Abstract
Background
Epstein-Barr virus-encoded small RNA (EBER) in situ hybridization is the primary biomarker assay employed to infer Epstein-Barr virus (EBV) involvement in nasopharyngeal carcinoma (NPC). However, discordance between EBER status and EBV DNA detection has been observed in certain NPC patients. This study aims to investigate the prognostic significance of EBER and EBV DNA status.
Methods
A total of 2,942 NPC patients with known EBV status, treated at our center between 2016 and 2022, were included in this analysis. Clinical characteristics and survival outcomes were compared between patients who had EBER-negative versus EBER-positive and who were matched in a 1:4 ratio. The Kaplan-Meier method was used to analyze survival data. The association between EBER-EBV DNA status and survival outcomes was assessed using multivariable analysis.
Results
Among the cohort, 51 patients (1.7%) were EBER-negative, while 2,891 patients (98.3%) were EBER-positive. EBV-negative NPC was significantly associated with the keratinizing subtype (15.7% vs. 0.3%, P < 0.001). EBER-negative patients exhibited a higher propensity for locoregional recurrence compared to their EBER-positive counterparts, with a 5-year locoregional failure-free survival rate of 76.5% (95% CI, 63.3%-92.5%) versus 86.8% (95% CI, 85.0%-88.6%), respectively (P = 0.012). Multivariable analysis further identified a significant association between double-negative status (EBER-/EBV DNA-) and an increased risk of locoregional recurrence (HR: 4.368, 95% CI, 2.058–9.269, P < 0.001), compared to the double-positive one. These associations remained robust after adjustment for confounding factors and in repeated analyses of the matched cohort.
Conclusions
This study highlights the potential prognostic value of EBER and EBV DNA combinations in NPC. However, given the limited number of EBV-negative cases, further investigations are warranted to substantiate these findings.
Supplementary Information
The online version contains supplementary material available at 10.1186/s12903-025-06733-5.
Keywords: Nasopharyngeal carcinoma, Epstein-Barr virus, EBER, EBV DNA, Prognosis
Introduction
Nasopharyngeal carcinoma (NPC) is a distinctive malignancy that differs from other epithelial cancers of the head and neck in its geographical and demographic distribution, with a marked prevalence in China and Southeast Asia [1, 2]. The endemic variant of NPC demonstrates a robust association with Epstein-Barr virus (EBV) infection and is predominantly characterized by an undifferentiated histological subtype [3, 4]. In contrast, in non-endemic regions, where the incidence of NPC is significantly lower (age-standardized incidence rate < 3 per 100,000 person-years), the disease tends to present with keratinizing squamous histomorphology [2]. Notably, EBV is frequently detected in NPC cases across worldwide, with a prevalence of EBV positivity in approximately 95% of cases in endemic regions, compared to around 86% in non-endemic regions [4–6].
Some evidence supports the involvement of EBV in the etiology of NPC, as evidenced by the detection of viral DNA within tumor cells and the display of viral antigens on the surface of neoplastic cells [7]. EBV-encoded small RNA (EBER), a key molecular marker, is universally expressed across all known latency states of EBV, providing a reliable target for viral detection in clinical specimens [8]. The detection of EBER through in situ hybridization (ISH) remains the gold standard for identifying latent EBV infection [9]. Recent advances in NPC treatment have been driven by insights from large-scale prospective clinical trials and retrospective studies exploring various therapeutic approaches [10, 11]. Notably, Du et al. proposed a refined staging system tailored to EBV-associated NPC in endemic regions [12]. However, its applicability to EBV-negative NPC remains to be determined.
Previous studies on EBV-negative NPC have primarily focused on its clinicopathological features and prognosis [13, 14]. EBER positivity has generally been associated with improved prognosis, particularly in endemic regions [15–17]. However, findings by Xiong et al. indicated that EBV-positive NPC patients are more likely to experience local recurrence compared to their EBV-negative counterparts [14]. Conversely, in low-incidence regions, no statistically significant associations were observed between EBER expression and overall survival or progression-free survival [6]. Additionally, clinical practice reveals inconsistencies between EBER and EBV DNA status exist in certain NPC cases. The prognostic significance of varying EBER and EBV DNA profiles in NPC remains uncertain and warrants further investigation.
In light of this, we conducted a retrospective analysis to explore the gaps regarding the prognostic implications of various EBER and EBV DNA combinations in NPC. These data have the potential to deepen the understanding of non-EBV infection NPC and contribute to strengthening clinical management of NPC in endemic areas.
Methods
Study design and participants
We conducted a retrospective analysis of NPC patients who were initially diagnosed at Fujian Cancer Hospital, China, between Jan. 2016 and Dec. 2022. The study cohort included patients with histologically confirmed primary squamous cell carcinoma of the nasopharynx, for whom data on EBER status (assessed by ISH) and EBV DNA quantification (measured by polymerase chain reaction) were available. All patients underwent cross-sectional imaging, biopsy for histological confirmation and received complete treatment with radical IMRT, with a subset also receiving chemotherapy. Exclusion criteria included the presence of distant metastasis at initial diagnosis, incomplete baseline data, prior anti-tumor treatment, treatment abandonment, or loss to follow-up (defined as no follow-up data available after therapy). The study was conducted in accordance with the principles of the Declaration of Helsinki and approved by the Ethics Committee of Fujian Cancer Hospital (No. K2024-311-01). Owing to its retrospective design, no additional informed consent from patients was required.
