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The plasma EBV DNA load with IL-6 and VEGF levels as predictive and prognostic biomarker in nasopharyngeal carcinoma

Virology Journal volume 21, Article number: 224 (2024) Cite this article

Abstract

Nasopharyngeal carcinoma (NPC) is often diagnosed at a very advanced stage due to its location and non-specific initial symptoms. Moreover, no clinically useful serological marker has been established so far for early detection of NPC. In this study, we have investigated the clinical significance of plasma Epstein–Barr virus DNA load along with interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) levels to evaluate if these three all together can be useful as a strong serological marker for early detection and prediction of treatment response in patients with NPC. Plasma EBV DNA load, IL-6 level, VEGF expressions were measured in 24 patients with NPC at presentation and various time points during and after treatment. There was a positive correlation between high plasma EBV DNA load with higher IL-6 and VEGF expression, which was closely associated with therapeutic response as well. Persistent or recurrent plasma EBV load with higher IL-6 and VEGF levels can potentially predict disease progression and may be useful to select patients for additional therapy and longer follow-up.

Introduction

Nasopharyngeal carcinoma is a complex disease, found to be associated with chronic infection by oncogenic gamma herpes virus Epstein–Barr virus [31]. Due to late diagnosis, the overall survival (OS) rate is less than 40% for 5 years in NPC patients [26]. Several clinico-pathological factors such as age, sex, tumor stage, grade, sensitivity to chemotherapy as well as radiotherapy affect overall survival. The severity of NPC is also closely associated with EBV DNA load due to chronic active EBV infection. Many biopsy specimens from nasopharyngeal carcinoma contain EBV [20]. Our previous studies have showed that the majority of NPC patients were EBV positive at presentation and the DNA load of plasma EBV (copy numbers per milliliter) was closely associated with the disease stage [22].

It is well established that EBV-encoded genes are involved in the regulation of various cellular signaling cascades including immune evasion, which protects infected cells from immunologic attack and trigger carcinogenesis. All EBV-infected NPC cells exhibit type II latency with the expression of EBV-associated nuclear antigen-1 (EBNA1) and EBV-encoded small RNA (EBER), which play important roles in immune evasion [15]. Since EBNA1 maintains the replication of EBV genome, it is constantly expressed in all EBV-related malignancies.

Generally, virus infection induces cytokine production during their interaction with the host cells. The surface glycoproteins and intracellular viral proteins have the capacity to activate signal transduction pathways leads to the expression of several cytokines and chemokines [17]. The direct EBV infection also induces the expression of certain growth factor and cytokine in NPC cells [14]. Among several cytokines and chemokines, IL-6 is a common inflammatory cytokine produced by most of the viruses during infection. The first wave of cytokines produced during EBV infection includes IL-6. In addition to EBV glycoproteins gp350, the latent membrane protein 1 (LMP-1) triggers the production of some cytokines such as IL-6, IL-8, and IL-10 [17]. The secretion of pro inflammatory cytokine in the blood is associated with an immune response. It has been reported that the patients with the highest levels of pro-inflammatory cytokines were found to have poor prognosis [11]. The highest levels of IL-6 in cancer tissue were also found to be associated with distant metastasis in patients with oral cancer [8].

The constitutive activation of signal transducer and activator of transcription 3 (STAT3), a major oncogenic transcription factor promotes tumor growth and proliferation through epithelial-mesenchymal transition, invasion, and angiogenesis [24]. In NPC pathogenesis also, STAT3 activation is well established though the exact mechanism of its activation remains poorly defined, especially in the context of EBV infection. It has been reported that stable EBV infection in immortalized NPC cells resulted into IL-6 driven STAT3 activation. Furthermore, STAT3 activation in these EBV-infected NPC cells potentiated their invasive properties in vitro [27].

It has been found in in-vitro studies that EBV-Infection in premalignant and cancerous nasopharyngeal epithelial cells enhanced IL-6/IL-6R signaling and promoted growth and malignant properties [27]. Upon binding with IL-6 receptor, IL-6 activates the receptor-associated kinases (JAK1, JAK2 and Tyk2), which subsequently activates STAT3 by phosphorylation and dimerization. On the other hand, STAT3 possesses regulatory abilities in angiogenesis through the transcription of VEGF [5]. VEGF is identified as a principal pro-angiogenic factor that enhances the production of new blood vessels from existing vascular network, plays crucial role in tumor growth and metastasis. Several studies and meta-analyses published recently indicated VEGF as a promising prognostic biomarker for several cancers like papillary thyroid cancer [7], cervical cancer [28], colorectal cancer [30], oral tongue squamous cell carcinoma [3] and NPC [23].

