Epstein-Barr virus (EBV) is associated with nasopharyngeal carcinoma (NPC) in an endemic area. The plasma EBV DNA concentration at the time of diagnosis and after treatment can be used as a biomarker for screening, monitoring and predicting clinical outcomes in NPC.1,2 Peng identified a pretreatment plasma EBV DNA (pre-EBV) cut-off value of 2010 copies/ml in predicting disease-free survival, overall survival, loco-regional relapse-free survival and distant metastasis–free survival when NPC patients were treated with intensity-modulated radiation therapy (IMRT).3 Others confirmed the prognostic value of pre-EBV in predicting clinical outcomes despite the different pre-EBV cut-off values.4,5,6,7,8,9,10,11 The persistent post-treatment EBV DNA concentration (post-EBV) has the strongest risk of disease relapse and distant metastasis4,7,12,13,14 and may help in treatment modification, such as the intensification of adjuvant chemotherapy regimens.15
In recent decades, the use of concurrent chemotherapy with IMRT has shifted the pattern of recurrence from locoregional recurrence toward distant metastasis alone.16,17,18,19 The mid-treatment plasma EBV-DNA may be useful in adaptive treatment, such as reducing treatment intensity in patients with early response or giving more intensified treatment to those without response. However, studies on the plasma EBV DNA during radiation treatment (mid-EBV) were scarce, and patients were not uniformly treated with IMRT or concurrent chemoradiation.20,21 Our primary endpoint was to identify the prognostic value of mid-EBV to predict the overall survival, progression-free survival and distant metastasis-free survival rates.
This study was a secondary analysis of a prospective randomized controlled trial that compared the utility of sequential (SEQ) or simultaneous integrated boost (SIB) IMRT in non-metastatic nasopharyngeal cancer.22 This study was approved by the institutional review board. Informed consent was obtained from every patient before entry into the study. One hundred and twenty-three patients had detectable pre-EBV. After excluding 18 patients because of missing blood samples at any of the three time-points, there were 105 patients in this study.
Chemotherapy consisted of weekly treatments of 40 mg/m2 cisplatin given concurrently with 70 Gy IMRT in 33-35 fractions to those with more than T1 or positive nodal disease for a maximum of 7 cycles. Adjuvant chemotherapy, consisting of 80 mg/m2 cisplatin and 1000 mg/m2/per day 5-fluorouracil (5-FU) over a 96-hour continuous infusion, was given at 4-week intervals for 3 cycles.
Plasma EBV DNA concentrations were evaluated before treatment (pre-EBV), at the 5th week of the radiation course (mid-EBV) and 3 months after the completion of radiation treatment (post-EBV). We elected to test the mid-EBV in the 5th week of IMRT because it was the best time to perform re-simulation and in accordance with Leung’s study which tested mid-EBV at completion of 4 weeks of radiation therapy.21 The EBV nucleic acids were purified from the plasma samples using the QIAsymphony SP in combination with the QIAsymphony DSP Virus/Pathogen Midi Kit (QIAGEN, Germany) using the manufacturer’s recommended protocol. After extraction, the eluates in the 96-microwell plates were transferred to the module for assembly with the master mix (QIAGEN artus EBV QS RGQ kit) by the instrument. The aliquoted reactions were subsequently put in a Rotor-Gene Q. The amplification parameters were as follows: 95°C for 10 min and 45 cycles of 95°C for 15 s, 65°C for 30 s, and 72°C for 20 s. The plasma DNA samples were quantified for EBV DNA using an RTQ-PCR system targeting the BamHI-W fragment region of the EBV genome. A plasma EBV DNA concentration of < 316 copies/ml was defined as an undetectable level in our institution. Note that in the following section, values of 0 represent an undetectable plasma EBV DNA concentration.
Local progression-free survival (LPFS), regional progression-free survival (RPFS), distant metastasis-free survival (DMFS), progression-free survival (PFS) and overall survival (OS) were analyzed using the Kaplan–Meier method and the log-rank test. Cox proportional hazard models with univariate and multivariate analyses were performed to identify the predictors for OS, PFS and DMFS. The factors, including age, sex, stage, pre-EBV, mid-EBV, post-EBV, WHO subtypes, and IMRT techniques, were included as covariates in this exploratory analysis. Factors with a
Between October 2010 and September 2015, 105 NPC patients who had detectable pre-EBV and completed blood sampling between RT and 3 months after RT were included. The median age was 50 years. Patient characteristics are outlined in Table 1. The median follow-up time was 45.3 months. Most patients were stage III-IVb. The majority had undifferentiated squamous cell carcinoma and male gender. Patients received a median of 6 cycles of concurrent weekly cisplatin (88.6% received ≥ 5 cycles) and a median of 3 cycles of adjuvant chemotherapy (76.2% completed 3 cycles).