Variables and outcome measures
Baseline and treatment-related characteristics were extracted from the medical records of each patient, including age, gender, TNM stage, histological subtype, EBER status, plasma EBV DNA load, treatment details, and clinical outcomes. To explore potential alternative biomarkers for patients with double-negative profiles, we included the immune inflammation index (III) and nutrition index (NI), derived from our previous research, which were identified as prognostic factors for EBV DNA-negative NPC in our previous research [18]. The methodologies for calculating the III and NI have been described elsewhere [18]. Clinical outcomes encompassed the date of last follow-up, recurrence, metastasis, or death. Staging for all patients was reassessed by Dr. Li and Dr. Huang from the Department of Oncology using the American Joint Committee on Cancer 8th edition staging system for NPC. All patients underwent radical IMRT, with treatment details provided in our earlier publication [19].
The study focused on two outcomes. The first outcome was the distribution of various combinations of EBER and EBV DNA status within the cohort. The second was locoregional failure-free survival (LRFFS), defined as the time interval from the completion of IMRT to the first occurrence of local or regional recurrence, or the last follow-up. Secondary outcomes included overall survival (OS), measured from the completion of IMRT to either death or the last follow-up. The final follow-up was conducted on July 20, 2024. Patients were regularly monitored through outpatient clinic visits or maintained contact via phone, with meticulous documentation of any cancer recurrences or mortality events.
Epstein-Barr virus detection
The tissue wax blocks were subjected to EBER in situ hybridization to assess the EBV status of the tumors [20]. ISH was performed using the EBER Probe ISH Detection Kit® (Taipu Bioscience Co., Ltd, China), in strict accordance with the manufacturer’s protocol, ensuring consistent application across all samples. Specimens underwent pretreatment with proteinase K, followed by the addition of a hybridization solution containing a fluorescein-labeled EBV nucleic acid probe. Subsequent to hybridization, an alkaline phosphatase-conjugated antibody targeting fluorescein isothiocyanate was applied, and the chromogenic reaction was developed using a mixture of 5-bromo-4-chloro-3-indolyl-phosphate and 4-nitro-blue tetrazolium chloride, with levamisole included to inhibit nonspecific staining. The slides were then counterstained with hematoxylin. EBER-positive NPC was identified by the presence of blue-purple granules within the nuclei, with the cytoplasm remained unstained. Conversely, samples without these nuclear granules were classified as EBER-negative NPC. ISH results were independently assessed by Dr. Hu, an experienced pathologist, using high-magnification microscopy. Representative pathological images of one EBER-positive and one EBER-negative patient are shown in Fig. 1.
Fig. 1.
In situ hybridization against EBER. A EBER-negative and (B) EBER-positive NPC tissues detected by hematoxylin-eosin staining and in situ hybridization. EBER, Epstein-Barr virus encoded small RNA; HE, hematoxylin-eosin; ISH, in situ hybridization; NPC, nasopharyngeal carcinoma
All NPCs in this study were classified as either keratinizing squamous cell carcinoma or non-keratinizing carcinoma. For EBER-negative patients, additional p16 immunohistochemical staining (a positive result indicating diffuse cytoplasmic and nuclear staining in > 70% of tumor cells) was performed to assess human papillomavirus (HPV) status, supplemented by real-time polymerase chain reaction (PCR) of DNA for high-risk HPV in some cases [21, 22].
Plasma EBV DNA quantitation
EBV DNA quantification was performed using a real-time quantitative PCR assay targeting the highly conserved BamHI-W region of the EBV genome [23]. This assay employed a test kit from Daan Gene Co., Ltd., Sun Yat-sen University (Guangzhou, China). Plasma samples were collected, and DNA extraction was carried out using magnetic bead technology (EA20160201, PerkinElmer) in conjunction with an automated nucleic acid extraction workstation (Pre-NAT, PerkinElmer). The BamHI-W region of the extracted DNA was then amplified to measure circulating EBV DNA concentrations. Patients were classified as EBV DNA negative if their plasma EBV DNA load was 0 copies/mL at all three time points assessed: upon admission, prior to radiotherapy, and after radiotherapy, as previously defined [18].
Statistical analysis
Descriptive statistics were computed for all collected variables, with comparisons made using the Chi-squared test and the Kruskal-Wallis test. Univariate and multivariate Cox regression analyses were conducted to identify potential prognostic factors. Survival rates were estimated using the Kaplan-Meier method, with differences between groups evaluated via the log-rank test. Propensity score matching (PSM) was employed to address selection bias in estimating EBER status, as reflected by a markedly low number of patients with negative EBER status compared to those with positive EBER status. Matching was conducted based on all relevant confounding variables using the nearest neighbor algorithm, with a caliper value of 0.25 and a 1:4 matching ratio. The balance of covariates before and after PSM was assessed using the standardized mean difference (SMD), with values < 0.1 indicating balance. Multivariable analysis was conducted to evaluate the impact of the EBER-EBV DNA combination on survival outcomes in both the entire and PSM cohorts, using three distinct regression models, each adjusting for distinct covariates to mitigate potential confounding biases. Model 1 adjusted for demographic variables, including age and gender; Model 2 further incorporated tumor characteristics, such as T category, N category, and histology; and Model 3 additionally accounted for treatment-related factors, including induction and concurrent chemotherapy. Analyses were performed using R software v4.3.0, Zstats v1.0 (www.zstats.net), and SPSS Statistics v25.0, with a significance threshold set at a two-tailed P-value < 0.05.