Generally oncogenic viruses such as EBV, lack their own angiogenic factors, rely on the recruitment of cellular genes for angiogenesis and tumor progression [2]. The secretion of IL-6 and subsequent STAT3 phosphorylation, up-regulate some angiogenic mediators such as VEGF, VEGFR2 and neuropilin 2 [12]. It has also been demonstrated that latent membrane protein 1 (LMP1), an EBV-encoded protein with oncogenic properties plays an essential role in tumorigenesis of NPCs through the activity of various signal pathways especially through the JNKs/c-Jun signaling pathway and VEGF over expression [25].

Despite the emerging biological importance of IL-6 induced STAT3 activation and up regulation of VEGF in NPC, their impact on disease severity or progression, especially in the context of EBV infection, has not yet been explored properly.

In this study, EBV DNA load was quantified by real-time polymerase chain reaction (RT-qPCR) and the level of IL-6 and VEGF expression was estimated at diagnosis as well as at different time points during therapy to find out if their levels are associated with the diseases burden and treatment response. Serial measurement of plasma EBV DNA levels during therapy showed a close correlation between changes in EBV DNA load with clinical response. Patients who responded well showed a drastic decrease in EBV load to low or undetectable whereas in patients with progressive disease, there was a rapid increase in EBV DNA even at the end of the treatment or during further follow up. Regular monitoring of plasma EBV load, along with IL-6 and VEGF expression during and beyond treatment may be potentially valuable for precise prediction and assessment of treatment response in patients with NPC.

Materials and methods

Clinical sample collection

Three milliliters of peripheral blood were collected in an EDTA tube from patients with NPC at baseline and at different time points during treatment such as post neo-adjuvant chemotherapy and post concurrent radiation therapy. For plasma separation, blood samples were centrifuged at 1600–2000 g for 15 min. The separate plasma removed carefully and collected into plain polypropylene tubes for further use. Plasma was stored at -80 degree centigrade.

Study design

This study was a single-center, retrospective, exploratory analysis on 24 patients with NPC, treated at All India Institute of Medical Sciences (AIIMS, New Delhi), India in between 16th Feb’2018–12th Aug’2022. Baseline evaluation included a detailed history and physical examination, full blood count, liver and kidney function tests, contrast-enhanced computed tomography (CT)/whole body 18F-FDG positron emission tomography-computed tomography (PET-CT), magnetic resonance imaging (of skull base, paranasal sinus, face, and neck), nasal endoscopy and biopsy with or without fine needle aspiration cytology (FNAC) from an enlarged lymph node. The tumors were staged by using the American Joint Committee on Cancer (AJCC) 8th edition TNM staging system. Patients with stage II-IV NPC (KSCC, NKSCC—differentiated/undifferentiated) of age in-between 15–62 and Eastern Cooperative Oncology Group (ECOG) performance status (PS) 0–3 were included in this study. Patients with any prior oncologic treatment such as surgery, chemo or radiotherapy or having immuno-compromised status like HIV sero-positivity, recipients of organ transplant, having any uncontrolled co morbidity were excluded in this study. The Institute Ethical Review Board at AIIMS New Delhi reviewed the protocol and approved this study (No. IEC-980/03.10.2020, RP-33/2020).

Treatment

All 24 patients received neo-adjuvant chemotherapy (NACT). The most common NACT regimen used in this cohort (18/24 patients) was a doublet of Injection Gemcitabine (1000 mg/m2 IV D1 and D8) and platinum, either Injection Cisplatin (80 mg/m2 IV divided over D1 and D2 in 14 patients) or Injection Carboplatin (target AUC5 IV D1 in 4 patients), repeated every 3 weeks. Four patients received a doublet of Injection Paclitaxel (175 mg/m2 IV D1) and Injection Carboplatin (target AUC5 IV D1), repeated every 3 weeks. The remaining 2 patients received a doublet of Injection Cisplatin (80 mg/m2 IV divided over D1 and D2) and Injection 5-fluorouracil (1000 mg/m2continuous IV infusion D1–4), repeated every 3 weeks. The median number of cycles of NACT administered in this cohort was 3 (range 1–6). Out of 24, 21 patients received 3 cycles of NACT.