Patient characteristics
N = 105
Age < 45
34 (32.4%)
Age ≥ 45
71 (67.6%)
Sex
Male
82 (78.1%)
Female
23 (21.9%)
T-stage
1
30 (28.6%)
2
28 (26.7%)
3
29 (27.6%)
4
18 (17.1%)
N-stage
0
1 (1.0%)
1
26 (24.8%)
2
57 (54.3%)
3
21 (20.0%)
Stage grouping (AJCC 2010)
II
14 (13.3%)
III
54 (51.4%)
IVa-b
37 (35.2%)
WHO subtypes
2A
11 (10.5%)
2B
94 (89.5%)
Mid-EBV
undetectable
90 (85.7%)
detectable
15 (14.3%)
Post-EBV
undetectable
98 (93.3%)
detectable
7 (6.7%)
Median pre-EBV was 6880 copies/ml. (interquartile range, IQR, 2555–14600 copies/ml). The corresponding values for stage II, III and IV were 3690 copies/ml (Interquartile range (IQR), 1462–8885), 6880 copies/ml. (IQR, 2407–16475) and 5620 copies/ml (IQR, 3735–17200), respectively. Fifteen patients (14.3%) had persistent mid-EBV, 4 of whom had residual post-EBV. Among the remaining 90 patients who had undetectable mid-EBV, 3 patients had rebound detectable post-EBV. A total of 7 patients (6.7%) had residual post-EBV.
During the follow-up period, a total of 24 (22.9%) patients died, 39 (37.1%) had progressive disease, and 27 (25.7%) developed distant metastases. The 3-year OS, PFS and DMFS for the patients were 83.2%, 77.4%, 84.7%, respectively. The overall survival rates for stages II, III, and IV were 85.1%, 88.7% and 75.0%, respectively (p = 0.253), while the PFS was 85.1%, 83% and 67.3%, respectively (p = 0.070). The corresponding DMFS was 100%, 88.5% and 74.2%, respectively (p = 0.102) (Figure 1).
Using a pre-EBV cut-off of 2010 copies/ml3, the 3-year OS, PFS and DMFS rates were 88.4%
Overall survival (OS), progression-free survival (PFS), distant metastasis-free survival (DMFS), local progression-free survival (LPFS) and regional progression-free survival (RPFS) rates among different plasma EBV time points
3-year OS(95%CI)
p-value
3-year PFS(95%CI)
p-value
3-year DMFS(95%CI)
p-value
3-year LPFS(95%CI)
p-value
3-year RPFS(95%CI)
p-value
Pre-EBV < 2010
88.4 (73.1–103.7)
0.360
82.9 (65.3–100.5)
0.114
82.9 (62.3–100.5)
0.386
100
0.328
100
0.475
Pre-EBV ≥ 2010
82.1 (73.9–30.3)
76.2 (67.0–85.4)
85.2 (77.0–93.4)
94.4 (89.1–99.6)
96.6 (91.9–101.3)
Undetectable mid-EBV
86.0 (78.5–93.4)
0.040
81.5 (73.3–89.7)
0.006
86.1 (78.5–93.7)
0.15
97.5 (94.0–101.0)
0.01
98.6 (95.9–101.3)
0.113
Detectable mid-EBV
66.7 (42.8–90.6)
52.5 (26.8–78.2)
76.6 (52.9–100.3)
79.5 (53.8–105.2)
87.5 (64.6–110.4)
Undetectable post-EBV
86.1 (79.0–93.2)
< 0.001
79.9 (71.9–87.9)
< 0.001
88.2 (81.3–95.1)
< 0.001
97.6 (94.3–100.9)
< 0.001
97.2 (93.5–100.9)
0.841
Detectable post-EBV
42.9 (6.3–79.6)
42.9 (6.3–79.6)
22.9 (15.7–61.5)
33.3 (20.0–86.6)
100
Among the 39 patients who had progressive disease, persistent mid-EBV and post-EBV were observed in 25.6% and 15.4% of these patients, respectively. There were 4 patients who had both persistent mid-EBV and post-EBV, and their 3-year OS, PFS and DMFS rates were 25%, 25% and 37.5%, respectively, which were significantly worse than those of the 11 patients who had detectable mid-EBV but undetectable post-EBV. The 3-year OS, PFS and DMFS rates in the latter group were 81.8%, 62.3% and 88.9%, respectively. Among the 3 patients who had undetectable mid-EBV but had rebound detectable post-EBV, the 3-year OS, PFS and DMFS rates were 66.7%, 66.7% and 33.3%, respectively. The best prognostic group was the patients who had both undetectable mid-EBV and post-EBV, and their 3-year OS, PFS and DMFS rates were 86.7%, 82.1% and 88.2%, respectively.