Results
Clinical characteristics
Our initial cohort comprised 4,925 non-metastatic NPC patients with well-defined staging who underwent radical IMRT. Of these, 1,983 patients were excluded: 1,862 lacked EBER or plasma EBV DNA data during treatment, 66 were not classified into keratinizing or non-keratinizing histological subtypes, 52 had missing follow-up data, and 3 experienced disease progression during treatment. Consequently, 2,942 patients were included for the final analysis (Fig. 2). The median age of the cohort was 49 years (range, 8 to 87) (Table 1). Among the 2,942 patients, 438 patients (14.9%) had early-stage disease (stage I-II), while 2,504 patients (85.1%) presented with locally advanced disease (stage III and IVa) at diagnosis. The non-keratinizing pathological subtype was predominant, observed in 99.5% of cases. In comparison to EBER-positive NPC patients (n = 2,891), those with EBER-negative were more likely to be female (41.2% vs. 26.4%, P = 0.018), had a higher incidence of the keratinizing subtype (15.7% vs. 0.3%, P < 0.001), and were more likely to be EBV DNA-negative (47.1% vs. 6.8%, P < 0.001) (TableS1). Of the 51 patients with EBER-negative NPC, p16 immunohistochemical staining was conducted on 39 tumor samples, while the remaining samples that PCR confirmed the absence of HPV DNA, revealing 3 p16-positive tumors (2 in double-nagetive group and 1 in EBER-/EBV DNA + group), classified as HPV-positive NPC. The overall median follow-up time was 32 months (range, 3 to 96 months). The EBER-negative group exhibited a significantly poorer 5-year LRFFS rate than the EBER-positive group (76.5% [95% CI, 63.3%−92.5%] vs. 86.8% [95% CI, 85.0%−88.6%], P = 0.012, Fig. 3A), with a marginal effect on OS (82.0% [95% CI, 70.3%−95.6%] vs. 86.6% [95% CI, 84.7%−88.7%], P = 0.066, Fig. 3B).
Fig. 2.
The flowchart of the patient inclusion
Table 1.
Baseline characteristics of patients with nasopharyngeal carcinoma according to each EBER-EBV DNA combination group
| Characteristic | EBER-/EBV DNA- (n = 24) |
EBER-/EBV DNA+ (n = 27) |
EBER+/EBV DNA- (n = 195) |
EBER+/EBV DNA+ (n = 2696) |
P value |
|---|---|---|---|---|---|
| Age, Median with range, years | 51.5 (31–67) | 50 (25–74) | 51 (23–78) | 49 (8–87) | 0.587 |
| Gender, No. (%) | 0.066 | ||||
| Male | 15 (62.5) | 15 (55.6) | 137 (70.3) | 1992 (73.9) | |
| Female | 9 (37.5) | 12 (44.4) | 58 (29.7) | 704 (26.1) | |
| Histology, No. (%) | < 0.001 | ||||
| Keratinizing | 7 (29.2) | 1 (3.7) | 0 (0.0) | 8 (0.3) | |
| Nonkeratinizing | 17 (70.8) | 26 (96.3) | 195 (100.0) | 2688 (99.7) | |
| T category, No. (%) | < 0.001 | ||||
| T0-1 | 3 (12.5) | 5 (18.5) | 65 (33.3) | 482 (17.9) | |
| T2 | 3 (12.5) | 5 (18.5) | 36 (18.5) | 432 (16.0) | |
| T3 | 13 (54.2) | 10 (37.0) | 68 (34.9) | 1150 (42.7) | |
| T4 | 5 (20.8) | 7 (25.9) | 26 (13.3) | 632 (23.4) | |
| N category, No. (%) | < 0.001 | ||||
| N0 | 2 (8.3) | 1 (3.7) | 46 (23.6) | 210 (7.8) | |
| N1 | 12 (50.0) | 13 (48.2) | 100 (51.3) | 1077 (40.0) | |
| N2 | 6 (25.0) | 11 (40.7) | 41 (21.0) | 822 (30.5) | |
| N3 | 4 (16.7) | 2 (7.4) | 8 (4.1) | 587 (21.8) | |
| Stage, No. (%) | < 0.001 | ||||
| Stage I | 0 (0.0) | 0 (0.0) | 17 (8.7) | 50 (1.9) | |
| Stage II | 3 (12.5) | 3 (11.1) | 53 (27.2) | 312 (11.6) | |
| Stage III | 11 (45.8) | 15 (55.6) | 90 (46.2) | 1194 (44.3) | |
| Stage IVa | 10 (41.7) | 9 (33.3) | 35 (18.0) | 1140 (42.3) | |
| EBV DNA, pre-treatment, copies/mL | 0 (0, 0) | 780 (120, 3395) | 0 (0, 0) | 2110 (392, 10800) | < 0.001 |
| EBV DNA, pre-RT, copies/mL | 0 (0, 0) | 83.65 (0, 512.25) | 0 (0, 0) | 30.7 (0, 355) | < 0.001 |
