Plasma EBV-miR BART7-3p and BART13-3p in NPC patients vs. non-NPC controls
The analysis included 483 NPC patients with a median age of 48 years (range, 18–83 years) and 243 control subjects (Table 1). EBV DNA was detected in 93.8% (453/483) of NPC patients, 7.2% (8/207) of healthy subjects, 16.7% (2/12) of subjects with chronic nasopharyngitis, and 12.5% (3/24) of HNSCC patients (Fig. 2A). MiR-BART7-3p and miR-BART13-3p were detected in 96.1% (464/483) and 97.9% (473/483) of NPC cases, but in only 3.9% (8/207) and 3.9% (8/207) of healthy controls (Figs. 2B and C). Neither miR-BART7-3p nor miR-BART13-3p was detected in patients with chronic nasopharyngitis or HNSCC.
Table 1
Demographic and clinical characteristics of nasopharyngeal carcinoma (NPC) patients and non-NPC controls.
Table 1
Demographic and clinical characteristics of nasopharyngeal carcinoma (NPC) patients and non-NPC controls.
Characteristic | Controls | NPC (n = 483) |
Healthy (n = 207) | Nasopharyngitis (n = 12) | HNSCC (n = 24) |
Age (years) | | | | |
Median | 45 | 49 | 55 | 48 |
Range | 18–75 | 19–69 | 26–72 | 18–89 |
Sex (n, %) | | | | |
Female | 68 (32.9) | 4 (33.3) | 12 (50.0) | 144 (29.8) |
Male | 139 (67.1) | 8 (66.7) | 12 (50.0) | 339 (70.2) |
Clinical stage (n, %) | | | | |
I | | | | 12 (2.50) |
II | | | | 126 (26.1) |
III | | | | 182 (37.7) |
IVA | | | | 145 (30.0) |
IVB | | | | 18 (3.70) |
miR BART7-3p (copies/mL) | | | | |
Median (range) | 0 (0-271) | 0 (0–0) | 0 (0–0) | 3860 (0-635720) |
miR BART13-3p (copies/mL) | | | | |
Median (range) | 0 (0-1265) | 0 (0–0) | 0 (0–0) | 10220 (0-1031760) |
EBV DNA (copies/mL) | | | | |
Median (range) | 0 (0-216) | 0 (0–84) | 0 (0-112) | 5430 (0-6610000) |
When compared with positive diagnosis by histology, miR-BART7-3p showed 96.1% sensitivity, 96.7% specificity and an area under the receiver operating characteristic curve (AUC) of 0.964 using the detection level as cut-off value; the corresponding values for miR-BART13-3p were 97.9%, 96.7%, and 0.973 (Fig. 1D and Table 2). Similarly, the sensitivity, specificity and AUC of EBV DNA were 93.7%, 91.4%, and 0.926, slightly inferior than those of miR-BART7-3p and miR-BART13-3p (Table 2). When the plasma levels of all three nucleic acid species were considered together, the sensitivity, specificity and AUC were 97.3%, 99.6%, and 0.997. In a subgroup analysis that included only the 138 patients with stage I-II NPC, the AUC was 0.921 for EBV DNA, 0.965 for miR-BART7-3p, and 0.980 for miR-BART13-3p (Table 2). When plasma levels of the three nucleic acid species were considered together, the AUC was 0.994, higher than any single biomarker (Fig. S1 in Additional file 2).
Table 2
Diagnostic performance of plasma biomarkers for detection of nasopharyngeal carcinoma (NPC) (cut-off = 0 copies/mL).
Table 2
Diagnostic performance of plasma biomarkers for detection of nasopharyngeal carcinoma (NPC) (cut-off = 0 copies/mL).