The unadjusted univariate analyses for the clinical parameters and the EBV-value associated with the OS, DMFS and PFS are shown in Table 3. Undetectable mid-EBV was significantly associated with better OS and PFS but not DMFS, while undetectable post-EBV was significantly associated with better OS, PFS and DMFS. T-stage was a significant prognostic factor for PFS only (p = 0.026). In the multivariate analysis (Table 4), only detectable post-EBV was significantly associated with a worse OS (hazard ratio (HR) = 6.881, 95% confident interval (CI) 1.699–27.867, p = 0.007), PFS (HR = 5.117, 95% CI 1.562–16.768, p = 0.007) and DMFS (HR = 129.071, 95%CI 19.031–875.364, p < 0.001)
Univariate analyses for the clinical parameters and EBV values associated with the overall survival (OS), progression-free survival (PFS), distant metastasis-free survival (DMFS
Hazard ratio for OS
Univariate
Hazard ratio for PFS
Univariate
Hazard ratio for DMFS
Univariate
95.0% CI
p-value
95.0% CI
p-value
95.0% CI
p-value
Lower
Upper
Lower
Upper
Lower
Upper
Age <45 vs. ≥45
0.497
0.185
1.331
0.164
0.655
0.319
1.346
0.250
0.807
0.353
1.845
0.611
Sex Male vs. Female
1.442
0.493
4.222
0.504
1.623
0.679
3.879
0.276
1.078
0.434
2.677
0.872
WHO type IIA vs. IIB
1.344
0.399
4.532
0.633
1.795
0.748
4.31
0.190
2.101
0.719
6.141
0.175
T Stage
0.152
0.026
0.119
T1 vs. T4
0.155
0.031
0.773
0.023
0.176
0.055
0.564
0.003
0.212
0.05
0.89
0.034
T2 vs. T4
0.73
0.257
2.073
0.555
0.523
0.224
1.222
0.134
0.617
0.204
1.862
0.391
T3 vs. T4
0.667
0.223
1.991
0.468
0.73
0.319
1.671
0.457
0.93
0.316
2.743
0.896
N Stage
0.740
0.501
0.188
N1 vs. N3
1.222
0.386
3.865
0.733
0.887
0.368
2.138
0.789
0.587
0.208
1.657
0.314
N2 vs. N3
0.844
0.297
2.4
0.750
0.651
0.302
1.406
0.275
0.445
0.187
1.063
0.068
Stage
0.253
0.070
0.102
Stage II vs. IVa-b
0.888
0.281
2.8
0.839
0.491
0.166
1.449
0.198
0.326
0.073
1.451
0.141
Stage III vs. IVa-b
0.484
0.2
1.17
0.107
0.472
0.242
0.92
0.028
0.47
0.214
1.034
0.061
Tech SIB vs. SEQ
1.619
0.707
3.71
0.255
1.309
0.693
2.472
0.407
1.461
0.674
3.167
0.336
Concurrent chemotherapy 0-5 vs. 6-7 cycles
1.172
0.525
2.616
0.699
0.795
0.418
1.514
0.486
0.632
0.287
1.393
0.255
Adjuvant chemotherapy 0-2 vs. 3 cycles
1.089
0.432
2.748
0.856
1.101
0.536
2.263
0.793
0.937
0.377
2.327
0.888
Pre-EBV ≥ 2010 vs. < 2010
1.760
0.524
5.915
0.360
2.309
0.819
6.512
0.114
1.595
0.550
4.625
0.390
Mid-EBV detectable vs undetectable
2.600
1.031
6.556
0.043
2.746
1.337
5.640
0.006
2.041
0.772
5.397
0.15
Post-EBV detectable vs. undetectale
5.923
1.989
17.638
0.001
5.961
2.457
14.465
<0.001
29.758
8.155
108.593
<0.001
Multivariate analyses for the clinical parameters and EBV values associated with the overall survival (OS), progression-free survival (PFS), distant metastasis-free survival (DMFS)
Hazard ratio for OS
95.0% CI for OS
P-value
Lower
Upper
Age < 45 vs. ≥ 45
0.409
0.140
1.190
0.101
T
0.305
T1 vs. T4
0.286
0.052
1.570
0.150
T2 vs. T4
1.214
0.361
4.078
0.754
T3 vs. T4
1.177
0.341
4.069
0.796
Mid-EBV undetectable vs detectable
1.620
0.617
4.251
0.327
Post-EBV undetectable vs. detectable
6.881
1.699
27.867
0.007
Age <45 vs. ≥ 45
0.422
0.180
0.988
0.047
WHO type IIA vs. IIB
1.354
0.441
4.159
0.597
T Stage
0.101
T1 vs. T4
0.497
0.104
2.375