| EBV DNA, post-RT, copies/mL | 0 (0, 0) | 0 (0, 0) | 0 (0, 0) | 0 (0, 0) | < 0.001 |
| Received induction chemotherapy, No. (%) | < 0.001 | ||||
| No | 4 (16.7) | 2 (7.4) | 72 (36.9) | 362 (13.4) | |
| Yes | 20 (83.3) | 25 (92.6) | 123 (63.1) | 2334 (86.6) | |
| Received concurrent chemotherapy, No. (%) | 0.314 | ||||
| No | 14 (58.3) | 9 (33.3) | 86 (44.1) | 1247 (46.3) | |
| Yes | 10 (41.7) | 18 (66.7) | 109 (55.9) | 1449 (53.8) | |
| 5-year outcomes: actuarial rate (95% CI), % | |||||
| LRFFS | 63.7 (44.4–91.3) | 87.8 (72.7–100.0) | 92.5 (87.9–97.4) | 86.3 (84.3–88.3) | 0.003 |
| OS | 75.5 (56.4–100.0) | 86.1 (72.3–100.0) | 94.6 (90.4–99.1) | 86.0 (83.8–88.1) | 0.012 |
| EBER, EBV-encoded small RNA; EBV, Epstein-Barr virus; LRFFS, locoregional failure-free survival; OS, overall survival; RT, radiotherapy | |||||
Fig. 3.
Prognostic outcomes in patients with nasopharyngeal carcinoma stratified by EBER status are presented. A The locoregional failure-free survival and (B) overall survival for the whole cohort between EBER-positive and EBER-negative groups. C The locoregional failure-free survival and (D) overall survival in the propensity score-matched cohort. E A representative patient with EBER-/EBV DNA-; more than 1 year after treatment, the patient developed local and regional recurrence. F A representative patient with EBER+/EBV DNA+; after a follow-up period of over 6 years, there was no evidence of locoregional recurrence or distant metastasis. EBER, Epstein-Barr virus encoded small RNA; EBV DNA, Epstein-Barr virus DNA
Subgroup analysis of EBER-EBV DNA combinations
Among the 2,942 patients with available EBER and EBV DNA test results, 2,696 (91.6%) were classified as EBER+/EBV DNA+ (double positive), 24 (0.8%) as EBER-/EBV DNA- (double negative), 195 (6.6%) as EBER+/EBV DNA-, and 27 (0.9%) as EBER-/EBV DNA+. Table 1 summarizes the clinical and demographic characteristics of patients stratified by EBER-EBV DNA status combinations. Notably, significant differences were observed in histological subtypes, with the double-negative subgroup demonstrating a substantially higher proportion of keratinizing carcinomas compared to other groups (P < 0.001). Differences were also apparent in TNM staging and the administration of induction chemotherapy (P < 0.001).
Patients in the double-negative subgroup exhibited a markedly increased risk of locoregional recurrence compared to the double-positive subgroup (5-year LRFFS rate: 63.7% [95% CI, 44.4%−91.3%] vs. 82.9% [95% CI, 80.4%−85.5%], P < 0.001), the EBER+/EBV NDA- subgroup (5-year LRFFS rate: 63.7% [95% CI, 44.4%−91.3%] vs. 87.8% [95% CI, 72.7%−100.0%], P = 0.023) and the EBER-/EBV NDA + subgroup (5-year LRFFS rate: 63.7% [95% CI, 44.4%−91.3%] vs. 92.5% [95% CI, 87.9%−97.4%], P < 0.001). Additionally, the double-negative group demonstrated a significantly higher risk of mortality compared to the double-positive group (5-year OS rate: 75.5% [95% CI, 56.4%−100.0%] vs. 86.0% [95% CI, 83.8%−88.1%], P = 0.033). Furthermore, patients in the EBER+/EBV DNA- subgroup displayed superior survival outcomes relative to the double-positive (5-year OS rate: 94.6% [95% CI, 90.4%−99.1%] vs. 86.0% [95% CI, 83.8%−88.1%], P = 0.016), double-negative (5-year OS rate: 94.6% [95% CI, 90.4%−99.1%] vs. 75.5% [95% CI, 56.4%−100.0%], P < 0.001) and EBER-/EBV NDA + subgroups (5-year OS rate: 94.6% [95% CI, 90.4%−99.1%] vs. 86.1% [95% CI, 72.3%−100.0%], P = 0.046). Similar findings were observed after excluding the three p16-positive patients from the EBER-negative cohort (see Supplementary Results).