Comparison | TP (n) | FN (n) | TN (n) | FP (n) | SE (%) | SP (%) | PPV (%) | NPV (%) | AUC |
EBV DNA |
Early stage vs. control | 128 | 10 | 222 | 21 | 92.8 | 91.4 | 85.9 | 95.7 | 0.921 |
NPC vs. control | 453 | 30 | 222 | 21 | 93.7 | 91.4 | 95.6 | 88.1 | 0.926 |
miR-BART7-3p |
Early stage vs. control | 133 | 5 | 235 | 8 | 96.4 | 96.7 | 94.3 | 97.9 | 0.965 |
All NPC vs. control | 464 | 19 | 235 | 8 | 96.1 | 96.7 | 98.3 | 92.5 | 0.964 |
miR-BART13-3p |
Early stage vs. control | 137 | 1 | 235 | 8 | 99.3 | 96.7 | 94.5 | 99.6 | 0.980 |
All NPC vs. control | 473 | 10 | 235 | 8 | 97.9 | 96.7 | 98.3 | 95.9 | 0.974 |
All three nucleic acid species combined |
Early stage vs. control | 137 | 1 | 242 | 1 | 99.3 | 99.6 | 99.3 | 99.6 | 0.994 |
All NPC vs. control | 471 | 13 | 242 | 1 | 97.5 | 99.6 | 99.7 | 94.9 | 0.997 |
Abbreviations: TP, true positive; FN, false negative; TN, true positive; FP, false positive; SE, sensitivity; SP, specificity; PPV, positive predictive values; NPV, negative predictive values; AUC, area under the receiver operating characteristic curve. |
EBV DNA levels correlated moderately with levels of both miR-BART7-3p (r = 0.453, p < 0.001) and miR-BART13-3p (r = 0.420, p < 0.001) (Figs. 2E and F). Levels of miR-BART7-3p correlated positively with those of miR-BART13-3p (r = 0.748, p < 0.001) (Fig. 2G).
Association between plasma miR-BARTs and NPC burden
Patients with advanced NPC stage had higher plasma levels of miR-BART7-3p (r = 0.354, p < 0.001), miR-BART13-3p (r = 0.329, p < 0.001) and EBV DNA (r = 0.316, p < 0.001) (Figs. 2H-J). There was a trend towards higher plasma levels of miR-BART7-3p, miR-BART13-3p and EBV DNA with advanced N- and T-classification (Fig. S2 in Additional file 2). In a majority of the 245 NPC study subjects with plasma samples after radiotherapy, miR-BART7-3p and miR-BART13-3p levels were undetectable or significantly reduced in comparison with pre-treatment levels (Figs. 2L and M). Similar results were observed for EBV DNA (Fig. 2K).
Prognostic performance of plasma levels of miR-BARTs
The prognostic performance of circulating miR-BARTs was first examined in the 465 NPC patients without distant metastasis at initial diagnosis. Clinicopathological features of these subjects are listed in Table 3. The median follow-up time was 55 (range 2–83) months. Median circulating levels at diagnosis were 4770 (range: 0-6610000) for EBV DNA, 3669 (0-635730) for miR-BART7-3p, and 9441 (0-1031760) copies/ml for miR-BART13-3p. The median expression value is used as the cut-off value for high and low expression of miR-BARTs and EBV DNA. The four-year DMFS rate was 81.3% in patients with high EBV DNA expression vs. 92.6% in those with low expression (p < 0.001; Fig. 3A), 80.9% in subjects with high miR-BART7-3p expression vs. 93.2% with low expression (p < 0.001; Fig. 3B), and 83.1% in subjects with high miR-BART13-3p expression vs. 90.8% with low expression (p = 0.005; Fig. 3C). In the multivariate analysis, short DMFS was independently associated with high levels of miR-BART7-3p (hazard ratio [HR] 2.94, 95% confidence interval [CI] 1.44–5.98, p = 0.003) and EBV DNA (HR 2.27, 95%CI 1.26–4.10, p = 0.006), but not high levels of miR-BART13-3p (HR 0.67, 95%CI 0.35–1.28, p = 0.227) (Table 4).
Table 3
Demographic and clinical characteristics of nasopharyngeal carcinoma patients before and after treatment.
Table 3
Demographic and clinical characteristics of nasopharyngeal carcinoma patients before and after treatment.