Subsequently all patients received definitive chemo-radiation (CRT) except 1, who had metastatic disease at baseline and received palliative loco-regional radiotherapy to a dose of 30 Gy in 10 fractions over 2 weeks. The radiotherapy prescription dose was 65 Gy, 60 Gy and 54 Gy to high risk PTV, intermediate risk PTV and low risk PTV respectively, in 30 fractions delivered over 6 weeks. This was delivered by simultaneous integrated boost (SIB)—volumetric modulated arc therapy (VMAT) technique. Concurrent chemotherapy was administered in 23 patients, Injection Cisplatin (40 mg/m2/week) in 19 patients and Injection Carboplatin (target AUC2/week) in the remaining 4 patients. Cisplatin was substituted by carboplatin in patients with compromised renal function (glomerular function rate < 50 ml/min) or moderately severe or greater sensorineural hearing loss. Treatment response was assessed by clinical examination and nasal endoscopy at 1 and 3 months and CECT/ PET-CT scan at 3 months after completion of CRT.

Histopathological analysis

Haematoxylin and eosin stained sections from primary tumor samples were used for histological classifications. The histological diagnosis was confirmed by the pathologist. The grading was performed according to WHO classification into keratinizing squamous cell carcinoma, (WHO Type I), non-keratinizing differentiated squamouscell carcinoma (WHO Type II) and non-keratinizing undifferentiated squamous cell carcinoma (WHO Type III).

Viral nucleic acid isolation

Viral nucleic acid was isolated from the plasma sample of patients as well as healthy controls (n = 5) by using High Pure Viral Nucleic Acid Kit (Roche, Cat No #11858874001). During nucleic acid extraction, all plasma samples were processed through a common set of steps involving cell lysis, inactivation of nucleases, nucleic acid binding in a column, washing and elution. Total nucleic acid extraction products purify both RNA and DNA from samples. Based on manufacture’s protocol, 200 µl plasma samples were processed for this isolation and eluted in 50 μl elution buffer from the extraction column [4].The quantity and quality of nucleic acid were measured using NanoDrop Spectrophotometers and saved for real time-PCR.

EBV load quantification by real time PCR

To estimate the EBV load, Qiagen artus EBV PCR Kits (QS-RGQ Kit) was used to do real-time polymerase chain reaction (RT-PCR). This kit is highly specific, based on the amplification and simultaneous detection of 97 bp, a highly conserved region of the EBNA-1 gene, optimized for diagnostic purposes. The sensitivity of the kit is around 95% [22]. The copy number of EBV DNA was calculated using Rotor-Gene Q Software 2.3.1.49 (Qiagen) using the standard curve based on CT value of the standards provided in the kit. Results were expressed as copies of EBV genome per ml of plasma (EBV DNA copy/ml). We also compared our data by performing real-time quantitative PCR using primer against EBNA-1 as described previously [16]. Similar trends in results were obtained using EBNA-1 PCR as recommended [16, 16, 16]. The analytical sensitivity of the test was determined by the lower limit of detection (LOD) and found 7–10 copies/ml of EBV DNA as described previously [22].

Quantitative estimation of IL-6

To determine the IL-6 concentration in plasma of patients in this cohort as well as healthy controls (n = 5), we have performed sandwich-ELISA by using human IL-6 ELISA kit (Cat. no# E-EL-H0102). The ELISA plate provided in this kit was pre-coated with an antibody specific to human IL-6. Standards or samples were added in the wells of ELISA plate and followed the recommended protocol. Only those wells that contain human IL-6 will bind with biotinylated antibody and Avidin-Horseradish Peroxidase (HRP) conjugate will appear blue in color, which further turns in yellow on addition of stop solution. The optical density (OD) was measured spectrophotometrically at a wavelength of 450 nm. The OD value will be proportional to the concentration of IL-6 present in the sample. The concentration of IL-6 in the sample was calculated by comparing with the standard curve based on the OD value of standards with known concentration.