Association between EBER-EBV DNA combination and survival outcomes in the entire cohort
To elucidate the association between different subgroups and survival outcome in NPC patients, a multivariable analysis was performed, accounting for potential confounders, including age, gender, histology, T category, N category, and the use of induction and concurrent chemotherapy. Compared to the double-positive group, patients in the double-negative subgroup demonstrated a significantly higher risk of locoregional recurrence (HR: 4.368, 95% CI, 2.058–9.269, P < 0.001 in the unadjusted model, Table 2). This elevated risk remained significant after adjusting for demographic factors (Model 1, HR: 4.713, 95% CI, 2.215–10.028, P < 0.001), tumor characteristics (Model 2, HR: 3.357, 95% CI, 1.401–8.039, P = 0.007), and further treatment-related confounders (Model 3, HR: 3.588, 95% CI, 1.486–8.659, P = 0.004). These results highlight a strong association between double-negative EBER and EBV DNA status and poor LRFFS in NPC patients. Additionally, the EBER+/EBV DNA- subgroup exhibited a reduced risk of mortality compared to the double-positive group in the unadjusted model (HR: 0.424, 95% CI, 0.209–0.862, P = 0.018) and Model 1 (HR: 0.404, 95% CI, 0.198–0.821, P = 0.012). However, this trend was attenuated and did not reach statistical significance after adjusting for tumor characteristics (HR: 0.522, 95% CI, 0.254–1.073, P = 0.077 in Model 2) and treatment-related factors (HR: 0.511, 95% CI, 0.248–1.050, P = 0.068 in Model 3).
Table 2.
Association of EBER-EBV DNA combination and survival outcomes in patients with nasopharyngeal carcinoma in the entire cohort
| Characteristic | Non-adjusted | Model 1 | Model 2 | Model 3 | ||||
|---|---|---|---|---|---|---|---|---|
| HR (95% CI) | P value | HR (95% CI) | P value | HR (95% CI) | P value | HR (95% CI) | P value | |
| LRFFS | ||||||||
| EBER+/EBV DNA+ | 1.000 (Reference) | 1.000 (Reference) | 1.000 (Reference) | 1.000 (Reference) | ||||
| EBER+/EBV DNA- | 0.554 (0.302–1.017) | 0.057 | 0.566 (0.308–1.039) | 0.066 | 0.669 (0.361–1.241) | 0.202 | 0.700 (0.378–1.297) | 0.257 |
| EBER-/EBV DNA+ | 1.123 (0.359–3.512) | 0.842 | 1.194 (0.381–3.738) | 0.761 | 1.297 (0.413–4.069) | 0.656 | 1.194 (0.380–3.748) | 0.762 |
| EBER-/EBV DNA- | 4.368 (2.058–9.269) | < 0.001 | 4.713 (2.215–10.028) | < 0.001 | 3.357 (1.401–8.039) | 0.007 | 3.588 (1.486–8.659) | 0.004 |
| OS | ||||||||
| EBER+/EBV DNA+ | 1.000 (Reference) | 1.000 (Reference) | 1.000 (Reference) | 1.000 (Reference) | ||||
| EBER+/EBV DNA- | 0.424 (0.209–0.862) | 0.018 | 0.404 (0.198–0.821) | 0.012 | 0.522 (0.254–1.073) | 0.077 | 0.511 (0.248–1.050) | 0.068 |
| EBER-/EBV DNA+ | 1.441 (0.534–3.885) | 0.471 | 1.349 (0.500-3.642) | 0.555 | 1.632 (0.601–4.431) | 0.336 | 1.649 (0.606–4.490) | 0.328 |
| EBER-/EBV DNA- | 2.654 (0.987–7.142) | 0.053 | 2.442 (0.906–6.585) | 0.078 | 1.604 (0.542–4.749) | 0.394 | 1.632 (0.550–4.842) | 0.377 |
Model 1 was adjusted for age and gender
Model 2 was adjusted for age, gender, T category, N category, and histology
Model 3 was adjusted for age, gender, T category, N category, histology, induction and concurrent chemotherapy
EBER, EBV-encoded small RNA; EBV, Epstein-Barr virus; LRFFS, locoregional failure-free survival; OS, overall survival
Association between EBER-EBV DNA combination and survival outcomes in the propensity-matched cohort
PSM analysis was employed to mitigate the bias associated with EBER status. Considering that the keratinizing subtype is exceptionally rare in NPC cases within our endemic region and is strongly associated with EBER-negative patients, we did not consider this variable in the PSM analysis. After adjusting for age, gender, T category, N category, EBV DNA status, and treatment modality, 199 EBER-positive patients were matched with 51 EBER-negative patients, achieving a well-balanced distribution of clinical and treatment characteristics between the groups (Table S2, Supplementary Fig. 1). The robustness of these results was maintained when analyses were confined to the PSM cohort. Survival outcomes varied significantly among the different EBER subgroups. The EBER-negative group exhibited a poorer 5-year LRFFS rate (76.5% [95% CI, 63.3%−92.5%] vs. 87.3% [95% CI, 81.4%−93.5%], P = 0.037, Fig. 3C). Meanwhile, OS also showed a trend toward worse outcomes in the EBER-negative group, albeit without significance (82.0% [95% CI, 70.3%−95.6%] vs. 89.2% [95% CI, 83.7%−95.1%], P = 0.071, Fig. 3D). Representative outcomes for patients with EBER-negative and EBER-positive status are depicted in Fig. 3E-F. Multivariable analysis further revealed that patients in the double-negative group faced a significantly higher risk of recurrence compared to those in the double-positive group, even after adjusting for confounding variables (Table S3). These findings underscore the association between double-negative EBER-EBV DNA status and poorer LRFFS in NPC patients.