Covariate | Pre-treatment (n = 465) (n, %) | Post-treatment (n = 245) (n, %) |
Age (year) | | |
≤ 45 | 189 (40.6) | 109 (44.5) |
> 45 | 276 (59.4) | 136 (55.5) |
Sex | | |
Male | 324 (69.7) | 172 (70.2) |
Female | 141 (30.3) | 73 (29.8) |
Pathology | | |
Keratinizing squamous cell | 2 (0.40) | 2 (0.80) |
Non-keratinizing, differentiated | 45 (9.70) | 22 (9.00) |
Non-keratinizing, undifferentiated | 418 (89.9) | 221 (90.2) |
T-category | | |
T1 | 120 (25.8) | 70 (28.6%) |
T2 | 86 (18.5) | 46 (18.8%) |
T3 | 158 (40.0) | 83 (33.9%) |
T4 | 91 (19.7) | 46 (18.8%) |
N-category | | |
N0 | 37 (8.00) | 22 (9.00) |
N1 | 226 (48.6) | 117 (47.8) |
N2 | 138 (29.7) | 73 (29.8) |
N3 | 64 (13.7) | 33 (13.5) |
Clinical stage | | |
I | 12 (2.60) | 7 (2.90) |
II | 126 (27.1) | 68 (27.8) |
III | 182 (39.1) | 95 (38.8) |
IVa | 145 (31.2) | 75 (30.6) |
Chemotherapy (cycles) | | |
≤ 3 | 155 (33.3) | 70 (28.6) |
> 3 | 310 (66.7) | 175 (71.4) |
Follow-up time (months) | | |
Median (range) | 55 (2–83) | 55 (2–82) |
Table 4
Univariate and multivariate analysis of distant metastasis-free survival (DMFS) according to pre-treatment levels of miR-BART7-3p, miR-BART13-3p and EBV DNA.
Table 4
Univariate and multivariate analysis of distant metastasis-free survival (DMFS) according to pre-treatment levels of miR-BART7-3p, miR-BART13-3p and EBV DNA.
Covariate | | Univariate analysis | | Multivariate analysis |
| 5-year DMFS (%) | p | | HR (95% CI) | p |
miR-BART7-3p | Low | 93.2 | < 0.001 | | 1.0 | 0.003 |
High | 80.8 | | 2.94 (1.44–5.97) |
miR-BART13-3p | Low | 90.8 | 0.005 | | 1.0 | 0.227 |
High | 83.1 | | 0.67 (0.35–1.28) |
EBV DNA | Low | 92.6 | < 0.001 | | 1.0 | 0.006 |
High | 81.3 | | 2.27 (1.26–4.10) |
Age (years) | ≤ 45 | 87.7 | 0.151 | | 1.0 | 0.204 |
> 45 | 82.1 | | 1.41 (0.83–2.40) |
Sex | Male | 81.9 | 0.004 | | 1.0 | 0.009 |
Female | 92.1 | | 0.40 (0.20–0.80) |
Chemotherapy cycles | ≤ 3 | 87.1 | 0.302 | | 1.0 | 0.620 |
> 3 | 83.1 | | 1.16 (0.65–2.05) |
Clinical stage | I-II | 95.1 | < 0.001 | | 1.0 | 0.018 |
III-IV | 81.0 | | 2.83 (1.19–6.72) |
Combining levels of miR-BART7-3p with those of EBV DNA levels improves prognostic performance
The four-year DMFS rate of subjects with both high levels of EBV DNA and miR-BART7-3p, subjects with high EBV DNA expression but low miR-BART7-3p expression, and subjects with high miR-BART7-3p expression but low EBV DNA expression were similar (78.7/ 83.3%/ 84.8%, all p > 0.05 for comparison between any two groups) (Fig. 3D). While the four-year DMFS of subjects with both low EBV-DNA and miR-BART7-3p expression (n = 150) was significantly better than other 3 groups (p < 0.001) (Fig. 3D). Based on these findings, we classified as “high-risk” those subjects with high levels of EBV DNA and/or miR-BART7-3p. This high-risk group showed a four-year DMFS rate of 82.3% (Fig. 3E) and four-year OS rate of 85.8% (Fig. 3F). The four-year LRRFS did not differ between the high- and low-risk groups (92.1% vs. 91.6%, p = 0.329, Fig. S3 in Additional file 2). In multivariate analysis, the abovementioned “high-risk” status was independently associated with short DMFS (HR 8.74; 95%CI 2.73–27.98; p < 0.001) and short OS (HR 2.45, 95%CI 1.16–5.19; p = 0.019), but not RFS (HR 1.24, 95%CI 0.61–2.50, p = 0.550) (Table 5).
Table 5
Multivariate analysis based on the pre-treatment levels of both miR BART7-3p and EBV DNA in nasopharyngeal carcinoma.
Table 5
Multivariate analysis based on the pre-treatment levels of both miR BART7-3p and EBV DNA in nasopharyngeal carcinoma patients.
