Dynamic change patterns of EBV DNA
A total of 900 NPC patients treated at SYSUCC between January 2010 and September 2013 were retrospectively enrolled. Median age was 44 years (range, 11–77 years) and 723/900 (80.3%) of patients were male. All patients were non-keratinizing undifferentiated squamous cell carcinoma. According to the clinical examination during the follow-up (median 55.78 months), there were 21 cases of nasopharyngeal locoregional recurrence and 97 cases developed metastasis (25 cases of bone metastasis, 31 lung, 26 liver, 2 lymph node, 2 bone and lung, 2 liver and lung metastasis, 2 bone and liver, and 7 multiple sites). The baseline clinical features of the patients were shown in Table 1.
At the end of treatment, 25.4% (229/900) of patients were EBV DNA-positive (median concentration 0 copies/mL; range 0–26,400,000 copies/mL). Pre-treatment, 72.0% (648/900) of patients were EBV DNA-positive (median concentration 3,205 copies/mL; range 0–49,100,000 copies/mL). The post-treatment EBV DNA load was significantly lower than the pre-treatment concentrations (P < 0.01).
Patients were then divided into three groups according to the follow-up results. sustained remission consisted of 778 patients without recurrence or metastasis at the end of the follow-up, locoregional recurrence consisted of 21 patients with recurrence at any time-point and metastasis consisted of 97 patients with metastasis at any time-point.
After radiotherapy, 39.18% (38/97) of the metastatic NPC patients were EBV DNA-positive (median 0 copies/mL; range 0– 26,400,000 copies/mL) and 33.33% (7/21) of the recurrent NPC patients were EBV DNA-positive (median 0 copies/mL; range 0–226,000 copies/mL). In the sustained remission group, 23.15% (181/782) of patients were EBV DNA-positive (median 0 copies/mL; range 0–843,000 copies/mL). At the end of radiotherapy, the EBV DNA load of some patients in sustained remission were still EBV DNA-positive, although the number was significantly lower than that in the other two groups (P = 0.01), with no significant difference between the recurrence and metastasis groups (P = 0.562).
We then evaluated the dynamic changes in EBV DNA load at each point-time. At the end of therapy, the EBV DNA load decreased rapidly in the sustained remission and metastasis groups (median 0 copies/mL). Subsequently, there was no marked change of the EBV DNA load in sustained remission group (<4,000 copies/mL), although the EBV DNA load in the metastasis group increased significantly (P= 0.025), reaching a peak at 3 months followed by a decrease to 1 year, and no marked change to 3 years (<5,000 copies/mL). The viral load in the recurrence group had increased rapidly by 3 months after treatment, and was lower than that in the metastasis group 1 year after treatment. This was followed by a gradual increase after 1 year, with a higher peak viral load in the recurrence group than that in the metastasis group, reaching 26,000 copies/mL at 12 months, 168,000 copies/mL at 24 months, and 375,000 copies/mL at 36 months, until the highest level at the time of relapse was 24,100,000 copies/mL (Fig.1).
Impact of EBV DNA levels on survival
According to the ROC curve analysis, the pre- and post-treatment cutoff values for EBV DNA were 4,000 copies/mL and 2,500 copies/mL, respectively. The 5-year OS, DMFS, and PFS rates for the high and low pre-treatment EBV DNA levels were 83.6% and 90.0%, 84.1% and 92.9%, and 87.6% and 94.1%, respectively. These data suggested that elevated EBV DNA before treatment was associated with a poorer OS (HR:1.647, 95%CI: 1.126–2.408, P = 0.009; Fig.2A). Similarly, elevated EBV DNA before treatment also indicated significantly decreased DMFS compared with lower EBV (HR: 2.377, 95%CI:1.558–3.626, P < 0.001; Fig.2B) and PFS (HR: 2.035, 95%CI:1.279–3.237, P = 0.003; Fig.2D), but not LRFS (P = 0.754; Fig.2C).
A total of 473 patients had pre-treatment EBV DNA loads below the cutoff value of 4,000 copies/mL. Among them, 21 patients had post-treatment EBV DNA loads higher than 2,500 copies/mL, there were18 cases with residual tumor, 32 developed metastasis, and 45 died.