Clinical characteristics and prognostic factors of EBER-/EBV DNA- patients
The treatment modalities and clinical outcomes of the double-negative cohort are summarized in Table S4. Given the poorer prognosis associated with this subgroup, we investigated potential prognostic factors. Univariate Cox regression analysis revealed significant associations between nonkeratinizing histology (HR: 5.170, 95% CI: 1.124–23.792, P = 0.035), and III (HR: 1.579, 95% CI: 1.113–2.240, P = 0.011), with decreased LRFFS, as presented in Table 3. However, these associations did not retain statistical significance in multivariate analysis. A similar trend emerged for OS, with nonkeratinizing subtype (HR: 11.490, 95% CI: 1.104-119.619, P = 0.041) and III (HR: 1.672, 95% CI: 1.057–2.645, P = 0.028) demonstrating prognostic significance in univariate analysis. In contrast, T category and N category showed no significant impact on LRFFS or OS (all P > 0.100).
Table 3.
Identification of prognostic factors in EBER-/EBV DNA- patients by univariate and multivariate Cox regression analysisa
| Characteristic | Univariate | Multivariate | ||
|---|---|---|---|---|
| HR (95% CI) | P value | HR (95% CI) | P value | |
| LRFFS | ||||
| Age (years) | 1.031 (0.959–1.109) | 0.407 | ||
| Gender | ||||
| Male | 1.000 (Reference) | |||
| Female | 0.441 (0.084–2.306) | 0.332 | ||
| Histology | ||||
| Keratinizing | 1.000 (Reference) | 1.000 (Reference) | ||
| Nonkeratinizing | 5.170 (1.124–23.792) | 0.035 | 2.480 (0.345–17.836) | 0.367 |
| T category | 0.983 (0.441–2.192) | 0.967 | ||
| N category | 1.375 (0.604–3.130) | 0.448 | ||
| III b | 1.579 (1.113–2.240) | 0.011 | 1.399 (0.917–2.134) | 0.120 |
| NI b | 1.174 (0.669–2.061) | 0.576 | ||
| OS | ||||
| Age (years) | 1.017 (0.923–1.120) | 0.740 | ||
| Gender | ||||
| Male | 1.000 (Reference) | |||
| Female | 0.333 (0.034–3.271) | 0.346 | ||
| Histology | ||||
| Keratinizing | 1.000 (Reference) | 1.000 (Reference) | ||
| Nonkeratinizing | 11.490 (1.104-119.619) | 0.041 | 6.060 (0.359–102.200) | 0.211 |
| T category | 0.861 (0.321–2.309) | 0.766 | ||
| N category | 1.979 (0.657–5.965) | 0.225 | ||
| III | 1.672 (1.057–2.645) | 0.028 | 1.389 (0.832–2.318) | 0.209 |
| NI | 1.288 (0.611–2.712) | 0.506 | ||
| aVariable exhibiting a P value less than 0.05 in the univariate Cox regression analysis were futher included in multivariate Cox regression analysis | ||||
| bThe calculation of III and NI is detailed elsewhere [18] | ||||
| EBER, EBV-encoded small ribonucleic acid; EBV, Epstein-Barr virus; III, immune inflammation index; LRFFS, locoregional failure-free survival; NI, nutrition index; OS, overall survival | ||||
Discussion
Although prior studies have explored the long-term outcomes of NPC based on EBV status within the Chinese population, research on the prognostic implications of combined EBER and EBV DNA statuses in endemic regions remains scarce. In this comprehensive cohort of NPC patients, we evaluated the prognostic impact of various EBER and EBV DNA combinations. Our findings indicated that EBER-negative patients are at an elevated risk of recurrence. Specifically, patients with double-negative status exhibited a significantly poorer LRFFS compared to the double-positive group. The findings from the PSM cohort further corroborated the heightened recurrence risk associated with the double-negative subgroup. These results highlight non-EBV-related NPC as a distinct entity, potentially requiring unique management strategy.
