The overall incidence was 93.6 per 100,000 person years and 32.1 per 100,000 person-years for NPC diagnosis within the first 5 years and beyond 5 years, respectively. Risk patterns by EBV sero-markers were similar to the overall pattern in analyses restricted to the first 5 years of follow-up, with the exception of anti-EBV EBNA1 IgA which yielded a risk ratio estimate of 12 in analysis restricted to the first 5 years of follow-up (RR = 12; 95% CI: 2.7–71; ). In our study population, there were only 4 NPC cases diagnosed beyond 5 years of follow-up, limiting our ability to formally evaluate risk in this subgroup stratified by EBV seromarkers. It should be noted, however, that of the 4 NPC cases diagnosed beyond 5 years, all were negative for both anti-EBV VCA IgA by ELISA assay and anti-EBV EBNA1 IgA based on the standard cutoff points suggesting that these 2 particular EBV markers may have limited long-term predictability for NPC.
The standard cutoff points used in to define positivity for each of the 4 seromarkers evaluated were defined based on published studies of sporadic NPC. To assess the appropriateness of these cutoff points for individuals from high-risk NPC families, ROC curves were evaluated. displays the ROC curve for each of the EBV biomarkers along with their area under the curve (AUC; anti-EBV VCA IgA IF, AUC = 0.55; anti-EBV VCA IgA ELISA, AUC=0.54; anti-EBV DNase, AUC=0.53; anti-EBV EBNA1 IgA, AUC = 0.73). Use of combinations of markers did not markedly improve AUC estimates (data not shown). The best 2-way EBV marker combination was observed for anti-EBV EBNA1 and anti-EBV DNase (AUC = 0.70), best 3-way EBV marker combination was observed for anti-EBV EBNA1, anti-EBV DNase, and anti-EBV VCA IgA (IF; AUC = 0.72). The 4-way EBV marker combination yielded an AUC of 0.73. When we restricted the data to the first 5 years, AUC scores for the best 2-way, 3-way, and 4-way combinations reported previously were 0.71, 0.90, and 0.91, respectively.
ROCs curve for each of the EBV biomarkers.
Using these ROC curves, we derived new cutoff points for each of the EBV biomarkers. Because our study population is known to be at high risk of NPC development, maximizing sensitivity over specificity was deemed desirable. Therefore, we defined the ideal cutoff points for our high-risk population by maximizing the Youden score conditional on a sensitivity estimate of more than 80%. By using this criterion, we were unable to identify a cutoff point that yielded a sensitivity of more than 80% for the anti-EBV VCA IgA by IF, so this marker was not evaluated further. For the remaining 3 EBV seromarkers examined, the following positivity cutoff points were identified: OD405 ≥ 0.10 for anti-EBV VCA IgA by ELISA, OD405 ≥ 0.10 for anti-EBV EBNA1 IgA, and 160 or more neutralizing units of DNase neutralizing activity for EBV anti-DNase antibody. The RR estimates that were observed using these new cutoff points for positivity are summarized in and are as follows: anti-EBV VCA IgA RR = 2.6 (95% CI: 0.39–109), anti-EBV EBNA1 IgA RR = 6.6 (95% CI: 1.5–61), and anti-EBV DNase RR = 1.4 (95% CI: 0.40–5.2). Patterns observed in analysis restricted to the first 5 years of follow-up were similar to those observed overall ().
Rate ratio of NPC (based on optimized cutoff points) for specific EBV biomarker profiles for which sensitivity levels of more than 80% could be achieved in high-risk families
Sensitivity ranged from 7.1% for VCA IgA (ELISA) to 50.0% for EBNA1 IgA using standard assay cutoff values compared with 85.7% for EBNA1 IgA, 92.9% for VCA IgA (ELISA), and 84.6% for DNase using the optimized cutoff values (). Specificity ranged from 73.8% for VCA IgA (IF) to 95.7% for VCA IgA (ELISA) and was 84% for EBNA1 IgA using standard cutoff values and were 16.0% for VCA IgA (ELISA), 51.2% for EBNA1 IgA, and 92.3% DNase using the optimized cutoff values (). The negative predictive values were greater than 99% in contrast to the less than 6% positive predictive value for all markers for the entire follow-up period irrespective of the cutoff values ().
Sensitivity, specificity, and negative and positive predictive values at different cutoff points for each of the EBV biomarkers