Quantitative estimation of VEGF

Human Vascular Endothelial Cell Growth Factor A (VEGF-A) ELISA kit (cat.no # E-EL-H0111) has been used to estimate the VEGF level present in the plasma sample of patients in this cohort as well as healthy controls (n = 5). In accordance with the manufacturer’s instructions, protocol has been followed and standard curves were made based on the OD values of the different dilution of the given standard. The concentration of the VEGF present in the sample was calculated by comparing with the standard curve. All the samples were tested in triplicates.

Statistical analysis

The median value with an interquartile range of the plasma EBV DNA copy number in this cohort was calculated. Log transformation was performed and student’s t-test was applied for comparison between patients and control groups. Where more than two groups were involved, comparisons were made using ANOVA and a p value of < 0.05 was considered statistically significant. We defined progression free survival (PFS) as the time between diagnosis and the first event. Overall survival (OS) was defined as the time between diagnosis and death or the last follow-up. The OS and PFS were assessed using Kaplan–Meier survival curves with 95% confidence intervals. The linear relationship between the number of EBV copies and the level of VEGF and IL6 levels was assessed by Pearson correlation coefficient. All analysis was performed using GraphPad Prism version 8.0.0 for Windows, GraphPad Software, San Diego, California USA and Stata 11.2.

Results

Demographic analysis

Baseline characteristics for all the NPC patients involved in this study are summarized in Table 1. Out of 24 patients, 9 patients (37.5%) were female whereas 15 patients were male (62.5%). The mean age was 28.5 (range: 15–62) years and 75% of the patients were younger than 30 years. The most common presenting symptoms were neck swelling followed by nasal obstruction. The mean duration of symptoms was 5 months (range: 1–18 months). Around two-thirds of patients had locally advanced disease (stage IV) at presentation and 75% patients had nonkeratinizing undifferentiated (WHO type III) histology. Out of 24 patients, 22 patients (91.7%) were EBV positive at diagnosis (plasma EBV DNA ˃ 50 copy/ml).

Table 1 Demographic features of patients at baseline (N = 24)
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EBV status in NPC patients VS healthy control

Latent EBV infection is a critical event in NPC tumorigenesis. In patients with NPC, the median EBV load was 1005 (IQR: 172–6327) copy/ml whereas in control, the median EBV DNA load was 7 (IQR 5–10) copy/ml, and the difference was statistically significant (p = 0.004). In addition, baseline plasma IL-6 (17 pg/ml) and VEGF (55 pg/ml) levels were also significantly higher in patients with NPC when compared with healthy controls (Table 2).

Table 2 The EBV status, IL-6 and VEGF expression in control (healthy) VS patients with NPC
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Correlation of EBV load (EBV DNA/ml) with disease burden and treatment

EBV plays an important role in the development of NPC and the EBNA-1 gene is essential for infection and stable persistence of the EBV genomes in the dividing cells for long time. To evaluate the direct relation of EBV load with the severity of disease, the EBNA-1 load was measured at base line and at different time points during treatment i.e., after neo-adjuvant chemotherapy (NACT) and after concurrent chemo-radiotherapy (CRT) in patients diagnosed with NPC. Patients with advanced disease (stage IV) had higher plasma EBV DNA load in comparison to patients with stage II-III NPC at baseline, and after NACT and CRT (Table 3). There was a drastic serial decrease in the plasma EBV DNA titer after the completion of NACT and CRT (Table 3).

Table 3 Correlation of EBV load (EBV DNA/ml) with disease burden and treatment
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Relations between plasma cell free EBV DNA with IL-6 and VEGF expression during therapy in NPC patients

Several studies have revealed EBV as an integral to the NPC pathogenesis and EBV DNA load is strongly associated with favorable prognosis [1, 18]. In this study, we have seen that there is a huge range in copy number of EBV DNA present in patient’s plasma at diagnosis and 91.7% NPC patients were EBV positive (≥ 50 copy/ml) at presentation (Fig. 1A). To assess the impact of plasma EBV load on the expression of IL-6 and VEGF, quantitative estimation of plasma EBV DNA along with IL-6 and VEGF expression were measured on the same set of patients at the same time points. Compared to baseline values, there was a significant (p = 0.0286) decrease in the plasma EBV DNA titer after the completion of NACT (median: 14 copies/ml) and post CRT (median: 0 copy/ml) (Fig. 1A). Similarly, there were significant reductions of plasma IL-6 and VEGF levels after the completion of NACT and CRT (Fig. 1B, C), suggesting a strong correlation in between plasma EBV load with IL-6 and VEGF levels at diagnosis, during and after treatment.