To our knowledge, this investigation constitutes the most extensive endeavor to date aimed at elucidating the prognostic significance of EBER and EBV DNA combinations in patients afflicted NPC. Previous research by Xiong et al. aggregated data from NPC patients with known EBV status across four hospitals in both endemic and non-endemic regions of China between 2013 and 2021, and identified 48 EBER-negative patients, though the total 48 EBER-negative patients was not disclosed [14]. In our cohort, the incidence of EBER-negative NPC was notably low, accounting for only 1.7% (51 of 2,942 patients). However, this rate should be interpreted with caution, as approximately one-third of patients were excluded due to missing EBER testing at enrollment. Furthermore, some studies have reported no significant differences in survival outcomes between EBV-negative and EBV-positive NPC patients [14, 24]. In contrast to Xiong et al., who observed a higher risk of local recurrence in EBER-positive NPC patients, our findings indicate that EBER-negative patients face an increased risk of recurrence and exhibit a trend towards shorter OS [14]. This discrepancy may partly attributable to regional variability. Xiong et al.’s cohort comprised patients from diverse geographic locations in China, including Guangdong, an area known for its endemic NPC incidence. However, the precise distribution of patients across these regions was not specified. Geographic and environmental variations, including variations in lifestyle, diet, and potentially racial or ethnic backgrounds, may contribute to these differing clinical outcomes [25–27]. Furthermore, factors such as smoking and HPV infection, aside from EBV infection, have been implicated in NPC pathogenesis [24]. A previous study reported a high attribution of HPV in the EBV-negative NPC subset (29 of 40 NPCs; 73%) in a western cohort [28]. In contrast, our cohort revealed only 3 of 51 (5.9%) EBER-negative cases attributable to HPV. Joint analyses of EBV and HPV have demonstrated worse outcomes for EBV/HPV-negative NPC compared to EBV-positive cases, lending partial support to our results [29, 30]. Additionally, Wang et al. identified higher tumor mutation burdens in EBV-negative NPC, suggesting a potential need for intensified local and possibly systemic treatment for EBV-negative NPC patients [31].
Comparative analysis of EBV-positive and EBV-negative NPC cases reveals distinct histological differences between NPC subtypes [2]. Non-keratinizing NPCs, predominantly observed in endemic regions, are almost universally EBV-positive, while keratinizing NPCs are exceedingly rare in these areas, accounting for less than 1% of cases [31, 32]. Our findings corroborate this trend, with only 16 cases (0.5% of the total cohort) identified as the keratinizing subtype. Racial variability in NPC histological subtypes is also evident; for instance, in the United States, a substantial proportion of White patients (64.2%) are diagnosed with keratinizing NPC [33]. Non-EBV-associated NPC has increasingly been recognized as a distinct pathological entity, which reflecting the complexity of NPC’s histological and biological heterogeneity. Consistent with prior studies, our results show a strong association between EBER-negative NPCs and the keratinizing subtype [14], underscoring the close relationship between EBV infection and NPC histological classification [34]. Additionally, studies have shown that these histological subtypes demonstrate divergent treatment responses; NPCs with nonkeratinizing subtype are notably sensitive to radiotherapy and platinum-based chemotherapy, in contrast to the less radiosensitive and more clinically aggressive keratinizing subtype [35]. Bossi et al. demonstrated that intensive treatment benefits disease-free survival predominantly in EBER-positive patients, indicating that additional therapeutic interventions may not offer advantages in EBER-negative cases within low-incidence regions [36]. However, due to limitations in sample size, our study did not evaluate survival differences across histological subtypes or optimal treatment strategies for EBER subgroups. Further research with larger cohorts is necessary to provide a more comprehensive comparison of survival outcomes and refine therapeutic approaches for these distinct NPC subtypes.
EBV DNA, released into peripheral blood from NPC cells through various mechanisms of cell death, serves as a crucial biomarker for assessing tumor burden, biological behavior, and the presence of occult metastatic lesions in NPC and has been also recognized as a unique biomarker for treatment evaluation and prognostic prediction in NPC [32, 37, 38]. In our study, we observed that 7.4% of histologically confirmed NPC patients were negative for plasma EBV DNA. In response to some studies indicated that 15.1–29.3% of NPC patients from endemic regions exhibit undetectable plasma EBV DNA at initial diagnosis [39, 40]. Le et al. proposed the possibility of non-EBV origins for some NPC cases [41]. Our findings provide additional support for this hypothesis, as we found that not all NPCs tested positive EBER ISH signals. However, upon supplementing the analysis with p16 testing for EBV-negative NPC, we found that 94.1% of these cases were not associated with HPV infection. This suggests that the origins of non-EBV-related NPC remain an area requiring further investigation, as additional factors may contribute to its pathogenesis, which may offer valuable insights into distinct molecular pathways and therapeutic strategies. In our study, patients classified in the EBER-/EBV DNA- group displayed poorer prognosis, characterized by a high risk of recurrence. For these patients, alternative biomarkers for treatment monitoring and prognostication are warranted. Several hematological biomarkers have been shown to hold prognostic significance across various malignancies [42]. Previous research from our group has highlighted the prognostic value of the immune inflammation index and nutritional index in NPC patients with negative EBV DNA [18]. We further investigated the prognostic factors influencing outcomes in double-negative patients. Our findings tentatively suggest that non-keratinizing subtypes and immunoinflammatory indices are associated with poorer LRFFS and OS. However, these factors were not identified as independent prognostic indicators, which may be due to the relatively small sample size in this cohort.