Covariate | | DMFS | | OS | | LRRFS |
| HR (95% CI) | p | | HR (95% CI) | p | | HR (95% CI) | p |
Combination | Low-risk | 1 | < 0.001 | | 1.0 | 0.019 | | 1 | 0.550 |
| High-risk | 8.74 (2.73–27.98) | | 2.45 (1.16–5.19) | | 1.24 (0.92–4.51) |
Age (years) | ≤ 45 | 1 | 0.239 | | 1.0 | 0.447 | | 1 | 0.512 |
| > 45 | 1.38 (0.81–2.35) | | 1.24 (0.71–2.15) | | 1.25 (0.64–2.42) |
Sex | Male | 1 | 0.014 | | 1.0 | 0.135 | | 1 | 0.572 |
| Female | 0.43 (0.22–0.85) | | 0.62 (0.33–1.16) | | 1.21 (0.63–2.31) |
Chemotherapy cycles | ≤ 3 | 1 | 0.337 | | 1.0 | 0.493 | | 1 | 0.058 |
| > 3 | 1.32 (0.75–2.33) | | 0.82 (0.47–1.43) | | 0.54 (0.28–1.02) |
Clinical stage | I-II | 1 | 0.010 | | 1.0 | 0.001 | | 1 | 0.078 |
| III-IV | 3.06 (1.31–7.16) | | 7.05 (2.19–22.71) | | 2.04 (0.92–4.51) |
Abbreviations: DMFS, distant metastasis-free survival; OS, overall survival, LRRFS, locoregional recurrence-free survival; HR, hazard Ratio; CI, confidence interval. |
Plasma miR-BART7-3p levels at the end of radiotherapy can predict outcomes
This analysis included 245 NPC patients (Table 3) followed up for a median of 55 months. At the end of radiotherapy, EBV DNA was detectable in 28.6% (70/245) patients; miR-BART7-3p, in 17.6% (43/245) patients; and miR-BART13-3p, in 54.7% (134/245) patients. The four-year DMFS rate was 73.0% in subjects with detectable miR-BART7-3p vs. 89.7% in those without (p < 0.001), 61.4% in subjects with detectable miR-BART13-3p vs. 90.0% (p < 0.001), and 82.7% in subjects with detectable EBV DNA vs. 89.5% (p = 0.035) (Figs. 4A-C). In multivariate analysis, independent risk factors for short DMFS included detectable levels of miR-BART7-3p (HR 4.13, 95%CI 1.89–9.01, p < 0.001) and EBV DNA (HR 2.14, 95%CI 1.04–4.42, p = 0.039) at the end of radiotherapy, but not of BART13-3p (HR 1.19, 95% CI 0.52–2.72, p = 0.672) (Table 6).
Table 6
Multivariate analysis of distant metastasis-free survival (DMFS) according to post-treatment levels of miR-BART7-3p, miR-BART13-3p and EBV DNA.
Table 6
Multivariate analysis of distant metastasis-free survival (DMFS) according to post-treatment levels of miR-BART7-3p, miR-BART13-3p and EBV DNA.
Covariate | | DMFS |
HR (95%CI) | p |
EBV DNA | Undetectable | 1.0 | 0.039 |
| Detectable | 2.14 (1.04–4.42) |
miR-BART7-3p | Undetectable | 1.0 | < 0.001 |
| Detectable | 4.13 (1.89–9.01) |
miR-BART13-3p | Undetectable | 1.0 | 0.672 |
| Detectable | 1.19 (0.52–2.72) |
Chemotherapy cycles | ≤ 3 | 1.0 | 0.762 |
| > 3 | 0.89 (0.42–1.90) |
Age (years) | ≤ 45 | 1.0 | 0.059 |
| > 45 | 2.10 (0.97–4.54) |
Gender | Male | 1.0 | 0.011 |
| Female | 0.25 (0.09–0.73) |
Clinical stage | I-II | 1.0 | 0.060 |
| III-IV | 2.78 (0.96–8.09) |
Abbreviations: miR, microRNA ; EBV, Epstein–Barr virus ; HR, hazard Ratio; CI, confidence interval. |
The four-year DMFS rate were similar between those with only EBV DNA detectable (83.6%) and those with only miR-BART7-3p detectable (71.8%, p = 0.293) (Fig. 4D). Based on these findings, we divided the patients into a low-risk group (neither EBV DNA nor miR-BART7-3p detectable), an intermediate-risk group (either detectable) and a high-risk group (both detectable). The groups showed respective four-year DMFS rates of 92.0%, 80.0% and 52.9% (p < 0.001) (Fig. 4E), and respective four-year OS rates of 94.6%, 79.8%, and 60.4% (p < 0.001) (Fig. 4F).