Fig. 1
figure 1

Estimation of cell- free EBV DNA (A), level of IL-6 (B), VEGF expression (C) in the plasma of patients with NPC at baseline, after NACT and after CRT. The horizontal lines represent median values for each group. Note: Post CRT, several patients had zero EBV load and hence could not be plotted in (A)

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Correlations between viral load with IL-6 and VEGF level

Viral infections are known to induce several pro-inflammatory cytokines and growth factors in the blood as an immune response. Serial measurements of plasma EBV DNA levels with inflammatory cytokines IL-6 were performed during the course of therapy. There was a correlation in between cell free EBV DNA in plasma with interleukin-6 (IL-6) level. Patients with high EBV DNA load (≥ 1000 copy/ml) also possessed higher levels of IL-6 (Fig. 2A). To understand the interplay between viruses and growth factor VEGF, also checked the expression of VEGF present in blood plasma of the patients at diagnosis and during the treatment. The scatter plot (Fig. 2B) also showed a strong correlation between EBV infection with VEGF expression (r = 0.7438, p =  < 0.001). Patients having high viral load also had higher VEGF level. In addition, a positive correlation between plasma IL-6 and VEGF levels was ploted (r = 0.5357, p = 0.003 (Fig. 2C). Patients with more plasma IL-6 level were also having enhanced expression of VEGF. Two patients with NPC, who had undetectable plasma EBV load at diagnosis and throughout the treatment, also had IL-6 and VEGF levels within the normal range. A positive correlation has been found in between inflammatory cytokines (IL-6) and growth factor (VEGF) during EBV infection.

Fig. 2
figure 2

Correlation of EBV DNA load with corresponding level of IL-6 (A), VEGF (B) and IL-6 VS VEGF (C) in NPC patients at baseline. The correlation coefficient (r) was calculated and found positive represent direct correlation

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Response assessment and treatment outcome

In patients, the overall response after NACT was 91.7% (complete response found in 8.3% and partial response was in 83.3% patients). After the completion of CRT, the complete and partial response rates were 75% and 16.7% respectively. Four out of 24 patients (16.7%) had progressive disease (distant metastases). The total mortality rate was around 8.3% (Table 4) in this study. The overall survival and progression free survival in this patient’s cohort were estimated by Kaplan–Meier Curve. The overall survival and progression free survival rates were 91.6% and 83.3% respectfully (Fig. 3).

Table 4 Response assessment at different time points after treatment (N = 24)
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Fig. 3
figure 3

Kaplan–Meier curve showing overall survival and progression-free survival in 24 patients with nasopharyngeal carcinoma

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Impact of high EBV DNA load with higher IL-6 and VEGF level on disease progression

Out of 4 patients with progressive disease, serial quantitative measurements of plasma EBV, IL-6 and VEGF were available in 2 patients. In these 2 patients, the baseline plasma EBV DNA load was high, the reduction in the same after NACT was nominal and the increase in the EBV DNA titer at progression was striking (Fig. 4A). Besides, there was a synchronous surge of plasma IL-6 and VEGF levels in both the patients even after NACT and at disease progression (Fig. 4B, C), further proved the positive correlation between plasma EBV DNA and pro-inflammatory cytokines (IL-6) and growth factor (VEGF).

Fig. 4
figure 4

Dynamic changes in cell-free EBV DNA load (A), IL-6 level (B) and the expression of VEGF (C) at baseline and during the treatment in two patients with NPC having progressive disease

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Discussion

Patients with NPC generally show a high titer of IgA antibody against EBV antigens. In many cases, the IgA antibody titer decreases when the tumor responds to radiotherapy and increases at disease progression or recurrence [21]. However, it is not very clearly known why antibody titer changes with the tumor size. It is also not clear why the EBV infected NPC cells proliferate faster in comparison to EBV-negative NPC cells. It has been found that after infection, oncogenic EBV expresses several genes such as EB nuclear antigen-1 (EBNA-1), latent membrane protein (LMP)-1, which can manipulate the signaling pathways related with tumorigenesis in the host cell [9]. We have reported in our previous paper that patients with NPC having circulating cell free EBV DNA load of more than 1500 copies/ml at baseline had a significantly worse outcome[22]. A similar prognostic impact of cell free plasma EBV DNA has also been observed in patients with NK/T cell lymphoma [13]. In this study, 91.7% of the NPC patients were EBV positive at presentation and had significantly higher circulating EBV DNA load (median, 1005 copies/ml: interquartile range, 172–6327 copies/ml) in comparison to healthy controls. There was also a strong association between plasma EBV DNA load with the stage and severity of the disease.