In the EBER-/EBV DNA + group, the presence of detectable EBV DNA is unlikely to be attributable to technical or sampling variations, as all cases were processed in a single laboratory using consistent EBV-targeting methods. However, the presence of plasma EBV DNA does not exclusively indicate active EBV-driven malignancy and could be detected in some cases without clear EBV-associated disease [43]. In our study, we focused on the specific population of patients with NPC, an EBV-associated malignancy, within an endemic region where EBV positivity is strongly linked to the pathogenesis of NPC [1, 2, 4]. EBER is typically a marker for active EBV infection in tumor cells, and its detection through ISH demonstrates the incorporation of the EBV genome into the tumor cells [8, 9]. However, in the EBER-/EBV DNA + subgroup, the EBV genome may not be efficiently incorporated into the tumor cells, resulting in diminished EBER expression. Nicholls et al. reported that EBER expression is often weak in patients with low plasma EBV DNA levels prior to treatment [39]. Moreover, some patients with advanced stage III to IVA disease may show reduced EBV genome incorporation into tumor cells, potentially leading to impaired EBER expression [39]. Additionally, issues related to biomarker sensitivity and lead-time effects may also contribute to these observations [44]. In light of these considerations, our data, within the context of an endemic population, suggest that the presence of EBV DNA in these patients may be biologically plausible. Nevertheless, we acknowledge the possibility that the EBER-/EBV DNA + subgroup could also represent EBV-negative NPC or cases with incidental EBV reactivation unrelated to tumorigenesis. Consequently, the EBER-negative while EBV DNA-positive subpopulation requires further investigation and a more cautious interpretation of its clinical implications, particularly in the context of NPC.
Despite our endeavors, it is imperative to acknowledge certain limitations in our study. Primarily, the retrospective design and limited sample size constrain the robustness of our findings. Although we have adjusted for potential confounders and employed PSM analysis to reduce the impact of these factors, the inherent limitations of retrospective data, such as residual confounding from unmeasured variables or missing patient data, may still influence the results. Future prospective studies with more comprehensive data collection and larger sample sizes are needed to validate and extend these findings. Secondly, variability in EBV testing methods (technique or detection time) may affect the replicability of presented results; our study was carried out at a single treatment center, serves to partially addresses this concern. Additionally, the reliance on a limited number of tumor slides from each patient’s formalin-fixed, paraffin-embedded samples for ISH may not fully capture the extent of EBV genome incorporation across all tumor cells, representing another limitation of our approach. We attempted to mitigate this by selecting the most representative slides containing the highest proportion of tumor cells to ensure reliable EBER scoring. Moreover, the absence of EBER test data for some patients may introduce selection bias, and the true incidence of EBER-negative NPC at our center may differ from the reported 1.7%. Future studies with complete testing on patients would be needed to more accurately reflect the true incidence of EBER-negative NPC in endemic regions. Finally, the limited cohort size restricts the ability to perform a robust analysis of optimal treatment strategies for EBV-negative NPC. Larger, adequately powered studies are essential to further investigate and refine treatment strategies, with the potential to offer more personalized and effective management approaches for these patients.
Conclusion
In conclusion, this study highlights the potential prognostic value of EBER and EBV DNA combinations in NPC. Given the established role of EBV infection in NPC, these findings provide a reference for enhancing the clinical management of non-EBV-infected NPC patients within endemic areas.
Supplementary Information
Abbreviations
- EBER
Epstein-Barr virus-encoded small RNA
- EBV
Epstein-Barr virus
- HPV
Human papillomavirus
- HR
Hazard ratio
- III
Immune inflammation index
- ISH
In situ hybridization
- IMRT
Intensity-modulated radiotherapy
- LRFFS
Locoregional failure-free survival
- NI
Nutrition index
- NPC
Nasopharyngeal carcinoma
- OS
Overall survival
- PCR
Polymerase chain reaction
- PSM
Propensity score matching
- SMD
Standardized mean difference
Authors’ contributions
Qiu S. had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: Qiu S., Hu D. Acquisition of data: Xu S., Wang J., Yu Y. Drafting of the manuscript: Li Y., Wu L. Statistical analysis: Huang Z., Cai S. Critical revision of the manuscript for important intellectual content: Qiu S. Obtained funding: Qiu S. Study supervision: Lai J. All authors of this paper have read and approved the final version submitted.
Funding
We thank the support of the grants of Science and Technology Program of Fujian Province, China (2018Y2003); Fujian Provincial Clinical Research Center for Cancer Radiotherapy and Immunotherapy (2020Y2012); Supported by the National Clinical Key Specialty Construction Program (2021); Fujian Clinical Research Center for Radiation and Therapy of Digestive, Respiratory and Genitourinary Malignancies (2021Y2014). National Natural Science Foundation of China (82473376); Major Scientific Research Program for Young and Middle-aged Health Professionals of Fujian Province, China (Grant No.2021ZQNZD010); Joint Funds for the Innovation of Science and Technology, Fujian province (2021Y9196, 2023Y9436); the Qihang Funds of Fujian Medical University (2023QH2050, 2022QH2048); and High-level Talent Training Program of Fujian Cancer Hospital (2022YNG07).
Data availability
Data of this study would not be shared. If necessary, data could be obtained from the corresponding authors.
Declarations
Ethics approval and consent to participate
The reporting of this study conforms to the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines. This study was approved by the Ethics Committee of Fujian Cancer Hospital (No. K2024-311-01). Due to the retrospective design, no additional informed consent from patients was required.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Ying Li, Lishui Wu and Zongwei Huang contributed equally to this work.
Contributor Information
Dan Hu, Email: hudan@fjmu.edu.cn.
Sufang Qiu, Email: sufangqiu@fjmu.edu.cn.
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Supplementary Materials
Data Availability Statement
Data of this study would not be shared. If necessary, data could be obtained from the corresponding authors.