0.381
T2 vs. T4
1.660
0.450
6.125
0.446
T3 vs. T4
2.099
0.646
6.825
0.218
Stage
0.099
Stage II vs. IVa-b
1.016
0.256
4.035
0.982
Stage III vs. IVa-b
0.416
0.170
1.021
0.055
EBV < 2010 vs. ≥ 2010
0.370
0.113
1.211
0.100
Mid-EBV undetectable vs. detectable
1.427
0.630
3.234
0.394
Post-EBV undetectable vs. detectable
5.117
1.562
16.768
0.007
WHO type IIA vs. IIB
1.653
0.470
5.810
0.433
T Stage
0.113
T1 vs. T4
0.945
0.068
13.158
0.966
T2 vs. T4
5.632
0.388
81.647
0.205
T3 vs. T4
4.628
0.384
55.737
0.228
N Stage
0.410
N1 vs. N3
4.484
0.305
65.955
0.274
N2 vs. N3
2.018
0.151
26.935
0.595
Stage
0.340
Stage II vs. IVa-b
0.090
0.003
2.435
0.153
Stage III vs. IVa-b
0.150
0.009
2.474
0.185
Mid-EBV undetectable vs detectable
1.583
0.517
4.843
0.421
Post-EBV undetectable vs. detectable
129.071
19.031
875.364
0.000
Zhang1 proposed the risk stratification into 4 groups based on the stage and mid-EBV as follows: (1) patients who had stage I-II with undetectable mid-EBV; (2) patients who had stage III-IV with undetectable mid-EBV; (3) patients who had stage II-IV with detectable mid-EBV; and (4) patients who had stage I with detectable mid-EBV. In our study, there were no patients in group (4). Thus, we analyzed the survival in group (1)-(3). The overall survival rates for group (1), (2), and (3) were 91.7%, 85.3% and 66.7%, respectively (p = 0.107), while the PFS rates were 91.7%, 80.2% and 52.5%, respectively (p = 0.010). The corresponding DMFS rates were 100%, 84.4% and 76.6%, respectively (p = 0.163) (Figure 2).
The plasma EBV DNA concentration has been identified as a prognostic biomarker and is correlated with the overall survival in NPC. Pre-EBV concentration has been evaluated in many studies. A higher concentration was associated with a higher mortality, poorer PFS and poorer DMFS.1,4,5,6,7,8,9,10,12,23 Leung
Other than the pre-EBV, mid-EBV may provide additional information on the risk of disease failure. To our knowledge, there is only one prospective trial that studied the prognostic value of mid-EBV. Leung
A meta-analysis confirmed the significant value of mid-EBV in terms of the OS, PFS and DMFS1; however, detectable post-EBV was a stronger prognostic factor than pre-EBV and mid-EBV. The HRs for the OS, PFS and DMFS of post-EBV were 4.26 (95%CI 3.26–5.57), 5.21 (95%CI 3.29–8.27) and 7.54 (95%CI 3.39–16.77), respectively. Peng
We further compared the patient groups according to the risk stratification proposed by Zhang
A limitation of our study is that the follow-up time was relatively short. The prognostic value of mid-EBV was not our primary objective in our randomized controlled trial comparing the different IMRT techniques, which may not have adequate power to detect the prognostic significance of mid-EBV on DMFS. However, the strength of our study was the uniform use of the IMRT technique and concurrent cisplatin-based chemotherapy followed by adjuvant cisplatin and 5FU in a prospective randomized trial.
In conclusion, detectable EBV during chemoradiation was an adverse prognostic factor for OS and PFS; however, it was not as a strong prognostic factor as post-EBV. Prospective clinical trials are warranted to allow evidence-based recommendations for adaptive treatment based on mid-EBV.