It has been observed that in-vitro EBV infection results in increase of certain growth factor and cytokine level in NPC cells [14]. The role of inflammatory cytokines in cancer pathogenesis is well established [29] but their direct link with EBV-infected premalignant and cancerous nasopharyngeal epithelium is poorly defined. IL-6, a major inflammatory cytokine, is a potent activator of STAT3 and is commonly implicated in the pathogenesis of many human cancers including NPC [32]. In this study, dynamic changes in plasma EBV DNA were assessed and correlated with the expression of IL-6 in patients with NPC during treatment and subsequent follow up. We have noted that patients with higher plasma EBV load also had high IL-6 level, especially at base line. But at the end of the treatment, the IL-6 level was decreased with the reduction of EBV copy number especially in those patients who had responded well. On the contrary, in patients with disease progression, simultaneous increments in the plasma EBV DNA load and IL-6 were observed, suggesting a strong correlation between EBV load (EBNA-1) with IL-6 expression. In the progressed patients, like EBV, IL-6 expression was also high throughout the therapy.

EBNA1 seems to play an additional role in EBV-associated malignancies by indirect enhancement of angiogenesis in NPC [19]. IL-6 has a number of tumor-promoting activities. IL-6 mediated activation of STAT3 possesses regulatory abilities in several pathological processes, especially angiogenesis through the transcription of VEGF [5]. VEGF is a powerful angiogenic factor, and its high levels correlate with that of higher IL-6 expression in several cancers. It has also been reported that IL-6 initiates VEGF action in gastric cancer [6].

It has been reported that viruses can utilize the vascular endothelial growth factor (VEGF) in diseases through different signaling pathways. EBV viral protein EBNA1 activates the transcription factor Hif-1α and AP-1, which enhance the transcription of VEGF. The EBV oncoprotein LMP-1 (Latent membrane protein-1) can also up-regulate the expression of VEGF through the phosphorylation of JNKs/c-Jun signaling pathways. On the other hand, LMP1 can up regulate COX-2 expression which further leads to the up regulation of VEGF expression [2]. Over expression of VEGF has been seen in 67% of NPC cases. Higher expression of VEGF in EBV positive tumors was related to higher rate of recurrence, nodal positivity and lower survival [10].

To establish the relationship between EBV infections with VEGF activation, we have monitored serial plasma VEGF levels at different time points before, during and after treatment. Our study demonstrated that plasma VEGF level was higher in patients with higher EBV DNA load and there was a strong association with IL-6 levels as well. Interestingly, in two patients, who had consistently high IL-6 level and VEGF expression even after treatment, had disease progression (mostly distant metastases).

Four out of 24 patients had progressive disease. Patient no. 18, had 1869 copy/ml EBV DNA at presentation with higher IL-6 (29 pg/ml, normal range: 0.01–10 pg/ml) and VEGF (121 pg/ml, normal range: 27–30 pg/ml) levels. After treatment, there was a transient reduction of the plasma EBV load (649 copy/ml) without any substantial reduction of IL-6 and VEGF levels. On disease progression, the plasma EBV load increased sharply to 257,095 copy/ml with concomitant increase of IL-6 level (87 pg/ml) and VEGF expression (283 pg/ml).Similarly patient no. 19 was presented with high levels of plasma EBV DNA (2845 copy/ml), IL-6 (27 pg/ml) as well as VEGF (98 pg/ml). Upon treatment with neo-adjuvant chemotherapy, there was no significant reduction of the plasma EBV load (2482 copy/ml), IL-6 and VEGF levels. At progression (distant metastases), simultaneous increase in the plasma EBV load (3916 copy/ml), IL-6 and VEGF expression were noted. In patient no. 21, the plasma EBV load was undetectable after the completion of treatment but increased precipitously (143,790 copy/ml) accompanied by increase in plasma IL-6 (68 pg/ml) and VEGF (327 pg/ml) levels 2.5 years after the completion of CTRT, coinciding with recurrence (bone metastasis). One patient with progressive disease died during treatment. Overall, better survival was observed in those patients who had rapid clearance of plasma EBV-DNA after NACT and undetectable EBV DNA after the completion of CRT. Thus, plasma EBV-DNA level before and after treatment and the dynamics of its clearance allows early discrimination between good and poor responders to treatment. Persistent levels or increase of IL-6 and VEGF expression in blood serum/plasma during and after treatment might be useful predictors of progressive disease and distant metastasis.