A total of 427 patients had pre-treatment EBV DNA loads higher than 4,000 copies/mL. Among them, 369 patients had post-treatment EBV DNA loads below 2,500 copies/mL, 46 patients developed metastasis, eight had recurrence, and 41 died. Among the 58 of 427 patients with post-treatment EBV DNA higher than 2,500 copies/mL, 19 patients developed metastasis, 4 locoregional recurrence, and 22 died, with a higher proportion. Thus, these data indicated that post-treatment EBV DNA load had a much more marked influence on the prognosis of patients with NPC than pre-treatment EBV DNA load.
The patients were then divided into two groups according to the post-treatment EBV DNA cutoff values. The prognosis in the high post-treatment EBV DNA group was poorer than that of the lower EBV DNA group. Patients with post-treatment EBV DNA <2,500 copies/mL achieved better OS (P < 0.001), DMFS (P = 0.040), LRFS (P = 0.004), and PFS (P = 0.001) than patients with higher post-treatment EBV DNA.
Other risk factors, including smoking, advanced tumor stage, elevated CRP before treatment and therapy were correlated with poor OS, DMFS and PFS. Multivariate analysis was also performed, incorporating patient factors (age, sex, smoking), tumor factors (TNM stage), treatment methods (radiotherapy and chemotherapy) and post-treatment EBV DNA load as covariates. As shown in Table 2, post-treatment EBV DNA load was confirmed as an independent prognostic factor for OS (HR: 2.019, 95%CI: 1.305–3.122, P = 0.002), DMFS (HR: 1.565, 95%CI: 1.051–2.515, P = 0.044), LRFS (HR: 5.363, 95%CI: 2.201–7.312, P = 0.008) and PFS (HR: 1.940, 95%CI: 1.153–3.264, P = 0.013). According to these data, post-treatment EBV load has a great impact on survival and was identified as an independent prognostic factor for OS, DMFS, LRFS, and PFS.
EBV DNA kinetics and survival
The 5-year OS rates for patients with sustained remission was 93.8%, which was significantly improved compared with that of the recurrence (47.6%) and metastasis (26.9%) groups (Fig.3), with a significant difference observed between the three groups (recurrence vs. sustained remission HR: 12.227; 95%CI: 6.568–22.760; P < 0.001, metastasis vs. sustained remission HR: 15.842; 95%CI: 10.494–23.916; P < 0.001).
According to baseline EBV DNA load and EBV kinetics, patients were further divided into three groups as follows: (1) continuously normal group: patients whose baseline EBV DNA normal and never elevated, 679 cases; (2) ever-elevated group: patients whose EBV DNA ever-elevated regardless time points, 97 cases; (3) continuously elevated group: patients whose baseline EBV DNA was elevated and never normalized, 124 cases. The 5-year OS rates in the three groups were 93.2%, 76.2% and 59.9%, respectively (Fig.4A); DMFS rates were 94.4%, 84.3% and 59.9%, respectively (Fig.4B); LRFS rates were 98.1%, 91.5% and 95.8%, respectively (Fig.4C); and PFS rates were 94.8%, 83.9% and 73.7%, respectively (Fig.4D). There were significant differences among the three groups in OS (HR: 2.542, 95%CI:2.077–3.111, P < 0.001), DMFS (HR:2.970, 95%CI: 2.392–3.687, P < 0.001), LRFS (HR: 1.699, 95%CI: 1.072–2.692, P = 0.013), and PFS (HR: 2.535, 95%CI: 1.987–3.233, P < 0.001).
Of 900 patients in the whole group, a total of 887 patients were alive and had follow-up for at least 12 months and therefore they were included in the 12-month landmark analysis for survival (Fig.5A). The 5-year OS of the 732 patients whose EBV DNA load sustained remission was 91.3%, and the elevated group was 74.4% (HR: 3.182, 95% CI: 2.114-4.788, P < 0.001).
Besides, a total of 854 patients were alive or had follow-up for at least 24 months and therefore were included in the 24-month landmark analysis for survival (Fig.5B). The 5-year OS of the 746 patients whose EBV DNA load sustained remission was 95.0%, and the elevated group was 62.9% (HR: 9.619, 95% CI: 6.027-15.353, P < 0.001).