Since measurement of EBV DNA, IL-6 and VEGF in the plasma is relatively easy, quick, and noninvasive, monitoring their serial levels before, during and after treatment would be helpful as a reliable marker to predict the emergence of recurrent or metastatic disease in patients with NPC. Patients having high plasma EBV DNA load at baseline or detectable load after treatment completion should be monitored more closely and can be selected for more intensive treatment under the banner of a clinical trial.

Limitations

The main limitations of this study include the small sample size (n = 24), retrospective exploratory nature, relative heterogeneity of NACT regimens and lack of correlation of the plasma EBV DNA titer with a comprehensive panel of pro-inflammatory cytokines and putative growth factors. In future larger prospective studies are needed to validate the role of serial plasma EBV load, IL-6 and VEGF expression as a predictive and prognostic biomarker in patients with locally advanced NPC. In addition, multiplex immunoassay would be a better option to explore the whole set of interacting partners with circulating plasma EBV DNA in the context of nasopharyngeal carcinogenesis.

Conclusions

The probable signaling pathways and key players involved in EBV-driven carcinogenesis have not been well characterized yet in NPC. Our study suggests that:

  1. (i)

    Assessment of plasma EBV DNA load in patients with NPC at baseline may be useful for correlation with disease burden and prognostication.

  2. (ii)

    The serial monitoring of plasma EBV DNA, IL-6 and VEGF levels before, during and after treatment would be helpful to predict the response to treatment and future disease progression.

  3. (iii)

    Patients with NPC having persistent EBV load with higher IL-6, and VEGF levels after treatment completion should be monitored closely for disease surveillance and may be offered additional or more intensive treatment.

Availability of data and materials

No datasets were generated or analysed during the current study.

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Acknowledgements

Authors sincerely thank all the patients who participated in this study, clinic personnel and laboratory technicians involved in this project.

Funding

This study was funded by Department of Biotechnology (BT/PR41087/Swdn/135/5/2020), India.

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Authors and Affiliations

  1. Department of Medical Oncology, All India Institute of Medical Sciences, New Delhi, 110029, India

    Sampa Ghose, Surender K. Sharawat & Raja Pramanik

  2. Department of Radiotherapy and Oncology, All India Institute of Medical Sciences, New Delhi, India

    Swarnaditya Roy, Vivek Ghosh & Ahitagni Biswas

  3. Amity Institute of Molecular Medicine and Stem Cell Research, Amity University, Noida, UP, India

    Subhrajit Biswas

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  1. Sampa Ghose
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  2. Swarnaditya Roy
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Contributions

Dr. SG: concept, supervision, methodology, data interpretation, funding acquisition, writing and editing of the manuscript and communication. Dr. SR: clinical data compilation, methodology preparation. Dr. VG: sample collection and clinical data acquisition. Dr. RP: discussion and reviewing of the manuscript. Dr. SKS: data preparation, statistical analysis and editing of the manuscript. Dr. SB: review and editing of the original manuscript. Dr. AB: investigation, treatment plan, review and correction of the manuscript.

Corresponding author

Correspondence to Sampa Ghose.

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Competing interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Patient’s consent

Based on the ethical clearance (approved by the Institute Ethical Board, No. IEC-980/03.10.2020, RP-33/2020), patients consent form was prepared. All the patients recruited in this study were given their consent to participate in this research.

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Ghose, S., Roy, S., Ghosh, V. et al. The plasma EBV DNA load with IL-6 and VEGF levels as predictive and prognostic biomarker in nasopharyngeal carcinoma. Virol J 21, 224 (2024). https://doi.org/10.1186/s12985-024-02473-0

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Virology Journal

ISSN: 1743-422X